Veterinary Mycology

Indranil Samanta
Veterinary
Mycology
Veterinary Mycology
Indranil Samanta
Veterinary Mycology
Indranil Samanta
Department of Veterinary Microbiology
West Bengal University of Animal & Fishery Sciences
Kolkata, West Bengal, India
ISBN 978-81-322-2279-8 ISBN 978-81-322-2280-4 (eBook)

DOI 10.1007/978-81-322-2280-4
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To Baba, Ma, Jhuma, Subhranil for love and support
and
To my beloved students for inspiration
Foreword
Dr. Indranil Samanta is a young scientist who has gotten very important
scientific achievements in his short carrier: Bachelor of Veterinary Sciences
and Animal Husbandry, Master of Veterinary Sciences, Ph.D. in Veterinary
Microbiology, and now he is Assistant Professor in the Microbiology Depart-
ment of West Bengal University of Animal and Fishery Sciences, in India.
He was also Assistant Professor, Division of Microbiology and Immunology,
S. K. University of Agricultural Sciences and Technology of Kashmir, India,
and Veterinary Officer of West Bengal Government. He received four awards
and six research grants. His experience as publisher is wide, he belongs to
the Editorial Board of six journals and acts as reviewer in other four journals,
and he published 22 research articles in international journals. Last year he
published a book entitled Veterinary Bacteriology.
Dr. Samanta kindly invited me to write the foreword of Veterinary
Mycology and it is a great pleasure for me to present this book.
Medical and veterinary mycology have suffered very important
transformations in the last three decades. The introduction of molecular
biology techniques in taxonomy, epidemiology and diagnosis procedures,
not based on cultures, was probably the most significant of them. The
acquisition of more precise knowledge about pathogenesis of fungal
infections was also very important in the management of these diseases.
New antifungal drugs were incorporated to the therapeutic arsenal to fight
against these infections, including new presentations of classic drugs as lipid
formulations of amphotericin B and new compounds as echinocandins and
second generation triazoles. These advances, as well as the increasing mor-
bidity and mortality generated by the mycoses, attracted the attention of a
great number of animal health professionals to veterinary mycology.
Those who are interested in this discipline will find in Veterinary Mycol-
ogy an excellent guide book to increase their knowledge of fungal infections
and their etiologic agents. This book is divided in three main parts: in the first
one the history of veterinary mycology, general aspects of morphology,
taxonomy and biology of fungi are considered. In the second part, the
etiologic agents of superficial, deep, systemic and opportunistic mycoses
are described with great detail. Biological aspects of these fungi, epidemiol-
ogy of these infections, the immunity response of the hosts and the modern
diagnosis techniques such as those searching for fungal antigens in organic
vii
viii Foreword
fluid and those which applied molecular biology are extensively exposed.
Clinical manifestations and therapy of the mycoses are presented in a more
synthetic way, using tables containing comprehensive information. The third
part is related to laboratory diagnosis including clinical samples collection
and their processing for fungal isolation, special stains for fungal micro-
scopic visualization and culture media composition. There are also special
chapters about very infrequent fungal ‘infection’ and a glossary.
This book is written in concise and clear English, very easy to read. I think
the readers will enjoy it very much.
Buenos Aires University School of Medicine Ricardo Negroni

Buenos Aires, Argentina
Hospital F.J. Muñiz
Buenos Aires City, Argentina
Preface
The study of fungi began during ancient times. Even in early Sanskrit
literature (Atharva Veda), scripts of Hippocrates and Lord Buddha and in
the Holy Bible, the importance of mycology was mentioned for prevention of
fungal diseases. Although it was little neglected both in medical and veteri-
nary sciences, studying mycology is gaining importance in recent times due
to emergence and re-emergence of fungal infection in human, animals and
birds. Emergence of black yeasts in poultry, Prototheca in pets, Laczia loboi
in human and marine animals, Lagenidium in pets, Emmonsia in human and
pets as well as re-emergence of brooder pneumonia in poultry, candidiasis in
human and animals, cryptococcosis in human and animals, and
dermatophytosis in animals is noted in recent times. The fungal infection
causes major economic loss in poultry and livestock related industry and it
poses zoonotic threat especially to the pet owners. Advancement of knowl-
edge helps in better understanding of the subject. Cumulation of the
advancements along with the conventional knowhow in the area of veterinary
mycology within the same cover was one of my best intentions. I have tried
to restructure the classification, genome characteristics, pathogenesis, immu-
nity, diagnosis and treatment of fungal diseases with the enlightened vision
of molecular biology and discovery of new antifungals. I hope the informa-
tion will be useful as reference text for undergraduate students, text for post-
graduate students, veterinary practitioners and in allied sectors. Any correc-
tion, modification and suggestion for improvement are most welcome by
the author.
During this auspicious occasion, I heartily acknowledge the mycology
faculty members and scientists throughout the world who are stalwarts in this
subject for evaluation of my chapters and their contribution of fungal
photographs. I offer my sincere thanks to Professor Dr. Ricardo Negroni
for his encouraging words. I also acknowledge friends and senior colleagues
of my department and university for their valuable suggestions. All the
schematic diagrams drawn by my wife Jhuma are duly accredited.
Kolkata, West Bengal, India Dr. Indranil Samanta

19 November 2014
ix
Contents
1 History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1 Medical Mycology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Veterinary Mycology . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2 General Characteristics of Fungi . . . . . . . . . . . . . . . . . . . . . . . 3
2.1 Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.1.1 Types of Hyphae . . . . . . . . . . . . . . . . . . . . . . . 4
2.1.2 Fungal Cell Structure . . . . . . . . . . . . . . . . . . . . 4
2.2 Nutrition and Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.3 Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3 Classification of Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
4 Cutaneous, Subcutaneous and Systemic Mycology . . . . . . . . . 11
4.1 Trichophyton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
4.1.1 Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
4.1.2 Classification . . . . . . . . . . . . . . . . . . . . . . . . . . 13
4.1.3 Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . 14
4.1.4 Susceptibility to Disinfectants . . . . . . . . . . . . . . 14
4.1.5 Natural Habitat and Distribution . . . . . . . . . . . . 14
4.1.6 Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
4.1.7 Isolation, Growth and Colony
Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . 15
4.1.8 Antigenic Characteristics . . . . . . . . . . . . . . . . . . 15
4.1.9 Virulence Factors . . . . . . . . . . . . . . . . . . . . . . . 16
4.1.10 Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . 16
4.1.11 Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
4.1.12 Disease Produced . . . . . . . . . . . . . . . . . . . . . . . 17
4.1.13 Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
4.1.14 Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
4.1.15 Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
4.1.16 Vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
4.2 Microsporum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
4.2.1 Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
4.2.2 Classification . . . . . . . . . . . . . . . . . . . . . . . . . . 22
4.2.3 Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . 23
xi
xii Contents
4.2.6 Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
4.2.10 Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . 25
4.2.11 Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
4.2.13 Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
4.2.14 Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
4.2.15 Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
4.3 Epidermophyton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
4.3.1 Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
4.3.2 Classification . . . . . . . . . . . . . . . . . . . . . . . . . . 29
4.3.5 Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
4.3.9 Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . 31
4.3.10 Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
4.3.12 Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
4.3.13 Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
4.4 Aspergillus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
4.4.1 Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
4.4.2 Classification . . . . . . . . . . . . . . . . . . . . . . . . . . 34
4.4.5 Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
4.4.8 Toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
4.4.10 Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . 37
4.4.11 Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
4.4.13 Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
4.4.14 Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
4.4.15 Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
4.5 Blastomyces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
4.5.1 Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
4.5.2 Classification . . . . . . . . . . . . . . . . . . . . . . . . . . 45
4.5.3 Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Contents xiii

4.5.6 Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
4.5.10 Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . 46
4.5.11 Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
4.5.13 Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
4.5.14 Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
4.5.15 Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
4.6 Coccidioides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
4.6.1 Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
4.6.2 Classification . . . . . . . . . . . . . . . . . . . . . . . . . . 51
4.6.3 Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . 51
4.6.6 Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
4.6.10 Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . 54
4.6.11 Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
4.6.13 Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
4.6.14 Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
4.6.15 Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
4.7 Histoplasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
4.7.1 Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
4.7.2 Classification . . . . . . . . . . . . . . . . . . . . . . . . . . 60
4.7.4 Natural Habitat . . . . . . . . . . . . . . . . . . . . . . . . . 61
4.7.5 Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
4.7.9 Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . 62
4.7.10 Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
4.7.12 Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
4.7.13 Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
4.7.14 Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
4.8 Rhinosporidium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
4.8.1 Morphology and Life Cycle . . . . . . . . . . . . . . . . 68
4.8.2 Classification . . . . . . . . . . . . . . . . . . . . . . . . . . 69
xiv Contents

4.8.5 Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
4.8.8 Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . 71
4.8.9 Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
4.8.11 Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
4.8.12 Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
4.8.13 Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
4.9 Rhizopus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
4.9.1 Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
4.9.2 Classification . . . . . . . . . . . . . . . . . . . . . . . . . . 74
4.9.3 Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . 74
4.9.6 Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
4.9.10 Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . 76
4.9.11 Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
4.9.13 Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
4.9.14 Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
4.9.15 Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
4.10 Mucor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
4.10.1 Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
4.10.2 Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . 80
4.10.3 Classification . . . . . . . . . . . . . . . . . . . . . . . . . . 80
4.10.6 Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
4.10.10 Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . 81
4.10.11 Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
4.10.13 Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
4.10.14 Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
4.10.15 Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Contents xv
4.11 Penicillium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
4.11.1 Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
4.11.2 Life Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
4.11.3 Classification . . . . . . . . . . . . . . . . . . . . . . . . . . 85
4.11.4 Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . 85
4.11.6 Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
4.11.9 Toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
4.11.11 Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . 88
4.11.12 Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
4.11.14 Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
4.11.15 Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
4.11.16 Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
4.12 Cryptococcus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
4.12.1 Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
4.12.2 Classification . . . . . . . . . . . . . . . . . . . . . . . . . . 92
4.12.3 Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . 93
4.12.6 Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
4.12.8 Biochemical Characteristics . . . . . . . . . . . . . . . . 95
4.12.11 Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . 96
4.12.12 Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
4.12.14 Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
4.12.15 Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
4.12.16 Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
4.13 Candida . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
4.13.1 Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
4.13.2 Classification . . . . . . . . . . . . . . . . . . . . . . . . . . 104
4.13.3 Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . 104
4.13.6 Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
4.13.8 Biochemical Characteristics . . . . . . . . . . . . . . . . 106
xvi Contents

4.13.11 Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . 106
4.13.12 Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
4.13.14 Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
4.13.15 Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
4.13.16 Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
4.14 Sporothrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
4.14.1 Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
4.14.2 Classification . . . . . . . . . . . . . . . . . . . . . . . . . . 114
4.14.3 Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . 114
4.14.6 Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
4.14.10 Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . 115
4.14.11 Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
4.14.13 Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
4.14.14 Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
4.14.15 Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
4.15 Mycetoma (Madurella, Pseudallescheria,
Scedosporium) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
4.15.1 Aetiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
4.15.2 Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
4.15.3 Classification . . . . . . . . . . . . . . . . . . . . . . . . . . 122
4.15.6 Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
4.15.10 Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . 125
4.15.11 Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
4.15.12 Disease Characteristics . . . . . . . . . . . . . . . . . . . 125
4.15.13 Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
4.15.14 Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
4.15.15 Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
4.16 Pythium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
4.16.1 Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
4.16.2 Life Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
4.16.3 Classification . . . . . . . . . . . . . . . . . . . . . . . . . . 128
4.16.4 Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . 128
Contents xvii

4.16.7 Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
4.16.11 Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . 129
4.16.12 Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
4.16.14 Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
4.16.15 Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
4.16.16 Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
4.17 Emerging and Uncommon Pathogenic Fungi . . . . . . . . . . . 132
4.17.1 Phaeohyphomycosis . . . . . . . . . . . . . . . . . . . . . 132
4.17.2 Pneumocystis . . . . . . . . . . . . . . . . . . . . . . . . . . 135
4.17.3 Prototheca . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
4.17.4 Lobomycosis . . . . . . . . . . . . . . . . . . . . . . . . . . 140
4.17.5 Lagenidium . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
4.17.6 Basidiobolus and Conidiobolus . . . . . . . . . . . . . 142
4.17.7 Adiaspiromycosis
(Adiaspirosis, Haplomycosis) . . . . . . . . . . . . . . 144
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
5 Collection and Transport of Clinical Material
for Isolation of Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
5.1 Transport of Clinical Materials . . . . . . . . . . . . . . . . . . . . . 157
6 Diagnostic Techniques for Fungi . . . . . . . . . . . . . . . . . . . . . . . 159
Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
Composition of Commonly Used Mounting Fluids/Stains . . . . . . 165
Composition of Commonly Used Media in Diagnostic
Mycology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
About the Author
Dr. Indranil Samanta obtained his Bachelor of Veterinary Sciences and

Animal Husbandry degree (B.V.Sc. and A.H.) from West Bengal University
of Animal and Fishery Sciences, Kolkata, India. He secured his Masters in
Veterinary Sciences (M.V.Sc.) in Veterinary Bacteriology and Mycology
from Indian Veterinary Research Institute, Bareilly, UP, India, and Doctor
of Philosophy (Ph.D.) in Veterinary Microbiology from West Bengal Uni-
versity of Animal and Fishery Sciences, Kolkata, India. He works currently
as Assistant Professor of Veterinary Microbiology in West Bengal University
of Animal and Fishery Sciences, Kolkata, India. Previously he has also
worked as Assistant Professor of Veterinary Microbiology in S. K. Univer-
sity of Agricultural Sciences and Technology-Kashmir, India, and as Veteri-
nary Officer, Government of West Bengal, India. He is actively engaged in
teaching of undergraduate, post-graduate and Ph.D. scholars of Veterinary
Microbiology and research related with animal health and zoonotically
important microbes. He has received six grants from national funding
agencies and he has supervised three post-graduate scholars till date. He
has published 70 research articles in reputed international and national
journals along with review articles in international journals. His current
total impact factor, h-index and total citations are 45, 10 and 280, respec-
tively. He has published a textbook entitled Veterinary Bacteriology
(ISBN13: 9789381450550, ISBN 10: 9381450552) from a reputed publisher.
He is editorial board member and reviewer of international and national
journals. He has delivered several talks in conferences, Government televi-
sion and radio channels regarding his research and animal, poultry health
related issues. He is the recipient of National Academy of Agricultural
Sciences (NAAS) Associate, Government of India.
xix
History
1
1.1 Medical Mycology of rice and sugarcane which can decrease their
production. In the Bible (Amos 4, 19)
The term ‘mycology’ was coined by instructions were provided to the priests for treat-
H.S. Berkley (1834) as a study of fungi. Medical ment of fungal infection such as dry rot.
or veterinary mycology is the study of medically The first description of dermatophytosis was
or veterinary important fungi and fungal diseases recorded by Celsus, a Roman encyclopaedist
in human and animals, respectively. The term who described a suppurative infection of the
‘mycosis’ (mykes ¼ mushroom) is used to scalp (‘porrigo’ or ‘kerion of Celsus’) in De Re
describe the infection of human, animal, birds Medicina (30 AD). Throughout the middle ages
and plants which is caused by numerous patho- several descriptions of dermatophytosis are pro-
genic fungi. The ‘mycotoxicosis’ describes the duced where it was described as ‘tinea’ (Latin
diseased condition produced by the ingestion of term). Micheli (1729) published Nova genera
mycotoxins (intoxication) present in the feed. Plantarum (written in copper plate) in which he
When the fungi produce the pathogenesis due to established several genera of fungi such as
in vivo toxin production after entry within the Aspergillus, Mucor, etc. However, the patho-
host, it is known as ‘mycetism’. genic potentiality of fungi in human or animals
In ancient Sanskrit writing of India (Atharva remained uncertain.
Veda, 2000–1000 BC), the first description of a Robert Hook (1665) first illustrated the patho-
fungal infection, i.e. mycetoma (Pada genic role of rose rust (Phragmidium
Valmikan), was documented. In seventeenth cen- mucronatum) in his book ‘Micrographia’. In
tury, a German physician (Engelbert Kaempfer) 1835, Agostino Bassi, an Italian lawyer and
working in India first reported clinical human farmer, first reported a fungal infection
cases of mycetoma which was followed by case (muscardine) of silkworm and illustrated that a
reports of French missionaries in Pondicherry, microbe can cause an infection. Robert Remak
India (ant-hill of worms, 1714). The fungal (1837–1841), a Polish physician, described the
plant diseases (smut, rust) were noted in other first human mycosis (tinea favosa). Gruby (1844)
Veda (1200 BC) also. In Rigveda crushing of first described the aetiologic agent of tinea
mushroom (an edible fungus) with the feet was endothrix, later became known as Trichophyton
illustrated as a punishment. Hippocrates tonsurans. The work of Remak and Gruby
(460–377 BC) first documented oral established the mycology as a separate branch
pseudomembranous candidiasis and he described of medical science. In 1892, Alejandro Posadas,
it with the name of ‘aphthae albae’ which was a medical student, and Robert Wernicke, a
later supported by Galen (130–200 BC). Lord pathologist, first described Coccidioides from a
Buddha (400 BC) observed the fungal diseases soldier with recurrent skin tumours in Argentina.
I. Samanta, Veterinary Mycology, DOI 10.1007/978-81-322-2280-4_1, 1

2 1 History
The histoplasmosis in human was first notified by Edwin John Butler (1901) started the systemic
Darling (1906), an American pathologist who study of fungi (Pythium, Phytopthora) and he is
observed it during an autopsy of a Martinique known as the ‘Father of Indian mycology’.
native person who died with tuberculosis-like
syndrome in Panama. Raymond Jacques Adrien
Sabouraud (France) compiled the description of 1.2 Veterinary Mycology
dermatophytes (Trichophyton) in his book Les
Teignes (1910) which was based on his observa- In 1749, Reaumur first described avian aspergil-
tion in artificial culture. This authentic book losis in birds which was followed by the report of
initiated the development of medical mycology. similar syndrome in a duck (Montagu, 1813).
Gomori (1946) first developed a stain for the Aspergillus fumigatus was first detected in the
microscopic observation of fungal cells in tissue lung of a great bustard (Otis tardaga) in 1863
which was later modified by Grocott in 1955. by Fresenius. He was also the first to use the term
Kligman (1951) used the periodic acid Schiff ‘aspergillosis’ for this respiratory disease.
stain for histological demonstration of fungi. The causative agent of Epizootic
Gridley (1953) later modified PAS stain by lymphangitis in horses (Histoplasma capsulatum
replacing periodic acid with chromic acid var. farciminosum) was first demonstrated in the
which can reveal both hyphae and yeast cells pus by Rivolta (1873). However, it was success-
in tissues. fully isolated in 1896 by Tokishiga in Japan.
In India, Lt. Col. Kirtikar (1885) established Smith (1884), a Veterinarian working in India,
the mycology with his collection and identifica- first described a chronic cutaneous granuloma-
tion of mushrooms from West Bengal. tous disease in horses known as ‘bursattee’ in
Cunningham (1872) exposed the grease covered local Indian language. Later the aetiology of the
slides to the air and collected spores of Rhizopus disease was correlated with the fungi (Pythium)
and rusts along with the cholera bacteria. Sir based on histology.
General Characteristics of Fungi
2
2.1 Morphology like filament called ‘pseudohyphae’. The examples

of pathogenic yeasts are Cryptococcus neoformans
The fungi are eukaryotic, heterogeneous, unicellular and Candida albicans. They are currently not
to filamentous, spore bearing, and chemoorga- considered as true yeast because they can exist in
notrophic organisms which lack chlorophyll. The other forms such as pseudohyphae and hyphae
fungi have three major morphological forms, (mycelial).
i.e. unicellular yeast, filamentous mould (mold) Mould: The whole body of a mould is called a
and yeast-like form (pseudohyphae form). The thallus (plural, thalli). The thallus is structurally
dimorphic fungi (Blastomyces dermatitidis, divided into two major parts, i.e. vegetative
Coccidioides immitis, Histoplasma, Sporothrix (mycelium) and reproductive portion (spores).
schenckii) are able to produce both the forms (yeast The mycelium is composed of filaments with or
and mould) depending on the temperature (thermal without branching known as hypha (plural,
dimorphism). The yeast form is produced within the hyphae). Each hypha (5–10 μm wide) is a tube-
body of the host (in vitro at 37 C) and the mould like structure containing a lumen surrounded
form is observed either in the environment or in externally by a rigid cell wall. The individual
artificial culture medium (at room temperature). hyphae are intertwined to make a mycelium.
The pseudohyphae form is chains of elongated ellip- Any part of the mycelium can absorb food. How-
soidal cells with constriction between them and it is ever, a specialised root-like structure (rhizoid)
produced by Candida albicans. attaches the substrate to absorb the food in
Yeast: Yeasts are unicellular, microscopic but some group of fungi (Zygomycetes).
larger (1–5 μm 5–30 μm) than most of the bac- The content of the hyphal lumen is proto-
teria (0.5–1 μm). They are pleomorphic and show plasm. A bilayer membrane (plasma membrane
spherical, elliptical and elongated forms. In artifi- or plasmalemma) separates the protoplasm from
cial culture media, the yeasts produce bacteria-like the outer cell wall. The elongation of the hypha
colonies which are moist or mucoid. The yeasts takes place near the tip (‘apical extension’) by
reproduce by budding. Both in the yeast and transverse cross wall formation from the existing
pseudohyphae form, the nuclear division and cell wall. The cross wall grows inward to form a
septa formation take place near the bud. The buds septum. A central pore is present in the septum
can elongate and are released as blastospores. which allows the movement of protoplasm along
Sometimes the buds elongate but fail to detach with the nuclei between the cells (protoplasmic
and they produce a chain of elongated hyphae- streaming).

4 2 General Characteristics of Fungi
2.1.1 Types of Hyphae
The following hyphal types are observed:

(a) Vegetative hyphae: They penetrate the
artificial medium to absorb the nutrients.
(b) Aerial hyphae: They grow above the surface
of the artificial medium.
(c) Reproductive (fertile) hyphae: They are
aerial hyphae carrying the reproductive
structure (spore).
(d) Coenocytic hyphae: Non-septate hyphae
which allow uninterrupted flow of proto-
plasm and nuclei through the lumen,
e.g. Phycomycetes.
(e) Septate with uninucleated cells.
(f) Septate with multinucleated cells (Fig. 2.1).
Several fungi (dermatophytes) produce
hyphae with specific appearance which helps in
their identification. The examples are mentioned
below:
(a) Spiral hyphae: Spirally coiled hyphae
(Trichophyton mentagrophytes)
(b) Pectinate body: Short, unilateral projections
from the hyphae resembling teeth of a comb
(Microsporum audouinii)
(c) Favic chandeliers (antler hyphae): Irregular
projections of the hyphae that collectively
resemble a chandelier or the antler of a deer
(Trichophyton schoenleinii, T. violaceum)
(d) Nodular body: Closely twisted hyphae resem-
bling a nodule (Trichophyton mentagrophytes,
Microsporum canis)
(e) Racquet hyphae: Chain of elongated hyphal
cells expanded at one end to produce a tennis
racquet-like arrangement (Epidermophyton
floccosum, Trichophyton mentagrophytes)
(Fig. 2.2)
2.1.2 Fungal Cell Structure

Fig. 2.1 Types of fungal hyphae; (a) coenocytic hyphae;
(b) septate hyphae; (c) septate, multinucleate hyphae of
Cell wall: All the morphological forms (yeast and Penicillium (Photograph courtesy: Prof. Sybren de Hoog
hyphae) of fungi are surrounded by a rigid cell and Dr. Marco van den Berg)
wall. The major component of the cell wall is
chitinous fibrils embedded in a matrix of a (β1, 4) linked polymer of N acetyl-D-glucos-
polysaccharides, proteins (acid phosphatase, amine (GlcNAc).It is synthesised by chitin synthe-
α-amylase, and protease), lipids and inorganic tase present in the chitosome (cell organelle). The
salts (calcium, magnesium, phosphorus). Chitin is matrix contains glucan (D-glucose polymer),
2.2 Nutrition and Growth 5
Fig. 2.2 Types of dermatophyte hyphae (schematic); a, spiral; b, pectinate body; c, favic chandelier; d, nodular organ;
e, racquet hyphae
mannan, chitosan (polymer of glucosamine) and also present in the cytoplasm. The microtubules
galactans. Among the glucans, polymers with are composed of tubulin protein and they help in
(β1, 3) and (β1,6) linked glucosyl units, known as the movement of chromosomes (during mitosis or
β glucan, is common. The β glucans are potent meiosis), nuclei, golgi vesicles, vacuoles and
immunomodulator and are used in poultry mitochondria. Disruption of tubulin synthesis by
industry. antifungal (griseofulvin) can prevent the fungal
In yeast form of dimorphic fungi (Histoplasma, mitosis. The cytoplasm contains membrane-
Blastomyces dermatitidis), α glucan makes the bound nucleus composed of chromatin and nucle-
cell layer, whereas the β glucan predominantly olus. The chromatin is composed of DNA and
make the cell wall of mycelium. The presence of associated proteins. The number, shape and size
α glucan is associated with virulence of the yeast of nuclei vary between the fungi. Majority of
cells than the mycelial form. Another major con- genetic material (80 %) is present in the chromatin
stituent of yeast cell wall is peptidomannan (man- and the rest (20 %) is associated with mitochon-
nan, galactomannans, rhamnomannans) which drial genome (Fig. 2.3).
helps in serological identification of the fungi.
The capsule is observed in Cryptococcus like
prokaryotes. The capsule consists primarily of two 2.2 Nutrition and Growth
polysaccharides, glucuronoxylomannan (GXM)
and glucuronoxylomannogalactan (GXMGal), The fungi are chemoorganoheterotrophic
along with smaller amounts of mannoproteins. It organisms. They use chemical compounds as a
is antiphagocytic and it protects the yeast cells. source of energy and organic compounds as elec-
Plasma membrane: The plasma membrane is tron and carbon source. They obtain their nutrition
present beneath the cell wall. Like other by absorption (osmotrophic) either from the envi-
mammalian cells, it is a phospholipid and ronment (saprophyte) or the host (parasite). Most
sphingolipid bilayer in which the proteins of the saprophytic moulds grow aerobically in
(peripheral and integral) are interspersed. The artificial culture medium at 20–30 C. The patho-
hydrophilic heads of phospholipids are towards genic yeasts and yeast phase of dimorphic fungi
the surface, and the hydrophobic tails are present prefer to grow at 37 C. High humidity, acidic pH
in the interior of the membrane. Major sterol of (3.8–5.6), high sugar concentration (4–5 %), car-
fungal plasma membrane is ergosterol. In con- bon, phosphorus, sulphur and traces of potassium,
trast, mammalian plasma membrane contains magnesium, iron and calcium are required for
cholesterol. The antifungals act on ergosterol optimum fungal growth. The peptone in the
(polyene) or biosynthetic pathway of ergosterol media and keratin in the skin act as a nitrogen
(imidazole, triazole) to inhibit the fungal growth. source. The nitrogen is required for synthesis of
Cytoplasm: The cell cytoplasm contains amino acids for building proteins, purines and
nucleus and organelles such as mitochondria, pyrimidines for nucleic acids, glucosamine for
endoplasmic reticulum, golgi vesicles, lysosomes, chitin and various vitamins. Most of the fungi use
vacuoles, etc. Long, hollow cylindrical structures nitrogen as nitrate which is reduced to nitrite and
(25 nm in diameter), known as microtubules, are further to ammonia. None of them can directly fix
Fig. 2.3 Fungal cell wall

(schematic); a, protein; b,
glucan; c, cytoplasmic
membrane
Table 2.1 Comparative growth requirements of bacteria and fungi

Characteristics Bacteria Fungi
Temperature 37 C 20–30 C (saprophyte)/37 C (parasite)
pH 6–7 3.8–5.6
Oxygen Aerobic, anaerobic Strictly aerobic
Sugar 0.5–1 % 4–5 %
Humidity Low High
Carbon Organic or inorganic Organic
Nitrogen Direct fixing by some bacteria (Rhizobacter) No direct fixing
Lysine synthesis meso-α,ε-diaminopimelic acid (DAP pathway) L-α-adipic acid (AAA pathway)
Growth rate Fast Slow
nitrogen. The growth is not dependent on light and reproduction of fungi produces spores which
ultraviolet and X-ray are mild inhibitory. The are considered as dispersal and survival unit of
growth rate of fungi is slower than bacteria and fungi without an embryo. The spores separate
the medium is easily contaminated with bacteria. from their mother fungi and develop into an
Antibiotics (e.g. chloramphenicol) and antifungal individual progeny.
(e.g. cycloheximide) are added in the media to In asexual reproduction, union of sex organ or
prevent the bacterial and saprophytic fungi con- nuclei does not occur. Instead, the following
tamination. The cycloheximide is inhibitory processes are observed:
against the growth of certain pathogenic fungi 1. Fission of mother cell: It produces two similar
and yeasts such as Aspergillus fumigatus, Candida daughter cells.
albicans and Cryptococcus. 2. Budding: The budding has three steps:
Inoculation of culture medium is done by a (i) Bud emergence: The outer cell wall of the
transfer needle with flattened tip which helps in parent cell thins and new inner cell wall
cutting of mycelium. The bacteriological inocu- and plasma membrane are synthesised at
lation loop is sufficient for inoculation of yeasts the site of bud emergence. The bud emer-
(Table 2.1). gence is regulated by the activation of the
polysaccharide synthetase (zymogen) for
the synthesis of new cell wall and turgor
2.3 Reproduction pressure of the parent cell.
(ii) Bud growth: Mitosis occurs and the
The fungi reproduce by two major ways, conidium (spore) possesses the daughter
i.e. asexual and sexual reproduction. The nucleus.
2.3 Reproduction 7
(iii) Conidium separation: The septum (c) Arthrospores (oidia): Disjoining of hyphal
between the developing conidium and cells produces single-celled arthrospores.
its parent cell is formed from a chitin (d) Chlamydospores: Thick-walled, single-
ring. The septum helps in the separation celled spores produced by aerial hyphae and
of conidia which leaves a bud scar on the they are highly resistant to adverse environ-
parent cell wall. Sometimes the conidia ment. Some fungi (Histoplasma) produce
are not separated and released from the chlamydospores with small spine-like
parent cells. Consequently, pseudohypha projections in their wall (tuberculate).
consisting of a filament of attached (e) Blastoconidia: The spores produced by bud-
conidia is produced (Fig. 2.4). ding are known as blastoconidia (Fig. 2.5).
Sexual reproduction occurs by the union of
3. Fragmentation of hyphae. two compatible thalli (homothallic or heterothal-
4. Spore formation. lic).In heterothallic fungi the male and female
Many kinds of fungal asexual spores are mating types exist in nature, designated as
detected. A and a (or + and ). The homothallic fungi do
(a) Sporangiospores: A specialised reproductive not require separate mating types. The sex organ-
hypha (sporangiophore) bears a sac-like struc- elle of fungi is called gametangium. Male and
ture known as sporangium. Each of the spo- female gametangia are known as ‘antheridium’
rangium contains numerous single-celled and ‘oogonium’, respectively. The union of com-
sporangiospores. Non-motile sporangiospores patible thalli is followed by fusion of gametangia
are called ‘aplanospore’ and motile (plasmogamy). In some fungi, ‘trichogyne’
sporangiospores are called ‘zoospore’. (a specialised hyphae) is present surrounding
(b) Conidia: Conidia are small asexual spores the female gametangium and it receives
attached directly with reproductive hyphae the male gamete. The male gamete is uninucleate
(conidiophore). Small single-celled spores spermatium (microconidium) or multinucleate
are called microconidia and large multicellu- macroconidium, formed either directly on
lar spores are called macroconidia. the male mycelium (+ type) or in a specialised
structure called ‘spermogonia’. The trichogyne
recruits a fertilising nucleus from the male gam-
ete which enters the female gametangium and
fusion of two haploid nuclei (karyogamy) occurs.
The karyogamy produces a diploid nucleus
which undergoes meiosis to reduce the chromo-
some number into haploid again. Some of the
progeny nuclei degenerate and in some fungal
Fig. 2.4 Steps of yeast budding (schematic); a, bud species post-meiotic mitosis occurs. Sexual
emergence; b, bud growth; c, conidium separation reproduction is typically controlled by genes
Fig. 2.5 Types of fungal

asexual spores (schematic);
a, sporangiospore; b,
conidia; c, arthrospores; d,
chlamydospores; e,
blastospores
Fig. 2.6 Types of fungal

sexual spores (schematic);
a, ascospore; b, ascus; c,
basidiospore; d, basidium;
e, zygospore; f,
gametangium; g, oospore;
h, oogonium; i,
antheridium
that reside in the mating-type locus. The sexual the female gametes (oospheres) to produce
spores occur less frequently and in smaller num- oospores (Fig. 2.6).
bers than the asexual spores. Different types of The sexual and asexual spores are protected
sexual spores are described below. from the environment by a highly organised
(a) Ascospores: These meiospores are single structure, known as ‘fruiting body’ or ‘sporo-
celled and are produced within a sac-like carp’. The fruiting bodies for sexual spores
structure, known as ascus (meiosporangium). include cleistothecia, perithecia, apothecia and
Each ascus contains eight ascospores. pseudothecia. The cleistothecium is a completely
(b) Basidiospores: These spores are also like closed fruiting body. In perithecium, a pore (osti-
ascospores but are produced within a club- ole) is present through which the spores can
shaped structure known as basidium. escape. The apothecium is a cup or saucer-like
(c) Zygospores: Zygospores are produced by the fruiting body. In addition to spores, some fruiting
fusion of two compatible thalli or their bodies contain sterile hyphae.
gametangia. The fruiting bodies such as pycnidium and
(d) Oospores: These special sexual spores are acervulus can protect asexual spores. Pycnidium
produced within female gametangium (oogo- is a spherical structure with a pore (ostiole) at the
nium). After fusion of two compatible top and it is arranged as separate or aggregate.
hyphae, the male gametes (produced in The acervulus is an open, saucer-shaped asexual
antheridium) enter the oogonium and fertilise fruiting body.
Classification of Fungi
3
The classification of fungi relies mostly on mor- proposal for classification of fungi is published
phological criteria such as the pigmentation, in official journal of International Association for
shape of hyphae, presence or absence of septa Plant Taxonomy (Taxon) and is discussed in
and types of spores. The taxonomy of mould annual meeting before acceptance. The classifi-
and yeasts is governed by International Code of cation of clinically relevant fungi is described in
Botanical Nomenclature (ICBN). Any new Table 3.1.

Table 3.1 Classification of clinically relevant fungi
Kingdom Phylum Class Order Family Genus Species
10
Fungi Zygomycota Zygomycetes Entomophthorales Basidiobolaceae Basidiobolus Basidiobolus ranarum

(previously Ancylistaceae Conidiobolus Conidiobolus coronatus
Phycomycetes)
Basidiomycota Tremellomycetes Tremellales Tremellaceae Cryptococcus Cryptococcus neoformans var.
neoformans, C. neoformans var.
grubii, C. gattii
Ascomycota Euascomycetes Onygenales Ajellomycetaceae Blastomyces Blastomyces dermatitidis
Onygenaceae Histoplasma Histoplasma capsulatum
Coccidioides Coccidioides immitis,
C. posadasii
Emmonsia E. crescens, E. parva
Arthrodermataceae Trichophyton T. tonsurans, T. equinum,
T. interdigitale
Microsporum M. audouinii, M. canis,
M. gypseum-fulvum complex
Epidermophyton E. floccosum, E. stockdaleae
Chrysosporium
Eurotiomycetes Microascales Microascaceae Aspergillus A. fumigatus, A. flavus, A. terreus,
A. niger, A. nidulans, A. ustus
Eurotiales Trichocomaceae Penicillium P. marneffei
Pyrenomycetes Ophiostomatales Ophiostomataceae Sporothrix S. schenckii
Saccharomycetes Saccharomycetales Saccharomycetaceae Candida C. albicans, C. tropicalis,
(Hemiascomycetes) C. glabrata
Dothideomycetes Capnodiales Davidiellaceae Cladosporium C. cladosporioides
Pleosporales Pleosporaceae Alternaria A. alternate
Bipolaris B. spicifera
Eurotiomycetes Chaetothyriales Herpotrichiellaceae Cladophialophora, C. bantiana, E. attenuate,
Exophiala, Fonsecaea, F. multimorphosa, P. verrucosa
Phialophora
Ochroconiales Ochroconis O. gallopava
Archiascomycetes Pneumocystidales Pneumocystidaceae Pneumocystis P. carinii
Mucoromycotina (subphylum) Mucorales Mucoraceae Rhizopus, Absidia, Rhizopus arrhizus (R. oryzae),
(incertae sedis, not assigned to Mucor Mucor circinelloides complex
any phylum)
Stramenopila Oomycota Oomycetes Pythiales Pythiaceae Pythium P. insidiosum
3 Classification of Fungi
(Chromista) Lagenidium L. giganteum

Cutaneous, Subcutaneous
and Systemic Mycology 4
4.1 Trichophyton named Achorion schoenleinii (Trichophyton

schoenleinii) in honour of his mentor. In 1844,
The first description of dermatophytosis was Gruby described the etiologic agent of tinea
recorded by Celsus, a Roman encyclopaedist endothrix, later became known as Trichophyton
who described a suppurative infection of scalp tonsurans. The genus Trichophyton was created
(‘porrigo’ or ‘kerion of Celsus’) in De Re and described by Malmsten (1845) with its
Medicina (30 A.D.). Throughout the middle representative species T. tonsurans. Charles
ages, several descriptions of dermatophytosis Robin identified T. mentagrophytes in 1847 and
were produced where it is described as ‘tinea’. T. equinum was identified by Matruchot and
The keratin-destroying moths which made circu- Dassonville in 1898. Raymond Jacques Adrien
lar holes in the woollen garments are known as Sabouraud (France) first compiled the descrip-
Tinea. Due to similarity in the structure of circu- tion of Trichophyton in his book (Les Teignes)
lar lesion of dermatophytosis on the smooth skin in 1910 which was based on his observation in
with the circular hole made by moth, Cassius artificial culture. The sexual state of dermato-
Felix introduced the term ‘tinea’ to describe the phyte was described by Nannizzi (1927).
lesions. In 1806, Alibert used the term ‘favus’ to Emmons (1934) first reported the classification
describe the honey-like exudate in some scalp of dermatophytes based on vegetative structures
infections. However, the fungal aetiology of and conidia. Gentles (1958) established the suc-
tinea was first detected by Robert Remak, a Pol- cessful treatment of tinea capitis with
ish physician who first observed the presence of griseofulvin.
hyphae in the crusts of favus. This detection is In India, Tewari (1962) first isolated
also a landmark in medical history because this is Trichophyton verrucosum, Trichophyton menta-
the first description of a microbe causing a grophytes and Trichophyton violaceum from
human disease. He himself did not publish his calves, dogs and poultry. T. rubrum was first
work, but he permitted the reference of his isolated from dog and calves at Bengal Veteri-
observations in a dissertation by Xavier Hube in nary college, Kolkata (currently West Bengal
1837. Remak gave all the credits of his discovery University of Animal and Fishery Sciences)
to his mentor Schoenlein who first published the (Chakrabarty et al. 1954). Tewari (1969) isolated
fungal etiological report of favus in 1839. He T. simii from chicken, dog and man for the first
observed the infectious nature of the favus by time in India. Trichophyton verrucosum infection
autoinoculation into his own hands and also suc- in camel and its handlers is also reported from
cessfully isolated the fungus later (1945) and India (Pal and Lee 2000).

12 4 Cutaneous, Subcutaneous and Systemic Mycology
4.1.1 Morphology The hyphae produce asexual spore known as

conidia which generally contains single nucleus.
The septate, branching hyphae are produced by Two types of conidia i.e. macroconidia
Trichophyton in the artificial culture and non- (macroaleuriospores) and microconidia (micro-
parasitic (environmental) state. Sometimes, aleuriospores) are detected. The macroconidia are
abnormal forms of hyphae such as spiral or large (up to 100 μm long), smooth-walled, slender
coiled hyphae, tennis racquet hyphae, pectinate club-shaped, blunt at the end, and with numerous
hyphae (like teeth of a comb) and ‘favic transverse septa. Their presence is variable,
chandeliers’ (irregular projections along one whereas the microconidia are abundant in pres-
side of the hyphae) are observed (Fig. 4.1). The ence, small, thin walled, hyaline, sub-spherical to
hyphae contain two genetically distinct nuclei club-shaped and borne singly or in grape-like clus-
(heterokaryon). So, the ploidy of the nucleus is ter (Fig. 4.2). In the parasitic state, Trichophyton
uncertain. produces hyphae and arthroconidia.
Fig. 4.1 Coiled hyphae

of Trichophyton
mentagrophytes
(Photograph courtesy –
Prof. Alexandro Bonifaz,
Head, Department of
Mycology and
Dermatology service,
General Hospital of
Mexico, Mexico City)
Fig. 4.2 Microconidia

of Trichophyton rubrum
Head, Department of
Mycology and
General Hospital of
4.1 Trichophyton 13
4.1.2 Classification The molecular study revealed that none of the

genera are monophyletic. The anthropophilic and
All the dermatophytes are classified into three zoophilic species in the genus Trichophyton are
ecological groups namely geophilic (soil), zoo- present in a single clad, whereas the geophilic
philic (animals) and anthropophilic (human). The species are present in another clad. It seems that
geophilic dermatophytes are saprophytes and they the ecological niche acted as a motivational force
derive their nutrients from keratinous substrates for evolution of Trichophyton. Further, within the
(hair, feathers, horns) present in the environment. same species complex such as Trichophyton
They may infect human and animals during direct mentagrophytes complex, both anthropophilic
contact with the contaminated soil. The examples (T. mentagrophytes var. interdigitale) and zoo-
include Trichophyton ajelloi and Trichophyton philic (T. mentagrophytes var. mentagrophytes)
terrestre. The zoophiles have a specific animal species are present.
host and they can infect human also. The The Trichophyton genus contains 25 species
examples include Trichophyton simii (monkeys), arranged under different teleomorphic species
Trichophyton mentagrophytes (rodents) and complex. The type species is Trichophyton
Trichophyton equinum (horses), whereas the host tonsurans. The teleomorphic species complexes
of anthropophilic species are human, but they can are Arthroderma vanbreuseghemii complex
infect animals also. The examples of (T. tonsurans, T. equinum, T. interdigitale),
anthropophilic species are Trichophyton rubrum, Arthroderma simii complex (T. simii, originally
T. kanei, T. schoenleini, T. concentricum and described from India and associated with
T. tonsurans. monkeys), Arthroderma benhamiae complex
All dermatophytes belonged to the phylum (T. mentagrophytes var. granulosum,
Ascomycota, class Euascomycetes, order Onyge- T. mentagrophytes var. erinacei) and
nales and family Arthrodermataceae. The family Trichophyton rubrum complex (T. rubrum,
Arthrodermataceae contains four morphological T. violaceum). The details of the anamorph spe-
anamorph genera such as Trichophyton, Micro- cies and their hosts under these complexes are
sporum, Epidermophyton and Chrysosporium. described in Table 4.1.
Table 4.1 Teleomorphic species complex, anamorph species and hosts of Trichophyton
Teleomorphic species
complex Anamorph Host
Trichophyton rubrum T. rubrum of Africa (including the old taxa of T. raubitschekii, Human
complex T. soudanense, T. gourvilii, T. megninii)
T. rubrum (including the old taxa of T. fischeri, T. kanei, T. raubitschekii) Human
T. violaceum (including the old taxa of T. yaoundei, T. glabrum) Human
Arthroderma simii T. simii Monkey
complex T. mentagrophytes (including the old taxa T. mentagrophytes var. Mice, camels
quinckeanum, T. langeronii, T. sarkisovii)
T. schoenleinii Human
Arthroderma benhamiae T. verrucosum Cattle
complex T. erinacei Hedgehogs
Trichophyton species of A. benhamiae (including T. mentagrophytes var. Rabbits, guinea
granulosum) pigs, rodents
T. concentricum Human
T. bullosum Horses
T. eriotrephon –
Arthroderma T. tonsurans Human
vanbreuseghemii complex T. equinum Horses
T. interdigitale (including zoophilic species as T. mentagrophytes var. –
mentagrophytes, var. granulosum, T. verrucosum var. autotrophicum)
T. mentagrophytes var. goetzii, interdigitale, nodulare, T. krajdenii Human
Table 4.2 Teleomorphs of Trichophyton 4.1.4 Susceptibility to Disinfectants

Anamorph Teleomorph
T. mentagrophytes var. A. benhamiae The experimental study revealed that among
mentagrophytes disinfectants such as chlorine, phenol, sodium
T. georgiae A. ciferrii dodecyl sulphate, and quaternary ammonium
T. flavescens A. flavescens salts, chlorine (1 %) exhibited the strongest fun-
T. vanbreuseghemii A. gertleri gicidal action against Trichophyton inoculum.
T. gloriae A. gloriae
T. terrestre A. insingulare
T. terrestre A. lenticularum
4.1.5 Natural Habitat and Distribution
T. terrestre A. quadrifidum
T. simii A. simii
The geophilic species of Trichophyton prefers
T. mentagrophytes var. A. vanbreuseghemii
interdigitale to reside in the soil. They can produce the
Unnamed A. curreyi antibacterial substances to survive from the soil
Unnamed A. tuberculatum bacteria. The zoophilic and anthropophilic spe-
cies can use the host keratin and prefer to reside
the hair, fur, and feathers of human or animals.
4.1.3 Reproduction They can reside in the soil if embedded in skin
scales, hair and feathers. It occurs in animal
The sexual reproduction cycle is observed in sheds where recurrent infection is common.
geophilic and zoophilic (except T. equinum) spe- The conidia can survive in the environment
cies of dermatophytes, whereas the anthro- with high moisture and low temperature. The
pophilic species do not possess such kind of conidia also remain alive in the skin and hair
sexual cycle and probably they reproduce clon- after the recovery of the animals.
ally. The mating competent species have an Trichophyton is worldwide in distribution.
asexual type (anamorph) and a sexual type The distribution matrix of different species
(teleomorph). The sexual stage (teleomorph) is changes with time. Before the Second World
known as Arthroderma (Table 4.2). War, prevalence of T. mentagrophytes and
The sexual reproduction of other ascomyce- T. verrucosum was highest in human causing
tous fungi such as Aspergillus is controlled onychomycosis and tinea pedis in North and
by MAT locus with two idiomorphs such as Central Europe. The prevalence of T. rubrum
MAT-1 and MAT-2. The heterothallic MAT-1 increased steadily among the human population
and MAT-2 can undergo sexual mating. The sim- after 1948–1950. In Southern Europe and Arabic
ilar kind of MAT locus is recently identified in countries, zoophilic dermatophytes, such as
three species of Trichophyton i.e. T. equinum, T. verrucosum are the most frequently isolated
T. rubrum, and T. tonsurans and two species of species. In the United States and Mexico,
Microsporum. All of them shared certain com- frequency of T. rubrum isolation from human is
mon features in MAT locus such as small size followed by T. mentagrophytes and T. tonsurans,
(around 3 Kb) and specific arrangement of SLA2, whereas T. simii was proposed to be endemic in
COX13, and APN2 genes at one side of the locus. Indian subcontinent as it was commonly isolated
In other Ascomycota these genes flank the MAT from soil, poultry and other animals in India.
locus. The transcriptional orientations of APN2 Recently it is also isolated from non-endemic
and COX13 genes in dermatophytes are also dif- areas such as Belgium, France and Ivory Coast.
ferent from other dimorphic fungi. Further, suc- In India, other studies indicated the prevalence
cessful mating is observed between T. rubrum of T. mentagrophytes var. mentagrophytes
and Arthroderma simii which suggests the possi- and T. rubrum in pet dogs which was further
bility of cross-species sexual recombination. transmitted to their owners.
4.1 Trichophyton 15
4.1.6 Genome The conventional solid medium includes

Sabouraud dextrose agar with cycloheximide,
Currently whole genome sequences of four chloramphenicol and gentamicin. For detection
Trichophyton species (T. equinum, T. rubrum, of sporulation, potato dextrose agar is preferred.
T. tonsurans, and T. verrucosum) are available T. schoenleinii are isolated in Sabouraud glucose
at Broad Institute’s dermatophyte comparative agar, BCP milk solid glucose agar. The cultures
database (http://www.broadinstitute.org). The are incubated at 25–30 C except T. verrucosum
dermatophyte genome is enriched in the InterPro which requires 37 C temperatures for optimum
domains (protease, kinase, secondary metabolite, growth. The incubation period is 2 weeks and the
LysM) and it contains 308 genes specific to the absence of growth after 3–6 weeks is considered
dermatophytes. Further, species-specific genes as negative.
are also present which play major role in host On Sabouraud dextrose agar, T. verrucosum
preference. colonies are slow growing, small, disc-shaped,
The size of T. rubrum genome is 22 Mb and it white to cream coloured, with a velvety surface,
contains five chromosomes of 5.8, 5.2, 4.6, 3.05, a raised centre and flat periphery. The colony
and 3.0 Mb sizes. The repetitive DNA constitutes surface pigmentation is white-grey, pink, buff
5–10 % of the genome, whereas the size of or pale yellow and the reverse pigment may
T. verrucosum genome is 22.6 Mb containing vary from non-pigmented to pink/yellow. The
8,024 predicted protein-encoding genes. Approx- surface and reverse colour of the colonies varies
imately 5744 genes of T. verrucosum genome with the species of Trichophyton which aids in
contain introns. The GC content is below 40 %. diagnosis.
The GC rich region is present as ‘long mosaic’ in Extensive pleomorphism is observed in
the genome separated by ‘AT islands’. The T. mentagrophytes cultures. The pleomorphism
genome encodes the enzymes for glycolysis, tri- converts the morphology of fungal colony from
carboxylic acid cycle, pentose phosphate shunt, granular texture to white fluffy tufts of aerial
and synthesis of all amino acids and the nucleic mycelium on the surface of colonies which
acid bases, proteases (235 protease encoding results in the loss of characteristic pigmentation
genes are present). The number of tRNA genes and conidia production.
is low (77) in comparison to other fungi.
4.1.7 Isolation, Growth and Colony 4.1.8 Antigenic Characteristics

Characteristics
The immunodominant antigens of Trichophyton
The standard isolation medium is dermatophyte are expressed during sporulation and early hyphal
test medium (DTM) containing antifungals and growth. Two types of keratinases are secreted
antibiotics such as cycloheximide, chloram- by them which belonged to subtilase (serine
phenicol and gentamicin that can inhibit the proteases) and zinc metalloprotease family.
growth of contaminant fungi and bacteria. Some The recombinant subtilase enzyme of T. rubrum
species have fastidious requirements. T. equinum (Tri r 2) is able to stimulate immediate and
requires nicotinic acid and T. verrucosum delayed-type hypersensitivity. The P5 peptide is
requires thiamine and inositol for their optimum identified as immunodominant epitope associated
growth. The DTM also contains phenol red indi- with delayed-type hypersensitivity. The heat
cator which changes the colour of the medium shock protein (hsp) of T. mentagrophytes is used
from yellow to red during growth of to formulate plasmid DNA-based vaccine in labo-
Trichophyton. The proteolysis occurs during the ratory animal model which offers some degree of
growth which liberates ammonium ion and the protection. It indicates the immunodominance
pH of the medium changes to alkaline. nature of T. mentagrophytes hsp.
Table 4.3 Virulence mechanisms and factors possessed by Trichophyton

Virulence factors Functions
Endoprotease They help to degrade the keratin
(a) Subtilisins (sub, alkaline proteases, pH 7–9 is required
for optimum activity)
(b) Fungalysins (Mep, Zn metalloproteases with HEXXE
motif. Their optimum pH of activity is between 7.0
and 8.0.)
Exoproteases The Dpp and Lap synergistically can degrade the large
(a) Serine proteases (DppIV, DppV) peptides into oligopeptides and free amino acids
(b) Leucine aminopeptidases (Lap1, Lap2)
(c) Carboxypeptidases (McpA, ScpA)
Kinase The kinases are involved in signalling necessary for
adaptation of Trichophyton in the skin
Mannan It can inhibit the macrophage-mediated phagocytosis of
conidia and inhibit the cell-mediated immune response
Sulphite In the presence of sulphite, disulphide bonds of the keratin
substrate are directly cleaved to cysteine and
S-sulphocysteine. The reduced proteins become accessible
to other proteases for further cleavage
LysM The LysM protein conceals the dermatophyte cell wall
components and carbohydrates and thus avoids the host
immune response to the fungi
4.1.9 Virulence Factors spread the infection by leaving infected hairs

around their barns.
The virulence factors of Trichophyton explored The zoophilic Trichophyton (T. verrucosum,
by different investigations till date are enlisted in T. equinum) are transmitted from animals to
Table 4.3. human by direct contact with infected animals or
through the environment contaminated with the
animal fomites. This kind of skin infection is an
4.1.10 Transmission occupational disease of farmers and their family
members, Veterinarians, abattoir and tannery
Most of the dermatophytes including workers and is common in pet owners especially
Trichophyton are transmitted into the host the children who handle the infected dogs and cats.
through the injured skin, scars and burns. The The human-to-human transmission occurs pri-
arthrospores or conidia are the major infectious marily through the indirect way such as loose
agents. The resting hairs do not possess essential strands of hair and desquamated epithelial cells
nutrients for the dermatophytes, so the resting (T. schoenleinii) than the direct contact. The
hairs are not infected with the organism. The transmission may occur via contaminated
infection after entry into a herd, kennel or cat- hairbrushes, combs and hats. Transmission of
tery, spreads from one animal to another by infection from mother to newborn child is possi-
direct transmission or indirectly via environmen- ble. T. schoenleinii can persist in the houses for
tal contamination or fomites. In farms, high ani- several generations without proper disinfection.
mal density and close contact promote direct
transmission between animals in the herd or
between the herds in pasture. Shedding of derma- 4.1.11 Pathogenesis
tophyte spores contaminates the farm environ-
ment which can last for several years depending The dermatophytes do not survive in the live
upon the moisture. The rodents also help to cells and areas of inflammation. So, they do not
4.1 Trichophyton 17
invade the skin epithelial cells. The spores, after different because the fungi can invade and grow
transmission through the skin trauma, can germi- in the moist living tissues. The trauma causing
nate in the non-living keratinised layer of the the breakage in the epidermis is a prerequisite for
skin (stratum corneum). The fungal metabolites the establishment of the infection in the dermis.
induce inflammatory reaction at the site of infec- During trauma, follicular disruption occurs
tion. The inflammation causes the pathogen to which causes the introduction of keratin and
move away from the site of infection in a circular necrosed materials into the dermis. The keratin
way with healing at the centre and papules at the and necrosed materials act as substrate for sur-
periphery which is responsible for classical ring vival of the organism. The growth of the organ-
shaped lesion. ism causes cellular destruction and inflammation
The invasion of stratum corneum is associated which raises the level of stromal acid mucopoly-
with the production of different virulence factors saccharides, lowering the dermal pH to make it
such as sulphite, keratinase and proteases as suitable for the fungal survival. The neutrophils
described in Table 4.3. The keratinous tissues and monocytes can ingest and kill the conidia of
are composed of insoluble proteinaceous com- T. rubrum. However during immunosuppression
plex made of a network of different cross-linked these neutrophils and monocyte mediated killing
proteins such as loricrin- and proline-rich is hampered and the infection may progress into
proteins which contain numerous cysteine the deeper part of the tissues.
residues to form disulphide bridges. The secreted
sulphite can break these disulphide bridges
and make the reduced proteins accessible by
different proteases and keratinases for further 4.1.12 Disease Produced
digestion.
The pathogenesis of anthropophilic T. rubrum The major animal and human diseases produced
causing Majocchi’s granuloma in human is little by Trichophyton are enlisted in Table 4.4.
Table 4.4 Major diseases of animal and human caused by Trichophyton

Fungi Host Disease
Trichophyton verrucosum, Cattle Bovine ringworm: The calves are most susceptible especially when kept
T. mentagrophytes, T. rubrum within the crowded pens. The lesions are circular, painless, thick, white and
scattered with occasional production of large plaques (‘asbestos’) with
thick scabs and crusts. The lesions appear in the head, neck and less
frequently in the back, flank and limbs. The secondary bacterial infection
may occur associated with pruritis. Other clinical signs are skin scaling and
loss of hair. Both T. verrucosum and T. mentagrophytes produce ectothrix
infection with the formation of spores on the surface of the hairs
T. equinum Horse Equine dermatophytosis: The dry, scaly and multiple lesions appear in any
part of the body especially in the groomed part. Sometimes the lesions
become larger to produce a ‘moth-eaten appearance’
T. verrucosum (‘club-lamb Sheep Ovine dermatophytosis: The lesions are scaly and appear in the hairless
fungus’) part of the face, ear and neck or it may soil the wool. The infection is
self-limiting, but the wool will not appear in the infected area for the next
4–5 months
T. simii Poultry The lesions are scaly, inflammatory and necrotizing which appear in
featherless part of the body. The disease is endemic in India
T. mentagrophytes Dogs The scaling to inflammatory lesions appears in the body with occasional
and cats secondary bacterial infection and suppuration. The later is known as
‘wet eczema’. Affected area become hairless and the vesicles appear at the
periphery
(continued)
Table 4.4 (continued)
Fungi Host Disease
T. rubrum, T. mentagrophytes Human Tinea corporis (tinea circinata): It is the classical ringworm infection in
human which is characterised by the formation of lesion in trunk and
extremities (non-hairy glabrous region of body). The lesions are pink-
to-red annular or arciform patches with scaly or vesicular borders that
expand peripherally with a tendency for central clearing. Sometimes
follicular papules are present at the active border. When it happens in the
face, it is known as tinea faciei, and when it affects the head, neck, upper
extremities but rarely the legs, it is known as tinea corporis gladiatorum
T. concentricum Human Tinea imbricata (Tokelau): It is a superficial mycosis restricted to South
Pacific islands (Polynesia and Melanesia), South Asia and South America.
The skin lesions are multiple concentric annular plaques with thick
adherent peripheral rims of scale
T. rubrum, T. mentagrophytes, Human Tinea pedis (athletes’ foot): It is the ringworm infection in which the
T. mentagrophytes var. lesions are found in feet. Clinically it has four types such as interdigital,
interdigitale moccasin, ulcerative and vesiculobullous. The erythema, scaling and
(ulcerative and maceration with fissures are observed in interdigital type which occurs
vesiculobullous) in the space between 4th and 5th toes. The diffuse scaling on the soles
extending to the sides of the feet is found in the moccasin type. In ulcerative
type, lesions are macerated with scaling border. Secondary bacterial
infection is a common feature in this type of tinea pedis. In vesiculobullous
type, vesicular eruptions occur at the side of the feet
T. rubrum, T. mentagrophytes Human Tinea manuum: It is a diffuse hyperkeratosis which is commonly found
in palmar creases of the palms and digits. White powdery scales along the
palmar creases are typically seen. It may occur in one hand along with
tinea pedis (two feet and one hand syndrome)
T. rubrum, T. mentagrophytes Human Tinea cruris (Jock’s itch): The lesions are found in the crural folds which
may extend to the thighs, buttocks and gluteal cleft area. Scrotal infection
alone is rare. It is more common in male
T. rubrum, T. mentagrophytes Human Tinea unguium (onychomycosis): Onychomycosis is a common nail ailment
(found in 90 % cases) associated with pain, discomfort and impaired/lost tactile functions. Toenail
dystrophy can interfere with walking, exercise or proper shoe fit. In addition,
it has both psychosocially and physically detrimental effects. Clinically four
types of onychomycosis are observed such as distal and lateral subungual
onychomycosis, proximal subungual onychomycosis, superficial white
onychomycosis and total dystrophic onychomycosis
T. tonsurans Human Tinea capitis: It is the dermatophytic infection of hair/scalp. It is more
common in children who remained in close contact with infected pet
animals. The infection produces kerion formation which is characterised by
a raised, tender mass of inflamed tissue
T. mentagrophytes, Human Kerion celsi-type tinea capitis (tinea profunda capillitii): Deep-seated
T. tonsurans, T. rubrum mycosis of scalp composed of tumorous mass with thick crusts
T. schoenleinii. T. violaceum, Human Tinea capitis favosa: It is a chronic inflammatory dermatophyte infection of
T. verrucosum, zoophilic the scalp and less commonly of the glabrous skin and nails. The classical
T. mentagrophytes var. lesion is concave, cup-shaped yellow crust on the scalp and glabrous skin
quinckeanum that is associated with severe alopecia (scutulum)
T. tonsurans Human Tinea gladiatorium: It affects the upper part of the body and is common
among wrestlers. It is transmitted by direct skin-to-skin contact
T. mentagrophytes, Human Tinea barbiae (Barbers’ itch): It is an uncommon dermatophyte infection
T. verrucosum, of the beard and moustache areas. In previous years, it was more frequently
T. rubrum observed due to use of common razor or blade in saloons. Clinically it
shows sycosis, iododerma, contact dermatitis and perioral dermatitis
T. rubrum Human Tinea incognito: It results from lack of diagnosis and continuous
corticosteroid application. The plaques with erythema and papules on the
neck and breast area are noted
T. rubrum (Aspergillus Human Majocchi’s granuloma: It is a folliculitic and perifolliculitic fungal
and Phoma) infection of the dermis in both healthy and immunocompromised hosts.
It presents as nodules, plaques and papules on areas that are prone to
trauma. It generally appears on the upper and lower extremities (forearms,
hands, legs, ankles) or on the scalp and face
4.1 Trichophyton 19
4.1.13 Immunity The activation of Th1 cells produces a cell-

mediated immune response with the cytokine
The innate and adaptive immune responses are profile such as IFN γ, IL12 and IL2 which can
generated against Trichophyton. The mammalian recover the animals. This cell-mediated response
immune system has pattern recognition receptors is characterised by production of positive
(dectin-2, chi-lectins) which identify fungal delayed-type hypersensitivity (DTH) reaction
pathogen-associated molecular patterns (PAMP) which occurs due to recruitment of cells into
such as mannan and chitin. The mannan–dectin the skin associated with deposition of fibrin. It
interaction can upregulate the pro-inflammatory is also associated with lower titres of IgG
cytokine (TNFα) production by macrophages. directed towards Trichophyton antigens and the
However, relationship between the chitin recog- absence of IgE or IgG4. The efficient vaccine
nition and development of innate or adaptive should activate the cell lineages which can pro-
immune response is not established. The anti- duce an effective cell-mediated immune
microbial peptide such as cathelicidin plays a response to clear the infection.
role to limit the invasion of dermatophytes. The balance of Th1 and Th2 immunity
During atopic dermatitis the level of cathelicidin determines the progress of Trichophyton infec-
is decreased which helps in invasion of tion, although, the dermatophytes prefer to
dermatophytes. The fungal hyphae can activate induce Th2 response with B cell stimulation
complement via the alternative pathway and and inefficient macrophage activation for estab-
generate chemotactic factors for neutrophils. lishment of the infection.
Further, during fungal invasion of keratin layer,
the keratinocytes release IL8 which also attracts
neutrophils in the stratum corneum. The 4.1.14 Diagnosis
neutrophils and monocytes can ingest and kill
the spores of T. rubrum. 4.1.14.1 Clinical Specimen
For development of adaptive immune The lesion area should be cleaned with 70 %
response, the dermatophyte antigens are trapped ethanol to remove the bacterial contamination.
and processed by the skin Langerhans’ cells in The scales from the active lesions should be col-
the epidermis. The Langerhans’ cells present the lected with a blunt scalpel (or toothbrush) and the
antigens in MHC class II restricted fashion to affected hairs should be plucked from the lesion
T cells in the lymph nodes draining the skin. without breakage. For suspected onychomycosis,
The migration of Langerhans’ cells for antigen the nails should be cleaned with alcohol and
presentation is initiated by the cytokine affected nail bed should be exposed by removing
granulocyte-macrophage colony-stimulating fac- the onycholytic nail plate with a nail clipper. The
tor (GM-CSF). In calves, experimentally subungual hyperkeratotic material should be col-
infected with T. verrucosum, influx of different lected with a scalpel or curette. They are wrapped
immune cells such as neutrophils, macrophages, in brown paper (or coloured paper) and are kept in
CD4+ and CD8+ T lymphocytes, and γδ T cells a tight container preferably without moisture for
were observed. The protective role of transport into the laboratory.
Th2-mediated immune response producing anti-
Trichophyton antibodies in calves is question- 4.1.14.2 Laboratory Examination
able. In human, Th2-mediated response produces 1. Direct examination: The skin scrapings, hair
IL4 which induces antibody class switching to including the hair bulb, nail fragments can be
IgG4 and IgE, associated with immediate hyper- observed under microscope by wet mount
sensitivity (IH) reaction (wheal and flare reac- with 10 % KOH preparation. The digestion
tion). The IH reaction is often detected in chronic of keratin with the KOH can be speed up with
infection which also indicates the inefficiency of heat. An alternative preparation is 20 % KOH
humoural immunity in offering protection. with 36 % DMSO that provides rapid
Table 4.5 Different species of Trichophyton associated atrophic epidermis. Inflammation with round-
with hair invasion cell infiltrate is seen in the adjacent dermis. In
Ectothrix invasion Endothrix invasion Noninvasion comparison to direct microscopy histopatho-
T. verrucosum T. schoenleinii T. rubrum logical investigation is much reliable,
T. equinum T. violaceum T. simii although it cannot detect the species of der-
T. mentagrophytes T. tonsurans T. concentricum matophyte. The diagnostic sensitivity can be
increased with biopsy which is not always
diagnosis without heating and the specimens possible to conduct especially in human
can be preserved for long time. The hyphae patients suffering with diabetes.
are observed under the microscope invading 4. Molecular biology: The conventional PCR
the hair and producing arthrospores. There have developed for identification of different
are two types of hair invasion by the Trichophyton species using nuclear ribosomal
dermatophytes, known as ectothrix and DNA (rDNA), mitochondrial DNA (mtDNA),
endothrix invasion. In ectothrix invasion, the chitin synthase 1 (chs1), topoisomerase II
arthrospores are observed on the surface of (TOP2) genes, small (18S rRNA) and large
the hair, whereas in endothrix invasion, the subunit (28S rRNA) of ribosomal RNA as
spores are found within the hair. There are major target genes. Further, randomly
some Trichophyton species also which cannot amplified polymorphic DNA (RAPD), PCR
attack the hair. The different Trichophyton fingerprinting, amplified fragment length poly-
species associated with hair invasion are morphism (AFLP) and sequencing of microsat-
enlisted in Table 4.5. ellite markers, have been successfully applied
The specimens can be stained with to species identification in dermatophytes but
chlorazol black or Parkers blue ink. Chlorazol were mostly unable to discriminate between the
helps in identification of hyphae by staining the strains. The sequencing of the internal-
carbohydrate rich cell wall without staining the transcribed spacer (ITS) region is commonly
contaminants such as cotton or elastic fibres. used for phylogenetic analysis and identifica-
The fine needle aspirates can be stained with tion of dermatophyte species. Although, the
the May–Grunwald–Giemsa method. ITS region shows high sequence similarities
Majority of Trichophyton species (except between the species of Trichophyton
T. schoenleinii) do not produce fluorescence (90–100 %), the discriminating polymorphisms
under Wood’s lamp. However, fluorescence (barcode sequence) exists in ITS1 and ITS2
microscopy (UVA, 365 nm) produces better region of the members of T. tonsurans and
resolution than wet mount with KOH. The T. equinum. In addition, non-transcribed spacer
fluorescent substance (calcofluor, acridinium (NTS) region of the rRNA genes is also consid-
orange or Blankophor) is added to the KOH. It ered suitable for strain typing of some
binds fungal cell wall and increases the visi- Trichophyton species, such as T. rubrum,
bility of fungal hyphae and spores. T. interdigitale and T. tonsurans.
2. Isolation and identification: Media and incu- Recent progress includes the use of
bation condition as described earlier will PCR-ELISA which can specifically identify
serve the purpose. the PCR amplified product with the help of
3. Histopathology: The histopathological slides enzyme labelled probe producing colour reac-
are prepared with periodic acid–Schiff (PAS), tion in positive cases. The uniplex-PCR-ELISA
Grocott methenamine silver and calcofluor can identify T. rubrum, T. interdigitale,
white (CFW) stains. The histopathological T. tonsurans and T. violaceum individually.
section shows the dead and degenerating The matrix-assisted laser desorption/ionisation
mycelium with cellular debris at the centre time-of-flight mass spectrometry (MALDI-
of the lesion with well preserved hyphae at TOF-MS) can identify the species of
the periphery of the lesion. In T. schoenleinii Trichophyton from the grown cultures which
infection, the ‘scutulum’ (concave, is very fast and specific method and the result
cup-shaped yellow crust) is observed on the shows 98–99 % similarity with PCR.
4.2 Microsporum 21
4.1.15 Treatment extracts showed antidermatophytic activity

against T. rubrum and T. violaceum in vitro.
The most of the ringworm infection in animals is
self-limiting; however, treatment is required for
its zoonotic potential. The infected animals 4.1.16 Vaccine
especially the pets and companion animals may
transmit the infection to the animal handlers or In several countries such as Russia, Hungary,
pet owners who come in direct contact. The hairs former German Democratic Republic, former
of the affected part should be clipped properly Yugoslavia, Norway and Bulgaria, the vaccine
before application of topical antifungals such as against Trichophyton is used since 1970. It is a
thiabendazole, miconazole, ecoconazole, ketoco- non-adjuvinated, live vaccine containing freeze
nazole, itraconazole, lime-sulphur solution, 5 % dried conidia and hyphal elements of
sodium hypochlorite solution, etc. If the topical T. verrucosum (LTF-130 strain). The vaccine is
application is ineffective, systemic antifungals prophylactic as well as curative in nature which
such as ketoconazole, clotrimazole, itraconazole, is inoculated intramuscularly at the side of the
terbinafine may be used. Among them, neck. In calves the recommended age for vacci-
terbinafine is most effective. Griseofulvin is nation is 2–4 weeks. The immunity is life-long
now a day avoided due to its acute toxicity. and no annual booster is required. If the vaccina-
In human, systemic treatment with antifungals tion is performed in the calves of 4–6 days old, a
is considered in tinea capitis, tinea unguium and lesion (1–2 cm) appears at the injection site with
during significant hyperkeratosis. In tinea capitis hair loss and moderate scaling which regenerates
favosa, systemic treatment with terbinafine or after several weeks probably due to residual vir-
itraconazole is preferred along with clearing ulence of vaccine strain. The bovine ringworm is
away the scalp debris, removing the crusts and eradicated from Norway by vaccination and con-
general improvement of scalp hygiene. In other tinuous monitoring.
cases, topical application of antifungals for 1–6 The formalin inactivated vaccine against
weeks is recommended. T. verrucosum is also produced which offered
The untreated patients of onychomycosis may intermediate protection under field trial. Experi-
act as reservoir and transmit the infection to other mentally, formalin inactivated conidia and
family members and neighbours during commu- mycelia elements of T. equinum along with adju-
nal bathing (ponds). In diabetic patients, it carries vant offered 75 and 87 % protection in horses,
the risk for development of diabetic foot. So, respectively.
treatment of onychomycosis is required not
only for cosmetic purpose but also for prevention
of severe menace. When the infection covers 4.2 Microsporum
more than 50 % of the distal nail plate and nail
matrix, the systemic treatment with ketocona- David Gruby (1843) described tinea capitis
zole, itraconazole, fluconazole and terbinafine (favosa) in human and isolated Microsporum as
is recommended. The new therapies such as causative agent with fulfilment of Koch’s postu-
ciclopirox hydrolacquer based on water soluble late. He coined the genus name Microsporum on
biopolymer technology, 1 % fluconazole and the basis of small spore production around the
20 % urea in a mixture of ethanol and water hair shaft. Gruby also first described the type
and terbinafine nail solution are also promising. species Microsporum audouinii in the name of
In more severe cases, surgical removal of the part his close associate Victor Audouin, the then
(debridement) or whole (avulsion) of the nail Director of the Museum of Natural History in
plate may be followed. Paris. In 1881, Megnin first described
Among plant-based antifungals, Neem M. gallinae. White (1899) reported 279 cases of
(Azadirachta indica A. Juss) seed and leaf human ringworm in Boston during the period
October 1895 to July 1898. Of the 279 cases, 1962; Gupta et al. 1970), pets (Pal et al. 1990)
139 (50 %) were caused by Microsporum and wild animal (Pal 1988) was also reported
audouinii in children. The existence of the sexual from India.
phase of certain dermatophytes was first
recognised by Nannizzi (1927) in Italy. He also
described the sexual form of Microsporum 4.2.1 Morphology
gypseum as Gymnoascus gypseum. Griffin
validated the Nannizzi’s investigations and con- The septate, branching hyphae are produced
firmed the existence of the sexual phase of by Microsporum in the artificial culture and
Microsporum. This sexual stage was renamed nonparasitic (environmental) state. Like other
as Nannizzia in the name of Nannizzi. In1934, dermatophytes, Microsporum also produce asex-
Emmons first classified the dermatophytes into ual spores known as conidia. Two types of
three genera, i.e. Trichophyton, Microsporum conidia, i.e. macroconidia (macroaleuriospores)
and Epidermophyton. This description is till and microconidia (microaleuriospores), are
date considered as the standard system of classi- detected. The size of macroconidia varies from
fication for dermatophytes. Bodin first described 6 to 160 μm 6–25 μm. The macroconidia are
two species of Microsporum such as M. canis and rough walled, spindle shaped, fusiform
M. gypseum. Kligman (1952) described the (M. vanbreuseghemii) or ovoid (M. nanum).
epidemics of tinea capitis due to Microsporum The wall of macroconidia is thin, moderately
audouinii which was known as the Atlantic City thick or thick in nature which can traverse inside
board epidemic among the school children. to produce 1–15 septa. They are generally
In India, Dey and Kakoti (1955) first reported arranged singly along the hyphae (Figs. 4.3 and
the isolation of Microsporum gypseum from ring- 4.4). The microconidia are sessile or stalked, less
worm lesion in a laboratory rabbit. Kandhari and abundant, clavate in shape and arranged singly
Sethi (1964) established the presence of M. canis along the hyphae.
infection in dogs in Delhi. The presence of
M. nanum in the Indian pigs was reported by
Gupta et al. (1968), whereas M. gypseum infec- 4.2.2 Classification
tion in other domestic animals such as cattle
(Gupta et al. 1970), sheep (Thakur et al. 1983), All dermatophytes belonged to the phylum
goat (Thakur and Verma 1984), horse (Tewari Ascomycota, class Euascomycetes, order
Fig. 4.3 Macroconidia of

Microsporum canis
Head, Department of
Mycology and
General Hospital of
4.2 Microsporum 23

Microsporum gypseum
Head, Department of
Mycology and
General Hospital of
Table 4.6 Ecological niche-based classification and host specificity of Microsporum

Zoophilic (animal associated)
Anthropophilic Isolated from other
(human associated) Fungi Major host animals Geophilic (soil associated)
M. audouinii M. canis Dog, cat Cattle, sheep, horse M. nanum (isolated from pigs)
(isolated from dogs also)
M. ferrugineum M. equinum Horses – M. gypseum
(isolated from dogs, cats)
M. gallinae Fowl Cattle, dog, cat M. amazonicum
M. persicolor Vole Dog M. boullardii
M. cookei
M. praecox
M. racemosum
M. ripariae
M. vanbreuseghemii
M. duboisii
Onygenales and family Arthrodermataceae. (human associated), zoophilic (animal associated)

The family Arthrodermataceae contains four and geophilic (soil associated) fungi like other
morphological anamorph genera such as dermatophytes. The examples of different
Trichophyton, Microsporum, Epidermophyton Microsporum species are enlisted in Table 4.6.
and Chrysosporium. The genus Microsporum
contains 18 numbers of species. The type of
species is Microsporum audouinii. The species 4.2.3 Reproduction
complexes are Arthroderma otae complex which
consists Microsporum canis (including the old The sexual reproduction is observed in geophilic
taxa M. equinum, M. distortum), M. audouinii and zoophilic species of Microsporum. The mat-
(including var. rivalieri and langeronii) and ing competent species have an asexual type
M. ferrugineum, and Microsporum gypseum- (anamorphs) and a sexual type (teleomorph).
fulvum complex which consists Arthroderma The sexual stage (teleomorph) of Microsporum
incurvata, A. gypsea and A. fulva. Clinically impor- and Trichophyton was previously known as
tant species are M. canis, M. gypseum (gardener Nannizia and Arthroderma, respectively. Due to
ringworm), M. gallinae, M. nanum and similarities in the properties of both genera,
M. audouinii. Nannizia is replaced with Arthroderma at pres-
The ecological niche-based classification ent. The teleomorphs of different Microsporum
system describes Microsporum as anthropophilic species are described in Table 4.7.
Table 4.7 Teleomorphs of Microsporum species in the soil. Some of the geophilic Microsporum
Microsporum species (anamorph) Teleomorph (M. gypseum) can survive in the environment
M. canis var. canis, Arthroderma otae in absence of keratinous materials also such
M. canis var. distortum as in sewage sludge, sediment/soil within the
M. nanum A. obtusum cave and cave wall. The infectious agent of
M. gypseum A. gypseum Microsporum (anthropophilic) is chlamydo-
M. gypseum A. incurvata spores or arthrospores which can survive in the
M. vanbreuseghemii A. grubyi environment for several years and are heat resis-
M. fulvum A. fulvum tant especially within the skin scales.
M. persicolor A. persicolor
Microsporum is worldwide in distribution,
M. racemosum A. racemosum
although some species are endemic in limited
M. amazonicum A. borelli
geographical location. M. canis is the major etio-
M. cookei A. cajetani
logical agent of tinea capitis and tinea corporis
M. boullardii A. corniculata
Unnamed A. cookiella
in some parts of Europe (Slovenia, Croatia
and Italy), the eastern Mediterranean, South
America, Asia (Iran, Yemen and Palestine) and
The sexual reproduction of other ascomy- Africa (Libya). Whereas M. audouinii, causing
cetous fungi such as Aspergillus is controlled tinea capitis in children, was restricted within
by MAT locus with two idiomorphs such as North America but currently is distributed in
MAT-1 and MAT-2. The heterothallic MAT-1 Asia and Africa. In Germany, it was considered
and MAT-2 can undergo sexual mating. The as major etiological agent of favus in humans.
similar kind of MAT locus is identified in two After introduction of griseofulvin (1958), they
species of Microsporum such as Microsporum are almost eradicated from Germany and other
gypseum (geophilic) and Microsporum canis parts of Central Europe. However, M. audouinii
(zoophilic) which indicates their mating compe- is still prevalent in African countries (Malawi,
tence. However, the teleomorph of Microsporum Western Kenya), causing tinea capitis in
(Arthroderma) isolates always exhibit only one human. M. gypseum was isolated from human
mating type which shows the possibility of inter- dermatophytic lesions in Asia (in Iran it shares
species sexual recombination as observed in mat- 4.1 % of the dermatophytic lesions) and Africa
ing between T. rubrum and Arthroderma simii. (Nigeria). Microsporum ferrugineum is now
restricted to a few rural areas of Asia and Africa.
M. canis is the predominant species causing
4.2.4 Susceptibility to Disinfectants dermatophytosis in dogs and cats in Italy which is
followed by geophilic species such as M. gypseum.
The spores of Microsporum are susceptible to In Iran, stray cats were reported to harbour
lime sulphur (1:33), 0.2 % enilconazole and chlo- M. canis without showing any symptom. In South-
rine bleach (1:10 to 1:100) ern India (Chennai), prevalence of M. gypseum
complex was detected among the stray dogs.

4.2.6 Genome
The anthropophilic and zoophilic Microsporum
primarily inhabits human and animals, respec- Currently whole genome sequences of two
tively, although they can infect vice versa. The Microsporum species (M. canis and M. gypseum)
geophilic Microsporum prefers to utilise the are available at Broad Institute’s dermatophyte
keratinous materials (hair, feathers, hooves, comparative database (http://www.broadinstitute.
horns) after dissociation from living animals org). The genome statistics of M. canis and
and which are in the process of decomposition M. gypseum is described in Table 4.8.
4.2 Microsporum 25
Table 4.8 Genome statistics 4.2.8 Antigenic Characteristics

Genome properties M. canis M. gypseum
Size (Mb) 23.1 23.2 The ribosomal fraction of M. canis showed
GC (%) 47.5 48.5 certain degree of immunodominance when
Number of protein-encoding 8,915 8,907 used as experimental vaccine in guinea pigs.
genes However, these antigens were not characterised
Mean coding sequence length (nt) 1,459 1,436 properly. Microsporum produces two types
Mean exon no. per gene 3.21 3.15 of proteases like other dermatophytes such as
Number of tRNAs 82 83
serine protease (Sub3) and zinc metalloprotease
(Mep3). The recombinant M. canis sub3 and
The mitochondrial DNA (mtDNA) of Mep3 can induce cell-mediated immune
M. canis and M. nanum is circular in nature and responses in experimental guinea pigs. The anti-
23,943 and 24,105 bp in size, respectively, which body response was observed against Mep3, not
contains 15 protein-encoding genes. The mito- against Sub3. Experimental vaccination in
chondrial genome size of M. canis is the smallest guinea pigs with the proteases did not offer any
genome of the studied dermatophytes. The GC protection.
value of mitochondrial genome is 24 % and Recent studies showed the presence of nine
it contains 2 rRNAs, 25 tRNAs and 1 intron. IgG-binding proteins (48–180 Kda) as immuno-
The coding region genes are highly similar dominant antigens in the sera of M. canis- and
(>90 %) between the species, whereas the M. gypseum-infected dogs.
differences between species exist in the introns
and intergenic regions.
4.2.9 Virulence Factors
The virulence factors of Microsporum explored
Characteristics
by different investigations till date are enlisted in
Table 4.9.
The conventional solid medium includes
Sabouraud dextrose agar with cycloheximide,
chloramphenicol and gentamicin. The cultures
are incubated at 25–30 C for 2 weeks. The der- 4.2.10 Transmission
matophyte test medium (DTM) is not suitable for
Microsporum isolation, as some of the species Transmission of M. canis arthrospores in animals
produce false-negative result in this medium. For occur through direct contact with the infected or
demonstration of macroconidia, potato dextrose carrier animals especially the cats and dogs. The
agar and other sporulation media are used. hair fragments containing the spores are major
The primary colonies are large, regular or source of infection. The risk factors include
irregular spreading types with varying pigmenta- introduction of new animals in house/shed, ani-
tion. For example, M. canis produces lemon yel- mal shows/market, mating, etc., where the
low pigment which may spread throughout the animals come in close contact with each other.
medium and M. nanum produces rusty colonies. Although indirectly, the spores are transmitted
The colonies later become velvety (M. audouinii) through brushes, collars and grooming utensils.
or powdery (M. gypseum) due to production of Sometimes the animals get infected with
white fluffy tufts of aerial mycelium on the sur- M. gypseum (geophilic) during their burrowing
face of colonies which results in the loss of or playing with contaminated soil.
pigmentation and conidiation. It is known as The direct contact with infected cats, dogs and
‘pleomorphism of dermatophytes’ which results rabbits may transmit the M. canis infection in
from single chromosomal gene mutation. human. Human-to-human transmission is rare
Table 4.9 Virulence mechanisms and factors possessed by Microsporum

Endoprotease [‘Ekase’ (keratinase) which is composed of They help to degrade the keratin
two following enzymes]
(a) Subtilisins (Sub3, serine proteases, 34 Kda, pH 7–9 is
required for optimum activity)
(b) Fungalysins (Mep3, 48 Kda, Zn metalloproteases with
HEXXE motif. Their optimum pH of activity is between
7.0 and 8.0.)
Pseudokinase (inactive kinase) They help in the adaptation of the fungi in diverse animal
hosts by increasing the tolerance against environmental
challenges. Specifically they compete for the substrates
with active kinase and act as inhibitory pseudo-substrates
Exoproteases The Dpp can degrade the large peptides into oligopeptides
(a) Dipeptidyl peptidases (DppIV, DppV) and free amino acids
(b) Carboxypeptidases
Sulphite In the presence of sulphite, disulphide bonds of the keratin
substrate are directly cleaved to cysteine and
S-sulphocysteine. The reduced proteins become accessible
to other proteases for further cleavage
LysM along with polysaccharide deacetylases (type I) (a) Probably LysM protein conceals the dermatophyte cell
wall components and carbohydrates and thus avoids the
host immune response to the fungi
(b) The polysaccharide deacetylase helps in chitin
catabolism and is involved in chitin scavenging in the soil,
defence from other fungi or cell wall modification
Polycyclic aromatic prenyltransferases (pcPTases) It causes prenylation of aromatic compounds which can
increase lipophilicity of the compounds and enhances their
interactions with biological membranes and proteins
although observed in neonatal and intensive care due to clipping, playing, aggressive behaviour,
units where an infected nurse was detected as the ectoparasite infestation, pruritus). After penetra-
source of infection. Among children no gender tion through the skin trauma, the Microsporum
predilection is observed, whereas in adults, spores germinate in the stratum corneum. The
women are infected more frequently than men, hyphae grow along the hair shaft to the follicles
with a ratio ranging from 3:1 to 6:1. where they produce spores. The spores remain
attached by forming a thick layer around the hair
shaft. As Microsporum cannot colonise the
4.2.11 Pathogenesis deeper part of the skin or hair follicles, the hairs
grow normally but break near the skin surface
Microsporum spores cannot invade the healthy causing alopecia. The fungal metabolites induce
skin of the animals after transmission. So many inflammatory reaction at the site of infection.
animals such as cats and dogs become carrier of The inflammation causes the pathogen to move
M. canis arthrospores. This carrier stage may away from the site of infection in a circular way
progress to infection which depends on certain with healing at the centre and papules at the
predisposing factors such as young age, immu- periphery which is responsible for classical
nosuppression (virus or drug induced), ringed lesion.
nutritional deficiency (protein, vitamin A) and The invasion of stratum corneum may occur
high environmental temperature with high in immunosuppressed animals with the help
humidity and skin trauma (injury or scratches of the virulence factors (Table 4.9). It produces
4.2 Microsporum 27
generalised skin illness with secondary bacterial restricted area (head in cats) which disappears
infection. Rarely, a marked inflammatory after several weeks or after proper treatment.
response against the hyphae produces a
nodular granulomatous reaction in dermis
(pseudomycetoma) in Persian cats. 4.2.12 Disease Produced
In immunocompetent animals which are kept
in hygienic conditions, the Microsporum- The major animal and human diseases produced
induced ringworm lesions are limited in a by Microsporum are enlisted in Table 4.10.
Table 4.10 Major diseases of animal and human caused by Microsporum

Fungi Host Disease
M. canis, M. gypseum Cattle Bovine ringworm: The calves are most susceptible especially when kept within the
crowded pens. The lesions are circular, painless, thick, white and scattered. The
lesions appear in the head, neck, paw and less frequently in the back, flank and limbs.
The secondary bacterial infection may occur associated with pruritis. Other clinical
signs are skin scaling and loss of hair
M. gypseum Horses Equine dermatophytosis: The dry, scaly and multiple lesions appear in any part of
the body especially in the groomed part. Sometimes the lesions become larger to
produce a ‘moth-eaten appearance’
M. gallinae Poultry Favus (white comb): Birds of all age groups kept under poor management are
susceptible. White patches are noticed in the comb of male birds. The lesions may
coalesce and the whole comb is covered with thick white coating. The infection may
progress into the feather, gastrointestinal tract and respiratory tract. Necrotic foci
with yellowish mass are observed in trachea. Emaciation followed by death may
occur
M. nanum, M. canis, Pigs Large breeds (Yorkshire) and all age groups are equally susceptible. Unhygienic
M. gypseum condition, high humidity, high stocking density favours the transmission. The
lesions are small, circular, mildly inflamed and are located throughout the body. The
skin becomes reddish with formation of crusts. No alopecia is observed. The disease
is self-limiting and resolves within 2–3 months
M. canis Cats Feline ringworm: The infection causes regular and circular alopecia (3–6 cm in
diameter) with desquamation, erythema in the margin and central healing. Lesions
are single or multiple, mostly confined in the head. Occasionally lesions are found in
legs and tails. Intensity of pruritis is variable and usually no fever or anorexia is
observed. ‘Miliary dermatophytosis’ affects dorsal trunk. In immunosuppressed
patients, larger area of alopecia, erythema, pruritis and crusts develops. Rarely
nodular granulomatous dermatitis (pseudomycetoma) develops. Feline otitis is also
produced which is characterised by waxy, ceruminous discharges from the ear canal
M. canis, M. gypseum Dogs The lesions are circular (2.5 cm in diameter) and are located in face, elbows, paws.
The skin becomes dry and scaly
M. audouinii, M. canis Human Tinea capitis: It is dermatophyte infection of the scalp and hair shaft, commonly
occurs in children. The transmission occurs due to close contact with infected pets.
The infection causes mild erythema, patchy area of scaling, grey hair stumps.
Sometimes the infection causes huge inflammation, folliculitis and kerion (boggy,
sterile, inflammatory scalp mass) formation, areas of scaring and alopecia. The
swollen hairs fracture a few millimetres from the scalp and produce ‘black dot’
alopecia. M. audouinii causes ectothrix type of hair invasion
M. canis, M. gypseum Human Kerion celsi-type tinea capitis (tinea profunda capillitii): Deep-seated mycosis of
scalp composed of tumorous mass with thick crusts
M. canis Human Onychomycosis: The clinical manifestation involves distal subungual
onychomycosis, onychodystrophy, proximal subungual onychomycosis. M. canis
generally does not invade the nails
M. canis Human Tinea incognito: It results from lack of diagnosis and continuous corticosteroid
application. The plaques with erythema and papules on the neck and face with hair
loss are noted
M. canis Human In South-Asian countries (Thailand), women suffer from chronic respiratory disease
which is associated with lung cancer
4.2.13 Immunity wrapped in brown paper (or coloured paper)

and are kept in a tight container, preferably with-
Innate immunity is the first line of defence against out moisture for transport into the laboratory.
Microsporum infection in animals or human. The brushes should be processed on the same
Dectin 2 acts as pattern recognition receptor day of collection.
(PRR) which can bind the hyphal fragments of For geophilic Microsporum (M. nanum,
Microsporum. The pro-inflammatory cytokines M. gypseum, M. cookie), the soil surface sam-
are required for activation of immune cells to pling is preferred. The top layer is removed and
produce an effective immunity. M. canis can the representative samples are scooped from
induce IL1β (pro-inflammatory cytokine) secre- the lower layer of the soil. The soil should be
tion through the activation of inflammasome processed on the day of collection, if stored,
(intracellular multiprotein complex, NLRP3). 0–4 C is adequate.
The activation of NLRP3 occurs through the
sequential stimulation of cathepsin B, K (+)
4.2.14.2 Laboratory Examination
efflux and reactive oxygen species.
1. Direct examination:
The pathways and the molecules involved in
The process of direct examination is same as
production of adaptive immunity (Th1 and Th2
described earlier (Sect. 4.1).
mediated) against Microsporum are not specifi-
Most of the Microsporum species produces
cally elucidated, although, the similarities in
ectothrix type of hair invasion. For example,
pathways with anti-Trichophyton immunity are
ectothrix invaders are M. canis, M. equinum,
assumed. The Th1 response (cell-mediated
M. audouinii, M. gypseum and M. nanum.
immunity) is associated with delayed-type
Majority of M. canis strains (>50 %)
hypersensitivity (DTH) and recovery from
produces green fluorescence due to trypto-
Microsporum infection. The study indicated the
phan metabolite under Wood’s lamp (λ ¼
presence of larger area in DTH reaction in the
366 nm). Sometimes medication, secretions
cats recovered from M. canis infection than
and artificial fibres may hinder the
the cats still having infection and which were
fluorescence.
never exposed. The antibodies produced in Th2
2. Isolation and identification: Media and incu-
immunity (humoural) can act as fungistatic due
bation condition as described earlier will
to opsonisation and complement activation
serve the purpose.
properties. Their exact role in recovery and
3. Molecular biology: A multiplex PCR is devel-
protection is unexplored.
oped which can simultaneously detect
Trichophyton spp., Microsporum canis and
M. audouinii from the clinical specimens.
4.2.14 Diagnosis The RAPD-PCR was developed for differen-
tiation of M. canis strains isolated from differ-
4.2.14.1 Clinical Specimens ent species such as dogs, cats, human, fox and
The hairs and scales can be collected from rabbit. Sometimes the band pattern generated
the margin of the lesion after gentle swabbing by this molecular technique produces ambig-
with alcohol for decontamination of the skin. uous results which can be resolved by geno-
In subclinically infected animals, brushing the mic in situ hybridisation (GISH). Using
suspected skin over the back, shoulders, sides, specific GISH probes, discrimination between
hindquarters and legs with an unused toothbrush Trichophyton interdigitale, Trichophyton
or hair brush can be performed. The brushes are rubrum and Microsporum canis has been
transported to the laboratory in brown paper or conducted. The uniplex PCR-ELISA can
plastic bags. The hairs and scales are also also individually identify M. canis.
4.3 Epidermophyton 29
4.2.15 Treatment
4.3 Epidermophyton
In cats, the mild lesions recover spontaneously,
although the treatment can reduce the course of The earlier report of Epidermophyton in the
the disease and transmission risk into the human. scientific literature was detected in 1870 when
Topical application of antifungals is not so effec- Harz described tinea cruris due to Acrothecium
tive due to undetected exact position of lesions, floccosum. Sabouraud (1910) classified the
poor penetration of drugs through the thick hair causal agents of dermatophytosis into four
coat and lack of tolerance in some breeds. The groups, i.e. Achorion, Epidermophyton, Tricho-
systemic treatment should be followed at least phyton and Microsporum. In 1923, Ota and
for 10 weeks, preferably up to when the lesions Langeron finally proposed the nomenclature of
heal and become culture negative. The Epidermophyton floccosum which was originally
antifungals used in systemic treatment of cats described by Harz (1870). Emmons (1934)
include itraconazole (better tolerance with less modernised the classification system and divided
reported toxicity except anorexia), terbinafine all dermatophyte species into three genera,
(side effects include vomition, pruritus), ketoco- i.e. Microsporum, Trichophyton and Epider-
nazole (major adverse effects are liver toxicity, mophyton, and declared that the genus Achorion
vomition, diarrhoea, anorexia, contraindicated in is unnecessary.
pregnancy) and griseofulvin (anorexia, vomition,
diarrhoea, bone marrow suppression especially
in Siamese, Himalayan, Abyssinian cats). The 4.3.1 Morphology
drug of choice is itraconazole which is
administered by pulse therapy. The pulse therapy Epidermophyton produces septate hyphae like
includes administration of ketoconazole at 5 mg/ other dermatophytes with production of
kg body weight/day for 1 week in every 2 weeks macroconidia. No microconidia are produced.
which should be continued for 6 weeks. Use of The macroconidia are smooth, thick or thin
M. canis-based vaccine produces poor immunity walled with 1–9 septa, club shaped (clavate)
against the pathogen in cats. and occur singly or in a cluster of 2–6 cells
In human, treatment of tinea capitis due (Fig. 4.5).
to M. canis infection is difficult than the
infection with Trichophyton probably due to
ectothrix type of hair invasion and small size
of the spores which make them inaccessible 4.3.2 Classification
to the antifungals. Commonly used oral anti-
fungals against M. canis are griseofulvin All dermatophytes belonged to the phylum
(microlyophilised form) and terbinafine. The Ascomycota, class Euascomycetes, order
fungistatic itraconazole can be used in children Onygenales and family Arthrodermataceae. The
to treat kerion celsi type of tinea capitis along family Arthrodermataceae contains four morpho-
with topical antifungals and antibacterials, even logical anamorph genera such as Trichophyton,
with the poor response of M. canis to topical Microsporum, Epidermophyton and
antifungals. Chrysosporium. The genus Epidermophyton
Among plant-based antifungals, Neem possesses 2 species which are E. floccosum and
(Azadirachta indica A. Juss) seed and leaf E. stockdaleae. The type species and only patho-
extracts, Curtisia dentata (triterpenoids) extracts genic species under the genus is E. floccosum.
showed antidermatophytic activity against The ecological niche-based classification system
M. nanum and M. canis, respectively, in vitro. describes E. floccosum as anthropophilic.

Epidermophyton floccosum
Head, Department of
Mycology and
General Hospital of
4.3.3 Susceptibility to Disinfectants 4.3.5 Genome
The dermatophyte spores are susceptible to The information regarding the whole genome of
benzalkonium chloride, 1 % sodium hypochlorite, E. floccosum is currently not available. Although
enilconazole (0.2 %), formaldehyde, iodophors, the complete sequence of the 30.9 kb mitochon-
glutaraldehyde, phenolic compounds, benzyl- drial (mt) genome is reported, the major genes
ammonium bromide and ethoxyllauric alcohol. present in the mitochondrial genome include
The last two are especially effective against the reduced nicotinamide adenine dinucleotide-
anthropophilic dermatophyte such as E. floccosum. ubiquinone oxidoreductase (nad1, nad2, nad3,
Recent finding indicated that Candida nad4, nad4L, nad5, nad6), cytochrome oxidase
albicans releases volatile compounds such as (cox1, cox2, cox3), apocytochrome b (cob), ATP
dihydrofarnesol (R-DHF) and (2E, 6E) farnesol synthase (atp6, atp8, atp9), the small and large
(F-ol) which can prevent the growth of dermato- ribosomal RNAs (rns and rnl) and 25 tRNAs.
phytes such as T. rubrum, T. mentagrophytes, The order of the genes present in the mtDNA
M. canis and E. floccosum in a concentration of E. floccosum is similar with Trichophyton
dependent manner. E. floccosum is completely rubrum mtDNA with the exception of some
destroyed by 12.5 mcg/mL dihydrofarnesol. tRNA genes. The phylogenetic analysis confirms
T. rubrum as a close relative of E. floccosum.

E. floccosum is associated with human due to Characteristics
its anthropophilic nature. Rarely it is isolated
from dogs or other animals. The other species No specific growth requirement of Epider-
(E. stockdaleae) is soil associated and non- mophyton is detected. The conventional solid
pathogenic. The arthrospores (asexual spore) of medium includes Sabouraud dextrose agar with
E. floccosum produced within the human body cycloheximide and chloramphenicol. The derma-
may survive in the environment for prolonged tophyte test medium (DTM) is also suitable for
period. their isolation. The cultures are incubated at
It is worldwide in distribution. 25–30 C for 2 weeks. Some authors suggested
4.3 Epidermophyton 31
30 C as an ideal temperature for growth of 4.3.10 Pathogenesis

Epidermophyton.
The colonies of E. floccosum in Sabouraud After transmission of the spores, E. floccosum
dextrose agar are velvety, greenish-brown or prefers to germinate at the stratum corneum
khaki coloured with orange-brown reverse of the skin without further penetration in the
pigmentation, radial grooves and folded centre. immunocompetent hosts. The sporadic invasion
The older cultures show white-coloured tufts of is reported in immunocompromised patients
mycelium. (Behcet’s syndrome). Further, E. floccosum is
not known to invade hair also. However, surface
erosion of hair cortex and rare report of hair
parasitism was observed.
4.3.7 Antigenic Characteristics
The study indicated the presence of 31 proteins

in SDS-PAGE profile of E. floccosum ranging 4.3.11 Disease Produced
between 12.5 and 97.5 KDa. Among them,
20.1 and 18.4 KDa proteins are identified as E. floccosum causes tinea corporis, tinea pedis,
immunodominant antigens. tinea unguium, onychomycosis and tinea cruris
No common antigens have been demonstrated (occasionally) affecting inguinal areas particu-
between T. mentagrophytes, M. canis and larly in males.
E. floccosum. E. floccosum causes rare infection in animals.
The sporadic reports of its isolation from
dermatophytic lesions of dogs with or without
immunosuppression (chronic hyperadreno-
4.3.8 Virulence Factors corticism) and other animals are available.
E. floccosum are able to grow at 37 C and

can produce proteinase enzymes in vitro which 4.3.12 Diagnosis
are not well characterised. However, the gene
encoding zinc metalloprotease (Mep1) is 4.3.12.1 Clinical Specimens
reported to be present in the isolates. The lesion area should be cleaned with 70 %
ethanol to remove the bacterial contamination.
The scales from the active lesions should be
4.3.9 Transmission collected with a blunt scalpel (or unused tooth-
brush). For suspected onychomycosis, the nails
The anthropophilic dermatophyte such as should be cleaned with alcohol and affected nail
E. floccosum produces arthrospores within the bed should be exposed by removing the
hyphae in a vertebrate host (human or rarely onycholytic nail plate with a nail clipper. They
in animals) and macroconidia outside the are wrapped in sterile brown paper (or coloured
hyphae in the environment or artificial culture. paper) and are kept in a tight container preferably
The viability of the spores in the environment without moisture for transport into the labora-
is longer than the other dermatophytes. tory. The loam soil is the most useful sample
E. floccosum is transmitted by these spores for isolation of soil associated Epidermophyton.
between the hosts or from environment into the
host. The fomites such as brushes and clippers, 4.3.12.2 Laboratory Examination
and the common items used in showers and 1. Direct examination: The skin scrapings can be
gymnasiums play major role in transmission observed under microscope by wet mount
among human. with 10 % KOH preparation as described
earlier (Sects. 4.1 and 4.2). As E. floccosum water in the churches. In 1749, Reaumur first
never invades hair in vivo, no fluorescence is described avian aspergillosis in birds which was
detected in wood’s lamp examination. followed by report of similar syndrome in duck
2. Isolation and identification: Media and (Montagu 1813). The pathogenic Aspergillus
incubation condition as described earlier will (A. candidus) was detected in air sac lesion of a
serve the purpose. bullfinch in 1842 by Rayer and Montagene.
3. Molecular biology: The conventional diag- Whereas A. fumigatus was first detected in the
nostic PCR is employed targeting the DNA lung of a great bustard (Otis tarda) in 1863
topoisomerase II gene for detection of by Fresenius, who was the first to use the term
E. floccosum from cultures. Further, the ‘aspergillosis’ for this respiratory disease. The
uniplex PCR-ELISA can also specifically major members of aflatoxins were first detected
identify E. floccosum directly from clinical and its association with Aspergillus flavus was
specimens. The dermatophyte-specific single established in 1961 during investigation of
tube real-time PCR assay is also developed mysterious ‘turkey-X disease’, causing high
which can identify the individual dermato- mortality in turkey poults (Sargeant et al. 1961).
phyte species including E. floccosum directly In 1962, the name ‘aflatoxin’, using first letter
from clinical samples such as nails. from ‘Aspergillus’ and the first 3 letters from
‘flavus’, was proposed.
In India, Datta (1938) first reported the asso-
4.3.13 Treatment ciation of Aspergillus with bovine haematuria
affecting the kidney and bladder. Asthana
In tinea pedis and tinea cruris, topical application (1944) first isolated Aspergillus from the lungs,
of terbinafine cream once daily for 1 week and caeca, intestine, kidney, ovary and testes from
butenafine 1 % cream applied once daily for poultry in India. Pal (1996) first reported guttural
2 weeks can produce effective result. Systemic pouch mycosis in horses.
(oral) antifungal drugs (as described in Sects. 4.1
and 4.2) may be necessary in severe cases, or if
the infection does not respond to treatment or 4.4.1 Morphology
reappears.
The leaves and stem powder prepared from The mycelium is composed of septate hyphae
Commelina cyanea (indigenous plant common in (3–6 μm) with dichotomous branching. The
Northwest Cameroon) and ethanolic extract of primary branch originating from the vegetative
propolis (wax-like substance) were detected hypha is known as foot cell which is further
to have in vitro antifungal activity against branched into the conidiophores (300 μm in
E. floccosum. length and 5–8 μm in diameter). The condio-
phores contain flask-shaped vesicles, 20–30 μm
in diameter. The distal half of the vesicle is
4.4 Aspergillus covered with a single series of phialide, 6–8 μm
in length, arranged upwards paralleling the axis
Aspergillus (L. aspergillus – a special type of of the conidiophore. From these phialides, the
brush) is a filamentous, most widespread fungal chains of conidia (asexual spores) originate
genus containing both the pathogenic and bene- (Fig. 4.6). The conidial chain length occurs up
ficial species producing antibiotics, antifungals to 400 μm (A. fumigatus). In the tissues, only
and antitumour drugs. In 1729, Micheli first mycelium is observed, and in the body cavities
described Aspergillus who found the similarity filled with air (air sac, nasal passage), the
between the spore chain of the fungi with the conidiophores with the phialides are found. Dif-
brush (‘Aspergillum’) used for sprinkling holy ferent species of Aspergillus can be identified on
4.4 Aspergillus 33
Fig. 4.6 Conidial

arrangement of Aspergillus
(schematic diagram).
a Hypha, b footcell,
c conidiophores, d vesicle,
e phialide, f conidia
the basis of their microscopic morphological The sialic acid helps in attachment of the conidia
appearances. with the extracellular matrix. The conidia are
The cell wall of Aspergillus (A. flavus) consists produced in large numbers, and due to their
of glycoproteins, β-(1, 3) glucan, β-(1, 6) glucan, small size (2–3 μm) and hydrophobicity, they
galactomannan and chitin. In A. fumigatus, the can remain viable in the air for prolonged
cell wall is composed of β-(1, 3) glucan, period. The mean concentration of Aspergillus
β-(1, 3/1,4) glucan, β-(1, 6) glucan, α (1, 3) glu- conidia in air is 0.2–15 conidia/m3 and is up to
can, chitin, mannan, β-(1, 5) galactofuranose, 106 conidia/m3 in some agricultural settings.
galactomannan (GM) and galactomannoprotein. In the favourable environment, the conidia can
These components are cross-linked with several swell and germinate into the hyphae.
other proteins. The cell wall is anchored with the Most of the Aspergillus does not have any
underlying cell membrane by glycosylphosphati- sexual stage of the growth (Fungi imperfecti)
dylinositol (GPI)-binding protein. Sometimes out- except A. fumigatus (teleomorph Neosartorya
side the cell wall, a hydrophobic layer is found fumigata), A. oryzae and A. nidulans which can
both in hyphae and conidia. produce sexual spores (ascospores). In both
The conidia or asexual spores are pigmented, A. fumigatus and A. oryzae, two opposite mating
echinulate, globose to subglobose. In addition to types (idiomorphs, MAT1-1 and MAT1-2)
the hydrophobic layer outside the cell wall, the are commonly found near close vicinity with
conidia also contain a melanin layer. The sialic the same frequency of occurrence (1:1). The
acid, composed of unsubstituted N-acetyl MAT1-1 idiomorph is associated with increased
neuraminic acid linked with galactose by α-2, virulence and higher resistance against
6 bond, is detected in the conidial surface. antifungals.
4.4.2 Classification A. flavus follows a life cycle consisting of a

saprophytic phase which occurs in soil, plant
Aspergillus belongs to Trichocomaceae family debris, tree leaves, decaying wood, animal
under the order Eurotiales and Phylum fodder, cotton, compost piles, dead insect and
Ascomycota. The family contains another impor- animal carcasses, outdoor and indoor air environ-
tant genus known as Penicillium. Currently, ment (air ventilation system), stored grains and
the Trichocomaceae family is proposed to even in human or animal patients. The fungi can
be further divided into three subfamilies also act as opportunistic pathogen of plants and
such as Aspergillaceae, Trichocomaceae and animals including human. In the saprophytic
Thermoascaceae. The genus Aspergillus contains phase, they are present either as mycelium or a
more than 250 species grouped into subgenera heavily melanised survival structures called
and species complex. There are eight such sub- sclerotia. In favourable environment, they pro-
genera, i.e. Aspergillus, Fumigati, Circumdati, duce conidiophores with conidia which are trans-
Candidi, Terrei, Nidulantes, Warcupi and mitted by air or any other means into the plants.
Ornati. The important species complexes are After the establishment of the infection in the
A. fumigatus, A. flavus, A. terreus, A. niger, susceptible host, aflatoxins are produced. It is
A. nidulans and A. ustus. Molecular studies most prevalent in warm regions, especially
revealed several cryptic species (hidden species between latitudes 35 N and 35 S. Other species
that are morphologically indistinguishable) of Aspergillus are worldwide in distribution.
within the Aspergillus species complex. For
example, Neosartorya peudofischeri, Aspergillus
lentulus, A. viridinutans and A. fumigatiafinnis 4.4.5 Genome
are cryptic species described under A. fumigatus
species complex. The genome of Aspergillus (A. flavus) contains
eight chromosomes. The genome size is 36.3
Mbp with 13,071 predicted genes. A 75 Kbp
4.4.3 Susceptibility to Disinfectants region in the chromosome consisting 29 gene
clusters is responsible for the aflatoxin biosyn-
Aspergillus is susceptible to disinfectants thesis. Although there are some genes present
such as 35 % ethanol (A. niger) and copper- outside the cluster which also helps in aflatoxin
8-quinolinolate (0.4 mcg/mL). A. niger is highly biosynthesis. The cluster is composed of a single
sensitive to radiation such as gamma and X-ray gene encoding polyketide synthases (PKS),
(500 and 100 Krad), whereas lower dose of radi- five genes encoding cytochrome P450 mono-
ation (1–5 Krad) can stimulate the sporulation oxygenases and genes for global regulation
of the fungi. [mitogen-activated protein kinase (MAPK),
MAPK kinase (MAPKK) and MAPKK kinase
(MAPKKK), signal transduction, pathogenicity,
4.4.4 Natural Habitat and Distribution oxidative stress and fungal development.
The genes encoding for different enzymes
Aspergillus is commonly found in soil, air, water such as amylase, cellulase, pectinases, proteases,
(fresh and marine), indoor environment (dust, chitinase, chitosanases and pectin methyl-
above the false ceiling in a room) vegetables and esterases have been also identified in the
feed as saprophytes. Some unusual habitats are genome.
also observed for Aspergillus (A. carneus, The genome of A. fumigatus is 29.4 Mbp
A. flavus, A. nidulans, A. niger, A. terreus) such containing 9630 genes, most of them have
as ‘microbial mat’ under the fresh water or hyper- unknown function. Deep sequencing study of
saline water (0–35 % salt concentration). They are A. fumigatus mRNA (RNA seq) revealed tens
common contaminants in the laboratory. of unannotated and hundreds of novel genes
4.4 Aspergillus 35
encoding small proteins which are involved in composed of mannose with side chains of
colony growth and establishment of different β (1, 5)-linked galactofuranosyl residues. It is a
clinical forms of aspergillosis. part of cell wall along with chitin. It is released
through the pores at the growing hyphal tips
during logarithmic growth phase of the fungi in
4.4.6 Isolation, Growth and Colony highest amount which helps in the detection
Characteristics of the antigen for diagnosis. GM is found in
other fungi including Penicillium, Fusarium,
Aspergillus can be isolated in Sabouraud Alternaria, Histoplasma and yeasts including
dextrose agar (SDA) with or without chloram- Cryptococcus which can produce antigenic
phenicol (0.05 g/L) and other common bacterio- cross-reaction.
logical media such as blood agar. The The cell wall of Aspergillus contains 1, 3 β D
cycloheximide can inhibit the growth. The plates glucan (BDG) which is also secreted during late
are incubated at 37 C for 4–5 days. The species logarithmic growth phase of the fungi. The BDG is
which cannot tolerate this temperature can be present in the cell walls of many pathogenic fungi
incubated at 25 C. Some species, such as including Candida, Cryptococcus, Fusarium and
A. fumigatus, is thermophilic which is able to Pneumocystis producing antigenic cross-reaction.
grow at 55–75 C. Heat shock protein (Hsp1/Asp f 12) is also
The colonies of A. fumigatus are white cot- detected as an immunodominant antigen in
tony which becomes velvety or granular with A. fumigatus which plays role in protective
green colouration after several days (Fig. 4.7). immunity.
Whereas A. flavus colonies are primarily cot-
tony and later become ‘sugary texture’ with
yellowish green colour, A. niger colonies are 4.4.8 Toxins
white in colour in the primary stage which later
becomes black due to production of black- The mycotoxins are generally produced during
coloured conidia. Similarly, A. terreus colonies hyphal growth and conidiogenesis which are
are initially white which later become cinnamon- secreted in the environment or released after the
buff coloured with ‘sugary texture’. death of the hyphae. Conidia may also contain
the preformed mycotoxin. A. fumigatus produces
many toxins as secondary metabolites.
4.4.8.1 Aflatoxins
Galactomannan (GM) is the major antigen of Aflatoxins are difuranocoumarin compounds
Aspergillus which is a carbohydrate molecule produced by different species of Aspergillus as
Fig. 4.7 Growth of

Aspergillus fumigatus
in SDA
secondary metabolite. It has six major types such characterised by a disulphide bridge across a
as B1, B2, G1, G2, M1 and M2. Aflatoxin M1 piperazine ring which is essential for their toxic-
and M2 are produced as metabolites of B1 and ity. It can inhibit macrophage and neutrophil-
B2, and they are commonly found in milk and to mediated phagocytosis, mitogen-activated
some extent in eggs. Among different species T cell proliferation, mast cell activation, cyto-
Aspergillus flavus is the chief producer which toxic T cell response and reactive oxygen species
can affect many agricultural crops such as production. Further, the gliotoxin causes mono-
maize, cotton, groundnuts (peanuts), Brazil cyte apoptosis, inhibition of epithelial cells cili-
nuts, pecans, pistachio nuts and walnuts. Both ary movement and epithelial cell damage. There
preharvest and postharvest contamination of is a putative 12-gene cluster which encodes
these crops with aflatoxins is common. However, the toxin. Among the gene cluster, gliZ and gliP
other species such as A. fumigatus can produce play major role.
aflatoxin B1 and G1.
Among the six types of aflatoxin, B1 (AFB1) 4.4.8.3 Ochratoxins
is the most potent toxin and carcinogen than In tropical countries, ochratoxins are mainly
others in human and animals including birds, produced by A. ochraceus and A. carbonarius.
fish and rodents. The chronic intoxication can A. ochraceus are mostly associated with dried
cause immunosuppression, malnutrition, and stored foods (cereals). Whereas A. carbo-
centrilobular necrosis and fatty infiltration of narius is commonly found in fruits (grapes) that
the liver including hepatomas and degeneration mature in sunlight and at high temperature, the
of respiratory system. Maximum permissible ochratoxin is considered as potent nephrotoxic,
limit of total aflatoxin and M1 (AFM1) is carcinogenic (human carcinogen of 2B group),
20 ppb in feed and food for human and 0.5 ppb genotoxic and immunotoxic.
in milk, respectively, as guidelined by the
US Food and Drug Administration (FDA). 4.4.8.4 Citrinin
European Commission has set the limit at It is a fungal metabolite which was first isolated
15 ppb for total aflatoxin in groundnuts. from Penicillium citrinum. Other fungi such as
The aflatoxin synthesis genes are located by A. terreus can also produce citrinin. It is fre-
making a cluster in the genome of A. flavus. The quently detected in feed stuffs or food along
major regulator gene is aflR, located in the centre with ochratoxin A, and it can increase the toxic-
of the cluster. Major transcription regulator gene ity of ochratoxin synergistically. The intoxica-
is aflS (aflJ) located adjacent to the aflR. Other tion with both of the toxins together may cause
unrelated genes such as laeA and veA are endemic nephropathy. Further, citrinin is also
involved in global regulatory role in aflatoxin embryocidal and foetotoxic.
biosynthesis. Many nutritional factors such as
carbon, nitrogen, amino acid, lipid and trace 4.4.8.5 Ribotoxins (Restrictocin,
elements control aflatoxin biosynthesis. Other Mitogillin)
regulatory factors required for aflatoxin synthesis The ribotoxins are produced by A. fumigatus
includes the temperature (28–35 C), acidic pH, which specifically targets the sarcin/ricin domain
hot weather and drought conditions, sporulation of 28S rRNA and inhibits the protein synthesis.
and sclerotia formation of the fungi and oxidative The toxins are encoded by asp f1/mitF/res gene.
stress. Certain plant metabolite such as n-decyl Mitogillin is highly cytotoxic causing cell death
aldehyde is inhibitory to aflatoxin production. even in low concentration.
4.4.8.2 Gliotoxin 4.4.8.6 Haemolysin

It is the major and most potent toxin produced A. fumigatus produces a haemolysin which is
by 95 % strains of A. fumigatus isolated from encoded by aspHS gene. The toxin can lyse the
both clinical cases and environment. It belongs rabbit and sheep red blood cells (RBC),
to the family of epipolythiodioxopiperazines, macrophages and endothelial cells.
4.4 Aspergillus 37
4.4.8.7 Helvolic Acid and Fumagillin 4.4.11 Pathogenesis

Helvolic acid belongs to a group of steroidal
antibiotic, known as fusidanes. It is produced 4.4.11.1 A. fumigatus
by A. fumigatus. It can inhibit the oxidative Healthy animals and human are exposed to the
burst of macrophages and metabolism of Aspergillus spores because the mean concentra-
low-density lipoproteins, and it can induce tion of conidia in air is 0.2 to 15/m3 and is up to
ciliostasis and rupture of epithelial cells. 106/m3 in some agricultural settings. In spite of
Fumagillin is an antitumour antibiotic which this constant exposure, the animals or human do
can inhibit angiogenesis, endothelial cell prolif- not suffer regularly which suggests that inhala-
eration and ciliary movement in the respiratory tion of spores in large numbers is required to
epithelium. establish the infection. After entry within the
host through inhalation, the conidia encounter
4.4.8.8 Fumitremorgin (A,B,C) and with the respiratory mucosa comprised of
Tryptoquivaline A mucus, proteins, lipids, ions, water and other
These are neurotropic toxins produced by cellular secretions that contribute to the
some strains of A. fumigatus. They can cause mucociliary clearance. The conidia are engulfed
tremor, seizure and abnormal behaviours in by tracheal epithelial cells, alveolar type II cells
experimental mice. and nasal epithelial cells. Most of them are killed
by the phago-lysosome, although the remaining
conidia are capable to germinate and exit the
phago-lysosome. Conidial sialic acids act as
4.4.9 Virulence Factors ligand for adherence with the alveolar epithelial
cells especially with fibrinogen and fibronectin,
The virulence factors possessed by Aspergillus commonly found in the wounded epithelial
are described in Table 4.11. surfaces. So lung injury acts as major
predisposing factor for causation of invasive
aspergillosis. Further, the gliotoxin, fumagillin
4.4.10 Transmission and helvolic acid produced by the fungi cause
damage in the respiratory mucosa and slow cili-
Inhalation of contaminated dust or ingestion of ary movement facilitating the attachment of the
contaminated feed with the fungal spores is the conidia.
major route of transmission in animals and poul- After penetration of the respiratory mucosa,
try. Intramammary inoculation of spores may the conidia are deposited into the alveolar space.
cause mastitis in cattle. In poultry farm, the In the poultry, air sac is the primary target organ
spores are introduced with the contaminated of the fungi because the inhaled air reaches the
feed or litter. In moist environment, they germi- air sacs prior to the epithelial surfaces of the
nate and produce more numbers of spores which lungs. Later the conidia reach the lung paren-
are inhaled by the birds. The poor ventilation and chyma and are embedded in the atria or infundib-
sanitation, humidity and long-term storage of ulum. The conidial maturation begins which
feed in a farm may increase the spore concentra- causes loss of hydrophobic layer and exposure
tion in the air. The predisposing factors are of the inner cell wall. The cell wall is composed
impaired immunity or stressed condition of the of galactomannan, chitin and β-glucan which act
birds due to administration of antibiotic or as ligand for the soluble and cell-associated pat-
steroids, vaccination, metabolic bone disease, tern recognition receptors (PRR). The soluble
inadequate diet resulting hypovitaminosis A, receptors are a family of C-type lectins including
overcrowding, shipping, starvation, thermal dis- lung surfactant proteins A and D and mannan-
comfort, inbreeding, toxicosis, traumatic injuries binding lectin (MBL). They act as opsonin and
and reproductive activity. can bind the A. fumigatus cell wall carbohydrates
Table 4.11 Virulence factors of Aspergillus

Virulence factors Function
Conidia Due to its small size, thermotolerance, resistance to
oxidative stress and high growth rate, they can easily
transmit and establish the infection within the host. The
pigmentation of the conidia can reduce C3 complement
deposition and neutrophil activation
Autophagy related gene (ATG) Autophagy helps the fungi to survive under the nutrient
stress condition by helping in conidiation, hyphal foraging
and maintenance of metal-ion homeostasis
α-1,2-mannosyltransferase (encoded by kre2 afmnt1) A putative virulence factor related with thermotolerance
(48 C) of A. fumigatus. In mutants, cell wall instability is
observed which causes leakage at the hyphal tips and
inhibition of growth at 48 C
cgrA (nucleolar protein, encoded by cgrA) It helps in ribosome biogenesis at 37 C. The mutants are
less virulent
Heat shock protein (Hsp1/Asp f 12) It acts as molecular chaperone and helps in protein
transport and growth at 37 C. It can produce stress
response during inflammation
Fks1 (encoded by fks1) It is the catalytic subunit of glucan synthase enzyme
involved in synthesis of β-glucan, essential for the fungal
growth
Chitin synthase (encoded by chsA, chsB, chsC, chsD, chsG is the only virulence factor involved in synthesis of
chsE, chsF, chsG) chitin. The mutants show reduced virulence
Galactomannan (GM) It helps in adhesion to host components such as fibronectin,
laminin, pentraxin3 and other surface receptors of
macrophages, dendritic cells and langerhans cells. The
released GM is used in commercial antigen detection kit.
The galactomannan along with the α-glucans forms a
polysaccharide matrix which helps in formation of
biofilms. The biofilms resist the antifungals and
concentrate the enzymes produced during the growth
which helps in further tissue invasion
Glycosylphosphatidylinositol (GPI) anchor The GPI anchor is required for cell wall integrity,
morphogenesis and virulence of the fungi
Phosphomannose isomerase enzyme (Pmi1) It is required for both cell wall synthesis and energy
production in A. fumigatus, but its actual role in virulence
is not elucidated
Rodlet Rodlet is a microfibril filled with proteins (hydrophobins),
encoded by rodA, rodB and others. These proteins help in
conidial dispersion, adhesion of the hyphae with the soil
surface or lung epithelium
Melanin It is a pigment which protects the genome of conidia from
ultraviolet light, enzymatic lysis and oxidation. It also
helps in survival of the conidia in the environment. In vivo,
the melanin can protect the conidia from complement-
mediated lysis and reactive oxygen species
Catalases (catA, Cat1/catB, catC, catE and cat2) and It helps in detoxification of reactive oxygen species (ROS)
superoxide dismutases (SODs cytoplasmic Cu/Zn SOD produced by macrophages and neutrophils
(Sod1),mitochondrial MnSOD (Sod2),cytoplasmic
MnSOD(Sod3) and (Sod4)
Protease (alkaline protease, elastase), aspergillopepsin They degrade the structural barrier (collagen, elastin,
(aspartic protease, Pep), Dipeptidyl peptidases (Dpp) fibrinogen, casein) in the host to obtain the nutrients
The Dipeptidyl peptidases (Dpp) can degrade collagen,
hormones and cytokines. They have T cell modulation
property
(continued)
4.4 Aspergillus 39

Virulence factors Function
Phospholipase (A–D) They can break the ester bond of phosphoglycerides and
thus destabilised the cell membrane to cause cell lysis
Trehalose biosynthetic proteins (encoded by tpsA, tpsB) They are associated with germination of conidia at 37 C
and inhibition of oxidative stress
Siderophores (fusaricine C, triacetylfusaricine C, They are low molecular weight proteins (Mr < 1,500),
ferricrocin and hydroxyferricrocin, encoded by different involved in iron uptake for the fungi. They are ferric iron
sid genes) specific and high affinity chelators. The siderophores are
also required for germ tube formation, asexual sporulation,
resistance to oxidative stress, catalase activity and
virulence
Zinc transporter protein (encoded by ZrfA-C) It affects the germination of conidia and growth capacity
under zinc-limiting conditions
cAMP-PKA (cAMP-protein kinase A) signalling pathway The mutants are hypovirulent having reduced growth,
(encoded by pkaC1 gene) germination rate and increased susceptibility to oxidative
stress
Ras proteins (RasA, RasB, RhbA) Ras proteins are monomeric GTPases which act as
molecular switches that transduce signals from the outside
of the cell to signalling cascades inside the cell. The rasB
mutants are hypovirulent having reduced growth and
germination rate
Histidine kinase (fos1, tcsB) It is involved in signal transduction under stress condition.
The fos1 mutants are remarkably hypovirulent
Calcium signalling pathway (calA/cnaA) It is involved in stress response, mating, budding and
tolerance to antifungal drugs. The cnaA mutant has
decreased filamentation, defective conidia and reduced
virulence
Cross pathway control (CPC, cpcA) signalling pathway This signalling pathway is involved in amino acid
homeostasis under environmental stress. The cpcA mutants
are hypovirulent
Transcription factor Ace2 It is associated with conidia formation, pigmentation and
virulence
Development-regulated protein (MedA) It is associated with adherence, host interaction and
virulence
Unfolded protein response (UPR) It is a stress response sensor associated with protein-
folding activities in endoplasmic reticulum. Among the
three sensors (IRE1, ATF6 and PERK) identified in
eukaryotes, IREA is so far reported from A. fumigatus,
A. niger and A. nidulans. It is regulated by ireA, hacA
genes. The mutants show increased cell wall fragility,
impaired secretory system that is unable to extract
nutrients from host tissues and lack of growth in hypoxic
condition (ireA mutant)
Dol-P-Man:protein O-mannosyltransferases (PMT) This enzyme helps in O-mannosylation of the proteins in
endoplasmic reticulum which is required for the
construction and maintenance of the cell wall. Loss of pmt
gene is lethal for A. fumigatus
Ubiqutin encoding genes (UBI1, UBI4) They help in stress and metabolic adaptation
in a calcium-dependent manner. This binding involved in recognition of conidia and hyphae.

enhances phagocytosis by the alveolar Most of the conidia are killed by reactive oxygen
macrophages, whereas the cell-associated species (ROS) or acidified phago-lysosome pro-
receptors such as toll-like receptors (TLR2, duced within the alveolar macrophages.
TLR4) and dectin-1(ligand β-glucan) are Non-oxidative mechanism may also contribute
in killing of the conidia. In immunosuppressed adaptation in hypoxic condition, etc. Production

animals having defective alveolar macrophage of different toxins such as gliotoxin, ribotoxins
function, the conidia are able to escape the and haemolysin also help in the establishment of
phago-lysosome-mediated killing. The virulence infection by preventing cellular effector function
factors of Aspergillus such as melanin, rodlets and causing cellular apoptosis.
and superoxide dismutase can protect the conidia A. fumigatus growing hyphae invades the
from reactive oxygen species. Other blood cells endothelial cell lining of blood vessels
such as platelets can also damage the (angioinvasion) from the albuminal side to the
A. fumigatus conidia by releasing serotonin. luminal side. The angioinvasion commonly
The conidia which survive from this first line occurs during neutropenia. During the invasion,
of defence can germinate. The germination part of hyphae may break off and invade the
involves conidial swelling (isotropic growth) endothelium of the opposite side. Haemato-
followed by protrusion of germ tube (polar genous dissemination of the hyphae into vital
growth). The polar growth occurs along with organs may take place. During angioinvasion,
mitotic nuclear division followed by a septum thrombosis and infarction occur creating a
formation at the base of the germ tube. Several necrotic focus (plaque) where the fungi can
such septi are formed after completion of each grow. The hyphae are not effectively phago-
mitotic cycle to create elongating hyphae which cytosed by the macrophages due to their larger
can invade the tissues. This germination depends size. The neutrophils aggregate around the
on nutrient sensing, acquisition and biosynthetic hyphae and damage them by reactive oxygen
pathways for obtaining the nutrients from the species and a variety of antimicrobial compounds
host. Under the nutrient-limited condition and such as proteases, defensins, lysozyme and
other stress factors, the protein-folding capacity lactoferrin released from their granules. The
of the fungal endoplasmic reticulum reaches its neutrophils can also inhibit the hyphal growth
maximum limit and the unfolded protein by the lactoferrin sequestration of iron, an essen-
response (UPR) initiates. The enzymes such as tial nutrient for fungal growth. The dying
elastase, aspartic proteinase, serine protease and neutrophils produce an extracellular trap with
metalloprotease produced by Aspergillus help in the nuclear DNA and several antifungal proteins
the breakdown of host tissue for nutrient acquisi- which can also inhibit the hyphal growth.
tion and invasion. For survival and virulence of
the fungi in vivo, there is a requirement for
biosynthesis of uracil/uridine, folate and lysine. 4.4.12 Disease Produced
Aspergilli can also utilise a wide range of nitro-
gen sources in which environmental nitrate is The major animal and human diseases produced
taken and converted into ammonium and subse- by different species of Aspergillus are enlisted
quently glutamine and glutamate. They degrade in Table 4.12.
the host proteins to obtain amino acids and other
nutrients or they can synthesise the amino acids
from carbon- and nitrogen-containing precursors. 4.4.13 Immunity
This synthesis is regulated by cross pathway
control (cpcA) locus. A. fumigatus can also The innate immune system recognises Aspergillus
uptake the iron with the help of siderophores or through either soluble receptors [C-type lectins,
through the reductive iron assimilation. Besides mannose-binding lectin (MBL), pentraxin
nutritional requirements, A. fumigatus also 3 (PTX3)] or the cell-associated pattern recogni-
adapts with the in vivo environment by the tion receptors (TLR 2, 4 and dectin1). Recently,
expression of different factors such as pH regu- intracellular NOD-like receptor (NOD2 and
lator (PacC) for adaptation in alkaline pH, sterol- NLRP3) is also found to recognise Aspergillus.
regulatory element-binding protein (SrbA) for Generally the cell wall carbohydrates of the fungi
4.4 Aspergillus 41
Table 4.12 Major diseases of animals and human caused by Aspergillus sp.
Fungi Host Disease
A. fumigatus, A. flavus, A. niger, Poultry (poultry are relatively more Avian aspergillosis/brooder pneumonia
A. glaucus, A. nidulans susceptible due to differences in The disease is characterised by rhinitis,
(A. fumigatus is the major cause anatomical, physiological and mycotic keratitis associated with
probably due to smaller size of the respiratory immune system blepharospasm, photophobia, periorbital
spores than other species which compared with other mammals) swelling, turbid discharge, swollen and
helps in easy transmission) adhered eyelids, cloudy cornea and cheesy
yellow exudates within the conjunctival sac,
lung congestion, unilateral drooping of the
wing due to infection of the thoracic air sac,
clavicular air sac or the proximal humerus,
repeated vomition due to lesions in the
anterior air sacs. The respiratory signs are
not regularly detected. In postmortem
examination, green/yellow/white necrotic
foci are observed in the lung and other
organs
A. fumigatus Horse (a) Guttural pouch mycosis
Necrotizing inflammation and formation of
diphtheritic membrane in guttural pouch
(auditory tube diverticulum). Clinical signs
include epistaxis, unilateral purulent nasal
discharge, abnormal head position.
It occurs most commonly in stabled horses
during summer
(b) Nasal granuloma
(c) Corneal ulcer
A. fumigatus Dogs (a) Canine sinonasal aspergillosis/canine
rhinitis
The disease is characterised by persistent
sneezing and nasal discharge which is
unresponsive to antibiotics. The infection
involves paranasal sinuses, nasal cavity,
turbinate bones and CNS. Golden Retrievers
and Collie breeds are more susceptible
(b) Otitis externa
(c) Chronic rhinitis
A. fumigatus Human Invasive aspergillosis
A disease of the respiratory tract which
involves the lung parenchyma, pleura,
trachea and bronchi. It is common in the
patients with haematological malignancy,
prolonged antibiotic users or stem cell
transplant recipients
A. fumigatus Cattle, horse Sporadic abortion especially during winter
which is associated with hay feeding. The
fungi invade the foetus and produce
cutaneous lesions
A. fumigatus Cattle (a) Mastitis (abscess in udder)
(b) Pneumonia
Aspergillus sp. Calves Mycotic gastritis
Aspergillus sp. Horses Keratomycosis
It is characterised by corneal opacity,
ulceration and formation of endothelial
plaques in the centre of the cornea
(β-glucan, GM, chitin) act as ligand (pathogen- receptor for advanced glycation end products
associated molecular pattern, PAMP). The soluble (RAGE) to activate the T cells. The epithelial
receptors act as opsonin which enhances phagocy- cells in addition express the pattern recognition
tosis by the alveolar macrophages. Pentraxin receptors for fungal recognition and as a conse-
(PTX3) specifically binds the conidia (not hyphae) quence induce the T cell tolerance.
and enhances their phagocytosis by dendritic The activated Th1 cells secrete IFN γ which
cells and alveolar macrophages. After the compat- helps in the clearance of the hyphae and produces
ible binding of the fungi and cell-associated a protective immunity. Whereas activation of
PRRs, the cytoplasmic tail attached with the Th2 cells causes the secretion of IL4, IL5 is
receptors which contains an immunoreceptor responsible for fungal allergy in the host and it
tyrosine-based activation (ITAM)-like motif gets prevents the clearance. Th17 cell activation
phosphorylated. This phosphorylation activates correlates with Aspergillus infection during
several pathways to stimulate the phagocytes immunodeficiencies. The Th17 cells secrete
for killing of the conidia through the produc- IL17 and IL22 which recruits neutrophils. Fur-
tion of NADPH-dependent reactive oxygen ther, fungal growth, inflammatory immunity and
species (ROS). The phosphorylation also acti- tolerance to Aspergillus are controlled by the
vates the NFκB and stimulates the secretion of activation of natural (n) Treg cells. This kind of
pro-inflammatory cytokines (IL12, TNFα). The Treg cells can suppress the neutrophils through
cytokines help in the recruitment of the immune the combined actions of IL10 and CTLA4 acting
cells at the site of the infection. Further, on dendritic cells to produce indoleamine
A. fumigates hyphae (not conidia), through the 2, 3-dioxygenase (IDO). This enzyme helps in
production of ROS, can stimulate NLRP3 controlling inflammation, infection and allergy.
inflammasome to release active IL1β which can Thus, protective immunity to Aspergillus is
recruit more amount of neutrophils. These dependent on balanced act of Treg cells over
neutrophils produce a neutrophil entrapment effector Th1 and Th17 cells, with the contribu-
traps (NETs) to engulf the larger hyphae which tion of IDO.
are not phagocytosed by the macrophages.
The innate immune system also influences
the subsequent development of T cell-mediated 4.4.14 Diagnosis
adaptive response. The adaptive response is
initiated with the dendritic cells which can engulf 4.4.14.1 Clinical Specimens
the conidia or hyphae to carry them into the The clinical specimens from animals or poultry
draining lymph nodes. The conidial engulfment include mastitic milk, foetal stomach contents,
occurs through the ‘coiling phagocytosis’ in ear swabs, skin scrapping and biopsies from
which the dendritic pseudopods rotate around nasal granuloma and plaques in the guttural
the conidia to form a layer following the binding pouch. The specimen from human is lower respi-
of the mannose receptor DC-SIGN and comple- ratory tract sample by bronchoalveolar lavage.
ment receptor 3 (CR3). Whereas the hyphal After postmortem, pneumonic lung, nodules
engulfment occurs through ‘zipper phagocytosis’ from vital organs may be collected. The small
after the binding of Fc receptor (as ligand) and tissue samples may be wrapped in a piece of
complement receptor 3 (CR3), this kind of paper before putting into the 10 % formalin.
phagocytosis occurs following the contour of
the fungi which further activates the 4.4.14.2 Laboratory Examination
IL4-producing CD4 T cells in the spleen and 1. Direct examination:
mediastinal lymph nodes. The dendritic cells The clinical specimens especially the tissue
can also recognise the damage-associated molec- scrapings should be cleared with 10 % KOH
ular pattern (DAMP) of the fungi through the and are observed under the microscope.
4.4 Aspergillus 43
Fig. 4.8 Vesicle

of Aspergillus flavus
(Lactophenol cotton blue,
100)
Histopathological staining can be performed 3. Detection of antigen:

with periodic acid–Schiff (PAS), Grocott’s Galactomannan (GM) is the predominant anti-
silver or methenamine silver stain for detec- gen released by A. fumigatus in the circulation
tion of tissue invasion. In the tissue section, during angioinvasion which can be detected by
Aspergillus hyphae are narrow and septate latex agglutination test, sandwich ELISA in
which are not easily distinguishable from serum or urine samples. The detection limit
other fungi. of both the tests is 15 ng/mL and 1 ng/mL,
Different species of Aspergillus has char- respectively. However, the GM detection assay
acteristic fruiting body structures through is not specific for Aspergillus as it is cross-
which they can be identified by an experi- reacting with other fungi such as Penicillium,
enced person. A. fumigatus conidiophores Fusarium, Alternaria and Histoplasma.
have a foot cell at their bases. The dome- Further, recent investigations revealed the
shaped vesicles contain phialides in their presence of GM in tazobactam antibiotic
upper portion. From the phialides, long chain preparations, nutritional supplements and
of conidia is borne which may sweep inwards, electrolyte solutions which may produce
whereas in case of A. flavus, the vesicles are false-positive reaction especially in human.
round in shape with the phialides containing Similarly, 1, 3 β D glucan (BDG) can be
spores present on the entire surface (Fig. 4.8). detected for identification of Aspergillus.
In A. niger, vesicles are spherical which However, it is produced by a lot of other
bear large metulae supporting small phialides. fungi such as Candida, Fusarium, Pneumo-
The conidia are black in colour. cystis, etc. So the test can predict the general
2. Isolation and identification: Media and incu- fungal infection rather than specifically asper-
bation condition as described earlier will gillosis. There are several BDG assay kits
serve the purpose. However, it is the most available for use in human such as Fungitell
common fungal contaminant in the laboratory kit using amoebocyte lysates from Limulus
and it is routinely isolated from respiratory polyphemus and Fungitec-G kit using reagents
tract of healthy animals. So repeated isolation from Tachypleus tridentatus.
from the clinical specimen is required, along 4. Molecular biology: Confirmation of
with correlation with the history, clinical A. fumigatus isolates by PCR-RFLP can be
signs and histopathological observations for performed using BccI, MspI and Sau3AI
proper diagnosis of clinical aspergillosis. restriction enzymes.
4.4.15 Treatment like superficial lesions at the border area of

Cooch Behar (West Bengal) and Bhutan
In poultry the treatment can be performed by (Ganguly 1925). Panja (1925) also diagnosed a
topical application of antifungal after removal case of generalised blastomycosis in scrapings
of thoracic granuloma followed by systemic anti- from the cutaneous nodular lesions. Further, the
fungal therapy. However, removal of granuloma blastomycosis is reported from insectivorous bats
from the respiratory tract is difficult. In that case, (Khan et al. 1982), male Mongrel dog (Iyer 1983)
only topical application can be performed by and migratory birds (Rawal et al. 1988).
nebulisation, nasal or air sac flushing, or surgical
irrigation of the abdominal cavities. For topical
application, amphotericin B (1 mg/kg body 4.5.1 Morphology
weight, 10–14 days, nebulisation), enilconazole
(0.1 mL/kg body weight, 5 days, nebulisation), Blastomyces dermatitidis is a thermally dimor-
miconazole and terbinafine can be used, whereas phic fungus exhibiting the mycelial form at room
for systemic application, amphotericin B temperature and yeast form at 37 C. The myce-
(1.5 mg/kg body weight, 3–5 days, intravenous), lial form consists of the branching hyphae which
fluconazole (5 mg/kg body weight, 7 days, oral or are 2–3 μm in diameter. The conidiophores car-
intravenous), itraconazole (5–15 mg/kg body rying single terminal conidia are present at the
weight, 5–21 days, oral), ketoconazole right angle of the hyphal branches which
(10–30 mg/kg body weight, 21 days, oral), resembles lollipops (Fig. 4.9). The conidia are
voriconazole or 5-fluorocytosine can be used. round or oval in shape, 2–10 μm in diameter and
Resistance to antifungals is increasingly prefer to enter the susceptible host through
reported especially from human patients since inhalation.
1997 when the first published case of The yeast cells are usually 8–15 μm in diame-
itraconazole-resistant A. fumigatus appeared. ter, encapsulated with a thick refractile cell wall
The itraconazole and other triazole compounds and contain 8–12 nuclei. They reproduce by a
bind lanosterol (encoded by CYP51) and prevent single broad-based bud, with the daughter cell
the synthesis of ergosterol from it. Point mutation often as large as the mother cell before detach-
in CYP51 is detected to be responsible for the ment (Fig. 4.10). The bud is attached with the
resistance. However, resistance to amphotericin mother cell with a persistent cell wall and a wide
B is limited among the Aspergillus isolates pore between them. This characteristic feature is
except A. niger. useful for identification of the yeast in the clini-
cal specimens.
The mycelium to yeast phase conversion
4.5 Blastomyces depends on increased temperature and hybrid
histidine kinase (DRK1) which acts as
In 1894, Thomas C. Gilchrist first recognised
blastomycosis in a patient with cutaneous lesion
in Baltimore, USA. In his honour, the infection
was named as Gilchrist’s disease. He first
described the causative agent as a protozoon
which was disproved by him and Stokes when
they isolated the fungus from another cutaneous
lesion in 1898 and named it Blastomyces
dermatitidis.
In India, Blastomyces dermatitidis was first
reported from tea garden workers having wart- Fig. 4.9 Conidial lollipop-like arrangement of
Blastomyces dermatitidis (schematic diagram)
4.5 Blastomyces 45
The mating begins with the formation of

dikaryon which is followed by the production
of ascogenous hyphae. These hyphae develop
into asci in which meiosis, recombination and
mitosis yield eight haploid ascospores.
It is not clear whether sexual reproduction of
B. dermatitidis occurs in the nature and whether
the generated spores help in transmission of the
infection.
Fig. 4.10 Budding characteristics of Blastomyces

dermatitidis in a FAT smear (Photograph courtesy: Prof. 4.5.4 Susceptibility to Disinfectants
P.P. Gupta, Ex-Director of Veterinary Research, Punjab
Agricultural University, Punjab, India)
B. dermatitidis are susceptible to 1 % sodium
hypochlorite, phenolics, glutaraldehyde, formal-
two-component signalling system. The conver-
dehyde, 10 % formalin.
sion results in an increased cell wall content of
α (1, 3)-glucan and a decreased β (1, 3)-glucan
which is associated with increased virulence.
Multiple changes occur in the lipid composition
of plasma membrane which causes remodelling
Blastomyces prefer to inhabit in warm, mild
and reorganisation of the membrane. The yeast-
acidic, moist, sandy soils with high organic con-
specific protein such as BAD1 (formerly WI-1) is
tent such as animal droppings and in close prox-
expressed.
imity to water bodies. The presence of moisture
helps in release of asexual conidia which can
enter the susceptible host to establish an infec-
4.5.2 Classification tion. Outbreaks of blastomycosis have also been
associated with disruptions of the soil, such as
The genus Blastomyces belongs to the phylum excavation that might lead to the increase num-
Ascomycota, class Euascomycetes, order ber of spores or hyphal fragments in the air. The
Onygenales and family Onygenaceae. The sole fungi are also isolated from the poultry house,
pathogenic species under the genus is mule stall and pigeon droppings, indicating their
Blastomyces dermatitidis. relationship with animal excreta.
Indian fruit bat (Pteropus giganteus) is a nat-
ural reservoir of B. dermatitidis. However,
4.5.3 Reproduction regarding other bat species as a reservoir of
Blastomyces is currently unknown.
B. dermatitidis can reproduce sexually and the The natural reservoir of the fungus is geogra-
sexual state (teleomorph) is known as phically restricted to a specific area (microfocus).
Ajellomyces dermatitidis. It is generally consid- So the outbreaks occur in that area or due to
ered as heterothallic species, and both the mating travel or fomite-mediated transmission from
types (plus and minus) are required to complete that area. In the United States, most clinical
the sexual cycle. The mating types occur in equal cases of blastomycosis occur surrounding the
frequency in the nature. However, the presence Mississippi and Ohio rivers such as Arkansas,
of homothallic mating system is also detected. Kentucky, Mississippi, North Carolina,
The mating results a thick-walled spore-bearing Tennessee, Louisiana, Illinois and Wisconsin in
structure (cleistothecia) and surrounding numer- addition to regions of Canada and United States
ous asci. Each ascus contains eight ascospores. bordering the Great Lakes and St. Lawrence
River. It is also endemic throughout Africa 4.5.8 Antigenic Characteristics

especially in 16 countries from Algeria to
South Africa. The sporadic infections are The yeast form of B. dermatitidis possesses the
observed in Israel, Saudi Arabia and India. antigen-A (135 KDa), composed of protein and
carbohydrate (37 %). On the basis of its presence,
B. dermatitidis has two major serotypes such
4.5.6 Genome as A-antigen positive and A-antigen negative.
Among them the A-antigen-negative serotypes
The sexual mating is governed by a single MAT are restricted within Africa.
locus with two idiomorphs. Each MAT idiomorph Klein and Jones (1990) identified another
encodes either an alpha domain or high mobility immunodominant antigen of B. dermatitidis
group (HMG) domain transcription factor. The yeasts (WI-1) which contributes in humoural
alpha domain and HMG domain genes determine and cellular immunity. It is a 120 KDa protein
plus and minus strains, and the correspond- which is present in the yeast cell surface but not
ing MAT locus is designated as MAT1-1 and the hyphal filaments or the conidia. It also acts as
MAT1-2, respectively. These genes that deter- adhesin and it contains the 24 amino acid tandem
mine the mating types are evolutionarily repeat which is 90 % homologous with the
conserved. The MAT locus is flanked by the Yersinia adhesin. The carboxy terminal of WI-1
SLA2, COX13 and APN2 genes. contains a cysteine-rich domain that is similar to
epidermal growth factor and it mediates binding
to extracellular matrix. Due to this binding prop-
4.5.7 Isolation, Growth and Colony erty, the protein is renamed as Blastomyces
Characteristics adhesin 1 (BAD1).
The carbohydrate moiety of B. dermatitidis
Both yeast and mycelial forms of B. dermatitidis A-antigen is shared with other fungi such as
can be isolated in the laboratory with suitable Histoplasma which creates cross-reactivity.
media and incubation temperature. The pre- However, due to very low level of carbohydrate
ferred culture media for the mycelial form are (less than 1.5 %), the WI-1 is not cross-reactive
Sabouraud dextrose agar, potato dextrose agar with others. So it is a better choice as a diagnostic
and potato flake agar. Media such as the antigen.
brain–heart infusion agar with blood and chlor-
amphenicol, yeast extract phosphate agar and
inhibitory mould agar are selective media which
can inhibit the growth of saprophytic fungi
and contaminating bacteria. Some strains of
The virulence factors possessed by B. dermati-
B. dermatitidis are able to grow in
tidis are described in Table 4.13.
cycloheximide-containing medium. So both the
media with and without cycloheximide can
be used.
At 25 C, B. dermatitidis can take up to 4–6 4.5.10 Transmission
weeks to form typical mould colonies that appear
white to off-white and glabrous or waxy and Inhalation of spores or mycelial fragments is the
becoming grey to brown as aerial hyphae major route of transmission of B. dermatitidis
develop with age. The aerial hyphae as spicules in animals and human. The zoonotic transmis-
may be visible after 1 week incubation. sion is rare and the person-to-person trans-
At 37 C, the hyphae convert slowly into yeast mission does not occur. However, cutaneous or
cells. The colonies are typically white to beige, percutaneous infection occurs in human through
creamy and 0.5–3 cm in diameter. the infected dog bite. The veterinarians may get
4.5 Blastomyces 47
Table 4.13 Virulence mechanisms and factors possessed by B. dermatitidis

α (1, 3)-glucan (i) It conceals immunostimulatory β-glucan layer of the cell wall from detection by host
phagocytic cells
(ii) It inhibits the production of the pro-inflammatory cytokine (TNFα) by phagocytes
BAD1(WI-1) adhesin (i) It helps in attachment of the yeast with macrophages which is mediated through
complement receptor (CR3)
(ii) The BAD1 suppress the production of TNFα by neutrophils and macrophages via a
mechanism that involves upregulation and secretion of TGFβ which can further
downregulate the Th1 response. It also impairs the complement cascades in the peripheral
blood. It helps in the progression of pulmonary infection
(iii) The BAD1 contains 35 copies of a 25 amino acid tandem repeat which can bind calcium
and helps the fungi to grow in calcium poor condition
Phospholipid The strains of B. dermatitidis lacking phopsholipids in the cell wall are less virulent than the
wild types. The presence of phospholipid produces granulomatous tissue reaction which is
not lethal and necrotizing
Alkali-soluble cell wall Lethal, tissue necrotising, endotoxin-like activity
fraction
DOPA melanin It is synthesised in fungi by polyketide synthase and/or phenoloxidases pathway which
(eumelanin) requires the presence of exogenous substrate such as O-diphenolic and P-diphenolic
compounds. It is produced by conidia and yeast phase, not the hyphae. It is observed
that melanin-producing yeast cells are less susceptible to antifungals
the infection during the postmortem examination 4.5.11 Pathogenesis

of infected dog without proper precaution.
Unusually human cases of blastomycosis are After transmission through the inhalation route,
reported, originated from sexual contact and the conidia are deposited in the lungs where
intrauterine transmission. natural resistance is mediated by neutrophils,
A number of occupational and recreational monocytes and alveolar macrophages that can
activities such as forestry, construction, garden- phagocytose and kill the conidia. The
ing, fishing, hunting and hiking have been macrophages can also inhibit the conidia to
associated with a risk of infection. These kinds yeast conversion. If the inhaled conidia over-
of activities increase the exposure to the natural come the natural resistance and are converted
reservoir of B. dermatitidis. Seasonal variations into the yeast form, they are difficult to phagocy-
of blastomycosis have been observed. The infec- tose due to thick capsule. The neutrophils are
tion is more common in the late summer, autumn also relatively inefficient in phagocytosing the
and winter months due to ideal temperature for yeasts. The proliferation of the yeasts occurs
mycelial growth, increased outdoor activities asexually by budding. The proliferating yeasts
and heat stress. All age groups are susceptible may produce the infection with the help of the
to infection; however, the mean age in human virulence factors as described in Table 4.13.
affected with blastomycosis is detected as 20–70 The infection may further progress through
which is consistent with the outdoor activities of the lymphohaematogenous route. In decreasing
this age group. order of frequency, the skin, bones and male
Sexually active male dogs aged between 2 and genitourinary system are the most common sites
4 years, larger breeds (Hounds) and immuno- where the extrapulmonary infection may dissem-
compromised dogs are more susceptible. Resid- inate. With the development of immunity inflam-
ing or travelling to the endemic zones act as matory pyrogranulomatous, reaction occurs
additional risk factors. either in the primary site of infection or in any
Table 4.14 Major diseases of animal and human caused by Blastomyces dermatitidis
Fungi Host Disease
Blastomyces Dogs Lung lesions occur in 65–85 % of cases and may be clinically silent or associated with
dermatitidis cough, tachypnea, exercise intolerance, cyanosis or respiratory distress. Other major
clinical signs are anorexia, focal bone infections causing lameness and osteolysis,
lymphadenopathy, skin lesions, corneal opacity, conjunctivitis, blindness, orchitis and
immunosuppression. Neurologic symptoms are uncommon in dogs reported in only 3–6 %
of cases. Reported neurologic signs include lethargy, neck pain, circling, cranial nerve
deficits, head pressing, seizures, hypermetria, ataxia and tetraparesis
Cats Pyrogranulomatous inflammation of different organs, nasal discharge, cough, weight loss
Horses Pyrogranulomatous pneumonia, pleuritis, peritonitis, cutaneous abscess
Human The clinical signs are highly variable. Common signs include weight loss, fever, fatigue
and other nonspecific complaints. Pulmonary infection produces either acute or chronic
pneumonia. The acute pneumonic patients show fever, chills and productive purulent
cough, with or without haemoptysis. The patients with chronic pneumonia show weight
loss, night sweats, fever and cough with sputum production. The extrapulmonary infection
is characterised by skin lesions which are either verrucous (raised lesion with sharp and
irregular border) or ulcerative, osteomyelitis, prostatitis, orchitis and meningitis
other organ-producing granulomas. These transfer of antibodies could not protect the exper-
granulomas do not caseate as occurs in case of imental animals infected with B. dermatitidis.
tuberculosis. After recovery from the pulmonary
infection, sometimes endogenous reactivation
occurs with or without previous therapy. 4.5.14 Diagnosis
4.5.14.1 Clinical Specimen

4.5.12 Disease Produced The clinical specimens for diagnosis of blasto-
mycosis includes bronchoalveolar lavage, saliva
The major animal and human diseases produced or sputum, skin scrapings, exudates from the
by Blastomyces are enlisted in Table 4.14. lesion, synovial fluid, urine, cerebrospinal fluid,
bone marrow, fluid from anterior chamber of the
eye and lymph node aspirates. For cutaneous
4.5.13 Immunity lesion, the samples should be collected from the
active part, and the biopsy specimen should
The cell-mediated immunity plays major role include the full thickness of the lesion.
in effective protection against B. dermatitidis.
It is primarily mediated by T lymphocytes 4.5.14.2 Laboratory Examination
(Th-1 response) and lymphokine-activated 1. Direct examination: The smear prepared from
macrophages. Experimentally successful transfer the clinical specimens can be observed under
of immunity occurs in mice against microscope by 10 % KOH preparation for
B. dermatitidis through the T cells. demonstration of yeast. The KOH dissolves
However, the antibodies against the tissue and cell debris present in the clinical
B. dermatitidis can also effectively kill the specimens especially in the saliva and skin
fungi through the activation of complements. scrapings to clear the visibility. The yeast
The BAD1 adhesin of B. dermatitidis is an cells with the broad-based bud are the diag-
immunodominant antigen that produces both nostic character. The KOH can be used along
the cellular and antibody-mediated responses. with calcofluor white, a fluorochrome com-
Experimentally the monoclonal antibody to pound that binds to chitin in the walls of
BAD1 significantly enhances the C3-binding fungal cells and make them visible under fluo-
capacity of B. dermatitidis, although the passive rescence microscope. Currently the yeast cells
4.5 Blastomyces 49
are also demonstrated by the negative staining and Coccidioides immitis. Blastomyces is
with the Diff-Quik (DQ) stain which demon- weakly positive, whereas Coccidioides
strates the unstained yeast cells present in immitis is negative. In situ hybridisation with
the stained background. Since they are not specific probe can diagnose Blastomyces
commensal and colonisation does not occur within the tissues.
so their detection in direct smear establishes 4. Detection of antigen: In human, the ELISA-
the diagnosis, and it is considered as most based kits are available to detect the
rapid diagnostic technique for blastomycosis. Blastomyces antigen (A-antigen) in urine,
However, negative finding is inconclusive as cerebrospinal fluid and bronchoalveolar
37–46 % of human blastomycosis patients lavage fluid. Sometimes it produces cross-
reveal the presence of Blastomyces in direct reaction with Histoplasma capsulatum var.
smear. capsulatum due to the presence of shared
2. Isolation and identification: Media and incu- carbohydrate fraction of A-antigen. Currently
bation condition as described earlier will BAD1 is the antigenic target having no cross-
serve the purpose. It is the most sensitive reaction with Histoplasma as it does not con-
and gold standard method for diagnosis of tain carbohydrate. Detection of β-D-glucan
blastomycosis. However, the mycelial form (BDG) is not so effective in blastomycosis.
is highly infectious, and the caution should 5. Serological tests:
be taken in handling of the culture in the (a) Complement fixation test (CFT) can be
laboratory and it is a time-consuming process performed with the yeast phase antigen
causing delay in diagnosis. The confirmation for detection of anti-Blastomyces
of culture can be done by conversion of antibodies. However, the sensitivity and
yeast phase into the mould phase, PCR and specificity is low (57 and 30 %,
commercially available DNA probe. respectively).
3. Histopathology: In the clinical specimens (b) Immunodiffusion using purified
such as saliva or sputum, fine needle aspirates B. dermatitidis A-antigen is more specific
and formalin-fixed tissues, the classical look and sensitive (65–80 %).
of the Blastomyces yeasts such as round to (c) Enzyme-linked immunossorbent assay
oval multinucleate cells with a single broad- (ELISA) and radio immune assay (RIA)-
based bud located intra or extracellularly based techniques are currently most
helps in diagnosis. The tissue sections are sensitive techniques for the detection
stained with haematoxylin and eosin, periodic of Blastomyces antibodies. The sand-
acid–Schiff stain (PAS), PAS with haema- wich and competitive-binding inhibition
toxylin counter stain and Grocott methena- ELISA are used for detection of antibodies.
mine silver (GMS). If the classical look 6. Skin test (delayed type of hypersensitivity):
is not detected, then the diagnosis becomes The crude fungal extract (blastomycin)
difficult as it produces confusion with can be inoculated into the susceptible animal
other yeasts. In such cases, the mucicarmine for detection of hypersensitivity (like tuber-
stain can differentiate the Cryptococcus culin test). However, the test is neither sensi-
neoformans and Blastomyces as it can specifi- tive nor specific. The swelling rapidly
cally stain the capsule present surrounding the diminishes and the test is negative during
Cryptococcus cells. Melanin stain can also repeat testing.
differentiate Blastomyces from other yeasts. 7. Molecular biology: PCR-based technique
The Alcian blue or acid-fast stain can be such as nested PCR can be used for detection
used for differentiation between Blastomyces of Blastomyces.
4.5.15 Treatment identified as the etiological agent of ‘Valley

fever’ at San Joaquin Valley of California.
In dogs and human, treatment with amphotericin He also described a milder form of infection
B lipid complex and itraconazole is safe and which occurred in his laboratory during acci-
effective. The amphotericin B is found effective dental opening of the Petri dish with the mould
in respiratory distress, CNS involvement and in by a medical student. Dickson coined the term
children with blastomycosis. However, ampho- ‘coccidioidomycosis’ to include all forms of
tericin B deoxycholate has relatively high rate of infection by Coccidioides.
toxicity such as infusion reaction, nephrotoxi-
city, hypokalaemia, hypomagnesaemia and
anaemia. Amphotericin B lipid complex is safer 4.6.1 Morphology
but is clinically as effective as the deoxycholate.
Itraconazole is the drug of choice for Coccidioides is a dimorphic fungus having two
non-meningeal, non-life-threatening blastomy- phases in the life cycle. The saprophytic phase
cosis in immunocompetent patients. However, occurs in the environment and artificial labora-
it has severe drug–drug interactions especially tory culture, and the parasitic phase occurs in
with antiretroviral and antirejection drugs. the host such as human, dogs, horses and other
Fluconazole drug–drug interaction is less, and animals.
it can easily penetrate the CNS with better The saprophytic phase consists of mycelia
bioavailability in oral route, but it has limited with septate hyphae which are 2–4 μm in
efficacy against Blastomyces. The second- diameter. In 5–7 days, the hyphae yield a chain
generation triazole compounds such as of multinucleate arthroconidia along the length
voriconazole and posaconazole are promising of the hyphae (enteroarthric development).
against blastomycosis. Initially, small, nonviable, brittle cells are pro-
Currently a primary treatment with duced, known as disjunctors. These cells degen-
amphotericin B lipid complex followed by any erate to release the buoyant arthroconidia which
azole compound preferably voriconazole is act as the source of infection especially during
recommended to treat blastomycosis. soil disruption and storm.
Within the body of the host, the arthroconidia
are converted into a different morphologic form,
known as spherule, in the presence of CO2 and
4.6 Coccidioides phagocytic cells. The arthroconidia shed the
outer wall and enlarge to produce the immature
In 1892, Alejandro Posadas, a medical student spherule containing one nucleus. The central
and pathologist Robert Wernicke, first described portion is occupied with a large vacuole. The
Coccidioides from a soldier with recurrent skin nucleus undergoes continuous division which
tumours in Argentina. They described it as is followed by partitioning of the cytoplasm
unknown parasite found in the lesion having surrounding the vacuole. It produces matured
similarity with Coccidia. In 1986, Rixford and spherule (60–100 μm diameter) containing
Gilchrist also identified it as a coccidial agent 800–1,000 endospores (2–5 μm in diameter
and designated it as Coccidioides immitis each). The spherule wall contains glucans, chitin,
(immitis ¼ not mild). Its fungal identity was mannose polymers, 3-O-methylmannose and
confirmed in 1900 by Ophüls and Moffitt. They galactose, whereas the wall of the endospores is
further showed the organism was dimorphic composed of glucan and chitin. The spherules
which switched from the mycelial phase in cul- release the endospores in vivo which again
ture to spherule forming phase in the tissues. enlarge to form a new spherule filled with
In 1937, Ernest Dickson established the clinical endospores to repeat the cycle. The mechanism
importance of Coccidioides immitis when it was of release is unexplored, although probable role
4.6 Coccidioides 51
Fig. 4.11 Life cycle of

Coccidioides immitis
(schematic representation).
1 Emergence of tubular
structure, 2 formation of
mycelia, 3 fragmentation of
hyphae, 4 release of
arthroconidia,
5 enlargement of
arthroconidia within the
host body, 6, 7 spherule
formation, 8 endospore
generation within spherule,
9 release of endospores
from spherule
of certain enzymes such as chitinase and There are minor differences in amino acid
glucanase is observed. The endospores may fur- sequence between two species, but they are
ther generate the mycelial form if they return to similar in antigenicity, virulence or morphology.
the soil or grow in artificial culture medium
(Fig. 4.11).
4.6.3 Reproduction
4.6.2 Classification No sexual stage (teleomorph) of the fungi is

detected.
The genus Coccidioides belongs to the
phylum Ascomycota, class Euascomycetes,
order Onygenales and family Onygenaceae like 4.6.4 Susceptibility to Disinfectants
Blastomyces and Histoplasma. Previously
Coccidioides immitis was considered as the C. immitis is susceptible to 70 % ethanol
sole pathogenic species under the genus. The for 1–20 min exposure, 37 % commercial
phylogenetic analysis using single nucleotide formaldehyde, chlorine dioxide gas (sporicidal),
polymorphisms and microsatellites has showed 1:10 dilution of bleach, 6 % hydrogen per-
the existence of two genetically different oxide and 3 % phenolics with a contact time of
C. immitis clades, California and non-California. 20 min or more. For fumigation of laboratory
Later the California clade was designated with paraformaldehyde, 0.3 g/ft3of room air
as C. immitis and non-California clade was is recommended to make it C. immitis free. The
designated as a new species, namely, mycelia form can be inactivated by heat at
C. posadasii, in honour of Alejandro Posadas. 120 C for 30 min.
4.6.5 Natural Habitat and Distribution species-specific ‘genomic islands’, located in

chromosomal sub-telomeric regions.
Moist alkaline soil rich in salt and carbonised The phylogenetic analysis with the ancestor
organic materials is required for growth of the and close relative of Coccidioides revealed the
hyphal phase. Following a subsequent dry gain of several virulence genes when they are
period, the hyphae will die leaving the diverged from their close relative. These acquired
arthrospores. Disturbance of the soil by different genes help the fungi to grow within the host even
human or animal activities such as construction, in the carcasses. One of such acquired gene
agriculture, excavation and archaeological encodes spherule-associated enzyme related with
exploration is required for transmission of the allantoin metabolism which acts as nitrogen
spores into the body of the host. source in the host and source of proteins for
There are a few endemic zones where haeme-/tetrapyrrole binding required for scav-
C. immitis are naturally found. The high endemic enging iron within a host. However, the analysis
zones include Southwestern United States and also detected reduced numbers of gene encoding
the bordering regions of northern Mexico. plant degrading enzymes (cellulases, cutinases,
In the United States, south central portion of tannases, pectinesterases) and proteins associ-
Arizona, particularly Tucson and Phoenix, the ated with sugar metabolism in the genome of
southern one-third of California, notably the Coccidioides in comparison to other Ascomy-
San Joaquin Valley, and southwestern Texas cetes. It indicated the association of the fungi
are most affected. The climate in this region is with live animals rather than the plants or soil.
arid with a yearly rainfall ranging from 10 to Comparative genomic analysis between the
50 cm, with extremely hot summers, winters two species of Coccidioides revealed that the
with few freezes and alkaline, sandy soil. 8 % of the genes in the C. immitis population
The sporadic human cases are reported from may be introgressed from C. posadasii. This
Central and South America, Utah, Nevada, introgression is driven by the natural selection
Brazilian states (Piau{’, Bahia, Ceara’, because the introgressed region is enriched with
Maranha˜o), Guatemala, Honduras, Nicaragua, coding sequences.
Argentina, Paraguay, Colombia and Venezuela.
In India, a few human cases of Coccidioido-
mycosis are reported which are imported in 4.6.7 Isolation, Growth and Colony
nature. The patients either travelled or lived for Characteristics
a certain period in the vicinity of Arizona, United
States. However, in animals, no authentic report Coccidioides can be isolated in ordinary fungal
of Coccidioidomycosis is available till date. media (Sabouraud dextrose agar, inhibitory
mould agar, brain–heart infusion agar, potato
dextrose agar, potato flakes agar) even in the
presence of cycloheximide as well as in bacterial
4.6.6 Genome media (blood agar, chocolate agar, selective
yeast extract medium used for Legionella or
The genome of C. immitis (28.9 Mbp) and Bordetella) when incubated at 25 C or
C. posadasii (27 Mbp) contain 10,355 and 30–37 C for 4–5 days (range is 2–16 days).
7,229 number of annotated genes, respectively. C. posadasii was reported to grow faster than
The mean GC% of genome is 46. The mean C. immitis at 37 C. The selective medium
intron and exon number per gene is 2.3 and along with cycloheximide is used to isolate
3.4, respectively. Coccidioides species are Coccidioides from mixed flora.
estimated to have four chromosomes by CHEF The spherule is formed at 40 C in the media
gel analysis. Like other Ascomycetes, the chro- containing casein hydrolysate, glucose, biotin,
mosome structure of Coccidioides also contains glutathione and salt.
4.6 Coccidioides 53
The young colonies are white and moist, to six copies of proline and aspartic acid-rich
glabrous and tenacious, and adhering to the sequences. The number of copies varies with
medium. At this young stage, no arthroconidia strain. However, the antigen is shared by geneti-
is detected. With maturity of the culture, discrete cally and geographically diverse strains.
concentric rings and the filamentous area The saprobic phase contains a heat-stable,
containing arthroconidia become visible. The 19 kDa antigen with proteinase activity which
pigmentation with tan, brown, pink, grey or is also considered as a Coccidioides-specific
yellow colour was also detected in matured antigen (CS-Ag). Recently, an in-house antigen
colonies. of Coccidioides is described containing
β-glucosidase (45–67 KDa) and glutamine syn-
thetase (67–97 KDa) as major immunoreactive
4.6.8 Antigenic Characteristics proteins which can react with serum samples of
the patients suffering from the infection.
The major immunodominant antigen of The carbohydrate antigen is detected in the
C. immitis is spherule outer wall glycoprotein mycelial extract (coccidioidin) along with some
(SOW Gp) which can produce both antibody- amino acid nitrogen.
mediated and cellular immune responses. This
glycoprotein consists of a signal peptide and
pro-peptide, a proline and aspartic acid-rich tan- 4.6.9 Virulence Factors
dem repeat motif, and a glycosylphosphatidy-
linositol (GPI) anchor. The repeat domain is The virulence factors possessed by Coccidioides
41–47 amino acids in length and contains three immitis are enlisted in Table 4.15.
Table 4.15 Virulence mechanisms and factors possessed by C. immitis

Extracellular The soluble conidial wall fraction (SCWF) is antiphagocytic, immunosuppressive for mice
proteinases lymph node proliferation. The fraction contains chiefly a serine proteinase enzyme, a dimer of
60KDa. It is released from the outer cell wall of the conidia after inhalation, to damage the
respiratory tract since elastin is the major structural component of the tissues. Two other
proteinase enzymes (56 Kda and 19 Kda) are also described. The 56 KDa proteinase is unable to
degrade immunoglobulin, whereas the 19 KDa fraction (Coccidioides-specific antigen, CS-Ag)
is able to degrade immunoglobulin
The proteinases help in the release of endospores from the spherule and local tissue damage.
The elastin degradation products attract inflammatory cells to produce damaging inflammatory
response
Oestrogen-binding Certain pregnancy-related hormones such as progesterone, testosterone, 17β-estradiol
proteins (E2) stimulate the rate of spherule maturation and endospore release of Coccidioides. The
oestrogen-binding proteins present in the cytosol of the fungi help in this process. In general,
men are more susceptible than women for the disseminated infection. However, during
pregnancy the risk of disseminated infection increases in women especially during late
pregnancy which is directly correlated with increased E2 level
Urease (a) It maintains the alkalinity of the host tissue to produce favourable condition for the fungal
(encoded by ure) growth
(b) It also makes the phagosome (containing endospores) alkaline to prevent its fusion with
lysosome
(c) It increases the expression of arginase I. The arginase I competes with the inducible nitric
oxide synthase (iNOS) produced by the host phagocytes for the same substrate (L-arginine).
Thus, arginase can inhibit the production of nitric oxide which is lethal to the fungi
Spherule The spherule is antiphagocytic. Due to its larger size, it escapes the phagocytosis by the
neutrophils and macrophages
Spherule outer It acts as adhesin in colonising respiratory tract
wall glycoprotein
4.6.10 Transmission
The infection is transmitted through inhalation

of arthrospores which are easily deposited into
the lung due to their smaller size. A single spore
is sufficient to produce an infection in human.
The chances of getting more than one spore are
associated with the disturbance of soil rich
in mycelial form especially during military
manoeuvres or archaeologic excavation. Rarely,
cutaneous lesions can develop as the result of Fig. 4.12 Spherule of Coccidioides immitis in stained
direct inoculation. tissue smear (Photograph courtesy: Prof. P.P. Gupta,
Ex-Director of Veterinary Research, Punjab Agricultural
Immunosuppressed individuals such as University, Punjab, India)
patients of acquired immunodeficiency syn-
drome (AIDS), Hodgkin’s disease, malignant
neoplasms, uraemia, collagen-vascular diseases after phagocytosis, they can inhibit the oxygen-
or those who had received recent immunosup- mediated killing. However, the spherule outer
pressive drug therapy and organ or bone marrow wall glycoprotein (SOWGp) can bind antibody
transplant recipients are more susceptible. Preg- to enhance opsonisation and subsequent phago-
nancy especially during the third trimester and cytosis. The peak of SOWGp production occurs
the peripartum period also increases the risk during isotropic growth of the spherule but then
of getting the infection. Rarely the neonatal suddenly decreases during endospore formation.
coccidioidomycosis has been reported which During the endospore forming phase, a 34 kDa
is maternal in origin. Certain human races metalloprotease (Mep1) that belongs to the
such as Filipinos, African Americans, Mexican metzincin superfamily is produced which can
Americans and Native Americans are more sus- digest the SOWGp. So, the Mep1 plays a crucial
ceptible to the infection associated with genetic role in the establishment of the infection.
factors. There is no known human-to-human or The endospores released from the spherule
animal-to-human transmission of this infection. can be phagocytosed, but many of them survive
due to antiphagocytic nature of the endospore
outer wall. Some of them, even after phago-
cytosis, can survive by employing various
4.6.11 Pathogenesis strategies. They can secrete the urease enzyme
which elevates the arginase I level within the
After transmission within the body of the host, phagosome. The arginase I can increase poly-
the arthroconidia are converted into spherule amine synthesis and can decrease the nitric
with numerous endospores as depicted earlier oxide (NO) production which is favourable for
(Fig. 4.12). The spherule can escape the phago- the fungal survival. The alkalinisation of the
cytosis by the host neutrophils, macrophages and microenvironment as a consequence of urease
dendritic cells due to their larger size activity prevents the fusion of phagosome with
(60–100 μm). The protease present in the spher- lysosomes.
ule can digest the antibody or other opsonins to Production of anti-Coccidioides Th1 immu-
evade phagocytosis. Further, the spherules nity (cell-mediated immunity) offers resistance,
showed moderate resistance against reactive whereas Th2 immunity (antibody mediated) is
oxygen species (ROS) such as hydrogen perox- associated with the establishment of infection.
ide produced by the host macrophages. The Experimentally the secreted endosporulation
spherules also inhibit the production of nitric antigens (EA) of Coccidioides in high concentra-
oxide (NO) by the host phagocytes. So, even tion can suppress the Th1 immunity, whereas in
4.6 Coccidioides 55
Table 4.16 Major diseases of animal and human caused by C. immitis

Fungi Host Disease
Coccidioides Dogs In dogs, primary pulmonary or disseminated coccidioidomycosis is developed. Signs of
immitis pulmonary form include coughing, weight loss, fever, lethargy and anorexia. The
pneumonia is either mild interstitial or extensive miliary pneumonia with consolidation of
the lung lobes or only hilar lymphadenopathy (enlargement of lymph nodes within the
lung hilum). The hilar lymphadenopathy is considered as a significant mark for diagnosis.
The bone involvement is restricted within appendicular skeleton and is characterised by
bone lysis as well as periosteal new bone formation. The CNS involvement is
characterised by seizure
Cats Like dogs, the cats also suffer from both the pulmonary and disseminated form. The CNS
form is characterised by incoordination, hyperesthesia, seizures and behaviour changes.
The skin lesions include dermatitis, ulceration or abscesses that do not respond to
antibiotics
Horse Disseminated coccidioidomycosis is observed. The female horses suffer more than male.
The infection is characterised by abortion, mastitis, osteomyelitis, nasal granuloma and
meningitis
Llama Pulmonary and disseminated Coccidioidomycosis is observed which is characterised by
respiratory distress, dermatitis, osteomyelitis, meningitis, polyperiarthritis syndrome
Cattle, Self-limiting infection is observed which is confined within the bronchial and mediastinal
sheep lymph nodes
Human California disease/desert fever/Posadas-Wernicke disease/San Joaquin fever/valley
fever: Primary infections affect respiratory system. Influenza-like, pneumonic and pleural
presentations are the most common. The disseminated form is noticed causing tissue
damage in the skin, bone, joints and central nervous system
low concentration stimulates the same. This 4.6.12 Disease Produced

finding shows that both inhibitory and stimula-
tory components are present in EA and the estab- The major animal and human diseases produced
lishment or clearing of the infection is dependent by Coccidioides are enlisted in Table 4.16.
on their ratio.
After establishment of the infection in
absence or reduced Th1 immunity, the 4.6.13 Immunity
endospores may disseminate via haematogenous
(fungaemia) or lymphatic drainage into the bone, Primary recognition of the fungi is mediated
skin, lymph nodes, central nervous system (CNS) through pattern recognition receptors (PRRs)
and heart. In dogs, the bone is the most common expressed by the host immune cells. Among
site of dissemination especially in the appendic- them, toll-like receptor (TLR2), dectin1, man-
ular skeleton. In human, vertebral osteomyelitis nose receptor (MR) and surfactant proteins A
is most prevalent. The CNS manifestation in (SPA) and D (SPD) are involved in recognition
animals is largely due to the production of gran- of Coccidioides ligands (pathogen-associated
ulomatous mass in the brain, not due to menin- molecular pattern, PAMP) (Table 4.17).
gitis which is typical in human. Pericardial The professional phagocytes especially the
dissemination is much common in dogs than polymorphonuclear leukocytes (PMN/neutrophils)
human. and macrophages are the first line of defence,
The disseminated infection may remain dor- recruited at the site of Coccidioidal infection.
mant for many years in human (25–35 % cases). They try to clear the pathogen through oxidative
The infection recurs rarely due to immunosup- or non-oxidative killing mechanism. However, it
pression, malnutrition, advancement of age and depends on fungal strain and morphotype. The
high dose of exposure. In animals, the recurrence arthroconidia and endospores are more susceptible
is not reported but cannot be ruled out. than spherule. The dendritic cells have the ability
Table 4.17 Pattern recognition receptors and their with endospores which is diagnostic of
ligands involved in recognition of Coccidioides coccidioidomycosis. The presence of imma-
Coccidioidal pathogen- ture spherule or endospores without spherule
Pattern recognition receptors associated molecular may be confused with other fungal infection.
(PRRs) pattern (PAMPs)
The calcofluor white (CFW) fluorescent stain
TLR2 Unidentified
offers best detection of the fungi by binding
Dectin1 β-glucan
the chitin and cellulose of the cell wall.
Mannose receptor (MR) Mannan
However, it can also nonspecifically stain the
Surfactant proteins A (SPA) Unidentified
Surfactant proteins D (SPD) Unidentified
plant and fatty substances.
2. Isolation and identification: Media and
incubation condition as described earlier will
to bind and internalise the spherules. It is followed serve the purpose. However, Coccidioides is
by production of a T cell-mediated response extremely hazardous to handle, and several
because they can elevate the T cell cases of infection from the laboratories are
co-stimulatory molecule production. documented through inhalation of arthro-
The PAMP–PRR interactions stimulate the conidia. It is also considered as a ‘select
production of IL12 and IL23 which can convert agent’ of bioterrorism. So, Coccidioides-
the naive T cells into antigen-specific T cell specific laboratory should have biosafety
populations. There are two functionally distinct level 3 containment maintained with negative
subsets of CD4+ T cells, i.e. Th1 and Th2 cells. air pressure. Further, expertise is needed in
Both clinical and experimental evidence have handling of the colonies because the
demonstrated that Th1-mediated immunity is arthroconidia are easily generated. However,
essential for defence against coccidioidomyco- the routine laboratory where suspected culture
sis. The activation of Th1 cells will increase the of Coccidioides is handled should have mini-
level of IFNγ, IL2 and other cytokines which mum biosafety level 2 facilities. All the
provides the signals for recruiting and activating cultures are handled under class II biological
immune effector cells, whereas the activation of safety cabinet containing vertical laminar air-
Th2 response increases the antibody level such as flow with HEPA (high efficiency particulate
serum IgG, IgE and IgA which does not offer any air) filter and exhaust air. The lid of the Petri
protection. dishes along with suspected culture should be
taped, if preserved for 5–7 days. After identi-
fication, the routine laboratory should destroy
4.6.14 Diagnosis the culture properly. The culture should not be
transferred without prior permission from the
4.6.14.1 Clinical Specimens authority.
The clinical specimens for diagnosis of coccidi- 3. Histopathology: The histopathological exam-
oidomycosis include pus or exudates from lesion, ination can be performed on the biopsy
transtracheal washes, skin scrapings and biopsies materials collected from the skin, bone,
from lymph node, bone, etc. In human, the lung, brain, etc., to confirm whether the infec-
specimens include sputum, bronchoalveolar tion is disseminated into different organs.
lavage, transtracheal aspirates and lung biopsies. In the tissues, endosporulating spherules,
All specimens should be labelled as potentially empty ruptured spherules and immature non-
hazardous during transport. endosporulating spherules can be detected by
staining. Sometimes, mycelia are observed in
4.6.14.2 Laboratory Examination the tissues which are indistinguishable from
1. Direct examination: The wet mounts with other common fungi. The Grocott methena-
10 % KOH are employed on the clinical mine silver stain is the best stain for detection
specimen for detection of the spherule of Coccidioides in histopathological sections.
4.6 Coccidioides 57
However, Grocott methenamine silver can higher IgG titres with Histoplasma and
also stain the tissue elements such as mucus, Blastomyces antigens than with Coccidioides
glycogen granules and some bacteria. It antigen.
causes overstaining of the fungi which can Currently ELISA-based tests are also
mask the endospores within the spherule. developed for detection of anti-Coccidioides
Other histological stains, such as periodic antibodies.
acid–Schiff (PAS), haematoxylin-eosin, 6. Skin test for detection of delayed-type hyper-
Giemsa, papanicolaou and mucicarmine, can sensitivity (DTH): The antigen used in classi-
also be used as a substitute. cal skin test is known as ‘coccidioidin’.
4. Detection of antigen: Detection of antigen is Primarily it was prepared as a soluble broth
useful in the very early stage of coccidioido- culture filtrate of mycelial cells grown for
mycosis when antibody is not developed 2 months in a synthetic asparagine–gly-
(within 1 week) and in the patients suffering cerol–salt medium. Later, an aqueous lysate
from hypogammaglobulinaemia or other of spherules was prepared from Coccidioides
immunodeficiency who are unable to produce strain (Silveira) that had been grown in Con-
the antibody. The tests such as radioimmuno- verse medium and then incubated in distilled
assay (RIA) and inhibition ELISA were water for up to 40 days at 34 C. The soluble
developed to detect the protein–carbohydrate aqueous lysate is designated as ‘spherulin’
conjugate antigen of the fungi. which can also be used as antigen in skin
5. Serological tests: The serological tests are test. The skin test can be performed in
not only diagnostic but also indicative about human, dogs, horses, llamas and nonhuman
the prognosis of the infection. Detection of primates. Reversion of positive result into
anti-Coccidioides IgM (within 1–3 weeks the negative indicates grave prognosis. How-
of infection) can be done by tube precipitin ever, negative result cannot rule out the
(TP) and immunodiffusion (ID) tests. The infection.
TP is based on heat-stable 120 kDa β glu- 7. Molecular biology: Primarily a polymerase
cosidase antigen, whereas detection of IgG chain reaction (PCR) was developed using
(after third week–several months) can be the primers of 18S rDNA of Coccidioides.
done by complement fixation test (CFT) Further, a real-time PCR is developed with
which is based on heat-labile chitinase anti- primers amplifying a 170 base-pair part in
gen. However, canine serum contains anti- the ITS2 (internal-transcribed spacer 2) region
complementary substances; so CFT cannot of the rDNA genome. Several studies also
be performed in the dogs. Serological IgG successfully validated about using PCR to
titre can indicate about the severity of the detect the unique coccidioidal gene [antigen-
infection in human. For example, increasing 2/proline rich antigen (Ag2/PRA)].
IgG titre such as 1:32 or more indicates about
the disseminated infection in human, whereas
IgG titre between 1:2 and 1:16 is considered 4.6.15 Treatment
as positive for infected dogs.
Sometimes, false-positive band is produced In dogs and cats, oral treatment with keto-
in ID test due to reaction between coccidioidal conazole, itraconazole and fluconazole with a
antigen and C-reactive substances present dose range between 2.5 mg/kg twice daily to
in the serum. This false band can be dissolved 20 mg/kg twice daily is usually performed to
with 1.5 % aqueous solution of sodium treat coccidioidomycosis. The adverse side
EDTA, whereas the authentic band will effects of oral azole therapy in dogs and cats
remain as such even after washing. are similar with human. It includes reduced
The cross-reaction is observed with other appetite, vomition and diarrhoea. Sometimes,
systemic fungi. The sera from mild and pri- a vasculitis causing suppurative skin lesions has
mary cases of coccidioidomycosis produce been observed as an adverse effect of itraconazole
in dogs. Similarly, polyuria/polydipsia, thinning whom had resided in Panama for a long period.
of hair coat is detected with fluconazole treatment Initially the fungal aetiology was not ascertained;
in dogs. Reduced fertility of male dogs is reported rather it was thought as ‘encapsulated protozoa
with ketoconazole treatment. residing within histiocytes’, so it was named as
As an intravenous infusion, amphotericin B Histoplasma. In 1934, William DeMonbreun
deoxycholate can be given. However, it has established the association of histoplasmosis
potent renal toxicity. Instead, currently ampho- with fungus. He first isolated the organism in
tericin B lipid complex is preferred to treat the artificial media, showed its dimorphism and
canine and feline patients who are not responding experimentally produced the infection in labora-
with the orally administered azoles. tory animals to meet the conditions of Koch’s
Administration of oral antifungals is not so postulate (De Monbreun 1934). In 1939, he
effective in llamas and alpacas where the drugs described the infection in a dog died from
are metabolised and lost without absorption hepatomegaly (De Monbreun 1939).
in the fermentative forestomach. Further, their
jugular vein is also not easily accessible for
intravenous application of antifungals. 4.7.1 Morphology
Histoplasma is a classical example of thermally

4.7 Histoplasma dimorphic fungus, which exhibits two different
morphotypes (i.e. yeast and mould) in two kinds
Histoplasma is an intracellular dimorphic fungus of temperature regime. The pathogenic yeast
causing severe pulmonary and systemic infection form is observed within the host tissue (37 C),
in man and animals. Histoplasma capsulatum whereas the invasive mould form prevails in the
var. farciminosum, causative agent of Epizootic environment (25–30 C).
lymphangitis in horses, was first demonstrated in In the infected tissues, the oval yeast cells of
pus in 1873 by Rivolta. However, it was success- H. capsulatum var. capsulatum are observed
fully isolated in 1896 by Tokishiga in Japan. The within macrophages, 3–5 μm in diameter. Their
infection histoplasmosis in human was first cell wall does not allow the easy passage of Gram
notified by Darling, an American pathologist or Giemsa stain, so the cell is observed to be
who observed it during autopsy of a Martinique surrounded with an empty areola in a stained
native person died with tuberculosis-like syn- smear, producing the impression of a capsule
drome in Panama (Darling 1906). He also (Fig. 4.13). They are rarely found in extracellular
described two similar cases observed in a Can- condition and they infrequently show the bud-
tonese and another Martinique native, both of ding. They can be stained by Gomori
Fig. 4.13 Yeast form of

Histoplasma capsulatum
within macrophages.
Arrow indicates the
presence of capsule like
material surrounding the
yeast cell (Photograph
courtesy: Prof. P.P. Gupta,
Ex-Director of Veterinary
Research, Punjab
Agricultural University,
Punjab, India)
4.7 Histoplasma 59
α-(1, 3)-glucosyl linear residues, while β-glucan

consists of a linear β-(1, 3)-glucosyl-linked back-
bone with β-(1, 6)-glucosyl-linked side chains
that vary in length and distribution. Especially
in the yeast cells of Histoplasma, the chitin forms
the inner layer with an outer fibrous covering
made of α-glucan, whereas the mycelial form is
predominantly made of β-glucan. Based on this
chitin and α-glucan concentration, H. capsulatum
is classified as chemotype I and II. The
chemotype II strains consist of less chitin and
more glucan than chemotype I. Further, concen-
tration of α-glucan is associated with virulence of
the strains. The virulent H. capsulatum strains
may possess 1,000-fold more α-glucan than non-
pathogenic strains. Sometimes H. capsulatum
produces nonpathogenic strains lacking the
α-glucan in their cell wall. They may produce
Fig. 4.14 Pear-shaped microconidia (schematic)
an unusual morphology and can persist within
the macrophages for prolonged period, observed
methenamine silver, periodic acid–Schiff (PAS), in chronic animal and human infections. The
Giemsa and Gram stain, haematoxylin and eosin α-glucan is found to be associated with multi-
stain, whereas H. capsulatum var. farciminosum plication of yeast cells within the macrophages,
yeast cells appear as large double-contoured oval maintenance of yeast cells within phago-
body in pus samples, measuring 2.5–3.5 μm lysosome and blocking of innate immune recog-
3–4 μm. The cytoplasm is granular and nition of H. capsulatum by a particular receptor.
sometimes budding is observed. In addition the cell wall contains mannan,
The mycelial form is characterised by the mannosylated proteins and low amount of lipids.
presence of aerial septate hyphae bearing The lipids are linked with saccharides and show
microconidia (2–3 μm in diameter, Fig. 4.14) structural heterogeneity. The glycosyl inositol
which is later converted into large chlamy- phosphoryl ceramides present in the mycelial
dospores (tuberculate macroconidia, 7–15 μm and yeast phases of H. capsulatum is required
in diameter, Fig. 4.15) having small spine-like for fungal survival.
projections in their wall. However, the tuber- The conversion from mould phase to yeast
culate macroconidia are not abundantly produced phase is known as ‘phenotypic switch’ which is
by H. capsulatum var. farciminosum. Instead required for virulence as the yeast phase is
other spores such as arthroconidia and blasto- protected from phagocytosis by the neutrophils,
conidia are detected. macrophages and monocytes due to its larger
The carbohydrate is the major constituent size to engulf. In addition, changes in chemical
(80 %) of the cell wall of both the morphotypes. composition of the cell wall are also noted.
The glucose (Glc), mannose (Man) and galactose In yeast phase, the α-glucan content of the cell
(Gal) are the most abundant monosaccharide in wall is increased than the mycelial phase as
the cell wall of Histoplasma capsulatum. The discussed earlier and also the changes occurred
glucan (Glc) and N-acetylglucosamine persists in lipid composition of the membrane due
as a polymer (β-1,4 linkage) known as ‘chitin’. to adaptation in higher temperature (37 C).
Two different glycosidic linkages (α and β) Some phase-specific virulence factors are
are present in the glucan structure of both expressed by H. capsulatum yeast cells such
yeast and mycelium. An α-glucan contains as CBP1, Yps 3 and Yps 21:E9. The sulphur
Fig. 4.15 Tuberculate

macroconidia (schematic)
metabolism can influence the morphotype in H. capsulatum var. duboisii. H. capsulatum

H. capsulatum. Addition of sulfhydryl-reducing var. farciminosum was previously designated
agent (dithiothreitol) fixes cells in the yeast as a separate species (H. farciminosum) under
phase independent of temperature, whereas the genus. Due to morphological similarities
the addition of sulfhydryl-oxidising agents in both mycelial and yeast phases presently,
(p-chloromercuriphenylsulfonic acid, PCMS) it is assigned as a subspecies of H. capsulatum.
fixes cells in the mycelial phase independent of Similarly, Vanbreusegham initially described
temperature. The enzyme hybrid histidine kinase H. capsulatum var. duboisii as a new species
(DRK1) regulates this phenotypic switch in of the genus (H. duboisii) which was later
H. capsulatum. It acts as a sensor in the change reassigned within the H. capsulatum group.
of environmental temperature and regulates the This H. capsulatum var. duboisii is more preva-
expression of the genes for yeast phase-specific lent in Western and Central Africa and in the
virulence factors like CBP1, AGS1 and Yps-3. island of Madagascar.
This phenotypic change is reversible and H. capsulatum is also divided into phylo-
blocking the transition may prevent the disease genetically distinct lineages/clades based on
progression. geographical distribution. The major clades are
North American class 1 (Nam1), North American
class 2 (Nam2), Eurasian, Latin American group
A (LamA), Latin American group B (LamB),
4.7.2 Classification
Australian, Netherlands (Indonesian) and
African.
The genus Histoplasma belongs to the phylum
Ascomycota, class Euascomycetes, order
Onygenales and family Onygenaceae like
Blastomyces and Coccidioides. The genus 4.7.3 Susceptibility to Disinfectants
possesses one major pathogenic species
H. capsulatum, subdivided into three sub- Histoplasma capsulatum var. capsulatum is sus-
species, i.e. H. capsulatum var. capsulatum ceptible to 1 % solution of sodium hypochlorite,
and H. capsulatum var. farciminosum and 2 % phenol, 2 % gluteraldehyde and isopropyl
4.7 Histoplasma 61
alcohol and 3 % formalin. However, for 4.7.5 Genome

decontamination of soil, only formalin is found
effective. Among the antifungals, it is suscep- Histoplasma capsulatum strains are extremely
tible to amphotericin B and the azole group diverse in terms of their number of chromosomes
of antifungal drugs, including ketoconazole, and genome size. For example, Downs strain
itraconazole, fluconazole, posaconazole and has seven chromosomes, G186B has at least
voriconazole. The spores and yeast phase can four, and G217B has at least three. The genome
be destroyed by exposure to more than 40 C is haploid, and the genome size ranges between
temperatures for an extended period of time, 23 Mbp (G186AS strain) to 32 Mbp (Downs
pH below 5 or above 10 and drying. strain), and the repetitive DNA content ranges
between 0.5 and 8.0 %, respectively. The
genome size is larger than ascomycetous yeast
4.7.4 Natural Habitat Saccharomyces cerevisiae (13 Mbp) and similar
to those of the filamentous ascomycetes such
Histoplasma is a noncompetitive soil saprobe. as Aspergillus nidulans (26 Mbp), Penicillium
It can grow in different soil types including paxilli (23 Mbp) and Neurospora crassa
hard clay and sandy loam. They are present (43–45 Mbp). The nuclear guanine and cytosine
within few centimetres from the soil surface (G + C) content is 45.4–49.8 %, with an
where a microbiota is formed with bacteria, observed mean of 47.3 %.
actinobacteria and other fungi. As they are non-
competitive saprobes, other soil microorganisms,
which are active organic matter decomposers, 4.7.6 Isolation, Growth and Colony
favour their growth with availability of nutrient Characteristics
sources and contribute to the maintenance of soil
structure, aeration and water content. Humid The mycelial phase of H. capsulatum var.
climate, temperature ranging from 25 to 35 C farciminosum can be isolated in Sabouraud
and neutral to alkaline pH are required for their dextrose agar enriched with 2.5 % glycerol,
optimum growth. Sometimes darkness favours mycobiotic agar, Hartley digest agar with 10 %
the sporulation and mycelial phase requires horse serum, brain–heart infusion agar with 10 %
Ca++ for their growth. The bird droppings and horse blood and pleuropneumonia-like organism
bat guano can enrich their growth with nitrogen (PPLO) agar enriched with 2 % dextrose and
supplementation. The birds act as carrier of 2.5 % glycerol. The addition of antibiotics to
Histoplasma, whereas in bats (including insectiv- the media is recommended to prevent the growth
orous bats such as Mormoops megalophylla, of contaminant bacteria. The recommended
Myotis californicus, Pteronotus parnellii) they antibiotics include cycloheximide (0.5 g/L) and
cause intestinal infection. Especially the bat chloramphenicol (0.5 g/L). The plates are kept
guano found in enclosed places, such as grottos, at room temperature (25–30 C) for 2–8 weeks.
caves, mines and abandoned buildings, acts For conversion into yeast phase, subculture of
as a major source of infection. Most of the mycelium can be performed in brain–heart infu-
human outbreaks occur when they disturb the sion agar containing 5 % horse blood or in Pine’s
soil of old bird roosting site or they visit medium at 35–37 C with 5 % CO2 tension
bat-inhabited caves for adventure or tour. The and high humidity. The complete conversion
bat guano represents a complex habitat of becomes possible after 4–5 repeated subcultures
mites, insects, bacteria and fungi. Among them into fresh medium every 7–8 days depending
the mites (Sancassania) are dependent on the upon the strain. The conversion is also possible
nutrients provided by the mycelial phase of by inoculation of the mycelium into cell mono-
H. capsulatum to complete their life cycle. layer (HeLa) or in experimental mice.
They are mycophagous and can be used as The mycelial phase appears as dry, grey-
biological control agent against the fungi. white, granular, wrinkled mycelium. The
colonies become brown with aging. The colonies 4.7.9 Transmission

chiefly contain sterile hyphae with rare occur-
rence of chlamydospores, arthospores and H. capsulatum var. farciminosum is transmitted
blastopsores. into susceptible hosts (horses) by wound infec-
The yeast colonies are small, grey, flaky tion through the nasal or ocular secretions,
composed of yeast cells and a few hyphae. contaminated saddlery, grooming utensils, soil
H. capsulatum var. capsulatum yeast cells pro- or harnesses, feeding and watering utensils, etc.
duce flat, raised, wrinkled, and white to greyish The flies and ticks can also help in transmission
brown colonies which often revert into its stable of the infection. Especially the conjunctival form
mycelial phase. of the disease is transmitted by flies (Musca or
Stomoxys). Rarely inhalation, ingestion of
contaminated feed act as possible ways.
H. capsulatum var. capsulatum is mostly
transmitted by inhalation, ingestion and rarely
by wound contamination. In human, males are
Cell wall mannans (Man-containing polysac-
found more susceptible especially those exposed
charides) and mannosylated proteins are major
with the area where the birds roost or the
antigens of H. capsulatum var. capsulatum which
bats inhabit. In such areas, infectious particles
help in host tissue adherence. Galactoman-
can exceed 105 particles per gram of soil. The
nan–protein complexes from the mycelial phase
people working as farmers, gardeners, poultry
cell wall of H. capsulatum can induce delayed-
farmers and cave explorers are at high risk of
type hypersensitivity in guinea pigs and inhibit
contamination, and their occupations can be
macrophage migration factor release. The
designated as ‘histo-hazard’ jobs. Recent evi-
filtrates of the mycelial culture (Histoplasmin)
dence suggests that exposure to a bamboo bonfire
are enriched with this protein complex. The
may also transmit the infection. The heating
galactomannan complexes may also protect the
increases the transmission efficiency of the
organism against its own serine-thiol protease, an
spores present in the bamboo, contaminated
enzyme associated with pathogen dissemination
with bird faeces.
through the extracellular matrix. In addition, heat
Inhalation is the probable route of entry for
shock protein (HSP60) of H. capsulatum var.
H. capsulatum var. duboisii in human. Rarely
capsulatum is considered as an immunodominant
transmission through transcutaneous route is
antigen; vaccination of mice with this protein
found.
protected them from lethal and sublethal histo-
plasmosis due to its stimulatory nature of CD4+
T cells. The M antigen (catalase B) is a major
4.7.10 Pathogenesis
diagnostic antigen as it can induce both the
cellular and humoral immune responses.
4.7.10.1 H. capsulatum var. capsulatum
Antigenically no difference is observed
H. capsulatum var. capsulatum is soil-based
between H. capsulatum var. capsulatum and
fungi and they do not require human or animals
H. capsulatum var. farciminosum.
to propagate. So mammals act as incidental host
in their life cycle. After inhalation of spores,
transition into the yeast phase occurs in higher
4.7.8 Virulence Factors body temperature of the host, required for
expression of virulence genes and pathogenicity.
The virulence factors possessed by The cellular entry of H. capsulatum
H. capsulatum are described in Table 4.18. var. capsulatum yeast phase occurs either by
4.7 Histoplasma 63
Table 4.18 Virulence factors of H. capsulatum

Virulence factors Location in fungal cell Function
α (1, 3)-glucan Outermost layer of yeast cell wall (i) It conceals immunostimulatory β-glucan layer of
the cell wall from detection by host phagocytic cells. It
blocks the recognition of β-glucan through its Dectin1
receptor. So it acts as ‘decoy ligand’ for Dectin1
receptor
(ii) It inhibits the production of the pro-inflammatory
cytokine (TNFα) by phagocytes
Heat shock protein Cell wall of both yeast and (i) It acts as ligand for CR3 (complement receptor 3)
(HSP60) mycelium in the macrophages. Specifically it binds with the
CD18 chain of CR3. Their attachment helps the fungi
to enter the macrophage and initiate the intracellular
phase. This kind of binding of carbohydrate ligand
(Man, GlcNAc and Glc) with CR3 repeals the release
of toxic oxygen metabolites. So it acts as safe portal
for entry into the macrophages
(ii) The binding of HSP60 with CR3 downregulates
host IL12 production, required for protection against
fungal infection
(iii) Chaperoning of proteins (associated with
oxidative stress response) to help their expression in
fungal cell surface
Calcium-binding protein Secreted by yeast cells It helps in survival of yeast cells in macrophages
(CBP1) in vitro, by acquiring calcium from the environment
Yeast phase-specific Yeast cell wall localised protein Absence of this protein in the cell wall hampers the
protein 3 (Yps3) and occasionally secreted (it is growth of the fungi in the lung and spleen. Exact role
localised with the help of in pathogenesis is uncertain
epidermal growth factor (EGF)-
like domain that can attach with
chitin of the cell wall)
(i) Catalase B Constitutively expressed protein of It prevents the lethal action of hydrogen peroxide
(M antigen; 70–94 KDa) yeast cells produced during oxidative burst defence mechanism
(ii) Catalase P (57 KDa) Monofunctional peroxisomal within the host phagocytes
(iii) Catalase A catalase, constitutively expressed
protein of yeast cells
Large subunit bifunctional
enzyme, induced upon H2O2 stress
Beta-glucosidases Secreted protein of yeast cells It helps in remodelling of the cell wall to acquire more
(H-antigen) nutrients
melanin Cell wall of the yeast and conidia It protects the fungi from host-derived free radicals,
microbicidal peptides and antifungal drugs
Dimerum acid, coprogen Secreted They act as siderophores in vitro under iron-limited
B and fusarinine condition. It scavenges for ferric iron (+3 redox state).
Exact mechanism of iron acquisition is unexplored
(a) Reduced glutathione- Secreted They can reduce Fe+++ to Fe++ and help in transport of
dependent enzymatic ferrous iron which is a way for acquisition of iron from
reductase inorganic or organic salts or siderophores, binding
(b) Low-molecular- with the ferric iron
weight nonenzymatic
reductants
Histone 2B (H2B) Cell surface of yeasts Exact role in virulence is uncertain, neutralisation of
H2B with monoclonal antibody reduced fungal
burdens, decreased pulmonary inflammatory damage
and prolonged experimental mice survival
70 KDa antigen Cell surface of yeast cells Role in virulence is uncertain
‘host-directed way’ or ‘fungi-directed way’. The unexplored. It causes suppurating lesions in

host-directed way requires opsonisation of the the skin of limbs and neck associated with the
yeast cells with antibody or complement to lymphatics followed by the development of ulcer
enter the macrophages. In fungi-directed way, and enlargement of the lymphatics with tortuous
they can directly bind the heterodimeric β2 or cord formation. In mucosal form of the disease,
CD18 integrins on the surface of host mononu- lesions are observed in the nasal passage, phar-
clear cells, including LFA-1 (with αL or CD11a), ynx, larynx, trachea, lung and genitalia with
Mac-1/CR3 (with αM or CD11b) and P150, possibility of venereal transmission. In ocular
95/CR4 (with αX or CD11c). The heat shock form, conjunctivitis, keratitis, papule formation
protein (HSP60) acts as major fungal ligand in conjunctiva or nictitating membrane is found.
for this kind of attachment. In addition to
the macrophages, the yeast phase of fungi can
also enter the dendritic cells through the inter- 4.7.11 Disease Produced
action between fungal adhesin (cyclophilin) and
fibronectin receptor (very late antigen 5). The major animal and human diseases produced
Within the host cells after internalisation, by different subspecies of H. capsulatum are
the yeasts enter into the phagosomes which enlisted in Table 4.19.
fuse with lysosomes to form phago-lysosome.
H. capsulatum var. capsulatum follows different
strategies to survive within the phago-lysosome. 4.7.12 Immunity
They can increase the pH of the phago-lysosome
with the help of urease and release of ammonia 4.7.12.1 H. capsulatum var. capsulatum
or bicarbonate, although it is not established The cell-mediated immunity (Th1 response) can
whether they inhibit or reverse the lowering of protect the host as the fungi (H. capsulatum
pH. It also prohibits the accumulation of vacuo- var. capsulatum) are intracellular in nature. The
lar ATPase/proton pump required for mainte- cell-mediated immunity develops 10 days post-
nance of H+. Thus, they elevate the pH of the infection, primarily with the production of IL12
phago-lysosome up to 6.5, optimum for iron followed by IFN γ which is crucial for protection.
acquisition from transferrin with the help of The macrophages are the primary target cells of
siderophores and lowering the action of lyso- the yeasts where they can replicate. However,
somal hydrolase. Calcium-binding protein for induction of protective cellular immunity,
released by the yeasts also lowers the lysosomal the fungicidal activity of the macrophages
enzyme capacity by chelation of calcium. Other should be enhanced. Phagocytosis of the yeasts
virulence factors such as catalase B, beta- with dendritic cells effectively clears them as
glucosidase and melanin help in the survival of they are fungicidal. Azurophilic granules of
the yeast cells within macrophages as described neutrophils have fungistatic activity in vitro.
in Table 4.18. The infected macrophages enter The IL12 is chiefly produced by dendritic
the lymph nodes and spread subsequently cells and neutrophils as the production capacity
through haematogenous route. In the absence of of macrophages are downregulated by the fungi.
a protective cellular immunity, the disease with The IL12 stimulates the IFNγ production by
dissemination may occur. Sometimes the yeasts CD4+ T cells, CD8+ T cells and NK cells.
remain dormant in the host throughout their life Among them CD4+ T cells are the predominant
and rejuvenate to produce disease during producers of IFN γ in H. capsulatum infection.
impairment of host immunity. The IFN γ can enhance the fungicidal activity of
macrophages with the production of nitric oxide
4.7.10.2 H. capsulatum var. farciminosum and can limit the accessibility of iron and zinc to
After transmission into the host, the exact mode the fungi required for their intracellular replica-
of dissemination especially at the cellular level is tion. The pro-inflammatory cytokine (TNFα)
4.7 Histoplasma 65
Table 4.19 Major diseases of animal and human caused by Histoplasma

Fungi Host Disease
H. capsulatum var. Horse, mule, donkey, Epizootic lymphangitis (African glanders/pseudo-glanders/pseudo-
farciminosum rarely in camel, farcy): It is characterised by suppurating, ulcerative pyogranulomatous
dog and cattle. It is dermatitis and lymphangitis especially in the limbs, neck and chest.
transmissible Nodular lesions appear in the skin, subcutaneous tissues and lymphatics.
to human (zoonotic) In other forms of the disease, conjunctivitis, keratitis, and multifocal
pneumonia are also observed. The disease is endemic in tropical
countries such as North, East and Northeast Africa and some parts of
Asia such as India, Pakistan and Japan. It occurs sporadically in other
parts of the world such as France, Russia, Egypt and Italy
H. capsulatum var. Human Two clinical forms, i.e. self-limiting mild respiratory tract infection and
capsulatum disseminated forms, are noted. The later form is found life-threatening
in elder or immunocompromised patients such as AIDS patients or
recipients of organ transplants. The fungus is endemic worldwide, but
there are regions with high incidences of infection, such as areas along
the Ohio and Mississippi River Valleys in the United States and in Rio
de Janeiro State in South eastern Brazil
Dog Three clinical forms, i.e. pulmonary, disseminated and clinically
inapparent form, are found. Appearance of clinical signs depends on
organ involvement. In general the signs include coughing, dyspnoea,
anorexia, ascites and ulceration of oral and nasal mucosa, fever and
diarrhoea
Cat Respiratory disease, fluctuating fever, weight loss, lethargy
H. capsulatum var. Human African histoplasmosis: This form is prevalent in western and central
duboisii Africa and in the island of Madagascar. The disease involves the skin,
subcutaneous tissue, lymph node, bone and rarely the lung or other
organs
also plays an important role in protection. IL23 (Th17 response) plays a vital role in
It can also stimulate the fungicidal activity of protection.
macrophages with the production of nitric oxide In contrast, Th2 type of immunity generated
and indirectly organises cellular migration by by IL4 delays the clearance of H. capsulatum
controlling chemokine production and adjusts from the lungs and spleen. IL4 enhances the
the emergence of regulatory T cells (Tregs). intracellular fungal growth by reducing nitric
Tregs in TNFα-neutralised mice dampen the pro- oxide production and increasing the availability
tective immune response in an IL10-dependent of iron, zinc and calcium.
manner. Other Th1-associated cytokines such as The antibody response to Histoplasma
granulocyte-macrophage colony-stimulating fac- capsulatum infection is characterised with an
tor (GM-CSF) stimulates macrophage fungistatic increase in IgM by 2 weeks, followed by rising
activity against H. capsulatum yeast. titres of IgA and IgG. The IgG fraction contains
Recently the role of Th17 response in complement-fixing and precipitating antibodies.
H. capsulatum infection is also explored. In However, the protective role of humoral immu-
pulmonary histoplasmosis, IL17, IL6 and IL23 nity is uncertain. Experimentally no significant
gradually increase the first week of infection and difference was observed between B cell-deficient
declines thereafter. CD4+ and CD8+ T cells and the wild-type mice in primary and secondary
expressing CD25 are the predominant source of histoplasmosis.
IL17 during infection. IL17 can enhance the
entry of inflammatory cells into the lungs; how- 4.7.12.2 H. capsulatum var. farciminosum
ever, it directly has minimal role in protection, Cellular immunity plays a protective role in
whereas in the absence of IL12 (Th1 response), epizootic lymphangitis. The antibodies develop
after appearance of the clinical signs. The in pulmonary histoplasmosis patients. In

infected animals produce a delayed hyper- South America, Tzanck cytodiagnosis tech-
sensitivity reaction after inoculation of mycelial nique provides a useful and rapid diagnosis
extract (histofarcin). method for AIDS-associated histoplasmosis,
due to the high frequency of mucocutaneous
lesions in these patients.
4.7.13 Diagnosis 2. Isolation and identification: Isolation of
H. capsulatum var. capsulatum from bone
4.7.13.1 Clinical Specimens marrow aspirates and blood samples suffering
For detection of H. capsulatum var. farci- from disseminated histoplasmosis is an
minosum, the exudate or biopsy samples can be important diagnostic tool. In blood culture,
collected from unruptured nodules with the help the lysis-centrifugation technique should be
of fine needle. The material should be kept employed, since it is sevenfolds more sensi-
in sterile peptone water with antibiotics and tive than the conventional techniques used for
transported to the laboratory in a flask with ice clinical sample preparation. Media and incu-
and proper label. The swabs of the material bation condition as described earlier will
should be prepared for direct examination. serve the purpose.
For histopathology, sections of lesion material 3. Animal inoculation test: Laboratory animals
should be placed in 10 % neutral-buffered such as mice, guinea pig and rabbits are used
formalin. for experimental inoculation of H. capsulatum
For detection of H. capsulatum var. var. farciminosum. Impression smear of the
capsulatum, urine, blood, bronchoalveolar liver and spleen from the inoculated animals
lavage (pneumonia patients), cerebrospinal fluid should show the yeast cells 2–4 weeks post
(meningitis patients) and bone marrow aspirates inoculation in positive cases.
can be collected from human patients. Pulmo- 4. Detection of antigen: ELISA and fluorescent
nary aspirate, gastric washings, peripheral blood antibody technique (FAT) are successfully
(febrile condition), faeces, sternal bone marrow, used for the detection of H/M antigen of
biopsied liver, spleen or lymph node can be H. capsulatum var. farciminosum from
collected from dogs. cultured mycelium. Enzyme immunoassays
are also used for the detection of H. capsu-
4.7.13.2 Laboratory Examination latum var. capsulatum circulating polysaccha-
1. Direct examination: The wet mount or gram- ride antigens in the immunocompromised
stained smear is prepared with the swab human patients with a disseminated form.
sample and observed under microscope for 5. Serological tests:
detection of typical H. capsulatum var. (a) Passive haemagglutination, immunodiffu-
farciminosum yeast cells, which will appear sion, complement fixation test (CFT),
as gram positive, pleomorphic or pear shaped, radioimmunoassay (RIA) and ELISA
double contoured, approximately 2–5 μm in were developed to detect H. capsulatum
diameter. They are intracellular within var. capsulatum antibodies from human
macrophages or neutrophils or occasionally and animal clinical samples.
they are extracellular. Budding may be visible Immunodiffusion test detects the pres-
from the pointed end of the yeast cells. The ence of two types of precipitin bands, i.e.
electron microscopy can be applied for detec- M and H bands. The M band develops in
tion of H. capsulatum var. farciminosum with both acute and chronic infection and may
minute cellular details in the skin biopsy be detected for years even after recovery,
samples. whereas the H band is rare and, if found, is
Direct examination provides best result for associated with chronic or severe acute
detection of H. capsulatum var. capsulatum infection. The assay is 80 % sensitive but
4.7 Histoplasma 67
more specific than CFT. It gives positive neck. The inoculation site is examined
results in the majority of progressive for the presence of swelling at 24, 48
pulmonary and disseminated forms, and and 72 h postinjection. In positive case,
its titres are related to the fungal burden, an indurated and elevated area at the
so they decrease with the successful treat- injection site will be observed.
ment. Immunodiffusion is not very useful 6. Molecular biology: A PCR assay was devel-
in HIV-positive patients with histoplas- oped for detection of H. capsulatum var.
mosis, since it gives positive results in capsulatum from both the clinical samples
only 35 % of mycologically proved cases and culture using the primers for the gene
of histoplasmosis. encoding M antigen. This assay had a sensi-
The CFT uses two types of antigens, tivity and specificity of 100 %.
i.e. yeast and mycelial (histoplasmin). The Real-time PCR showed promising result by
diagnosis is based on fourfold rise in CF detection of H. capsulatum var. capsulatum
antibody titre; a single titre of 1:32 from tissue biopsies and bronchoalveolar
is suggestive but not diagnostic. The CF lavage fluid from human patients who had
antibody persists for more than 1 year. documented histoplasmosis.
The test is cross-reactive with other fungal
infection and granulomatous diseases
such as tuberculosis and sarcoidosis. 4.7.14 Treatment
Histoplasma antibody was detected in
the urine of 89 % of human patients with In human suffering from pulmonary and
disseminated form of histoplasmosis and disseminated form of histoplasmosis, liposomal
37 % of human patients with other forms amphotericin B (for 2–3 weeks) followed by
of histoplasmosis by RIA and ELISA itraconazole can be administered. In chronic pul-
methods. The specificity was noted as monary histoplasmosis, itraconazole and keto-
99 %. However, false-positive result was conazole are effective. The course of treatment
noted in transplant patients receiving is 3 months to 1 year depending on the severity.
rabbit antithymocyte globulin. The sero- Due to prolonged therapy, careful monitoring for
logical response is strongly detectable nephrotoxicity (amphotericin B) and drug inter-
in pulmonary histoplasmosis than diss- action (itraconazole) is required. Itraconazole
eminated and asymptomatic forms due to is also used as prophylactic drug in patients of
lower intensity of exposure resulting in endemic zone having less CD4+ T cells. In AIDS
reduced fungal burden and antigenic sim- patients, bioavailability of itraconazole is
ulation or due to immunosuppression. decreased. Oral posaconazole and voriconazole
(b) Skin hypersensitivity test (histofarcin test) are currently considered as alternative treatments
The mycelial form of H. capsulatum for non-severe disseminated histoplasmosis.
var. farciminosum is grown on polysty- Fluconazole is less effective but can be used in
rene discs floating PPLO media the patients who are allergic to itraconazole.
containing 2 % glucose and 2.5 % glycer- In horses suffering from epizootic
ine at 23–25 C for 4 months. The fungus- lymphangitis, surgical removal of lesion along
free culture filtrate is mixed with acetone with amphotericin B administration is
(2:1) and held at 4 C for 48 h. The super- recommended.
natant is decanted and the acetone is In dog and cats, drug of choice is itraconazole
allowed to evaporate. The precipitate is due to low level of toxicity. Other drugs such as
suspended to 1/10 original volume in dis- ketoconazole and fluconazole are also effective.
tilled water. The animals are inoculated Pulmonary form is curable with proper treatment.
intradermally with 0.1 ml of antigen in the The prognosis of disseminated form is grave.
and granular cytoplasm containing mitochondria.

4.8 Rhinosporidium The cytoplasm also contains endoplasmic reticu-
lum, lipid-rich globules and vacuoles. The cell
In 1900, Seeber first published the report of wall is unilamellar in nature with a fibrillar cap-
rhinosporidiosis which occurred in an sule of electron-dense material. This juvenile
Argentinean youth suffering from laboured sporangium undergoes the sequential morpho-
breathing due to nasal polyp. Seeber although logical changes to produce immature, intermedi-
noted that Malbram (1892) was the first to diag- ate, early mature and mature sporangia. The
nose the disease in human in Argentina which intermediate sporangia (70–150 μm) contain
was unpublished. Seeber described the thickened bilamellar cell wall made of chitin
similarities of the organism with Coccidioides (thinner electron-dense outer wall and thicker
immitis. Accordingly, Robert Wernicke, advisor translucent inner wall), several nuclei, several
of Seeber, named the organism as Coccidioides flat cristae mitochondria and an early pore. The
seeberia or Coccidium seeberi in 1900. In 1903, cytoplasm condenses around each of the nucleus
O’Kineley first described the histological to develop endospores (4–10 μm in diameter),
changes caused by the organism which were and the sporangium with such endospores is
isolated from India. Minchin and Fantham known as early mature sporangia. The mature
(1905) further studied O’Kineley’s Indian sporangium is a large (up to 400 μm) spherical
isolates and named the organism as body containing hundreds of mature and imma-
Rhinosporidium kinealyi. The earliest evidence ture endospores and a pore or operculum. It is
of animal infection was noted by Theiler (1906) enclosed within a thin cell wall with three prom-
in South Africa in a horse. Later, similar organ- inent inner layers. The mature endospores are
ism from the horses was reported by ZSchokke located near the centre and towards the pore,
(1913) in South Africa, who also named them as whereas the immature endospores are located at
Rhinosporidium equi. Ashworth (1923) revised the opposite pole of the sporangium (germinative
all the nomenclature proposals and finally zone) (Fig. 4.16).
concluded Rhinosporidium seeberi as the name
of the pathogen. Thus, the different names such
as Rhinosporidium ayyari (isolated from buf-
falo), Rhinosporidium hylarum and
Rhinosporidium amazonicum are currently
known as R. seeberi.
In India, animal rhinosporidiosis was reported
by several workers such as Ayyar (1927) and Rao
(1938, 1951). Ayyar (1927) reported for the first
time about the occurrence of rhinosporidiosis in
cattle in India.
4.8.1 Morphology and Life Cycle
The morphology and life cycle study of

Rhinosporidium seeberi is based on its appear-
ance in the tissue section, as the organism cannot
be grown in vitro. Seeber (1900) first described
Fig. 4.16 Matured sporangium of Rhinosporidium
the life cycle in the infected tissues. Primary
seeberi in a nasal polyp stained with PAS (Photograph
juvenile sporangium (trophocyte) is a thin- courtesy: Prof. P.P. Gupta, Ex-Director of Veterinary
walled structure with a single nucleus, nucleolus Research, Punjab Agricultural University, Punjab, India)
4.8 Rhinosporidium 69
The mature endospores contain a bilaminated After release, the endospores increases in size
thin cell wall (1–3 μm), 15–20 spherical (1–3 μm (10–70 μm) and lose all the EDBs to become a
in diameter) electron-dense bodies (EDB), juvenile sporangium and the cycle is repeated.
nucleus with nucleolus, Golgi apparatus, endo- The life cycle of Rhinosporidium seeberi is
plasmic reticulum and mitochondria. The shown in Fig. 4.17.
electron-dense bodies either act as nutrient reser-
voir or precursor of endosporulated sporangia
and they are known as ‘spore-morula’.
The mature endospores are released from the 4.8.2 Classification
sporangium through the pore. There are two
explanations of the endospore release mecha- The taxonomical status of Rhinosporidium
nism. In contact with watery material, lytic seeberi is controversial because it could not be
enzymes are accumulated at the pore site of the isolated in artificial culture. Previously, it was
mature sporangia. The inner osmotic pressure of considered as a cyanobacterium under the genus
the mature sporangia is also increased which Microcystis probably due to morphological
facilitates the release of endospores through the resemblance between coccal-shaped Microcystis
pore. Another explanation states that due to nanocytes and spherical endospores of
high antigenicity of the sporangia, anti-sporangia Rhinosporidium. However, 16S rRNA sequence
antibodies are developed by the host defence analysis revealed only 79–86 % similarity with
system. The immune complexes are produced Microcystis. The ultrastructural studies revealed
by the interaction of the antigen and antibody. that the nanocytes do not possess a true cell wall
The complexes aid in expulsion of endospores by and are held together with an amorphous matrix,
producing external pressure during expansion of whereas the R. seeberi endospores are
the sporangia through increased water intake. surrounded with a thin cell wall.
Fig. 4.17 Life cycle of

Rhinosporidium seeberi
(schematic); a release of
endospores from matured
sporangium, b released
endospores become large
(10–70 μm), c formation of
juvenile sporangium
possessing single or double
nuclei with nucleolus and
thick cell wall, d formation
of intermediate sporangium
(70–150 μm), e early
matured sporangium
(<150 μm)
Protists
The total period of exposure of endospores to
the studied disinfectants was for 7 min which
Plant Animal produced metabolic inactivation.
Jellyfish
Fungi
Sponge 4.8.4 Natural Habitat and Distribution
Choanoflagellate
DRIP
Aquatic environment is detected as major eco-
logical niche of R. seeberi because most of the
Fig. 4.18 Phylogenetic position of DRIP group
patients are observed in close proximity with
same water source in a locality such as lake,
Further studies revealed its eukaryotic nature pond, etc. However, the fungi are also present
and claimed its morphological similarities with in the dry areas of North India, Middle East
Chytridiales group of fungi. However, compara- countries and Argentina probably through the
tive studies by other workers showed certain formation of a resistant structure.
morphological dissimilarities especially in the The fungus is reported from more than
cell wall structure between R. seeberi and proto- 70 countries with highest incidence (95 %) in
type member of Chytridiales. The most impor- India and Srilanka which are considered as
tant dissimilarity is the compulsive intracellular endemic zones. India and Srilanka is followed
nature of Chytridiales which is not possible in by South America, Brazil, Argentina and Mexico
R. seeberi due to their larger size. and a few reports are available from Nepal,
Later, phylogenetic and taxonomic analysis Columbia, Venezuela, United States, Uganda,
placed it under the new cluster of aquatic Madagascar, Ghana, Iran, Russia, Europe and
pathogens, known as the DRIP group Southeast Asia. Most of the cases in human and
(Dermocystidium, rosette agent which is pres- animals such as cattle, buffalo, goat, dog, cat,
ently known as S. destruens, Ichthyophonus mule, duck, geese and water fowl are sporadic.
and Psorospermium). The DRIP group was The sole human and swan outbreak of ocular and
renamed as Mesomycetozoa class which is nasal rhinosporiodiosis was reported so far from
located at the divergence between animals Serbia and Florida, United States, respectively.
and fungi (Fig. 4.18). The class is composed
of two orders such as Dermocystida and
Ichthyophonida. The morphological and phylo- 4.8.5 Genome
genetic analysis of R. seeberi revealed its simi-
larity with the members of Dermocystida. The The juvenile sporangium of R. seeberi contains
presence of synchronised nuclear division with four chromosomes. Other genome characterisa-
the formation of endospores in the matured stage tion of R. seeberi is poorly known because most
confirms its association with Mesomycetozoa of the sequences deposited in the genbank
class. (NCBI) are rDNA sequences rather than the
protein-coding sequences. Further no informa-
tion is also available about the genome of closely
4.8.3 Susceptibility to Disinfectants related mesomycetozoeans.
Experimentally metabolic inactivation with or

without altered structural integrity of R. seeberi 4.8.6 Isolation, Growth and Colony
was observed with hydrogen peroxide, glutaral- Characteristics
dehyde, chloroxylenol, chlorhexidine, cetrimide,
thimerosal, 70 % ethanol, 10 % formalin, Since the time of discovery, attempts have made
povidone–iodine, sodium azide and silver nitrate. to isolate R. seeberi in artificial and live cell
4.8 Rhinosporidium 71
culture without any success. Although a few these factors. There is no clear evidence of
workers have claimed about isolation of transmission between human, animals, human
R. seeberi in artificial culture and in human rectal to animals and vice versa.
epitheloid cell line, further studies failed to sup-
port their statement. The confirmational studies
revealed that the isolates were environmental 4.8.9 Pathogenesis
contaminant. In crushed rhinosporidial tissues,
formation of endospores and sporangium was The primary lesion develops in the nasal passage
noticed when kept at low temperature or room of the host after transmission of the infection.
temperature. However, the sustained propagation The rhinosporidial lesions are polypoidal, less
with repetition of the whole life cycle was not than 3 cm in diameter, pedunculated or sessile,
found in any culture system. granular, multilobed and red in colour due to
marked vascularity. Due to pronounced vascular-
ity, the lesion bleeds easily. The surface of the
4.8.7 Antigenic Characteristics lesion contains yellowish small spots underlying
mature sporangia. The polypoidal lesions devel-
In the mature sporangium of R. seeberi, an oped in the face and trunk are converted into
immunodominant antigen is detected which is verrucous warts.
absent in juvenile or early mature sporangium The dissemination of infection may occur
and the endospores. The antigen is located in through the blood circulation into the distant
the inner electron lucent cytoplasmic layers of viscera. The local spread may occur into the
the mature sporangium. Another study revealed adjacent area of the polypoidal lesions by spill-
the presence of a circulating antigen in the sera of age of the endospores during trauma or surgery
the patients infected with R. seeberi. However, of the polyp. It is known as ‘autoinoculation’.
further characterisation is needed to detect their The disseminated lesions have been detected
chemical structure. in the conjunctival sac, ear, vagina, limbs, trunk
and other external part of the body where it
appears as subcutaneous lump with intact skin.
4.8.8 Transmission The lesions are ulcerated growths resembling
sarcomas and carcinomas. The involvement of
In human, nasal form is the most common form the limbs often causes the destruction of under-
of rhinosporidiosis which occurs due to frequent lying bones. The dissemination of the infection
bathing or working in stagnant fresh water. The into the central nervous system has fatal
skin injury is the most frequent predisposing consequence.
factor. It is commonly observed in river-sand
workers in India and Srilanka. The sand particles
cause skin abrasions through which the infection 4.8.10 Disease Produced
can occur during direct contact with the
contaminated river water. However, in arid There are four clinical forms of rhinosporidiosis
zones such as in North India and Middle East detected in human patients. In nasal form, the
countries, ocular form predominates and the lesions (polyp) develop at the anterior nares,
resistant endospores are considered as major nasal cavity, septum and floor. The nasopharynx,
infectious agent. The endospores are transmitted larynx and soft palate are sometimes affected.
through air especially during sand storms. The formation of polyp is painless until it
In animals, the infection takes place through becomes large enough to obstruct the nasal
traumatised skin from the contaminated passage or to put pressure on the nerve and vas-
water bodies. The hunting or street dogs are culature endings. In ocular form, the lesions
in increased risk due to more exposure to develop in the bulbar and palpebral conjunctiva.
The ocular lesion begins with a sessile growth macerated polyp to detect the sporangia
which later becomes pedunculated, degenerating (350–400 μm in diameter) with endospores
to friable. If the lesion becomes large, there is or the empty sporangia and free endospores
redness of eyes, tearing, photophobia, lid ever- in positive samples. It may produce confusion
sion and conjunctivitis. The cutaneous form is with the spherules of Coccidioides immitis
characterised by formation of papule initially which are smaller (60–100 μm diameter;
which later becomes wart-like growth with a Fig. 4.12) than the mature sporangia of
crenulated surface. In the disseminated form, R. seeberi.
the sporangia are detected from the bone, liver, 2. Histopathology: The histological inspection
spleen, lung and brain. of the lesion detected hyperplastic epithelium
In dogs, the clinical signs such as visible mass with sporangia containing endospores below
within the nares, wheezing, sneezing, unilateral the surface. The area is highly vascularised
purulent nasal discharge and epistaxis are and surrounded by a connective tissue layer.
noticed. Different immune cells such as polymorpho-
nuclear cells, lymphocytes, plasma cells, giant
cells and histiocytes infiltrate into the area.
4.8.11 Immunity Different stages of R. seeberi can be observed
with special fungus stains such as the Gomori
The cell-mediated immune (CMI) response (Th1 methenamine silver, Gridley’s, periodic
mediated) is generated against R. seeberi along acid–Schiff stains and haematoxylin–eosin
with suppressor reaction. The major cell popula- stain.
tion involved in CMI response are macrophages 3. Molecular biology: The polymerase chain
(CD68), NK cells (CD57+), numerous CD8+ T reaction can detect R. seeberi by amplification
lymphocytes and less numbers of CD4+ T of the 18S rRNA gene directly from the
lymphocytes. In chronic and disseminated infec- infected tissues.
tion, the immune response is converted into
Th2-mediated response. The antibodies are
generated without any protective role against 4.8.13 Treatment
infective endospores. The antibodies are detected
to bind the soluble antigen of R. seeberi which is Spontaneous regression of the nasal growth is
released by sonication of the sporangia in vitro. observed in human and animals which is rare in
Probably it acts as an immune evasion mecha- occurrence. So, surgical intervention or treat-
nism which protects the endospores from the ment with drugs is required. Radiotherapy has
antibodies in vivo. no effect in curing of the rhinosporidial polyp.
The surgery by hot or cold snare techniques is
the treatment of choice; however, recurrence of
4.8.12 Diagnosis polyp may occur sometimes (10 %).
In animals, berenil (diminazine aceturate) and
4.8.12.1 Clinical Specimens imizol (imidocarb dipropionate) can be used.
The clinical specimens for rhinosporidiosis The human and animal drugs such as ampho-
include nasal scrape, fine needle aspirates from tericin B, dapsone, ketoconazole, trimethoprim–-
the lesion and tissue biopsy specimens. The nasal sulphadiazine and sodium stibogluconate
scrape is preferred over the fine needle aspirates showed effective in vitro anti-rhinosporidial
because the lesions bleed easily. activity. However, in vivo trial report is available
with amphotericin B which was effective topi-
4.8.12.2 Laboratory Examination cally. Instead, dapsone may be a better choice for
1. Direct examination: The KOH mount can be treatment with a dosage of 100 mg/kg body
prepared from the clinical samples such as weight orally for 6 months to 1 year.
4.9 Rhizopus 73
4.9 Rhizopus
The genus Rhizopus was first established in 1820

by Ehrenberg, and the fungus was chiefly known
for the production of fermentation products like
ethanol, lactic acid, fumaric acid and malic
acid. The pathogenic potentiality was recognised
later when the human infection (rhinocerebral
zygomycosis) due to Zygomycetes was detected
in Germany by Platauf (1885). He described the
infection as ‘mycosis mucorina’. The sketches
drawn by Paltauf to describe the etiological
agent were more similar with Rhizopus than Fig. 4.19 Sporangiophore of Rhizopus (Photograph
Mucor. In 1943, similar kinds of human cases, courtesy: Andrii Gryganskyi, Lambert Spawn)
affecting meninges and central nervous system, Rhizopus. The branching usually takes place at
were reported from patients with diabetes right angle which may vary from 45 to 90 . The
mellitus. Bauer et al. (1955) first isolated the root-like structure (rhizoid) and the unbranched
major etiologic agent of rhinocerebral zygomy- sporangiophore in clusters (umbels) arise from
cosis, i.e. Rhizopus arrhizus (R. oryzae). the opposite side of the hyphae. The horizontal
Baker (1957) coined the term ‘mucormycosis’ aerial hypha connecting two adjacent umbels is
to describe Zygomycetes infection. Clark known as stolon. The stolon grows laterally and
(1968) restricted the term ‘mucormycosis’ for contacts the growth medium in vitro to expand
the infection produced by Mucorales and the colony, whereas the sporangiophore is pro-
introduced the term ‘Entomophthoromycosis’ to duced erect to the mycelia. The sporangiophores
describe subcutaneous phycomycosis caused by are dry and easily blown with the wind. They
Entomophthorales fungi. expand at their tip to generate columnella (colu-
Chick et al. (1958) first described ampho- mella) which bears the sporangium, containing
tericin B as a therapeutic agent for human numerous asexual sporangiospores. The broad
zygomycosis experimentally produced in rats area below the columnella is known as apophy-
with the inoculation of Rhizopus arrhizus sis, which is inconspicuous (not noticeable) in
(R. oryzae). Rhizopus (Fig. 4.19).
In India, Rhizopus was first detected as an Differentiation of genera is possible on the
etiological agent of bovine mastitis (Monga and basis of presence or absence and location of
Kalra 1971). Other earlier reports on detection of rhizoid, shape of the columnella, size and shape
Phycomycetes (Rhizopus or Absidia) in pigs are of sporangia and appearance of apophysis.
available which is based on histopathology In Rhizopus, the rhizoids are found directly
(Sadana and Kalra 1973; Chauhan and Sadana opposite the base of aerial sporangiophore. In
1973). Entomophthora was also detected histo- Absidia the rhizoids are also present in opposite
pathologically in the tissues of a mare suffering but between two adjacent sporangiophores.
from fatal cutaneous mycosis (Chauhan In Mucor, the rhizoids are absent. Further, the
et al. 1972). stolons are found in Rhizopus only. The globose
sporangium (round) is produced by both Rhizo-
pus and Mucor, whereas Absidia produces pyri-
4.9.1 Morphology form (pear shaped) sporangium. The apophysis
is inconspicuous (not noticeable) in Rhizopus,
The branching, wide (10–50 μm) and sparsely absent in Mucor and prominent in Absidia
septate or coenocytic hyphae are produced by (Fig. 4.20).
4.9.3 Reproduction
Most of the Rhizopus is heterothallic except

R. sexualis and R. homothallicus which are
homothallic in nature. Both heterothallic and
homothallic Rhizopus can undergo sexual repro-
duction to produce zygospores. Two sexually
compatible hyphae (zygophore) attach with
each other by conjugation. The zygophores
enlarge to produce progametangium which
Fig. 4.20 Morphology of Rhizopus (schematic);
matures into gametangium. The gametangia
a stolon, b sporangium, c columella, d apophysis, are attached with zygophores by a suspensor.
e sporangiophore, f rhizoid There is cytoplasmic continuity between the
suspensor and gametangia through the pores
4.9.2 Classification (plasmodesmata) for supply of the nutrients.
Soon after conjugation, plasmogamy, karyogamy
Before the introduction of fungi kingdom, all the [between (+) and () mating types] and meiosis
agents causing zygomycosis and entomoph- take place which generates zygospores
thoromycosis were classified under the class (meiospores). In Rhizopus, some nuclei fuse,
Phycomycetes of the subdivision Thallophyta while others degenerate and meiosis is delayed
in the plant kingdom. Later, the phylum Zygo- until zygospore germination (‘Rhizopus pattern’
mycota was introduced under the kingdom of zygospore production).
Fungi which consists of class Zygomycetes. However, germination of zygospores for
The Zygomycetes class is composed of two the development of new progeny is a rare
orders, i.e. Mucorales (currently elevated to the event in Rhizopus (except in R. stolonifer,
subphylum Mucoromycotina) and Entomophtho- R. microsporus). In R. stolonifer, two types
rales (entemon ¼ insect). The order Mucorales of zygopsore germination occur. If the
contains 8 genera such as Rhizopus, Absidia, immatured zygospores germinate, they produce
Mucor, Rhizomucor, Apophysomyces, Cunning- a vegetative mycelium which is either (+) or ().
hamella, Saksenaea and Cokeromyces, whereas When the matured zygospores germinate,
the order Entomophthorales contains two genera, they form a germosporangium that produces
i.e. Basidiobolus and Conidiobolus. Molecular spores with equal proportions of (+) and ()
phylogenetic analysis also confirmed the mating types.
phylum Zygomycota to be polyphyletic. Certain The sexual mating is regulated by diver-
taxa conventionally classified in Zygomycota gent alleles of a single gene, i.e. SexM ()
are now placed under the new phylum and SexP (+). The sex locus is a member of
Glomeromycota and subphyla incertae sedis the high mobility gene (HMG) family and
(uncertain placement). is located between two flanking genes that
Major species under the genus Rhizopus code for a triose phosphate transporter homolog
are R. arrhizus (R. oryzae), R. rhizopodiformis, (TPT) and an RNA helicase. In R. oryzae,
R. azygosporus, R. stolonifer, R. schipperae, the (+) allele of sex locus is significantly
R. microsporus var. microsporus, R. microsporus larger than () allele due to additional
var. rhizopodiformis and R. microsporus var. gene insertion. However, like the pathogenic
oligosporus. Further, R. oryzae can be function of MAT locus in Cryptococcus, the
subdivided into two cryptic species groups, role of sex locus in Rhizopus virulence is not
designated as R. oryzae s. s. and R. delemar. confirmed.
4.9 Rhizopus 75
4.9.4 Susceptibility to Disinfectants dehydrogenase (LDH) encoding genes, i.e. ldhA

and ldhB. Type I strains of R. oryzae produce
Rhizopus is detected to be susceptible to lactic acid only and contained both ldhA and
benzalkonium chloride. ldhB, whereas type II strains produce fumaric
and malic acids and possess ldhB gene only.
The mitochondrial genome size of
R. stolonifer is 54.2 kbp.
The Mucorales are ubiquitous in nature,

thermotolerant and are usually found in decaying
Characteristics
organic matter, soil (cultivated grassland, forest
and volcanic mud), dung, sewage, municipal
Rhizopus prefers to grow in standard fungal and
waste, saw dust, wood pulp, different foods
bacterial isolation medium such as Sabouraud
stuffs, cereals and vegetables such as old bread
dextrose agar, malt agar, potato agar, blood
(‘bread mould’), barley, sorghum, wheat, corn,
agar and chocolate agar at 25–30 C. It is able
oat, rice, onions, cotton, groundnuts, sweet
to grow well at a wide temperature (up to 40 C)
potatoes, pecans, brazil nuts and tomatoes espe-
and pH range (4–9). The virulent strains are able
cially which are rich in sugar and nitrogen. The
to grow at 37 C. The media should contain
skin scrapings of healthy fowl are found to har-
antibiotics to prevent the growth of contaminants
bour R. microsporus. Some nonpathogenic
except cycloheximide. Most of the Zygomycetes
strains of R. oryzae are used as fermented starters
are sensitive to cycloheximide. Isolation of Rhi-
or alcoholic beverages in Asian countries.
zopus from tissue specimens is difficult if the
Mucormycosis is worldwide in distribution
isolation process involves tissue maceration
in both developed and developing countries.
which causes destruction of fungi. As there are
R. oryzae has been identified in India, Pakistan,
no septa, the cytoplasm is expelled out under
New Guinea, Taiwan, Central and South Amer-
pressure which prevents the fungal isolation in
ica, Africa, Iraq, Somalia, Egypt, Libya, Israel,
the media.
Turkey, Spain, Italy, Hungary, Czechoslovakia,
Rhizopus produces fluffy white, grey-
Germany, Ukraine and the United States,
coloured colonies in the beginning which later
whereas R. stolonifer is detected in tropical and
becomes yellowish or brownish colonies dotted
subtropical environments.
with black sporangia. The colonies rapidly fill
the plates within 1–7 days.
4.9.6 Genome
The genome of R. oryzae is 45.3 Mbp in size and
20 % of the genome is composed of transposable The heat-stable, extracellular polysaccharide
elements (repetitive DNA sequences). There are (EPS) antigen of Rhizopus has an immuno-
approximately 13,895 protein-encoding genes dominant carbohydrate epitope constituted with
which do not overlap with transposable elements. 2-O-methyl-D-mannose residue. This is a com-
The phylogenetic analysis revealed ancestral mon compound detected in Rhizopus, Mucor,
whole genome duplication which increases the Absidia and Rhizomucor which is responsible
expression of genes related with virulence, fungi- for the cross-reactivity between these genera.
specific cell wall synthesis and signal transduc- Rhizopus also secretes rhizoferrin, a
tion. This genome duplication also allows the siderophore that belongs to the polycarboxylate
fungi to grow under various adverse conditions. family. This siderophore can elicit immune
The genome contains two types of lactate response.
Table 4.20 Virulence mechanisms and factors possessed by Rhizopus

Thermotolerance It allows Rhizopus to grow at host body temperature
Ketone reductase It helps the fungi to grow in acidic pH and glucose-rich condition such as
diabetes mellitus/grain overload
Protease, lipase They help in tissue invasion by the fungi
Iron acquisition system: The system allows to uptake the iron from the host as in most of the cases, the iron is bound
with carrier protein (transferrin) and become unavailable to the growing fungi. So Rhizopus grows poorly in the
presence of serum which contains these carrier proteins
High-affinity iron permease It helps in iron uptake from the host which is required for survival of the fungi. It
(encoded by FTR1 gene) is a part of a reductive system containing redundant surface reductases that
reduce ferric into the more soluble ferrous form. The reduced ferrous iron is
captured by a protein complex consisting of a multicopper oxidase and a ferrous
permease
Rhizoferrin It is a siderophore which also provides iron to Rhizopus through a receptor-
mediated, energy-dependent process. It can chelate the ferric iron. Rhizopus can
also utilise siderophores secreted by other organisms (xenosiderophores) such as
deferoxamine (a bacterial siderophore)
Haeme oxygenase It helps in obtaining iron from host haemoglobin and also promotes the
angioinvasion by Rhizopus
SreA It is a transcriptional regulator which is required for adaptation to the ambient
iron availability
Toxin
Rhizoxin (R. microsporus, It is an antimitotic macrocyclic polyketide metabolite and is involved in
R. chinensis) pathogenesis in plants. Recent studies indicated that instead of Rhizopus, this
toxin is synthesised by intracellular symbiotic bacteria (Burkholderia)
Agroclavin (R. arrhizus) Toxic for ruminants
Rhizonin A (R. microsporus) The toxin causes hepatitis in ducks and rats. The toxin is produced by
Burkholderia, associated with the fungi Rhizopus microsporus
4.9.9 Virulence Factors insect bite, intramuscular injection are also

reported in human.
The virulence factors of Rhizopus are enlisted in The predisposing factors for establishment
Table 4.20. of infection include malignant haematological
disorder (especially acute myelogenous leukae-
mia) with or without stem cell transplantation,
4.9.10 Transmission neutropenia (<0.10 109/L in human), diabetes
mellitus (type 1, type 2 and secondary) or
The transmission of Rhizopus occurs by grain overload in animals, iron overload
inhalation, ingestion and percutanoeus route. (deferoxamine therapy in human), trauma, use
The sporangiospores, produced by Rhizopus, are of corticosteroids (producing defect in macro-
easily aerosolised due to small size (3–10 μm in phage or neutrophil function) and intravenous
diameter) and are dispersed throughout the envi- drug, neonatal prematurity and malnourishment.
ronment. They can enter through inhalation, The iron overload helps in growth of the
damaged skin due to trauma, burn or perforation fungi. Rhizopus can utilise deferoxamine as
produced by venous catheters. Even there is a siderophore for iron uptake. The prolonged
report of human infection which occurred from use of certain antifungal (voriconazole) in
the spore-contaminated adhesive bandages. haematological disorder patients receiving
Ingestion route of transmission by mouldy stem cell transplants is also associated with
feedstuffs is observed in animals with trauma in mucormycosis. In India, majority of human
buccal cavity. Sporadic cases of transmission by mucormycosis cases (74 %) are associated with
4.9 Rhizopus 77
prolonged uncontrolled diabetes mellitus, and a The hyphae can penetrate the blood vessels
proportion of cases are detected in renal trans- especially the arteries (angioinvasion), causing
plant recipients. thrombosis and necrosis. The endothelial
The seasonal variation in Rhizopus infection glucose-regulated protein (GRP78/BiP/HSPA5)
in human is also detected. The studies in Asian acts as receptor for R. oryzae, although the fungal
countries (Israel, Japan) identified the autumn ligand is still not identified. The interaction
season (August–September) as most suitable for between the ligand and receptor helps in endocy-
transmission of Mucorales infection. tosis of hyphae. The endocytosis of both live and
killed hyphae produces endothelial cell damage,
indicating the presence of a toxic chemical in the
4.9.11 Pathogenesis hyphae which is not identified. As a conse-
quence, the vascular smooth muscle cells are
The entry of sporangiospores into the immuno- exposed releasing tissue factors and producing
competent host cannot ensure the establishment intravascular thrombosis.
of infection. If the host is immunosuppressed due The proliferation and dissemination of the
to prolonged use of steroids or the host is fungal hyphae depends on production of viru-
suffering from diabetes and satisfy the other lence factors such as ketone reductase, proteases,
predisposing factors, there is every possibility lipases and iron acquisition system (Table 4.20).
of establishment and progress of infection. The angioinvasion helps in haematogenous
Under favourable conditions such as low pH, dissemination of the fungi into the different tar-
increased iron content and high glucose con- get organs such as the uterus of the immuno-
centration, the sporangiospores germinate suppressed dam causing mycotic abortion.
and produce hyphae. The low pH (acidosis)
impairs the iron-binding capacity of serum trans-
ferrin and increases the level of free iron which 4.9.12 Disease Produced
favours the fungal growth. So, diabetic
ketoacidosis (DKA) patients suffer more from The major animal and human diseases produced
mucormycosis. by Rhizopus are enlisted in Table 4.21.
Table 4.21 Major diseases of animal and human caused by Rhizopus

Fungi Host Disease
Rhizopus Ruminants Mycotic abortion and placentitis
Pigs Gastric ulcer, submandibular granuloma, abscess in the stomach, lymph node,
liver
Dogs and A rare infection in dogs and cats and is associated with use of steroids, intestinal
cats trauma, diabetes, malnutrition and feline panleukopenia infection. The fungal
menace causes vomition, diarrhoea, necrotic lesions in the intestinal wall,
peritonitis, intestinal obstruction
Chicken, The vital organs such as the lungs, heart, aorta, spleen, liver, air sacs, kidney and
duck vertebrae are involved. White nodules are observed in the affected tissues
Rhizopus stolonifer Horse Equine invasive pulmonary mycosis
(with Aspergillus niger)
Human In human, mucormycosis produces six major clinical forms such as
rhinocerebral, pulmonary, cutaneous, gastrointestinal, disseminated and rare
forms, like endocarditis, osteomyelitis, peritonitis and renal infection. The most
common sites of invasive mucormycosis are the sinuses (39 %), lungs (24 %)
and skin (19 %). The skin and gastrointestinal tract are also commonly infected
in children. The mortality rate is 66 % in patients with malignancy, 44 % in
patients with diabetes and 35 % in the patients with no other menace. In India,
rhino-orbito-cerebral manifestations are most common feature followed by
cutaneous disease, and most of the infections are associated with diabetes
mellitus
4.9.13 Immunity is vital because the Zygomycetes spores

are common in the environment and may pro-
The ciliated bronchial cells and mucus in the duce contamination in the artificial culture.
respiratory tract are the first line of defence. The typical wide, coenocytic hyphae,
The ciliary movement and the cough attempt to branching at right angle, are seen when potas-
expel the sporangiospores of Rhizopus during sium hydroxide with Parker ink or calcofluor
inhalation. The pulmonary alveolar macrophages white is added to the material.
act as second line of defence which can phago- 2. Isolation and identification: Media and incu-
cytose the sporangiospores before their germina- bation condition as described earlier will
tion. The intact skin and mucous membrane also serve the purpose.
act as barrier to prevent the entry of the spores. 3. Histopathology: The haematoxylin and eosin
The toll-like receptors (TLR 2) and other pat- (H&E) or Grocott stains are used for the dem-
tern recognition receptors (PRR) present in the onstration of Rhizopus in the tissue section.
phagocytes can interact with the fungal patterns The Grocott stain produces better resolution
(PAMP) in the spores or hyphae. The interaction due to high contrast with minimal background
can transmit the intracytoplasmic signal for impregnation. The tissue alterations include
pro-inflammatory cytokine release and activa- neutrophilic infiltrate, necrosis, thrombosis,
tion of antifungal activities of the phagocytes. septic infarction and angioinvasion.
The phagocytes can damage the spores or 4. Molecular biology: A molecular diagnostic
hyphae through oxygen-independent or oxygen- technique based on real-time PCR was devel-
dependent pathways. The study indicated that oped for the simultaneous detection of Rhizo-
in comparison to Aspergillus fumigatus, the pus oryzae, Rhizopus microsporus and Mucor
R. oryzae spores are less killed by healthy in both culture and clinical samples. Further,
human neutrophils, whereas the magnitude of the amplification of the internal-transcribed
cytokine release (IL6, IL8, TNFα) from blood spacer (ITS2) region by a semi-nested PCR
monocytes is more in R. oryzae-induced cells and followed by the hybridisation of the
than A. fumigatus. The probable reason is that amplicons to the probe using Luminex tech-
the cell wall of R. oryzae contains more chitin nology was also performed for simultaneous
than other fungi which is considered as a potent detection of major medically important fungi
PAMP for the phagocytes. Further, the antifungals including Rhizopus.
such as echinocandins and amphotericin B 5. Matrix-assisted laser desorption/ionisation
formulations exert potent immunomodulatory time-of-flight mass spectrometry (MALDI-
effect on the phagocytes against Rhizopus. TOF-MS): Recent development in diagnostic
technique includes MALDI-TOF-MS-based
system which shows higher accuracy than
microscopic methods at reduced time. The
4.9.14 Diagnosis
differentiation of filamentous fungal species
including Rhizopus has been established with
4.9.14.1 Clinical Specimen
this cutting edge technique.
The clinical specimens from animals include vagi-
nal discharge, aborted foetal content, faeces and the
vital organs collected after postmortem, whereas
4.9.15 Treatment
the specimens from human include nasal aspirates,
sputum, bronchoalveolar lavage, pleural fluid,
In human, treatment with amphotericin B lipid
transbronchial biopsy, blood and skin scrapings.
formulations is the drug of choice against
Zygomycosis. If the treatment does not produce
4.9.14.2 Laboratory Examination satisfactory result, the combined antifungal ther-
1. Direct examination: The direct examination in apy consisting liposomal amphotericin B with
clinical specimens for detection of Rhizopus either caspofungin or posaconazole is preferred.
4.10 Mucor 79
The antifungal voriconazole is contraindicated in

the treatment of Zygomycosis. Sometimes, the
surgical intervention (lobectomy or pneumonec-
tomy) and immune reconstitution with colony-
stimulating factor (CSF) to increase the blood
neutrophil level is required for the survival of
the patients.
In animals, systemic antifungal therapy with
amphotericin B is preferred to cure the Rhizopus
infection, although the prognosis is grave even
after early diagnosis of the infection. Decreased
exposure to the sporangiospores can prevent the Fig. 4.21 Sporangiophore of Mucor (schematic)
infection in animals with adequate ventilation
in the barns and reduced feeding of moldy hay M. genevensis, M. bacilliformis and
or silage. M. subtilissimus (certain strains). The yeast
cells can develop from sporangiospores,
arthrospores and hyphae but not directly from
4.10 Mucor the zygospores. The Mucor yeasts are large
(20 μm in diameter), spherical, multinucleate
The first description of Mucor was observed cells, and they produce several buds at random
in Robert Hooke’s Micrographia (1665) and in locations. The yeast cell wall is much thicker,
Marcello Malpighi’s Anatome Plantarum (1679). more diffuse and more fibrous than the cell wall
Louis Pasteur noted the fermentative capacity of hyphae. The yeast cell wall contains two sep-
of Mucor and described the dimorphism of arate layers, whereas the hyphal cell wall is
Mucor racemosus. In 1918, Ernst first reported uniform and single layered. There are several
Mucor corymbifer (Absidia corymbifer) as an environmental factors influencing dimorphism
etiological agent of human vocal cord infection such as anaerobic condition which favours the
and established the pathogenic status of Mucor. generation of yeast cells.
In India, Barua and Ahmed (1963) first Mucor produces both asexual and sexual
detected Mucor from the stomach content of spores such as arthrospores, sporangiospores
aborted foetus in cattle as an etiological factor and zygospores for different purposes. The
of mycotic abortion. Mucor was also isolated arthrospores are produced under adverse climatic
from the cases of bovine gastritis affecting all and nutritional condition after cessation of loga-
the compartments of stomach (Damodaran rithmic growth. The arthrospores are produced
et al. 1976). through septation of coenocytic hyphae and the
deposition of a new three-layered wall beneath
the original hyphal wall. The arthrospores pro-
4.10.1 Morphology duced by dimorphic Mucor are able to germinate
into hyphae or yeast cells. The zygospores are
The hyphae are wide, coenocytic, branched and spiny, thick-walled, black-coloured structure
usually tapering. The branching occurs at fre- produced by the hyphal fusion of either hetero-
quent but unpredictable interval along the hyphal thallic or homothallic mating types.
length. The sporangiophores are hyaline and The Mucor sporangiophores expand at its tip
rhizoids are usually absent (Fig. 4.21). to generate columnella (columella) which bears
The property of dimorphism separates the sporangium (15–80 μm in diameter),
Mucor from other members of Zygomycetes. containing numerous asexual sporangiospores
The dimorphism is produced by some species (like Rhizopus). The apophysis is absent in
of Mucor such as M. racemosus, M. rouxii, Mucor. After maturity, the sporangial wall
deliquesces, and the spores remain attached Like Rhizopus, a similar sex locus was
with the columella, so that they could not be reported in the M. circinelloides and M. mucedo
carried away with the wind. The sporangiospores genome. The putative sex locus contains a single
are ellipsoidal, uninucleate (except M. mucedo) high mobility group (HMG) transcription factor
in nature, but they vary in size according gene which is flanked by an RNA helicase and a
to the species. M. bacilliformis produces the triose phosphate transporter gene. The HMG
smallest and M. mucedo produces the largest proteins are designated as SexP for the (+) and
sporangiospores. The cell wall of the sporangio- SexM for the () mating type, respectively. The
spores is enriched with protein, lipid, melanin transcription of SexM is highly stimulated than
and glucan. The melanin and glucan are absent SexP with the pheromone (trisporic acid) in
in hyphae, whereas the galactose and fucose M. mucedo cultures kept in vitro. The higher-
present in hyphal cell wall are not detected in transcriptional stimulation is correlated with dif-
sporangiospores. The sporangiospores help in ference in the promoter sequences of SexM and
the transmission of infection. They germinate SexP. However, whether the similar kind of
to produce hyphae or yeast cells. The spores higher-transcriptional stimulation of SexM and
first undergo a logarithmic growth phase (spheri- simultaneously repression of (+) mating type
cal growth) due to macromolecular synthesis. occurs under in vivo condition is yet to be
During this growth phase, a new vegetative cell elucidated.
wall is synthesised under the spore cell wall. The The () mating types of M. circinelloides can
chemical constituents of the vegetative cell produce larger spores and are more virulent than
wall are specific for either yeast or hyphae in the (+) mating types.
dimorphic Mucor.
4.10.2 Reproduction
The genus Mucor is placed under the order
Most of the Mucor species are able to reproduce Mucorales (described in details in Sect. 4.9).
sexually by heterothallic (M. racemosus, The major species under the genus Mucor are
M. mucedo, M. hiemalis) or homothallic mating M. circinelloides complex, M. hiemalis,
(M. rouxii, M. genevensis). In heterothallic mat- M. racemosus, M. ramosissimus and
ing, fusion of gametangia occurs between oppo- M. rouxianus. The M. circinelloides complex
site mating types. In homothallic mating, fusion consists of three subspecies, i.e. M. circinelloides
occurs between the gametangia located at the f. lusitanicus (Mcl), M. circinelloides f. cir-
different sites of the same thallus. The cinelloides (Mcc) and M. circinelloides
zygophores develop at the end of the hyphal f. griseocyanus (Mcg).
branches. In response of trisporic acid secreted
by the opposite mating types from prohormone
precursors, the zygophores are attracted to each 4.10.4 Susceptibility to Disinfectants
other. After direct contact between two
zygophores, they swell and form progametangia. Mucor are susceptible to sodium hypochlorite
The progametangia fuse to produce the gametan- (2.4 %), phenol, chloroxylenol and isorophyl
gium, which undergoes plasmogamy and kary- alcohol.
ogamy. The wall becomes thickened with
multiple layers and forms the zygospores. How-
ever, the generation of zygospores with karyog- 4.10.5 Natural Habitat and Distribution
amy is rare in nature. The zygospores germinate
to produce haploid sporangiospores with unipa- Mucor is saprophyte and ubiquitous in nature.
rental genetic makeup. They are commonly detected in soil, hay, dog
4.10 Mucor 81
fur, cereals, nut and flour (M. circinelloides, Table 4.22 Colony characteristics of Mucor species
M. hiemalis), wheat grains (M. racemosus), Mucor species Colony characteristics
soyabean seeds, stored cabbage (M. hiemalis), M. circinelloides The growth takes place at 25–37 C
oranges (M. circinelloides) and environmental and the colonies are pale grey or
samples throughout the world. yellowish. The colonies become
brownish at 37 C
M. hiemalis Optimum growth temperature is 32 C.
Greyish colonies are produced and
reverse pigmentation is pale
4.10.6 Genome
M. rouxianus Optimum growth temperature is 37 C
and the required temperature range is
The nuclear genome of Mucor (M. miehei) 9–45 C. The colours of the colonies
contains about 100 copies of rRNA gene, each are white, yellow or grey. The colonies
of which is 7.7–12 kbp in size and is arranged in are 4 mm in height and delicate in
nature
tandem. The genes encoding the 25S, 18S and
M. racemosus The colonies are low to moderately
5.8S rRNAs are closely linked within the raised and are brownish in colour due
repeated unit which also contains the 5S gene. to formation of sporangia
This 5S gene appears to be transcribed in the M. ramosissimus Rapid growth occurs at 25 C. The
opposite direction. In M. racemosus genome, colonies are grey to buff in colour
the 35S rDNA cistron and the 5S rDNA are
linked but are transcribed from opposite strands.
The transposable element is also detected in
nuclear genome of Mucor racemosus and other
Mucor (M. racemosus) possesses a heat-stable,
Mucor species. In eukaryotes, most of them
extracellular polysaccharide (EPS) antigen
influence gene expression and promote genetic
which is shared with Rhizopus, Absidia and
diversity by mediating genetic rearrangements.
Rhizomucor.
Such kind of function is yet to be elucidated
in Mucor.
The mitochondrial genome of Mucor contains
a circular chromosome. The approximate size 4.10.9 Virulence Factors
is 63.8 kbp in circumference (M. racemosus).
The average size of M. piriformis mitochondrial The virulence factors of Mucor are enlisted in
genome is 33.6 kbp containing the mitochondrial Table 4.23.
genes such as cob, nad2 and SrRNA. Excep-
tionally, the M. racemosus mitochondrial
genome is found to exist in the form of two
4.10.10 Transmission
flip-flop isomers with inverted repeat sequences
encoding rRNA genes.
Like other Mucorales, the transmission of Mucor
occurs by inhalation, ingestion and percutanoeus
route. Inhalation of spores results rhinocerebral
4.10.7 Isolation, Growth and Colony and pulmonary form of the infection in human.
Characteristics The percutaneous infection may occur during
injection, insect bite and trauma. The nail infec-
Mucor can be isolated in selective or nonselec- tion with Mucor in human was reported to be
tive fungal media. They are rapid grower, and the originated from handling with the contaminated
mycelia can fill and reach up to the lid of the Petri objects (e.g. orange).
dish within 1–7 days (‘lid-lifters’). The colony Immunosuppression due to bone marrow
characteristics of major Mucor species are transplantation, aplastic anaemia, diabetes,
described in Table 4.22. asthma, burns, hepatitis and human
Table 4.23 Virulence mechanisms and factors possessed papules, nodules and plaques without superficial
by Mucor necrosis are noted in the skin. In the nails,
Virulence punctate erosions are observed.
factors Functions
Thermotolerance Mucor does not show any
thermotolerance. Only M. rouxianus
grows optimally at 37 C, whereas 4.10.12 Disease Produced
M. ramosissimus and M. circinelloides
grow slowly and M. hiemalis does not The major animal and human diseases produced
grow at all at this temperature
by Mucor are enlisted in Table 4.24.
Spore size Larger spore size of M. circinelloides
complex is associated with virulence,
and generally () mating types
produce abundant larger spores than
the (+) mating types. The larger spores
4.10.13 Immunity
are more virulent probably due to
shorter time requirement for germ tube The ciliated bronchial cells and mucus in the
generation and subsequent hyphal respiratory tract are the first line of defence.
growth
The ciliary movement and the cough attempt to
Fungal It is produced by M. hiemalis and it can
metabolite suppress cell division in germinating
expel the sporangiospores of Mucor during inha-
grains lation. The pulmonary alveolar macrophages act
Calcineurin The calcineurin pathway helps in the as second line of defence which can phagocytose
dimorphic transition from yeast to the sporangiospores before their germination.
hyphae, correlated with virulence The spores of Mucor ramosissimus,
Sialic acid The surface sialic acids contribute to
M. plumbeus and M. circinelloides are also
the negative charge of Mucor yeasts
and spore cells and protect them from observed to activate alternative complement
phagocytosis pathway. The intact skin and mucous membrane
act as barrier to prevent the entry of the spores.
immunodeficiency virus (HIV) infection may act
as predisposing factors for Mucor infection.
4.10.14 Diagnosis
4.10.11 Pathogenesis 4.10.14.1 Clinical Specimens

The clinical specimens from animals include
Most of the Mucor species are opportunistic mastitic milk, skin scrapings and swab from tho-
fungi and can establish the infection during racic cavity, etc., depending upon the clinical
breakdown of the host immunity. Under the low symptom, whereas the specimens from human
pH, high glucose concentration and increased include nasal aspirates, sputum, bronchoalveolar
iron content, the spores germinate and produce lavage, pleural fluid, blood, skin scrapings and
hyphae. The hyphal extension can invade the nail specimens.
blood vessels, nerves and fascial planes. Some-
times haematogenous dissemination of the 4.10.14.2 Laboratory Examination
spores occurs to establish the infection in differ- 1. Direct examination: The typical wide, coeno-
ent organs such as udder in cattle. In immuno- cytic hyphae, branching at right angle, are
compromised hosts, Mucor produces invasive seen when potassium hydroxide with Parker
diseases in solid organs such as pulmonary, ink or calcofluor white is added to the
rhinocerebral, rhino-orbital and gastrointestinal material.
illness. 2. Isolation and identification: Media and incu-
In immunocompetent hosts, the infection is bation condition as described earlier will
restricted within the skin and nails. Erythema, serve the purpose.
4.11 Penicillium 83
Table 4.24 Major diseases of animal and human caused by Mucor

Fungi Host Disease
Mucor spp. Human Pulmonary, rhinocerebral, rhino-orbital, gastrointestinal cases predominate among
immunocompromised patients. Gastrointestinal diseases include abdominal pain with
hepatic abscesses, necrotic ulcerations of the stomach wall, diarrhoea and paralytic
ileus. Skin lesions and nail infections are observed in immunocompetent patients
Cattle Mastitis, thromboembolic encephalomyelitis (rare)
Cat Non-painful, subcutaneous swelling at nasal dorsum associated with scratch injury
Duck Thoracic cavity infection
Pig Papular dermatitis (rare)
Marine (a) Mucor is associated with fungal granuloma in cardiac muscles of killer whale
animals coinfected with Herpesvirus and Aspergillus
(b) M. circinelloides is detected to produce cardiac tissue damage in juvenile zebra
shark
(c) Systemic mucormycosis was detected in a captive hooded seal comprising muscle
necrosis over the left flank, necrotic iliac lymph node and necrotic bronchial lymph
node
Toad Dermatitis
M. ramosissimus Canary Dermatitis and feather loss
birds
M. amphibiorum Platypus Cutaneous lesions ranging from raised red nodules or plaques with purulent material to
ulcerated lesions with central cavitation, red exuding centres and raised epidermal
margins are observed
3. Histopathology: The haematoxylin and eosin 4.10.15 Treatment

(H&E) stain is used for demonstration of
Mucor in the tissue section. In subcutaneous Amphotericin B is the drug of choice for treat-
lesions, coenocytic hyphae are immersed in ment of Mucor infection in human and animals.
inflammatory exudate of neutrophils, M. ramosissimus is detected to be susceptible to
eosinophils, epitheloid and multinucleated miconazole in vitro. The subcutaneous lesions
giant cells. In dermis, spores and coenocytic and the nail infection can be treated with potas-
hyphae are observed. The superficial and deep sium iodide and mercuric chloride (1: 10,000).
nodular granulomatous dermatitis consisting
of lymphocytes, histiocytes, plasma cells and
giant cells is detected in epidermis. 4.11 Penicillium
4. Molecular biology: There are no serological
tests available for detection of Mucor, so Link (1809) introduced the name Penicillium
PCR-based molecular biology techniques are which was derived from the word ‘penicillus’
better options for detection in clinical (little brush).
specimens. The broad range real-time PCR, Penicillium marneffei was first isolated from a
DNA microarray is developed for simulta- hepatic lesion of bamboo rats (Rhizomys
neous detection of several pathogenic fungi sinensis) in 1956 which were maintained in
such as Mucor, Aspergillus, Candida, Crypto- captivity for experimental infections at Pasteur
coccus, dermatophytes, etc. For rapid detec- Institute of Indochina, South Vietnam (Capponi
tion of circulating DNA from serum of et al. 1956). The fungus was named Penicillium
patients, quantitative PCR (qPCR)-based marneffei in the honour of Hubert Marneffe,
approach was developed. Subsequently, PCR the then director of Pasteur Institute of
followed by high-resolution melt analysis Indochina. The first reported human case of
(PCR/HRMA) is used for the detection of P. marneffei was accidental when the scientist
Mucor in bronchoalveolar lavage samples. Dr. G. Segretain (France) pricked his own finger
with a needle filled with the culture. He devel-

oped small nodule at the site of inoculation
which was followed by axillary lymphadenopa-
thy. The infection was treated successfully
with the antifungals (Segretain 1959). In 1973,
first naturally occurring human infection was
reported which occurred in an American patient,
suffering from Hodgkin’s disease and living in
Southeast Asia (DiSalvo 1973). Its potentiality as
an opportunistic pathogen was observed during
human immunodeficiency virus (HIV) pandemic
(1988 onwards) in Southeast Asian countries.
The maximum number of P. marneffei infection Fig. 4.22 Hyphae of Penicillium chrysogenum (Photo-
in human with AIDS was diagnosed in Thailand graph courtesy: Marco van den Berg, DSM Biotechnol-
along with other countries such as Cambodia, ogy Centre, The Netherlands)
China, Hongkong and India.
Sir Alexander Fleming (1929) discovered the
first antibiotic penicillin during accidental con-
tamination of bacterial cultures with Penicillium
notatum at Saint Mary’s Hospital, United
Kingdom.
4.11.1 Morphology
The mycelium of Penicillium consists of highly

branched, hyaline and septate hyphae (Fig. 4.22).
Sometimes the hyphae interweave with each
other to form rope-like structure. The conidio-
phores are branched. The secondary branches
of conidiophores are known as metulae which Fig. 4.23 Conidial arrangement of Penicillium (sche-
bear the phialide from which the smooth or matic); a conidia, b phialide, c metulae
rough and round conidia (2.5–5 μm) are borne
(Fig. 4.23). intracellular fission cells within macrophages
Peroxisomes are morphologically simple which represent the parasitic phase.
organelles present in Penicillium (e.g.
P. chrysogenum) and other filamentous fungi
which produce secondary metabolites such as 4.11.2 Life Cycle
penicillins, polyketides and terpenes. The
organelles are produced from endoplasmic retic- The conidium of P. marneffei germinates (25 C)
ulum and are detected predominantly at the to produce germ tube. The germ tube grows
hyphal tips. The formation of peroxisomes is apically to make hypha. The subapical cells
controlled by peroxins, encoded by PEX gene. grow at a new point in the hypha to make its
P. marneffei is a dimorphic species under the branches. After formation of the mycelia, the
genus Penicillium showing filamentous growth specialised vegetative hyphal cells ( foot cell)
and asexual reproduction (conidia) at 25 C differentiate to produce multinucleate aerial
and generation of yeast cells at 37 C. The stalk cells. The tips of these stalk cells grow by
yeast cells are characterised by the formation of budding to generate uninucleate metulae and
4.11 Penicillium 85
phialides. The first uninucleate bud is a metula divided into two major clades, i.e. Penicillium
which subsequently buds to produce more spo- sensu stricto (previously Penicillium subgenus
rogenous cell type, known as phialide. These of Pitt’s classification) and Talaromyces (previ-
phialides produce conidia in basipetal mode, ously Talaromyces and Penicillium species under
i.e. old spores are replaced with new spores. the subgenus Biverticillium). Penicillium sensu
Thus, a chain of conidia is produced having stricto currently includes the genera Eupeni-
green colouration. Under carbon-limited condi- cillium, Chromocleista, Eladia, Hemicarpen-
tion, short unbranched stalks with conidia are teles, Thysanophora and Torulomyces. Some
produced or sometimes the metulae appear authors prefer to treat this clade under the
directly from the foot cells. singular genus Penicillium. There are total
The conidia can germinate and produce highly 225 accepted species under the genus Penicil-
branched hyphae at 37 C. These hyphal cells are lium. Major important species are Penicillium
shorter (20 μm) than vegetative hyphal cells marneffei, P. chrysogenum, P. commune,
(40 μm) produced at 25 C and are separated by P. cyclopium, P. lilacinum and P. oxalicum.
double septa. This is a transitory phase and these Penicillium is pleomorphic fungi detected
hyphal cells are known as ‘prearthroconidial either separately as anamorph (asexual),
cells’. These prearthroconidial cells generate teleomorph (sexual) or the holomorph (both
arthroconidia (uninucleate) by dissolving the phases). A few species have only one known
double septa within 48 h. The arthroconidia state. However, some authors segregated sexu-
divide by fission to generate true yeast cells ally reproducing Penicillium (teleomorph) into
within 72 h. This kind of arthroconidiation the genus Talaromyces, Hamigera and
followed by yeast formation is unique in dimor- Eupenicillium.
phic P. marneffei. Within the body of the host, Modern taxonomy is based on fungal genome
the yeast cells may remain as intracellular sequencing and genealogical concordance phylo-
(within macrophages) or extracellular. The intra- genetic species recognition (GCPSR)-based
cellular yeast cells are oval or spherical and analysis of sequence data. It changed nomencla-
smaller (2–3 μm in diameter). The extracellular ture of certain important Penicillium species.
yeast cells are elongated and larger (13 μm in For example, the major pathogenic species
diameter). (Penicillium marneffei) is currently designated
The yeast cells can undergo coupled nuclear as Talaromyces marneffei. The original strain
and cytoplasmic divisions. When the yeast cells of Fleming was later identified as Penicillium
are shifted at 25 C, the nuclear division becomes chrysogenum. Recent taxonomy showed that
uncoupled. The elongated multinucleate cells Penicillium chrysogenum is a complex of
divide only by septation without cell separation, five species, i.e. P. chrysogenum, P. rubens,
and they produce branched hyphal network P. vanluykii, P. tardochrysogenum, P. allii-
(yeast to hyphae transition). sativi. Fleming’s strain and the classical strain
The dimorphic switch in P. marneffei is used for the penicillin production (Wisconsin
regulated by temperature which causes cellular strain) are actually P. rubens.
changes such as coupling and uncoupling of There are several genera having Penicillium-
cellular and nuclear divisions, septation with or like conidiophores including Hamigera,
without cell separation, etc. Paecilomyces, Rasamsonia, Sagenomella,
Talaromyces and Trichocoma which are also
known as Penicillium-like anamorphs.
Penicillium belonged to Trichocomaceae family 4.11.4 Reproduction

under the order Eurotiales (class Eurotiomycetes
and phylum Ascomycota). The Penicillium is Penicillium produces asexual spores (conidia) by
a polyphyletic genus and the species can be a process known as ‘conidiation’. There are four
Fig. 4.24 Conidiation stages of Penicillium (schematic)
stages of conidiation. In the first stage, the mechanism. Nutrient starvation is detected by
extension of apically growing hyphae is seized several protein sensors in the hyphae which can
which is followed by the formation of septum to transmit a positive signal for conidiation. There
separate the apical cell. The apical cell is swelled are certain endogenous inducers such as
and there is subapical branching (stage 2). The conidiogenone, conidiogenol, sporogen (PF1)
apical cell differentiates into phialide (stage 3) which may induce the conidiation through quo-
which buds at the tip to generate the conidium rum sensing, a communication network detected
(stage 4) (Fig. 4.24). Upon the first conidium, in bacteria. The sporogen can act as inducer in
new conidia appear at hourly interval to produce darkness also. The conidiation signalling path-
a chain of conidia. The whole procedure up to way is regulated by GasA G protein α-subunit in
stage 4 takes approximately 7 h time. From the P. marneffei. The signalling involves a cAMP-
subapical branches, formation of phialides and dependent protein kinase A cascade.
conidia takes place to generate the typical brush- Majority of Penicillium species (73 %)
like structure (‘penicillus’). including P. marneffei do not have any sexual
Certain environmental factors regulate state. However, the presence of stlA gene (homo-
conidiation in Penicillium such as exposure to logue of conserved STE12) suggests a cryptic
air, light, high osmolarity, calcium and nutrient sexual cycle in P. marneffei. The sexual cycle is
starvation. The hydrophobin coating of aerial apparently not visible probably due to heterothal-
hyphae prevents the drying during exposure lic nature of P. marneffei. Heterothallic fungi
to air. Light is not a major requirement for require two compatible mating types for pairing
conidiation as some species of Penicillium can which may not be available in the environment
produce conidia without light. The calcium can simultaneously. Majority of other Penicillium
bind the Penicillium hyphae extracellularly to species having sexual reproduction are homo-
trigger the induction of conidia by an unexplored thallic in nature. The sexual reproduction is
4.11 Penicillium 87
noted in P. chrysogenum and P. dipodomyis, (ITS) region contains very high GC mol%. The
and experimentally sexual cycle is induced mitochondrial genome (35 kb) has similarity with
in P. pinophilum, previously considered as an the mould (Aspergillus nidulans) than the yeast.
asexual species. The abaA gene present in P. marneffei
genome controls the generation of conidia prob-
ably through the control of cell cycle during
4.11.5 Natural Habitat and Distribution sporulation. The expression of abaA gene is
increased during conidiation. The abaA deletion
Penicillium and Aspergillus are considered as mutants produce phialide-like cells but are
the most prevalent fungi in the world including unable to produce conidia. Another asexual
temperate, tropical as well as arctic locations. reproduction regulatory gene is stuA which is a
Many species of penicillia are soil saprophytes member of the APSES group of transcription
except P. marneffei. The saprophytic penicillia factors possessing a basic helix-loop-helix
can be isolated from rotting fruits, plant roots and (bHLH) DNA-binding motif. The expression
indoor environments such as archives, compost of stuA is also increased during conidiation.
heaps, damp buildings, etc. They can liberate However, stuA deletion mutants are unable to
different enzymes (phosphatase), chelators, produce metulae and phialides, but the conidia
organic anions and siderophores (trihydroxy- are generated directly from the conidiophores.
mates, coprogen, ferricrocin) to survive in the The other regulatory genes for asexual develop-
soil. Association of penicillia with the plant ment and secondary metabolite formation include
roots stimulate the plant defence system leading Gα-subunit gene (gasA). There is a CDC42 homo-
to improved seed germination and growth. For logue (cflA) detected in P. marneffei which is
pathogenic penicillia (P. marneffei), different required for correct morphogenesis of yeast cells
species of bamboo rats (Rhizomys sinensis, and accurate polarisation of hyphae. However,
R. pruinosus, R. sumatrensis, reddish-brown these regulatory genes (stuA, gasA, cflA) have no
subspecies of Cannomys badius) are considered role in dimorphic switch of P. marneffei.
as enzootic reservoir. In India (especially in MAT gene was identified in several Penicillium
Manipur, a Northeastern state), Cannomys species with both MAT1-1 and MAT1-2 genotypes.
badius acts as major reservoir. Other sex-related genes such as stlA (homologue of
The epidemiological study of P. marneffei in conserved STE12), pheromone precursor and
human identified Southeast Asia as an endemic receptor genes were detected in P. marneffei
zone. The infection was reported from North- (within RST) and P. chrysogenum, respectively.
eastern India, Thailand, China, Taiwan, Hong Comparative genomic analysis between
Kong, Laos, Cambodia, Malaysia, Vietnam and penicillin-producing strains (P. chrysogenum)
Myanmar. Soil exposure especially in rainy sea- revealed higher transcription of penicillin biosyn-
son was the critical risk factor associated with thesis genes (pcbAB, pcbC, penDE) in high amount
P. marneffei infection. of penicillin-producing strains. Due to high geno-
mic plasticity and ability to grow in different media
and conditions, strains of P. chrysogenum are the
4.11.6 Genome first choice for the production of penicillin
enzymes in biotechnology industry.
The genome of P. marneffei contains three
chromosomes (2.2, 4, 5 Mbp); however, the size
of the genome is 17.8–26.2 Mbp. A section of 4.11.7 Isolation, Growth and Colony
genome (9 %) is occupied by random sequence Characteristics
tags (RST) containing genes for ribosomal pro-
tein, tRNA synthetase, translation initiation, Penicillium can be isolated on Czapek dox agar,
elongation factor, metabolism and multidrug potato dextrose agar and 25 C. Most species
resistance. The internally transcribed spacer sporulate within 7 days, whereas the mycelial
phase of P. marneffei can be isolated in ripening (meat processing) or from the animals
Sabouraud glucose agar without cycloheximide fed with contaminated cereals. The ochratoxin is
at 25 C. They are converted into the yeast phase considered as potent nephrotoxic, carcinogenic
at 37 C in brain–heart infusion agar (thermal (human carcinogen of 2B group), genotoxic and
dimorphism). immunotoxic.
The colonies of Penicillium are bluish-green
and velvety. The colour and texture of the 4.11.9.2 Citrinin
colonies vary with the species. For example, Citrinin is a fungal metabolite which was first
P. marneffei produces diffusible wine red or isolated from Penicillium citrinum. Other species
brownish red pigment in the media. such as P. expansum and P. verrucosum can also
produce citrinin. It is frequently detected in feed
stuffs or food along with ochratoxin A and it can
4.11.8 Antigenic Characteristics increase the toxicity of ochratoxin synergisti-
cally. The intoxication with both of the toxins
Galactomannan (GM) is the major antigen of may cause endemic nephropathy. Further, citri-
Penicillium which is a carbohydrate molecule nin is also embryocidal and foetotoxic probably
composed of mannose residues with side chains due to the production of oxidative stress or
of β (1, 5)-linked galactofuranosyl residues. increased permeability of mitochondrial
It is released through the pores at the growing membranes. Oral LD50 for rats is 50 mg/kg
hyphal tips during logarithmic growth phase of body weight, while subcutaneous LD50 is
the fungi in highest amount which helps in the 67 mg/kg body weight. The heat-decomposed
detection of the antigen for diagnosis. GM is products of citrinin (CTN H1 and CTN H2) are
found in other moulds such as Aspergillus, more toxic than the parent citrinin.
Fusarium, Alternaria, Histoplasma and yeasts
including Cryptococcus which can produce anti- 4.11.9.3 Ribotoxin (RNase T1)
genic cross-reaction. Certain Penicillium species can produce a
Pen c is a protective antigen and allergen ribotoxin known as RNase T1. Like other
detected in P. citrinum and it is the homologue ribotoxins, RNase T1 can also cleave the
of Asp f commonly found in Aspergillus. The phosphodiester bond located within a universally
Asp f is a 19 KDa protein and it has two T cell conserved sequence of the large rRNA gene
epitopes (11mer and 13mer) which offer protec- (sarcin–ricin loop). The cleaving inhibits protein
tion against the fungi. synthesis and causes cellular death. The virus-
The yeast phase of P. marneffei contains three infected or virus-transformed cells having altered
immunodominant proteins (50 KDa, 54 KDa and cellular membrane are more susceptible to
61 KDa) which are specific for the fungi and can ribotoxin. The ribotoxins are used in preparation
be used in serodiagnosis. of immunotoxin due to their cytolytic property.
4.11.9 Toxins 4.11.10 Virulence Factors
4.11.9.1 Ochratoxin The other virulence factors of Penicillium are

The ochratoxin in feed stuffs in temperate enlisted in Table 4.25.
countries is mainly produced by Penicillium
verrucosum and P. nordicum. Growth of
P. verrucosum occurs in cereals in North and 4.11.11 Transmission
Central Europe and Canada. P. nordicum prefers
to grow in meat and meat products such as pork. In human and animals, traumatic implantation of
The meat contamination takes place either during the fungi into the skin results infection.
4.11 Penicillium 89
Table 4.25 Virulence mechanisms and factors possessed by Penicillium marneffei

Catalase-peroxidase protein The high expression of cpeA contribute to the survival of P. marneffei within the host cells
(encoded by cpeA) at 37 C
Acid phosphatase The enzyme produced by P. marneffei helps in survival of the fungi within host
macrophages
Vacuolar serine Vacuolar serine proteases (cerevisin) are produced by P. citrinum (Pen c),
proteases (cerevisin) P. chrysogenum (Pen ch) and P. oxalicum (Pen o). They act as fungal allergen by
such as Pen c2, Pen c18, activating other proteinaceous allergens present in the inoculum and make the whole
Pen ch18, Pen o18 organism a potent allergen (bystander effect)
Additionally P. marneffei is also transmitted of a sialic acid-containing receptor before phago-

by inhalation of the contaminated dust. cytosis. The phagocytic cells act as primary line
of defence against the fungi. The engulfment of
the fungi by the macrophages is a cation-
4.11.12 Pathogenesis independent process, and it largely depends on
the glycoprotein receptor containing N-
After entry within the body of the host, acetyl-β-glucosaminyl groups.
P. marneffei is converted into the yeast cells The CD4+ T cells producing Th1 type
and spreads by haematogenous route throughout of immune response play major role in protec-
the body. The survival within the phagocytes tive immunity against P. marneffei. The T cells
such as macrophages depends on the production activate the macrophages with the help of IFNγ
of acid phosphatase and iron availability. The and other cytokines. The activated macrophages
severity of infection depends on immune status can kill the intracellular yeast cells through
of the host. In immunocompromised patients, the L-arginine-dependent nitric oxide pathway. The
yeast cells can multiply and kill the macrophages neutrophils after activation with the granulocyte-
with dissemination throughout the body and pro- macrophage colony-stimulating factor (GM-CSF)
duce fatal necrotising reactions and histiocyte can lyse the yeast cells (not conidia) with the
infiltrations. Severe P. marneffei infections can secretion of different granular cytolytic
affect different tissues and organs such as the molecules.
bone marrow, liver, spleen, kidney, lungs,
lymph nodes, skin and soft tissues, whereas in
immunocompetent patients, granulomatous and
4.11.15 Diagnosis
suppurative reactions are detected in the lung,
skin, liver and subcutaneous tissues with the 4.11.15.1 Clinical Specimen
The human clinical specimens for diagnosis
formation of multiple abscesses.
of P. marneffei infection include bone marrow
aspirate, blood, skin scrapings, sputum, broncho-
alveolar lavage, pleural fluid, cerebrospinal fluid,
4.11.13 Disease Produced pharyngeal ulcer scrapings, palatal papule
scrapings, urine, faeces and lymph node biopsies,
The major human and animal diseases produced skin biopsies, liver biopsies, kidney, pericar-
by Penicillium are enlisted in Table 4.26. dium, stomach or intestine.
The animal clinical specimens include mas-
titic milk, skin scrapings, aborted foetus.
4.11.14 Immunity
4.11.15.2 Laboratory Examination
The conidia of P. marneffei are attached with 1. Direct examination: Detection of Penicillium
the respiratory epithelial cells with the help is possible by direct examination of the
Table 4.26 Major diseases of animal and human caused by Penicillium

Fungi Host Disease
Penicillium Human The infection is confined in Southeast and Eastern Asia, including northeast
marneffei India, Thailand, China, Hong Kong, Laos, Cambodia, Malaysia, Myanmar,
Vietnam and Taiwan. The syndrome includes fever, weight loss, cough,
anaemia (haemoglobin level of 10 g/dl or less), lymphadenopathy and
hepatosplenomegaly. In HIV-infected patients, skin lesions appear having
similarity with molluscum contagiosum in most of the cases (60–85 %).
The skin lesions appear on the face, upper trunk and extremities. The lesions
are necrotic or generalised papules or nodules and with central umbilication.
The osteoarticular lesions are sometimes observed in the ribs, long bones,
vertebrae and skull. Without the skin lesions, clinically it is difficult to diagnose
because lots of opportunistic pathogens produce a similar kind of syndrome.
The bone lesions are also detected in tuberculosis, cryptococcosis,
blastomycosis and African histoplasmosis
Penicillium sp. Ruminants and Mastitis, keratitis, dermatitis, abortion and other reproductive disorders
other animals
Penicillium Cattle Mycotic abortion (rare in occurrence)
vermiculatus
P. decumbens Human Invasive mycosis in HIV-infected patients
smears prepared from the clinical specimens 4. Detection of fungal antigen: The fungal anti-
(blood, bone marrow aspirate, biopsies) and gen can be detected with immunodiffusion
stained with Wright’s stain. test, latex agglutination test, monoclonal
2. Isolation and identification: Media and antibody-based sandwich ELISA.
incubation condition as described earlier 5. Serological tests: The antibodies developed
will serve the purpose. The ideal clinical in patients against P. marneffei infection
specimens for culture are bone marrow, can be detected by immunodiffusion, indirect
blood and skin biopsy. fluorescent antibody test (detects IgG) and
3. Histopathology: The haematoxylin and eosin ELISA with a recombinant mannoprotein
(H&E), Grocott methenamine silver and (Mp1p). Western blot analysis detected two
periodic acid–Schiff (PAS) stains are used yeast phase-specific immunodominant protein
for the demonstration of P. marneffei in the of P. marneffei (50KDa, 54 KDa).
tissue section. Within the macrophages or 6. Molecular biology: The PCR can detect
histiocytes, round or oval yeast cells are P. marneffei targeting the genes for 5.8S
detected. Sometimes they appear as fission rRNA and internally transcribed spacer
arthroconidia which will produce yeast cells (ITS1). The detection of 18S rRNA gene is
by cross wall formation which is considered possible by PCR hybridisation assay having
as differentiating criteria from other yeasts. sensitivity of 0.1 pgm/μL of DNA and one
Extracellular elongated or sausage-shaped tube semi-nested PCR. A TaqMan real-time
cells are also observed. PCR is also developed to detect P. marneffei
In histological sections, indirect fluores- 5.8S rRNA gene in clinical samples and
cent antibody test can detect P. marneffei. In culture.
paraffin-embedded formalin-fixed tissues,
P. marneffei can be detected with monoclonal
antibody against the galactomannan antigen 4.11.16 Treatment
(EBA1). Immunoperoxidase staining can
detect the fungi in deparaffinised tissue P. marneffei is susceptible to miconazole,
sections of skin biopsies. itraconazole, ketoconazole and flucytosine
4.12 Cryptococcus 91
in vitro. The amphotericin B showed inter-

mediate activity against the fungi.
4.12 Cryptococcus
Cryptococcus is a haploid-encapsulated yeast

causing primarily central nervous system
(CNS)-related disorders most commonly in
dogs and cats and less commonly in ferrets,
horses, cattle, goats, sheep and llamas and
Fig. 4.25 Narrow-based buds of Cryptococcus
among nondomestic animals such as elk, koalas
neoformans (schematic)
and dolphins. The yeast was first isolated
from peach juice in Italy, and it was named as
Saccharomyces neoformans (Sanfelice 1894) growth phase. The sexual phase (teleomorph)
and also subsequently isolated from the infected of C. neoformans is known as Filobasidiella
tissues of a German patient (Busse 1894). Two neoformans. Two major morphological forms
years later, encapsulated bacilliform yeast, which exist in the vegetative growth phase. The pre-
was named Saccharomyces subcutaneous dominant form found in the environment and
tumefaciens, was isolated from a healthy man in animal hosts is unicellular budding yeast. The
France, later identified as Cryptococcus gattii yeast cells are thin walled, spherical to oval
(Curtis 1896). Vuillemin examined several of with widely varying diameter (2–20 μm) and
these cultures, and due to lack of Saccharomy- they are reproduced by mitotic division
ces-specific characteristics, he placed these (Fig. 4.25). The buds are present in the narrow
species in the genus Cryptococcus in 1901. base. C. gattii yeast cells are oval and larger.
In 1905, von Hansemann reported the first case Another vegetative form is pseudohyphae, pro-
of meningoencephalic cryptococcosis. Emmons duced by joined yeast cells due to incomplete
isolated C. neoformans from pigeon nests with separation after mitotic division. This form is
their droppings and clinical cases of bovine mas- sometimes found in the environment or the clini-
titis (Emmons 1951, 1952). The first published cal samples produced to be protected from the
report of Cryptococcus as an aetiology of disease natural predators.
in cats appeared in 1952 (Holzworth 1952). The morphological transition from the yeast
Cryptococcus neoformans var. gattii was for- phase to hyphal phase is noticed during sexual
mally proposed as a new taxonomic entity in mating. However, they are not considered as
1970 (Gatti and Eeckels 1970). Ellis and Pfeiffer dimorphic fungi probably due to their predomi-
(1990) isolated C. gattii from the bark and fruits nant existence as yeast form in the environment
of Eucalyptus camaldulensis in Australia. and hosts and the lack of involvement of this
In India occurrence of C. neoformans was transition in the pathogenesis. Further, both at
recorded from cutaneous lesion of cats (Pal and 25 and 37 C, they can produce yeast-like
Mehrotra 1983b), mastitis in dairy cattle (Pal and colonies in the isolation media. Recently unusu-
Mehrotra 1983a) and meningitis in cats (Pal ally large yeast-like morphological form
1991). (30–100 μm) is also detected in clinical samples,
known as ‘giant’ or ‘titan’ cells. These cells are
produced as a response against host defence
4.12.1 Morphology molecules, temperature, oxygen and CO2, and
the availability of specific macro- and
Cryptococcus life cycle is predominantly divided micronutrients (e.g. glucose and iron). The G
into two phases, i.e. vegetative and sexual protein-coupled receptors (GPCRs) Ste3a and
influenced by the cell wall composition present

beneath it. The cells without α (1, 3)-glucan in
their cell walls do not display the surface capsule,
whereas the cells lacking cell wall β (1, 3) glucan
form larger capsules than those of wild-type
cells. Two major transcription factors, namely,
cryptococcal Nrg1 and Cir1, play central role in
regulation of the capsule formation. Recently
seven more transcriptional regulators that affect
capsule size are also elucidated (Gat201, Tup1,
Gcn5, Rim101, Hap3, Hap5 and Ada2). The cap-
sule size is increased with iron deprivation,
increased CO2 concentration and the presence
of serum in vitro and increased age of the organ-
ism or duration of infection in vivo.
Fig. 4.26 Demonstration of Cryptococcus neoformans Like other eukaryotic organism, Cryptococ-
capsule in India ink stained smear (Photograph courtesy: cus also possesses mitochondria which serves as
Prof. P.P. Gupta, Ex-Director of Veterinary Research, a source of energy and is involved in different
Punjab Agricultural University, Punjab, India)
processes such as aging, calcium homeostasis,
apoptosis and regulation of virulence (exclusive
Gpr5 have been shown to regulate this gigantism property of Cryptococcal mitochondria). Within
of cryptococcal cells via the activation of G the macrophages, the mitochondria may undergo
protein Gpa1 and its downstream signalling path- fusion to generate ‘tubular mitochondria’ which
way (Gpa1-Pka1-Rim101signaling cascade). are more effective in repair of mitochondrial
The phospholipids can also stimulate the giant DNA (mtDNA) damage caused by reactive
cell formation. oxidative species and increase the survival of
Another unique morphological feature of the yeast. The organelle cannot be synthesised
Cryptococcus is the presence of capsule, like de novo; it is inherited directly from the single
prokaryotes (Fig. 4.26). The capsule can be best parent (MATa) during sexual reproduction prob-
observed in fresh preparations by staining with ably due to migration of the nucleus from the
diluted India ink or phase contrast microscopy. MATα cell unidirectionally to the MATa cell,
Giemsa can also partially stain the capsule. leaving behind its mitochondria. Whereas in
In tissue section, it can be viewed with other fungi [e.g. black ink mushroom (Coprinus
mucicarmine or Alcian blue stain. The capsule cinereus)], this nuclear movement between two
of Cryptococcus neoformans consists primarily mating cells is bidirectional, resulting biparental
of two polysaccharides, glucuronoxylomannan mitochondrial DNA inheritance.
(GXM) and glucuronoxylomannogalactan
(GXMGal), along with smaller amounts of
mannoproteins. In addition to being associated 4.12.2 Classification
with the cell, these molecules are also shed as an
exopolysaccharide. It is noticed that the Ca++ Cryptococcus belongs to the family
acts as the bridge between the negatively charged Tremellaceae under the order Tremellales, class
glucuronic acid residues of neighbouring GXM Tremellomycetes and phylum Basidiomycota.
fibres, mediating capsule assembly through The genus Cryptococcus possesses over 37 spe-
GXM-GXM aggregation. So in absence of cies, majority of which do not cause disease
Ca++, there is increased GXM shedding, and in in mammals. The most important pathogenic
the presence of EDTA the capsule size is species are C. neoformans–C. gattii species
decreased. The assembly of capsule is also complex, which includes C. neoformans var.
neoformans, C. neoformans var. grubii and and rounder. This type of mating generates
C. gattii (C. bacillisporus). Sometimes inter- diversity in C. neoformans populations and
species hybrids of C. neoformans and C. gattii also it contributes to the global spread of
are observed. The teleomorphs of C. neoformans cryptococcosis.
var. neoformans and C. gattii are Filobasidiella
neoformans and Filobasidiella bacillispora,
respectively. Other closely related species such 4.12.4 Susceptibility to Disinfectants
as Cryptococcus amylolentus, Tsuchiyaea
wingfieldii and Filobasidiella depauperata are The replication of Cryptococcus stops above
isolated from insects. 40 C. Alkaline pH is detrimental for the yeast.
Among chemical disinfectants, C. neoformans is
susceptible to 1 % sodium hypochlorite, iodine,
4.12.3 Reproduction phenolics, glutaraldehyde and formaldehyde.
The mating of Cryptococcus is not common in

nature or within the host. In nature the proportion 4.12.5 Natural Habitat and Distribution
of two compatible yeast cells (α and a) are not
equal which is the cause behind the absence of C. neoformans chiefly inhabits the avian excreta
mating. The α-mating type predominates in such as pigeon (Columba livia), parrot and
nature and clinical specimens. Further, certain canary excreta and in soil, decaying woods. The
factors such as temperature (37 C), high humid- pigeon excreta are rich in creatine, urea and uric
ity and 5 % carbon dioxide (CO2) can inhibit the acid which act as major nitrogen source for the
mating which is commonly found within the fungi. Transmission of the agent from the pigeon
host. In the laboratory, the specific stimulants excreta to immunocompromised patients is also
(myo-inositol, indole acetic acid, copper ions noted. Whereas the soil is the major reservoir
and nitrogen starvation) can induce mating. The of C. gattii, including the trees especially Euca-
pigeon faeces can also favour the mating of lyptus, almond (Terminalia catappa), pottery
C. neoformans var. neoformans and Cryptococ- trees (Moquilea tomentosa), etc. The decaying
cus neoformans var. grubii but not C. gattii. hollows in trunks and branches of a number of
During bisexual mating (opposite sex), two other tree species (>50 species) are also the
compatible yeast cells fuse to produce a hypha preferred habitat of the fungus. However,
containing two parental nuclei (dikaryotic C. gattii is generally not isolated from the pigeon
hypha/zygote). There will be a development of faeces. The inositol produced by the plants can
a specialised cell called basidium, in which the stimulate sexual reproduction of Cryptococcus
fusion of the parental nuclei and meiosis takes which explains the utilisation of the environmen-
place. The daughter cells of the meiotic division tal niche by the fungi. The organism was also
undergo rounds of further mitotic division, and isolated from the air, water bodies, car wheel
the mitotic nuclei are packaged into spores that wells and footwear sampled from high traffic
bud from the apical surface of the basidium to location.
form four spores in a basidium. After germina- C. neoformans var. grubii is the most preva-
tion, the spores develop into new daughter yeast. lent organism among C. neoformans group in
Same sex mating (unisexual mating/haploid human throughout the world. Human infections
fruiting/monokaryotic fruiting) is also detected with C. neoformans var. neoformans are more
in Cryptococcus generating monokaryotic common in France, Denmark and Italy. Previ-
hypha, basidium and spore. The unisexual mat- ously it was observed that C. gattii was prevalent
ing involves fusion between two haploid cells of in tropical and subtropical regions such as South-
the same mating type, and it does not produce east Asia (New Guinea, Thailand), Australia,
dikaryotic hyphae, and the spores are smaller South America, and parts of Africa and the
United States (California). Currently the preva- locus regulating the sexual growth phase
lence area is expanding throughout the world spanning over 100 kb with more than 20 genes.
especially in North America. It is quite larger than other MAT loci of different
In India, the reports of C. neoformans var. fungi. This locus can encode one of the two
neoformans isolation from HIV patients and idiomorphic alleles which determines whether
pigeon droppings in Tamil Nadu and C. gattii the cell will be MATa or MATα. In general two
isolation from the trees (Eucalyptus types of genes in a MAT loci, one encoding
camaldulensis) in Punjab are noticed. In cattle pheromone and pheromone receptor and the
and buffalo, C. neoformans was detected from other encoding homeodomain transcription
cases of mastitis in other states such as Madhya factor, are required for sexual mating. Initially
Pradesh. In cats, C. neoformans was isolated during mating of C. neoformans, compatibility
from meningitis and cutaneous lesion. of the pheromone and pheromone receptor
genes is required for cellular fusion, followed
by dikaryon formation which is dependent on
4.12.6 Genome compatible homeodomain transcription factors
(Sxi1α/Sxi2a). Further, CLP1 gene products
Cryptococcal genome is rich in introns also have regulatory role in dikaryon formation
(5.5 introns/gene) and antisense messages which is Sxi-dependent. Mutation in Sxi1α gene
(endogenous antisense transcripts) in comparison can modify the mitochondrial DNA inheritance
to other Ascomycota yeasts. The reported from uniparental to biparental.
genome sequence of two related strains of Comparative genome analysis of C. neofor-
C. neoformans serotype D (JEC21 and mans var. neoformans and C. neoformans var.
B-3501A) revealed a 19-Mb genome sequence grubii revealed the transfer of a nonreciprocal
(excluding the ribosomal RNA repeats) which 40Kb segment (‘identity island’) from
spans 14 chromosomes from 762 kb to 2.3 Mb. C. neoformans var. grubii to C. neoformans
A total of 6572 protein-encoding genes were var. neoformans nearly 2 million years ago. At
identified, which contain an average of 6.3 present the ‘identity island’ is widely prevalent
exons of 255 bp and 5.3 introns of 67 bp. There among the C. neoformans var. neoformans
was no evidence of whole genome duplication. isolates in nature.
Overall 65 % of the genes of C. neoformans are C. neoformans also contains small noncoding
conserved, 10 % of the genes are unique to RNAs (sRNAs) like other eukaryotes such as
C. neoformans and the remaining 25 % have small interfering RNAs (SiRNA) and micro
non-fungal sequences. There are total 11 gene RNAs (miR1 and miR2). The micro RNAs are
families, among them 2 are exclusive for 22 (miR1) and 18 nucleotides (miR2) in length
C. neoformans, one encodes protein required for and are derived from 70 nucleotide RNA
capsule formation and the other encodes nucleo- precursors. In the genome, miR1 and miR2 are
tide sugar epimerases associated with cell wall distributed in 4 (chromosome 1,4,9,12) and
formation. Approximately 5 % of the genome 7 chromosomes (chromosome 1,3,4,6,8,9,13),
is constituted with transposons clustered in respectively. These sRNAs actively take part in
single blocks (40–100Kb) that may represent gene silencing process via RNA interference
sequence-independent regional centromeres. mechanism.
Each block contains Tcn5 and Tcn6 transposons. The mitochondrial DNA (mtDNA) size varies
The transposons are also clustered adjacent to the from 24 to 34 Kb. The genome is present in high
rDNA repeats and within the mating-type (MAT) copy number and shows a higher mutation rate
locus. The genome shows the evidence of alter- than the nuclear DNA. The genes associated with
native splicing (e.g. exon skipping, truncation, mitochondrial function (ND1, ND2, ND3, ND4,
etc.) and antisense transcripts which have no ND4L, ND5, ND6, ATP6, ATP9, COX1, COX2
coding potentiality. There is a single mating and COB) and protein synthesis (SsrRNA and
LsrRNA) are localised in the mitochondrial colony type. The smooth colonies are round with
genome. The number of introns in some genes smooth-domed surface and smooth edges,
(COX1, COB, LsrRNA) vary between different whereas the mucoid colonies have a shiny
species (C. neoformans var. neoformans and mucoid surface due to excess production of vis-
C. neoformans var. grubii) of Cryptococcus. In cous polysaccharide. These mucoid varieties
several introns of the COB and COX1 genes, show slower growth rate and produce the larger
LAGLIDADG motifs are present. The intergenic capsule. The major constituent of the capsule
regions are highly conserved in mtDNA. (GXM) is more viscous, can accumulate in the
meninges and cerebrospinal fluid (CSF) and is
more resistant to phagocytosis by the macro-
4.12.7 Isolation, Growth and Colony phages which enhance the virulence of the
Characteristics mucoid varieties. Further, the mucoid varieties
can increase the intracranial pressure in chronic
Cryptococcus can be isolated in corn meal agar, experimental cryptococcosis as observed in rat
Sabouraud dextrose agar, blood agar, honey agar, model.
brain–heart infusion agar and malt agar. The
specific medium is birdseed agar/Staib medium
(niger seed agar)/sunflower seed extract agar 4.12.8 Biochemical Characteristics
with antibiotics to eliminate the bacterial con-
tamination. They are sensitive to cycloheximide. C. neoformans and C. gattii can hydrolyse urea
The plates are incubated at 28–37 C for (within 4 h) and assimilate inositol and creati-
2 days–2 weeks. The pathogenic strains prefer nine, do not ferment carbohydrates and produce
to grow at 37 C. The colonies are initially small, melanin which differ it from other pathogenic
convex, mucoid, creamy in colour and increases yeasts. They cannot reduce nitrate but can obtain
in diameter up to several centimetres after their nitrogen from urea, creatinine and peptone.
prolonged incubation. In birdseed agar, the C. gattii can also use D-proline, glycine and
colonies appear as brown coloured in the centre tryptophan as a nitrogen source.
of the plate due to the production of melanin. The
optimum capsule production is detected in choc-
olate agar after incubation at 37 C with 5 % CO2 4.12.9 Antigenic Characteristics
tension. The L-canavanine glycine bromothymol
blue media can differentiate C. neoformans and The capsular polysachharide is the major antigen
C. gattii by the formation of distinctive blue of Cryptococcus. The serotyping is based on the
colouration with the growth of C. gattii. variations in the O-acetylation of the capsular
In Cryptococcus and a few other yeasts polysachharide. C. neoformans was initially
(Candida albicans), ‘phenotypic switching’ of divided into five serotypes, A, B, C, D and
the colony is observed. It is defined as spontane- AD. Currently, C. neoformans var. neoformans
ous emergence of colonies with altered colony has the serotypes D, AD; C. neoformans var.
morphology at rates higher (102 to 105 per grubii has the serotypes A, AD; and C. gattii
generation) than the somatic mutation (107 to has the serotype B, C. Further, molecular tools
108 per generation). It is a reversible process such as PCR-based fingerprinting, amplified
and occurs only in a small fraction of the patho- fragment length polymorphisms, restriction frag-
genic population both in vitro and in vivo espe- ment length polymorphism, random amplifica-
cially during chronic infection. It is detected in tion of polymorphic DNA and multilocus
Cryptococcus neoformans serotype A, D and in sequence typing have generated several molecu-
C. neoformans var. gattii. The switching results lar types. C. neoformans var. grubii (serotype A)
the change of the smooth colonies into mucoid isolates belong to molecular types VNI and
Table 4.27 Serotypes and molecular types of Cryptococcus

Cryptococcus neoformans Cryptococcus neoformans
var. neoformans var. grubii Cryptococcus gattii
Serotype D, AD A, AD B, C
Molecular type VNIV, VNIII hybrid VNI, VNII, VNIII hybrid VGI, VGII, VGIII, VGIV
VNII, whereas C. neoformans var. neoformans is 4.12.12 Pathogenesis

molecular type VNIV and the hybrid serotype
AD is molecular type VNIII. C. gattii isolates 4.12.12.1 C. neoformans var. neoformans
are classified as VGI, VGII, VGIII and VGIV. The spores lodge in the lung alveoli of the
The B and C serotypes of C. gattii are not specific animals or human after inhalation. Initially the
to any molecular types (Table 4.27). infection is asymptomatic and dormant. The dor-
mancy of the yeast cells is associated with sev-
eral adaptive and immune evasion mechanisms.
Calcineurin, produced by C. neoformans
dephosphorylates a group of proteins that allow
for their growth at physiological body tempera-
The virulence factors possessed by
ture (37 C), elevated CO2 and alkaline pH. It
C. neoformans are described in Table 4.28.
also induces metabolic and oxidative stress genes
required for their survival within the host tissues.
The capsule of the fungi helps in evasion of
4.12.11 Transmission immune system and survival within the host.
The capsule is constituted with mannan (polysac-
Cryptococcus is non-contagious, transmitted charide) which is highly hydrophilic. It makes a
chiefly through inhalation or sometimes through gelatinous zone surrounding the yeasts that
percutaneous route in animals and human. The conceals the pattern recognition receptors
basidiospores, produced by sexual reproduction, (PRR) from the immune system. Further, the
are major infectious particles, small in size mannan component can induce the production
(2–3 μm) which can easily invade the lung of IL10 (anti-inflammatory) and reduces the pro-
alveoli than the encapsulated yeast (10–60 μm). duction of pro-inflammatory cytokines from the
The desiccated yeast form may become a source antigen-presenting cells and T lymphocytes.
of infection, although they have limited viability It also reduces the surface expression of acti-
in the environment. Rarely ingestion of large vation markers and thus downregulates the
number of organisms may produce gastrointesti- macrophages, dendritic cells and neutrophils.
nal lesion. The capsule also prevents antibody binding
The children are often exposed to C. neofor- with the yeasts. Other capsular characteristics
mans before the age of 5 years. In adults such as antiphagocytic property, shedding of
also antibodies to Cryptococcus is common, L-selectin from the neutrophil surface associated
indicating their carrier state. In immunocompe- with reduced adhesion and chemotaxis of
tent hosts, the organism is eliminated or it neutrophils into the inflammatory site and altered
becomes dormant, whereas in immunocompro- phagocytosis (binding of capsular GXM with
mised hosts, they spread into other organs with FcRγIIB of host cells produces IL10 which
fatal consequences. However, human-to-human decreases the immune response) also help in
transmission is not recorded, and it seems that immune evasion. However, persistence within
human/animals are the dead-end host from where the host for the longer period depends on survival
the cells can be recycled in the nature. of the yeasts within the macrophages and
Table 4.28 Virulence factors possessed by Cryptococcus neoformans var. neoformans

Location in
Virulence factors fungal cell Function
Capsule Outside the (a) Prevention of phagocytosis by macrophages and
cell wall neutrophils
(b) Adherence with the host cell surface
(c) Depletion of complements
(d) Circulating capsular antigens help in the removal of
selectins from endothelial cell surfaces and prevent the
neutrophil migration into the tissues. Increased size is
correlated with increased resistance against
phagocytosis and antifungal therapy
(e) Glucuronoxylomannan (GXM) component of the
capsule interferes T cell activation and proliferation and
disrupts cell-mediated immunity
Calcineurin (encoded It helps the yeast to grow at body temperature (37 C)
by CCN1gene) and alkaline pH. The thermal tolerance is controlled by
both calcineurin and the protein kinase C1 (PKC1)-
activated MAP kinase (Mpk1) pathway
Melanin Cell wall It protects the organisms from oxidative damage by the
scavenging host antioxidants, and it maintains the
capsule structure or negative charge. Possession of
melanin is also directly correlated with CNS invasion,
as the dopamine is used as a substrate for melanin
production
It is catalysed by laccase enzyme which otherwise also
decreases the hydroxyl radical level and offers
protection to the organism. Both capsule formation and
melanin production are controlled by Gpa1 G protein
signalling pathway via regulation of cellular cAMP
levels
Phospholipase B1 (Plb1) The enzyme Plb1 is multifunctional containing the
activities of phospholipase B, lysophospholipase and
lysophospholipase transacylase. It helps in initiation
and dissemination of Cryptococcus in the CNS and
survival and replication within the macrophages. The
Plb1 produces arachidonic acid from the host cellular
phospholipid and the enzyme laccase produces
prostaglandin E2 (PGE2) from arachidonic acid
The PGE2 activates prostanoid EP2 receptor in the
macrophages and inhibits microbicidal activity of the
macrophages by increasing the cAMP level. The Plb1
mutants have reduced capability to escape macrophages
(vomocytosis)
Urease The enzyme urease can breakdown urea to generate
ammonia which can produce a local damage in the
endothelium, helping transmigration of the yeasts into
the CNS
Superoxide dismutase [SOD1, They are components of Cryptococcal antioxidant
SOD2 (mitochondrial)], glutathione system which helps in the survival of the organism
peroxidases (GPX1 and GPX2), within the phagocytes
cytochrome C peroxidase (CCP1)
ATPase (encoded by ENA1 gene) It helps in the survival of the organism in extreme pH
acetoin and dihydroxyacetone Cell They inhibit the host neutrophil function
metabolites
Inositol sphingolipid Biosynthesis and breakdown of inositol sphingolipid by
different enzymes are required for enhancing
phagocytosis resistance and survival, invasion in CNS
(continued)

Location in
Virulence factors fungal cell Function
CnRAM [Regulation of Ace2 and Mutants lacking CnRAM pathway signal transduction
Morphogenesis (RAM) network] showed slow growth at 37 C and virulence
Vesicle (‘virulence factor delivery bags’) It helps in carriage of virulence factors to the cell
surface
Cir 1 It is GATA-type zinc finger protein which helps to
maintain capsule size and integrity and enhance the
growth at 37 C. In addition, Cir 1 has negative control
on melanin production with the help of Gat201. Further,
they act as sensor for the iron level
Nrg1, Gat201, Tup1, Gcn5, Rim101, They help to maintain capsule size and integrity,
Hap3, Hap5, Ada2 enhance the growth at 37 C. Tup1, Nrg1 also act as
transcriptional regulator to influence iron uptake
Sre1 [homolog of sterol-regulatory element- They help to maintain the melanin activity. Sre1 also
binding protein (SREBP)], Tup1, Vad 1 regulates the genes encoding ergosterol biosynthetic
enzymes and the proteins involved in iron and copper
uptake, stress-related functions, various transport and
metabolic functions under the low-oxygen condition
(1 %). These measures are required for Cryptococcus to
disseminate into the brain tissues, where oxygen tension
is low than the atmosphere
Ferric reductase Cell surface They reduce ferric iron to its ferrous state which helps
in iron uptake
Ferrooxide permease (Cfo1, Cft1) They are essential for ferric iron uptake, iron
acquisition from transferrin and full virulence in mice
Sit1 They are specific transporter of the siderophore
ferrioxamine B. However, they are not required for
virulence in mice model of cryptococcosis
Hap proteins (Hap3, Hap5, HapX) They act as regulator for iron acquisition especially
from the environment (HapX)
Copper regulatory It controls melanin production, filamentation and
transcription factor (Cuf1) growth at high temperature, dissemination to the brain
(not in the lung)
Tubular mitochondria It increases fungal survival within the macrophages
Dol-P-Man:protein O-mannosyltransferases This enzyme helps in O-mannosylation of the proteins
(PMT) in endoplasmic reticulum which is required for
construction and maintenance of the cell wall. Loss of
pmt gene can attenuate the virulence of Cryptococcus
cAMP-protein kinase A signalling (cAMP- It is a signalling pathway involved in protein
PKA) phosphorylation which is required for mating, capsule
formation and melanin production
Histone acetyltransferase (Gcn5) The Gcn5 mutants show defective growth at high
temperature, sensitive to oxidative stress and defective
capsule attachment with the cell surface
Ubiquitin-conjugating proteins They are associated with oxidative stress response
(encoded by UBC6-2 and UBC8)
F-Box protein ( fbp1) It is required for spore formation and the mutants are
less virulent in experimental mice
endothelial cells. The survival strategy includes intervention with lipid metabolism, antioxidant
increased capsule synthesis, enzyme production system (superoxide dismutase, glutathione per-
(laccase, phospholipaseB1), production of mela- oxidase), interference with nitric oxide synthase
nin, Ssa1 (heat shock protein homolog), (NOS) induction leading to inhibition of nitric
oxide production and capability to exit the cells they are present in a membrane-bound vacuole,
(expulsion or phagosome extrusion). Further, and they can exit from the cells with minimal
Cryptococcus can modulate the adaptive immune damage. Thus, the endothelial cells are used as a
response by several ways such as inhibition of vehicle by the yeasts to penetrate the barrier
T cell activation and induction of T cell apopto- (transcytosis). Occasionally it is observed that
sis, induction of nonprotective Th2 response, occludin (tight junction maker protein) is
interference with dendritic cell maturation and degraded during the Cryptococcus and BMEC
interference with antibody function that also interaction which suggests the partial disruption
helps in dormancy of the yeasts. of tight junction to allow the passage of the
During immunosuppression of the hosts, the yeasts. Further, the ‘Trojan horse mechanism’
spores reactivate and disseminate into the differ- through the infected macrophages or other
ent organs such as the skin, eyes, myocardium, phagocytes is also used by the yeasts to penetrate
bones, joints, lungs, prostate gland, urinary tract the blood–brain barrier.
and the central nervous system (CNS). The dis-
semination into the blood circulation can occur 4.12.12.2 C. neoformans var. gattii
through the infected macrophages (‘Trojan The pathogenesis described for C. neoformans
horse mechanism’). They can multiply within var. neoformans is also applicable for C. gattii
the macrophages to produce ‘cryptococcal with some distinctions. C. gattii can arrest the
phagosome’ which lyses the macrophages to neutrophil migration both in vivo and in vitro
release the daughter yeast cells. Occasionally which explains the reason behind the susceptibil-
the daughter cells exit by extrusion without ity of healthy individuals to this fungus. Further
lysis. Sometimes they are again engulfed by it can produce extracellular fibrils which can
fresh macrophages to avoid the contact with the prevent the phagocytosis by the neutrophils and
host defence. After development of fungaemia, help to establish the initial pulmonary infection.
through the blood circulation, they can reach the Trehalose is detected as major antioxidant in
blood–brain barrier (BBB) and penetrate the bar- C. gattii. The genes encoding for trehalose
rier to enter the CNS and cause rapid multiplica- synthase (TPS1p and TPS2p) are required for
tion and meningoencephalitis as a consequence. thermotolerance, virulence and expression of
The human or animal cerebellum is a rich source the capsule and melanin in C. gattii.
of inositol which maintains the normal neurolog-
ical responses. Cryptococcus can use the inositol
as a sole carbon source which explains their 4.12.13 Disease Produced
affinity for the brain.
Penetration of the barrier is crucial for the The major animal and human diseases produced
development of meningitis. They can induce by different species of Cryptococcus are enlisted
the formation of microvilli-like protrusions by in Table 4.29.
the reorganisation of the host cell cytoskeletal
structures. These protrusions help the yeast cells
to enter brain microvascular endothelial cells 4.12.14 Immunity
(BMECs). Sometimes the yeast cells utilise
CD44 protein present in the lipid rafts of the Innate immunity offers first line of defence
host endothelial cells as receptor for invasion of against Cryptococcus by recognising the Cry-
BMECs (hyaluronic acid of the yeast acts as ptococcal patterns through toll-like receptor
ligand). After close attachment with the host (TLR), β-glucan receptor, mannose receptor
endothelial cells, the activation of host protein and the complement system. The professional
kinase C α-isoform occurs followed by cytoskel- phagocytes especially the bronchoalveolar
eton rearrangement that contributes to the intra- macrophages engage in complement-mediated
cellular transport. Within the cell cytoplasm, phagocytosis of the yeasts. The dendritic cells
Table 4.29 Major diseases of animal and human caused by Cryptococcus

Fungi Host Disease
Cryptococcus Cats It is the most common fungal infection in cats occurring in 3–7 years of
neoformans var. age. The infection is characterised by formation of nasal proliferative
neoformans lesion, granuloma in lymph node and the skin around the head and neck,
fever, sneezing, head shaking and blindness. The fungi may invade the
facial bone to cause distortion of nasal cavity. Sometimes the CNS
involvement with meningitis is observed. Siamese cats are more
susceptible than other breeds
Dogs Dogs 1–6 years old are susceptible. The infection is disseminated
within the respiratory tract, ocular tissue, skin, lymph nodes, CNS. The
subcutaneous granuloma surrounding the ears, face and feet is
observed, although not so common like cats. The renal involvement is
observed. The clinical signs include the respiratory signs (epistaxis,
sneezing and nasal discharge) and neurological signs (stumbling, partial
paralysis, ataxia, hyperesthesia, often along the dorsum or cervical area,
severe seizure)
Horse Nasal granuloma, meningitis, granulomatous pneumonia, bowel or
draining lymph node involvement (rare)
Cattle Mastitis with severe swelling and firmness of the udder, milk is mucoid
and rarely the infection may be transmitted into the lung
Human In human the patients with suppressed immunity (HIV infected,
suffering from lymphoma, haematological malignancy, using
corticosteroid for prolonged period) are mostly susceptible to the fungal
infection. It causes meningitis in human. One million cases of
cryptococcal meningitis occur globally per year in AIDS patients,
leading to approximately 6,25,000 deaths (majority of them in Africa)
Cryptococcus Cat Infection in the nasal cavity
neoformans var. grubii
Cryptococcus gattii Cats, dogs, goat, The infection is restricted within the respiratory tract and central
sheep, horse nervous system. The infection with VGII genotype involves several
organs with grave prognosis, whereas infection with VGI genotype is
restricted within the nasal cavity. The VGII genotype has hypervirulent
strains
Parrot Mycotic rhinitis/lower respiratory tract infection, CNS involvement
Pheasant Enterohepatitis
Pigeon Opportunistic infection (lesion in face, lateral elbow, preen gland area
and ankle, conjunctivitis)
Human Meningoencephalitis [in both the immunocompetent as well as
immunocompromised patients (including AIDS patients)]
(DC), natural killer cells (NK) and neutrophils effective clearance of the infection, regulatory
can also be lethal for the nonpathogenic Crypto- T cell (Treg) response develops for the suppres-
coccus. The capsule can activate the alternative sion of the response through the production of
complement pathway and the binding of C3 with tumour growth factor (TGFβ) and IL10. How-
the yeasts. This C3-mediated binding is absent in ever, pathogenic yeasts can also induce Treg
CNS explaining another reason for their CNS response for their survival. The antibodies are
affinity. Most pathogenic strains produce differ- also part of the immune response because the
ent virulence factors (Table 4.28) which can antibodies against the capsule component
evade the innante response. The innate response glucuronoxylomannan (GXM) can lyse the
helps to develop the adaptive immune response. fungi in vitro.
The protective anti-Cryptococcal immunity is The mucoid colony producers of Cryptococ-
T cell-mediated (CD4+ Th1 and Th17). After cus after switching generate a different type of
immune response as observed in experimental 2. Isolation and identification: The centrifuged

pulmonary infection in mice. They produce urine precipitate produces better isolation of
more vigorous inflammatory response and a dif- the yeasts than the other clinical samples
ferent cytokine profile than the smooth colony as most of the animals suffer from renal
producers. They upregulate the production of impairment. Media and incubation condition
monocyte chemoattractant protein (MCP) and as described earlier will serve the purpose.
macrophage inflammatory protein (MIP1α) 3. Animal inoculation test: Mice are inoculated
chemokines and downregulate the production of intracerebrally or intraperitoneally or into the
IL10, IL4, IL2 and tumour necrosis factor tail vein, and after 2 weeks they are sacrificed
(TNFα) which is correlated with massive lung following the standard animal ethics protocol.
damage and rapid death of the mice. In positive cases, gelatinous lesions are
observed in the abdominal cavity and lungs.
Cryptococcus neoformans var. neoformans
is the only pathogenic species in mice.
4.12.15 Diagnosis
Rabbits are naturally resistant to cryptococco-
sis. This test is not considered as a standard
4.12.15.1 Clinical Specimens
practice nowadays.
The samples include nasal swabs, tissue
4. Detection of antigen: Detection of Cryptococ-
specimens collected by fine needle aspiration,
cal antigen in the CSF (antigenaemia titre)
blood, cerebrospinal fluid (CSF), urine, pleural
can be performed by latex particles coated
and abdominal fluids, mastitic milk and lymph
with polyclonal serum and ELISA. Both of
node, skin, lung and bone marrow (after
the tests are specific and sensitive. The detec-
postmortem).
tion of antigen is possible from both the live
and dead organisms. During initial phase of
4.12.15.2 Laboratory Examination therapy, disintegration of the yeast cells
1. Direct examination: The smear can be releases the capsule which produces high
prepared from the tissue samples and from titre (>1: 60,000). So, the tests should not be
the precipitate after centrifugation of the done within 6–8 weeks after initiation of the
CSF collected from the suspected animals. therapy. The titre can be observed even after
The smear is stained with India ink or successful treatment as the dead organisms
Nigrosin or Romanowski for demonstration also have the intact capsule. However, nega-
of capsule. The Romanowski stain produces tive titre is inconclusive because the relapse of
clearer capsule against the lightly stained the infection may occur if the organism
background. The tissue biopsies can be persists in the nasal cavity. So, nasal culture
stained with periodic acid–Schiff base (PAS) should be done as a supplementary test for
– haematoxylin stain – which will outline the confirmation.
yeast cell and the capsule will appear as clear 5. Serological tests: The serological tests for
zone surrounding the cell. In Mayer’s detection of Cryptococcal antibodies in
mucicarmine stain, the capsule and cell wall serum are not very useful because the healthy
appears as red. animals also produce antibody titre due to
Another characteristic feature of Crypto- carriage or persistent infection. Latex particle
coccus is narrow base budding (Fig. 4.25) in coated with monoclonal rabbit anticapsular
comparison to other yeasts (Blastomyces) antigen and indirect fluorescence antibody
having broad base budding (Fig. 4.10). Some- test (FAT) can be used for the detection of
times false-positive results are produced due serum antibody. The false-positive reaction
to confusion with globules of myelin, lysed may occur in the patients infected with
cells, lymphocytes and dead yeasts after Trichosporon, Mucor, Penicillium and
successful treatment. Histoplasma.
6. Molecular biology: Polymerase chain reaction flavonoids, tannins), Curtisia dentata (triter-
(PCR) is developed to detect Cryptococcal penoids), Polygonum acuminatum (sesquiter-
CAP 59 gene in biopsy samples collected penes) and Daucus carota (sesquiterpenes) have
from suspected animals. antifungal activity against C. neoformans.
4.12.16 Treatment 4.13 Candida
Treatment in the early phase of the infection in Candida (previously known as Monilia) is
animals is successful. However, diagnosis with- imperfect unicellular dimorphic fungus which
out the laboratory help is difficult as most of the often reproduces by budding, and it can produce
clinical signs are vague. Further, prolonged anti- hyphae or pseudohyphae depending on the envi-
fungal therapy (1–2 months) is required for ronmental condition. Hippocrates (460–377 BC)
effective clearance of the yeasts which may not first documented oral pseudomembranous candi-
be a cost-effective option for the owners. diasis, and he described it with the name of
Amphotericin B (0.5 mcg/mL minimum inhibi- ‘aphthae albae’ which was later supported by
tory concentration) is the suitable antifungal for Galen (130–200 BC). In modern era, Berg and
the animals especially with the CNS involve- Wilkinson separately described oral candidiasis
ment. Other antifungals such as ketoconazole, (1841) and vaginal candidiasis (1849), respec-
itraconazole, albaconazole and fluconazole may tively, for the first time. Candida was first
be used. Itraconazole is more effective in the described as a separate genus in 1923 by
lung and bone involvement. Periodic assessment Berkhout. Candida parapsilosis was first isolated
of liver enzymes is required with prolonged keto- by Ashford from the stool of a diarrhoeic patient
conazole therapy in animals. Further, manage- in Puerto Rico in 1928.
ment of increased intracranial pressure is In India, Pathak and Singh (1962) reported for
required especially during the first 3–4 weeks the first time the occurrence of Candida albicans,
of the infection. Nonsteroidal anti-inflammatory C. tropicalis and C. paratropicalis from the
drug is recommended as the corticosteroids crops of the fowls. Candida was also isolated
are not useful in Cryptococcal infection. For from empyema (Pal 1987), mastitis in buffaloes
evaluation, fungal antigen detection should be (Pal 1997) and pneumonia in goats (Pal and
performed just after completion of therapy and Lee 1999).
1 month later to know whether there is any
relapse. The titre should be either undetectable
or decreased by twofolds. Surgical intervention, 4.13.1 Morphology
cryotherapy and administration of amphotericin
B and sodium iodide are recommended for There are three major morphological forms of
treatment of Cryptococcal rhinitis. Candida such as unicellular yeast, hyphae and
In human, second-generation triazoles such as pseudohyphae. The yeast form is oval (3.5–6 μm
voriconazole and posaconazole are being used 6–10 μm) with axial or bipolar budding. The
nowadays. The voriconazole has better penetra- hyphae are long tubes consisted of the cells with
tion capacity of blood–brain barrier than parallel sides, uniform width and true septum
posaconazole. However, antifungal therapy espe- without any constriction (Figs. 4.27 and 4.28).
cially with amphotericin B can promote the The pores are present in septa for cell-to-cell
selection of more virulent mucoid variety which communication.
often causes treatment failure. The pseudohyphae are chains of elongated
The extracts of certain plants such as ellipsoidal cells with constriction between them.
Blepharispermum subsessile (desmethyl isoence- In the yeast and pseudohyphae, the nuclear divi-
calin), Anacardium occidentale (phenolic lipids, sion and septa formation takes place near the
4.13 Candida 103
Fig. 4.27 Morphological forms of Candida albicans

(schematic). a Yeast form, b pseudohyphae, c hyphae
Fig. 4.29 Pulmonary candidiasis in Gram-stained

sputum smear (Photograph courtesy: Prof. P.P. Gupta,
Ex-Director of Veterinary Research, Punjab Agricultural
University, Punjab, India)
The thick-walled chlamydospores remain

attached with hyphae or pseudohyphae by a
suspensor cell.
The cell wall is present outside the cell mem-
brane composed of inner and outer layer. The
inner layer is a meshwork of chitin, β-(1, 3) glu-
can and β-(1, 6) glucan which is more electron
translucent. The outer layer is 150 nm width
Fig. 4.28 Budding yeast, hyphae and pseudohyphae of composed of mannoprotein. Three types of cell
Candida albicans in GMS stained udder tissue smear wall proteins (CWP) are present in the cell wall.
(Photograph courtesy: Prof. P.P. Gupta, Ex-Director of The most abundant type is GPI-CWP which is
Veterinary Research, Punjab Agricultural University, covalently attached with β-(1, 6) glucan through
Punjab, India)
glycophosphatidylinositol (GPI) anchor. Another
class, known as Pir protein (Protein with internal
bud, whereas in hyphae both the procedures repeats), is also covalently attached with β-(1, 3)
occur within the germtube. Both the yeast and glucan. The third category of proteins (Pra1p)
pseudohyphae can grow along with the cell lacks the covalent attachment and is heteroge-
cycle, but the hyphal cells remain arrested in neously distributed throughout the cell wall.
G1 phase after completion of first cell cycle Some of them are secreted into the environment.
until they can accumulate sufficient cytoplasmic The expression of morphological forms varies
mass to enter the second cycle. So, hyphae with the species of Candida. All the three forms
are less branched in comparison to pseudo- are expressed by C. albicans and C. tropicalis,
hyphae (Fig. 4.29). The hyphal cells contain a whereas other species such as C. parapsilosis can
specialised organelle, known as spitzenkorper, express the yeast and pseudohyphae forms. Some
which helps in growth at the hyphal tips. The species such as C. glabrata can express the yeast
intermediate morphological forms such as elon- and pseudohyphae forms, but primarily a yeast
gated single yeast cell, pseudohyphal cells with form both in the environment and host tissue
parallel sides and minor septa are also observed. without any morphological switch.
The morphological switch between the yeast form flat and grey or opaque colonies. Certain
and filamentous form is correlated with the viru- phenotype-specific genes (WH11, EFG1) are
lence. Several external (environmental cues) expressed by white cells, while OP4 and SAP4
and internal factors regulate the morphological are expressed by opaque cells. White cells are
switch of Candida. The environmental cues more virulent and can easily colonise the host
include presence of serum, temperature (37 C), internal organs, whereas the opaque cells are
low levels of oxygen, high levels of CO2 and associated with cutaneous infection probably
poor nutrition. Bacterial peptidoglycan present due to expression of SAP4 gene. The stable
in serum, N-acetylglucosamine found in mucus opaque type and either a or α-mating-type cell
of gastrointestinal tract, 5 % CO2 as a product of can undergo efficient sexual mating. Master
host cellular respiration and poor nutrition regulator of this complex switching system
can induce the filamentous growth through the is WOR1 which can convert the white cells into
activation of cAMP/PKA pathway (regulated by opaque cells. This WOR1 expression produces a
Candida Ras protein). The pH change response direct positive feedback loop by binding its own
is mediated through Candida Rim101 pathway. promoter and turning ‘on’ its own expression.
The contact-dependent response and osmotic The a/α-heterodimer cells (mating type) cannot
sensing are regulated by Mkc1 and Hog1 switch from white to opaque because they
MAPK pathway, respectively. The internal directly repress the WOR1 promoter. The host
factors include the filament-induced gene environmental factors regulating white–opaque
(HGC1) which encodes a cyclin-related protein transition are CO2 and N-acetyl glucosamine
required for septin phosphorylation and inhibi- (present in commensal bacterial cell wall and
tion of cell separation. Several members of gastrointestinal tract mucus) which can produce
secreted aspartyl proteinase (SAP) gene family stable opaque phenotype. Other factors, such
(SAP4, SAP5, SAP6) are also expressed during as oxidative stress, UV light and adenine, also
morphological switch required for invasion of regulate white–opaque transition.
host tissues. The transition from yeast to hyphal
phase in Candida is regulated by the activation of
mitogen-activated protein (MAP) kinase and 4.13.2 Classification
cAMP-protein kinase A (PKA) signal transduc-
tion pathways which can also coordinately regu- The genus Candida belongs to the class
late the virulence gene expression associated Saccharomycetes (Hemiascomycetes). It has
with this transition. Three sensor histidine more than 200 species. Approximately, 20 spe-
kinases (Sln1, Chk1, Nik1) also regulate the cies have been associated with causing candi-
phase conversion and mutants are unable to pro- diasis in human and animals among which
duce hyphae. Quorum-sensing (microbial com- seven species are of major importance. Candida
munication) molecules such as farnesol, tyrosol albicans, Candida tropicalis and Candida
and dodecanol are also associated with the glabrata are the most frequently isolated
transition. from clinical specimens. The other pathogenic
Another type of morphological transition species are C. parapsilosis, C. stellatoidea,
observed in C. albicans is known as C. guilliermondii, C. krusei and C. pseudo-
white–opaque transition. In the laboratory, this tropicalis. In dogs C. natalensis is the most fre-
switching is rare (1 in every 104 cell division) quently identified species.
and it is regulated by host environmental factors.
These morphological types are genetically sta-
ble, i.e. white or opaque cells always produce 4.13.3 Reproduction
white or opaque type of progeny, respectively.
The ‘white’ cells are relatively round and Recent evidence suggests the presence of sexual
form smooth colonies on solid media, while reproduction in several Candida species espe-
‘opaque’ cells are large and elongated and cially in C. albicans (not yet detected in
4.13 Candida 105
C. parapsilosis and C. tropicalis). Two mating quaternary ammonium compounds (1:10,000) is

types (a and α) commonly exist in nature. For lethal at short contact time.
mating the cells have to be switched into opaque Among the physical agents, Candida is sus-
state. Once in opaque state, the a- or α-type of ceptible to heat (more than 50 C) and ultraviolet
cells secrete the pheromones (MFa and Mfalpha) light and resistant to freezing.
that are sensed via the cell surface receptors, Ste3
and Ste2, respectively. Pheromone signalling
produces projections at the pole of the opposite 4.13.5 Natural Habitat and Distribution
type of cells (a and α). Subsequently fusion of the
cells and karyogamy occur, producing mononu- Candida normally inhabits in the mucosal layer
clear tetraploid cells. These tetraploid cells of animals and human such as alimentary, upper
can undergo mitosis several times. In Candida, respiratory, genital tract and oral mucosa such as
meiotic division is not observed to reduce the posterior dorsum of tongue. They can invade the
ploidy (formation of diploid from tetraploid deeper part of the tissues to establish the infec-
cells). Instead, the concerted chromosome loss tion under immunosuppressed conditions caused
occurs to reduce the ploidy even in vivo, by prolonged antibiotic or steroid use, inflamma-
generating many aneuploid progeny cells. tion and breakage of epidermal layer by injury.
These cells have different filamentation, growth From the environment, Candida is commonly
rate, white––opaque transition and increased isolated even from hypersaline niche.
antifungal resistance. The aneuploid cells with C. albicans is distributed throughout the
higher fitness are selected. world.
The a/α-heterodimer cells cannot directly par-
ticipate in the mating as they cannot switch to
opaque state. However, decreased expression of 4.13.6 Genome
HBR1 (haemoglobin response gene 1) causes the
a/α-heterodimer cell to behave phenotypically Each Candida species contains 6–9 numbers of
like a cell. They are able to switch into the chromosomes. The genome is diploid in most of
opaque state and participate in the mating. pathogenic Candida species such as C. albicans,
C. albicans also shows the evidence of homo- C. tropicalis and C. parapsilosis. The haploid
thallic mating (a-a or α-α-mating) occurring genome is detected in C. guilliermondii and
between the same type of cells. The a cells can C. lusitaniae. The size of C. albicans genome is
initiate the mating even if the α-cell is not avail- 10.6–15.5 Mbp with the number of genes varying
able in the vicinity through autocrine pheromone from 5,733 to 6,318. The genome contains the
signalling. The a cells release α-pheromone major repeat sequence (MRS) elements where
(Mfalpha) which binds with the Ste2 receptor the genetic recombination can take place.
either in the same or neighbouring a cells. It is Another feature of C. albicans genome is the
followed by a–a fusion of the cells. High level of presence of one single nucleotide polymorphism
Bar1 protein can inhibit the homothallic mating (SNP) in every 330 bp (SC5314 strain) to 390 bp
between the opaque a cells. (clinical strain WO-1).
At the transcription level, the sexual mating
of Candida is regulated by MTL (mating-type
4.13.4 Susceptibility to Disinfectants locus) loci present in the genome. The trans-
cription factors a2, α1 and a1/α2 are required
Crystal violet is an effective disinfectant against for the expression of specific genes and
C. albicans. Among antiseptics, 4 % chlor- generation of a-, α- and a/α-heterodimer cells.
hexidine gluconate in alcohol and 10 % The locus also contains the additional genes
povidone–iodine have potential antifungal activity such as gene for phosphatidylinositol kinase
against Candida, whereas among disinfectants, (PIK), oxysterol-binding proteins (OBP) and
poly A polymerases (PAP) with unknown role in C. dubliniensis isolates exhibited a hyphal fringe
mating. In some species of Candida, homothallic surrounding the colonies.
mating between same sexes is detected where
either the MTL is absent or both the loci are
fused into a single locus. 4.13.8 Biochemical Characteristics
Production of urease and carbohydrate fermenta-

tion pattern (Table 4.30) helps in identification of
4.13.7 Isolation, Growth and Colony Candida species.
Characteristics
Candida can be isolated in general fungal 4.13.9 Antigenic Characteristics

or bacteriological media such as Sabouraud
dextrose agar (with penicillin, streptomycin, Candida has two major groups of antigens such
chloramphenicol), potato dextrose agar, blood as heat-stable polysaccharides (glucan, mannan)
agar and brain–heart infusion agar. They are and heat-labile glycoproteins. The mannan is a
obligate aerobe and they can grow within a major antigen of Candida that circulates during
wide range of temperature and pH. The plates infection and it comprises 7 % dry weight of
are incubated at 25–30 C for 2–3 days. Pigmen- the cell wall. It is resistant to heat, proteinase
tation is detected in corn meal tween agar. and acidic pH. The antigenicity varies with the
Currently the media with resins are in use length of the polysaccharide side chain and
which can absorb the residual antifungal or position of the glycosidic linkages (α-man and
other inhibitory substances present in the clinical β-man indicating the α-/β-linked oligomannose
samples and significantly improves the recovery residues). C. albicans has two serotypes (A and
of Candida. Further, C. albicans can grow in the B) based on these variations.
presence of 0.04 % cycloheximide.
The colonies are circular, white or opaque in
colour (white–opaque transition is required) and 4.13.10 Virulence Factors
creamy in consistency.
For identification of Candida at species The virulence factors possessed by C. albicans
level, a chromogenic medium (CHROMagar) are described in Table 4.31.
can be used in which different species such as
C. albicans, C. tropicalis and C. krusei produce
green/bluish-green, blue/purple with halo and 4.13.11 Transmission
pink-coloured/ruffled colonies, respectively.
Similarly, Oxoid Chromogenic Candida Agar Candida causes the opportunistic infection in
(OCCA) can also differentiate the species with most of the clinical cases during immunosup-
the colour of the colonies. The sunflower seed pression. In bovine udder, they may be transmit-
agar (Pal’s medium) can differentiate between ted by the contaminated milker’s hands during
C. albicans and C. dubliniensis where only milking or administration of medicines.
Table 4.30 Carbohydrate assimilation pattern of different Candida species

Carbohydrate C. albicans C. tropicalis C. pseudotropicalis C. krusei C. parapsilosis
Glucose + (acid and gas) + (acid and gas) + (acid and gas) + (acid and gas) + (acid and gas)
Maltose + (acid and gas) + (acid and gas) – – –
Sucrose + (acid only) + (acid and gas) + (acid and gas) – –
Lactose – – + (acid and gas) – –
4.13 Candida 107
Table 4.31 Virulence mechanisms and factors possessed by C. albicans

Yeast–hyphae transition The hyphae are more invasive due to application of mechanical force and can
easily penetrate both the individual cells and the space between them, whereas the
yeast cells are easily disseminated into the blood circulation. The Candida hyphae
can damage endothelial cells, lyses the macrophages after phagocytosis
Thigmotropism (directional hyphal It is the contact sensing mechanism of Candida hyphae by which they can identify
growth through contact sensing) the crevices, grooves present in the host tissues for effective penetration. The
contact sensing also triggers the biofilm formation over the solid surface. It is
regulated by extracellular calcium uptake through the calcium channels Cch1,
Mid1
Biofilm Candida is able to produce biofilm in host mucosal cell surfaces and medical
devices along with some bacterial species. Within biofilm the cells have reduced
growth rate due to limited nutrition. The CDR and MDR genes encoding two types
of efflux pumps, i.e. ATP-binding cassette (ABC) transporters and major
facilitators, are upregulated in biofilm cells which are associated with antifungal
resistance along with other factors such as complex architecture and metabolic
plasticity
B cell mitogenic protein (ISM p43) It causes hyper stimulation of B cells and immunosuppression and helps in
survival of the fungi within the host
HYR1 (hyphal protein) It is associated with antifungal resistance
pH-sensitive protein (PHR1 and They are cell wall β-glycosidases. The PHR1 and PHR2 are expressed in neutral-
PHR2) alkaline and acidic pH, respectively. So they are associated with systemic and
vaginal infection, respectively
Extracellular pH modulation Candida can modulate the extracellular pH towards alkaline and auto-induce the
hyphae formation. It is associated with starvation and uptake of amino acids,
polyamine, etc. The amino acids are cleaved intracellularly by fungal urea
amidolyase (Dur1,2) to generate ammonia which is exported into the external
environment through the Ato (ammonia transport outward) export proteins. The
export of ammonia makes the environment alkaline which promotes the hyphal
growth
Metabolic plasticity In the hostile environment (within macrophage), Candida promptly switches
glycolysis to gluconeogenesis where lipid and amino acids serve as a nutrient
source
Iron acquisition Candida can acquire iron through different mechanisms such as reduction of host
ferritin (Als3 mediated), acquisition of iron from the siderophores produced by
other organisms (xeno-siderophore) and uptake from haemoglobin and other
haeme proteins
Zinc acquisition It is mediated by zinc-binding protein (Pra1: pH-regulated antigen 1) which acts
as a zincophore by binding extracellular zinc
Enzymes
Secreted aspartyl proteinases The enzyme degrades immunoglobulin and complement of the hosts. Its help in
(Sap 1–8; Sap 9,10 cell bound) penetration of host tissues is controversial as detected in the recent studies
Phospholipase A, B, C, D; They are associated with host cell membrane damage (PLB), adherence and
lysophospholipase penetration
and lysophospholipase-
transacetylase
Haemolysin (mannoprotein) They lyse the erythrocytes to release iron required for survival of the fungi
Catalase and superoxide dismutase It provides protection against oxidative stress
Adhesins/invasins
Glycolytic proteins (GAPDH, (a) They act as adhesion factors and modulators of host antifungal immune
PGK, ADH, enolase), heat shock response
proteins (Hsp104, Hsp90, Hsp78, (b) The heat shock proteins can further act as chaperones and prevent protein
Hsp70, Hsp60) unfolding and aggregation under stress such as high temperature, starvation and
Small Heat shock proteins (sHsp) oxidative stress
[Hsp31, (c) The sHsp proteins are low molecular weight chaperones that prevent protein
Hsp30, Hsp21, Hsp12, Hsp10] aggregation
(continued)

Lectin-like molecules They help in adhesion of Candida with L-fucose, N-acetylgalactosamine or
N-acetylglucosamine containing glycosides present in the epithelial cell receptors.
They also recognise salivary protein (mucin) and bacterial cell surface
(Staphylococcus) which helps in oral colonisation
Fimbriae It helps in the adhesion of Candida with the erythrocytes mediated through the
fimbrial protein (66 Kda) and glycosphingolipids present in the erythrocyte
surface
Integrin analogue [a protein that (a) They help in the adhesion of Candida with the proteins having RGD motifs
can bind RGD (arginine-glycine- such as extracellular matrix protein, complement (C3), fibrinogen, heterodimeric
aspartic acid) motif] transmembrane proteins
(b) They play major role in yeast–hyphae transition.
Mannan They help in adhesion of Candida with epithelial and endothelial surfaces,
erythrocytes, salivary components
ALS protein [encoded by ALS gene The hyphae-associated ALS protein (ALS3) adheres with host laminin, fibronectin
(agglutinin-like sequence)] and collagen through the recognition of certain amino acids (threonine, serine or
consisting of eight members (ALS alanine). The ALS3 also acts as invasin and is associated with induced endocytosis
1–7, ALS 9) by the host epithelial cells
Hwp1 (a hypha-associated It acts as a substrate for mammalian transglutaminases and this reaction may
GPI-linked protein) covalently link Candida hyphae to host cells
Ssa1 (cell surface-expressed It acts as invasin and is associated with induced endocytosis by the host epithelial
protein cells
belonged to HSP70 family)
Fibronectin adhesin They help in the adhesion of Candida with fibronectin and vitronectin receptors
Flucoside-binding adhesin They help in the adhesion of Candida with glycoside receptor
4.13.12 Pathogenesis environment and remain as commensal without

any cell damage and further cytokine response a
4.13.12.1 C. albicans few strains express the virulence factors which
Adhesion to the host epithelial cells is a prereq- also depend on host conditions such as immuno-
uisite step for both commensal and pathogens. suppression due to prolonged use of gluco-
The adhesion is a complex process as both the corticoid, breaching of the mucosal surface,
cells (Candida and host cells) are negatively concurrent infection, neutropenia associated
charged which will repeal each other. To over- with chemotherapy, etc. These strains are either
come this repulsive force, some other attractive eradicated by the host immune system or they
forces also act such as Van der Waals force, become able to establish a systemic infection.
hydrophobic interaction and Brownian move- Following adhesion, C. albicans changes
ment force. Successful adherence depends on morphologically from the yeast phase to the
the interaction between these forces [extended hyphal phase required for tissue penetration.
Derjaguin–Landau–Verwey–Overbeek (DLVO) They use two different mechanisms such as
theory]. Overcoming the repulsive forces, ‘induced endocytosis’ or ‘active penetration’ for
C. albicans detects the host environment either invasion into the host epithelial cells. For
by contact sensing due to body temperature, pH induced endocytosis, the fungi express different
change or any other unexplored mechanism to proteins in their surface (‘invasins’ such as
express different invasins (Table 4.31) on their ALS3, Ssa1) which can bind the ligands present
surface for attachment with the host cells. in the host cells (E-cadherin on epithelial
Majority of the C. albicans strains with cells and N-cadherin on endothelial cells). This
hidden pathogen-associated molecular pattern binding stimulates the host cells for engulfment
(PAMP) can adapt the epithelial surface of the hyphae (both live and dead) by a
4.13 Candida 109
clathrin-dependent mechanism or macropino- 4.13.14 Immunity

cytosis. Active penetration is conducted by live
hyphae only with the help of mechanical force The innate immune system is activated through
and damaging factors which can disrupt the host the recognition of Candidal pathogen-associated
cell membrane, although the detailed mechanism molecular pattern (PAMP) by the pattern recog-
is still unknown. The damaged epithelial cells in nition receptors (PRR) expressed by different
turn stimulate the release of pro-inflammatory immune cells (Table 4.33).
cytokines which attract neutrophils and macro- The PAMP molecules are present both in
phages in the area for destruction of the invading outer and inner layer of Candida which is
hyphae. exposed during budding or during exposure
Within the macrophages, Candida uses sev- with f antifungals and host enzymes.
eral survival strategies such as production of Recognising the specific Candidal PAMP, the
catalase and superoxide dismutase, providing TLR4 induces pro-inflammatory signals in
protection against oxidative stress. C. albicans monocytes, macrophages and dendritic cells
also delays phagosome maturation and induces (DCs) through the MyD88-Mal-mediated NFκB
recycling of late maturation markers like and mitogen-activated protein kinase (MAPK)
LAMP-1. Soon the hyphae disrupt the macro- pathways. They also stimulate TH1 responses
phage membrane and escape. Even recently through IRF3-dependent mechanisms which pro-
non-lytic escape mechanism of C. albicans is vide protection against disseminated Candidia-
also detected as observed in Cryptococcus. sis, whereas the TLR2 stimulates the production
However, C. glabrata not only survives, but of moderate amounts of pro-inflammatory cyto-
replicates inside the phagosome until the phago- kines and strong IL10 and TGFβ responses. The
cyte bursts due to the modification of normal dectin1 induces IL2, IL10 and TH17 responses
phagosome maturation process. Their phago- through a Syk-CARD9 cascade and increases
some lacks any lysosome marker such as cathe- the effects of TLR2. The mannose receptor
psin D and remains non-acidified. (MR) induces pro-inflammatory effects in
For the establishment of infection, C. albicans monocytes and macrophages. Other pathways
requires macro- and micronutrients (iron, zinc) include production of tumour necrosis factor
from the host. They can metabolise different (TNF) and IL1Ra by dectin 2 and production of
sugars and can use all amino acids as nitrogen the immunosuppressive cytokine (IL10) through
sources. In addition, C. albicans possesses sev- DC-SIGN.
eral families of secreted hydrolases and trans- The mucosal surface of healthy individuals is
membrane transporters (Table 4.31) which help often colonised with C. albicans. The mucosal
in the macro- and micronutrient acquisition. Dur- cells provide a structural barrier and they can
ing disseminated candidiasis, C. albicans gains recognise the fungi. The epithelial cells can
access to the bloodstream, relatively rich in glu- respond to C. albicans in a two-phase MAP
cose (6–8 mM) which is the preferred nutrient kinase (MAPK) pathway that enables them to
source of most fungi. discriminate between the commensal yeast and
the invasive pathogenic form. The second-phase
MAP kinase (MAPK) pathway can be induced by
4.13.13 Disease Produced the invasive hyphae, not the yeast. This second-
phase induction leads to activation of the tran-
The major animal and human diseases produced scription factor cFos and the MAPK–MKP1
by different species of Candida are enlisted in which is necessary for the production of
Table 4.32. cytokines (IL1, IL6). The hyphae can activate
Table 4.32 Major diseases of animal and human caused by Candida sp.
Fungi Host Disease
Candida albicans Poultry Thrush (crop mycosis): It is observed in chicken, turkey, guinea fowls. In acute cases,
the birds may die without any syndrome. The crop, mouth, oesophagus,
proventriculus and gizzards are commonly affected. The lesions are white plaque,
ulcer and/or pseudomembrane adherent with the mucosal surface. The crop mucosa
produces characteristic ‘Turkish towel’ appearance. The disease is associated with
coccidiosis, overcrowding
Candida Cattle Mycotic mastitis: Usually prevalence is low in cattle herd (10 %) and the infection is
parapsilosis, mild and self-limiting in nature. It has been associated with using contaminated
C. krusei, syringes, canulars and antibiotic preparations. Teat injuries may act as predisposing
C. tropicalis, cause. It is transmitted either from contaminated udder skin through the milker’s
C. rugosa hand, milking machine or from the contaminated floors, straw, feed, sanitary agents
and other equipments
Candida albicans, Foals, Gastroesophageal candidiasis and gastric ulceration
C. krusei calves
Candida albicans Pigs Alopecia, circular skin lesion covered with exudates, pseudomembrane formation
Candida albicans, Dogs Exfoliative dermatitis in the muzzle, scrotum and feet along with pruritis and
C. parapsilosis, alopecia, otitis externa, genital tract infection
C. guilliermondii
C. albicans, Dogs Canine peritonitis with marked pyogranulomatous inflammation
C. glabrata
Candida albicans, Cats Localised and disseminated infection associated with feline panleukopenia and feline
C. parapsilosis immunodeficiency virus, granulomatous rhinitis associated with chronic use of
immunosuppressive drug, enteritis
Candida albicans, Human (a) Invasive candidiasis: It is associated with immunosuppression, neutropenia due to
C. glabrata, stem cell or solid organ such as liver transplantation. Candida is among the important
C. parapsilosis, pathogens in the patients admitted to the intensive care unit. It causes high rate of
C. tropicalis morbidity and mortality without specific syndrome
(b) Oral candidiasis: The patients receiving broad spectrum antibiotics, steroid or
cytotoxic therapy and having diabetes mellitus, xerostomia, nutritional deficiency
and AIDS are susceptible to oral candidiasis. It is characterised by white patches on
the surface of oral mucosa (thrush); erythematous lesion on the dorsum of the tongue,
palate or mucosa (antibiotic sore mouth); formation of leukoplakia (chronic lesion);
denture-induced stomatitis; glossitis; etc.
the inflammasome and induce IL1β production in 4.13.15.2 Laboratory Examination

immune cells such as macrophages and stimulate 1. Direct examination: The smear prepared from
TH17 cells to produce cytokines (IL17, IL22). the clinical specimens can be observed under
The IL17 activates neutrophils, while IL22 microscope either by 10 % KOH preparation
induces the release of defensins from epithelial or by staining with Gram stain method. In the
cells; both are crucial components of mucosal tissue section, Candida can be observed by
antifungal defence. PAS-haematoxylin or methenamine silver
stain. They appear as unicellular budding
yeast or pseudohyphae.
4.13.15 Diagnosis 2. Isolation and identification: Media and incu-
bation condition as described earlier will
4.13.15.1 Clinical Specimen serve the purpose.
The clinical specimens include skin scrapings, 3. Demonstration of germ tube formation: A sin-
centrifuged milk samples, blood, serum and tis- gle Candidal colony is picked up and mixed
sue samples in 10 % formalin for biopsy. with 0.5 ml of rabbit or sheep or bovine or
4.13 Candida 111
Table 4.33 Pathogen-associated molecular pattern (PAMP) of C. albicans and their pattern recognition receptors
(PRR) expressed by different immune cells
PRR Immune cells PAMP
Toll-like receptors (TLR)
TLR2 Macrophage, neutrophil, Phospholipomannan
dendritic cells, CD4+ T cells
TLR4 Macrophage, neutrophil, dendritic Mannan
cells, CD4+ T cells
TLR9 Macrophage, neutrophil, dendritic cells CpG oligodeoxynucleotides
C-type lectin receptors (CLR)
Mannose receptor Macrophage, dendritic cells Mannan
Dectin-1 Macrophage, neutrophil, dendritic cells β-1,3-glucan
Dectin-2 Macrophage, neutrophil, dendritic cells High-mannose structures
(Man9GlcNAc2)
DC-SIGN Macrophage, dendritic cells High-mannose structures
(Man9GlcNAc2)
Galectin-3 Macrophage β-1,2-mannosides
Mincle (Clec4e and Clecsf9) Macrophage Unknown
Complement receptor 3 (CR3) Neutrophils β-glucan
Langerin Langerhans cells Mannose, fucose
NOD-like receptors (NLR)
NLRP3 (NLR Unknown
family pyrin domain containing 3)
human serum and incubated at 37 C for stained smear can be identified. A monoclonal
1–2 h. After the incubation, a drop of prepara- antibody (3H8) is developed against
tion is observed under the phase contrast or C. albicans cell wall mannoprotein which
dry objective of the microscope. In positive can specifically recognise C. albicans in cul-
case, small tubes projecting from the yeast ture and in paraffin-embedded tissue sections
cells without any constriction at the point of using immunofluorescent and immunohisto-
attachment are observed. This is a character- chemical staining. This antibody specifically
istic of C. albicans and C. tropicalis (after detects the mycelial forms and to a lesser
prolonged incubation for 3 h). extent blastospores of C. albicans and does
4. Demonstration of chlamydospore production: not react with any other Candida spp. Fluo-
For demonstration of chlamydospore, single rescent in situ hybridisation (FISH) using oli-
isolated Candidal colony can be inoculated gonucleotide probes directed against 18S
into the cornmeal tween 80 agar or chlamydo- rRNA has been used to differentiate
spore agar by making the parallel cuts in the C. albicans from C. parapsilosis in tissues.
media. Sometimes below the surface inocula- 6. Immunological assays: The ELISA-based
tion is preferred for better chlamydospore pro- tests are developed to detect the Candidal
duction in low-oxygen tension. The plates are antigens such as secreted aspartyl proteinase,
incubated at 30 C for 2–4 days. A coverslip is mannan and β-D glucan. Dissociation of
placed over the surface, and the plate is antigen–antibody complexes is necessary for
observed under the high dry objective for the the optimal detection of mannan in the circu-
detection of thick-walled chlamydospores lation. This antigen is heat stable and resists
(8–12 μm) at the tips of pseudohyphae. boiling, proteinase treatment and acidic
5. Histopathology: The presence of blastospores pH. So antigen–antibody complexes are rou-
and pseudohyphae in the histochemically tinely dissociated by boiling with EDTA or by
enzymatic treatment. The sensitivity and 4.13.16 Treatment

specificity of the mannan-based test is
58 and 93 %, respectively, whereas the sensi- Copper sulphate in drinking water or nystatin in
tivity and specificity of β-D glucan (BDG) feed is the traditional treatment of crop mycosis
detection test is 77 and 85 %, respectively. in poultry. Currently amphotericin B and flucon-
These immunological assays are useful in azole are also in use. The amphotericin B is
diagnosis of Candida in immunocompro- effective against wide species range of Candida
mised patients who produce negligible or and is relatively cheap. However, nephrotoxicity
undetectable amount of antibodies. is detected as side effect in human patients.
7. Serological tests: The ELISA-based sero- Experimentally Linum usitatissimum (linseed/
logical tests are developed for detection of flaxseed)-fixed oil and the herb (Persicaria
antibodies against Candidal aminopeptidase, senegalense) are found effective against cattle
mannan and secreted aspartyl proteinase in mastitis causing strains of C. albicans.
the suspected serum. Indirect immunofluores- The cutaneous infections in animals are
cence assay is also developed for the detection treated by removal of crusts which is followed
of anti-Candidal antibody (IgG). However, by topical application of povidone–iodine, cop-
serological tests for detection of Candida are per sulphate or chlorhexidine or selenium sul-
not so much reliable. The false-positive reac- phide in the area.
tion occurs due to superficial colonisation, and Other antifungals such as voriconazole,
false-negative reaction occurs in immuno- caspofungin and micafungin are also effective
compromised patients producing low or unde- against Candida with minimal side effects.
tectable level of antibodies. They are either expensive or their veterinary
8. Molecular biology: Several types of PCR such preparation is not always available.
as semi-nested and nested PCR, multiplex
PCR followed by DNA sequencing or
pyrosequencing are developed for detection 4.14 Sporothrix
of Candidal DNA. The target DNA includes
rRNA (5.8S, 18S, 28S) gene, internal- Sporotrichosis was first described by Linck in
transcribed spacer (ITS) and intergenic spacer 1809 and Lutz in 1889, but they could not isolate
(IGS) region. However, liberation of DNA the fungi. In 1896, Sporothrix schenckii was first
from the cell is challenging due to the pres- isolated by Benjamin Schenck, a medical student
ence of a highly rigid cell wall which often at Johns Hopkins Hospital in Baltimore, USA.
produces false-negative result. The false- He isolated the organism from a male patient
positive result is produced when highly with cutaneous lesion in the right arm. The iso-
conserved rRNA (5.8S, 18S, 28S) or other late was confirmed by Erwin Smith, a mycologist
genes are targeted and airborne fungal con- who described the fungus as Sporotrichum. In
tamination occurs. Confirmation of PCR 1900, Hektoen and Perkins further reported spo-
amplicon by enzyme immune assay (EIA) rotrichosis from cutaneous lesion of a patient in
using colorimetric substrate is considered as the Unites States. The dimorphism of Sporothrix
an easy and cheapest method. was described by Howard in 1961. However,
Real-time PCR method is also developed the erroneous classification of the fungi under
using Taqman probe or light cycler system the genus Sporotrichum continued until 1961.
for the identification of Candida. They also Carmichael (1962) first recognised the differ-
lack the post amplification detection costs; ences of the Sporothrix isolates from the
however, the initial cost for setup of the labo- Sporotrichum genus, and finally the nomencla-
ratory and instruments is high. ture Sporothrix schenckii was accepted.
4.14 Sporothrix 113
In 1907, the first natural animal infection with

Sporothrix schenckii was reported by Lutz and
Splendore in rats from Brazil.
In India, the first authentic report of human
sporotrichosis was published in 1986. The isolate
was obtained from a 55-year-old housewife with
multiple lymphocutaneous lesions on her arm,
residing at Ranikhet, Uttar Pradesh. Further
human sporotrichosis was reported from
Himachal Pradesh, Manipur, Uttarakhand and
Bangalore (Sharma et al. 1990). In 2009, feline
transmission of lymphocutaneous sporotrichosis
in human was also reported from Karnataka,
India (Yegneswaran et al. 2009). Fig. 4.30 Daisy flower appearance of Sporothrix
schenckii (Photograph courtesy- Prof. Alexandro Bonifaz,
Head, Department of Mycology and Dermatology service,
4.14.1 Morphology General Hospital of Mexico, Mexico City)
Sporothrix schenckii is a dimorphic fungus with

a saprophytic mycelial and parasitic yeast phase.
The mycelium is composed of slender (1–3 μm),
hyaline, septate and branched hyphae bearing
thin conidiophores. The apex of conidiophore
carries a small vesicle with symmetrically
arranged denticles. Each denticle bears one
conidium. Each of the conidium is pear shaped
to clavate, approximately 2–4 μm in measure-
ment and do not yield any chain. These conidia
are arranged like the petals of flower forming a
‘peach flower’ or ‘daisy flower’ appearance Fig. 4.31 Cigar-shaped bud of Sporothrix schenckii
(Fig. 4.30). Occasionally, dark, thick-walled yeast cells (Schematic)
conidia are directly attached with the hyphae
which are arranged bilaterally side by side in a the plasma membrane. The cell wall is composed
row (aleurioconidia or raduloconidia). These of alkali-soluble and alkali-insoluble glucans.
dark cell-walled conidia are specific for The alkali-soluble glucans of yeasts contain β
Sporothrix schenckii and are not detected in non- (1, 3), β (1, 6) and β (1, 4) linkages in a propor-
pathogenic species. The yeast cells are pleomor- tion of 44 %, 28 % and 28 %, respectively,
phic showing oval or spindle-shaped cells whereas the proportion of same linkages differs
measuring 2–5 μm in diameter. The cells contain in alkali-insoluble glucan [66 % β (1, 3), 29 % β
elongated or ‘cigar-shaped buds’ on the narrow (1, 6) and 5 % β (1, 4)]. The β-glucan composi-
base which are diagnostic (Fig. 4.31). Some- tion of cell wall remains the same in the mycelial
times, fragments of hyphae are attached with phase. In addition, the yeast cell wall contains a
the yeast cells. peptide rhamnomannan complex composed of
Sporothrix schenckii contains an amorphous 50 % D mannose, 33 % L rhamnose, 1 % galac-
microfibrillar material in the outermost layer of tose and about 16 % peptides, whereas the myce-
the cell wall which resembles capsule. Both the lial cell wall contains large amount of lipids and
mycelial and yeast cells contain a cell wall protein and a lower quantity of mannose. Mela-
beneath the capsular material and surrounding nin and other proteins are also an integral
component of yeast cell wall which is involved in 4.14.3 Reproduction

fungal adhesion and pathogenesis.
The plasma membrane is present beneath the Sporothrix does not undergo any sexual repro-
cell wall in yeast, mycelia and also in conidia. duction. Although the evidences suggest that it
The structure of plasma membrane varies produces recombination in the nature, some stud-
between these three morphological forms. In ies related with 18S rDNA analysis identified
yeast, the plasma membrane contains scarce but Ophiostoma stenoceras as the sexual form of
longer invaginations, whereas in mycelia, the S. schenckii. Later, due to differences in morpho-
invaginations are absent and in conidia, they are logical, physiological and virulence properties, it
abundant but short. There is a vesicle-like struc- was concluded that Ophiostoma stenoceras and
ture present in the cell wall which helps in trans- S. schenckii are two different species.
fer of periplasmic molecules and pigments from
the plasma membrane to the extracellular space.
The mould-to-yeast phase transition requires
4.14.4 Susceptibility to Disinfectants
higher temperature (35–37 C) (except S. globosa
which grows well in low temperature) and
S. schenckii is sensitive to 70 % ethanol, sodium
nutrient-rich culture medium. The transition
hypochlorite (500–1,000 ppm), hydrogen perox-
occurs by formation of buds at the tips and
ide (6,000 ppm) and formaldehyde.
along the hyphae. After septation of the hyphae,
oidial cell formation takes place. There is no
direct budding of yeast from the conidia. The
reverse transition (yeast to mould phase) is 4.14.5 Natural Habitat and Distribution
mostly regulated by calcium which induces
RNA and protein synthesis in the yeast cells. It Sporothrix is commonly found as a saprophyte
is followed by nuclear division and germ tube on living and decaying vegetation, woods, leaves
formation. The germ tubes become elongated to and branches of the plants, animal excreta and
produce hyphae, and a septum is produced to soil. Sphagnum moss, rose thorns (infection in
separate them from the mother cells. human is sometimes known as rose gardeners’
disease) and hay are the most common plants
which can harbour the fungi. The organic
4.14.2 Classification materials present in the soil such as cellulose,
pH range between 3.5 and 9.4, temperature of
Sporothrix belongs to the division Ascomycota, 20–25 C and humidity (more than 90 %) are
class Pyrenomycetes, order Ophiostomatales required for the survival of Sporothrix in nature.
and family Ophiostomataceae. Previously, The humidity requirement varies between the
Sporothrix schenckii was considered as sole spe- species of Sporothrix. Earlier it was thought
cies under the genus. Recent study showed exis- that armadillo (Dasypus septemcinctus) is a
tence of five new species under Sporothrix potent reservoir of Sporothrix as the armadillo
species complex on the basis of similarities hunters often suffer from sporotrichosis. How-
in macroscopic characteristics, sucrose and ever, it was detected that armadillos do not har-
raffinose assimilation, ability to grow at 37 C bour the fungi in their intestine or skin, whereas
and the nucleotide sequence (calmodulin). the grasses used by the armadillos to make the
The six sibling species under the complex are nests are the major source of the fungi. Other
S. schenckii, S. albicans, S. globosa, S. brasi- living creatures related to Sporothrix transmis-
liensis, S. mexicana and S. luriei. Other non- sion are rodents, cats, dogs, squirrels, horses,
pathogenic species include S. cyanescens birds especially parrots, insects and reptiles.
(isolated from the human blood and skin), Sporothrix causes the most widespread subcu-
S. stylites, S. humicola and S. lignivora. taneous mycosis throughout the world. The
4.14 Sporothrix 115
endemic zones are found in India, Japan, Peru, The production of conidia is better detected
Mexico, Colombia, Uruguay, Guatemala, Brazil in potato dextrose agar and corn meal agar.
and African countries. The largest human out-
break was detected in South Africa which was
originated from wooden beam used in the mines. 4.14.8 Antigenic Characteristics
Cases are also reported from United States,
France, Spain, Italy, China and Thailand. The major antigen of S. schenckii is cell wall
In India, West Bengal and North Eastern glycopeptides (peptidorhamnomannan). The
states such as Assam and Manipur are considered antigenicity varies with the rhamnose/mannose
as endemic zone of Sporotrichosis. Sporadic molar ratio depending on the cultural conditions.
cases are reported from Himachal Pradesh, The peptide portion is composed of threonine,
Sikkim, Tripura, Meghalaya and Nagaland. serine, aspartic acid and glutamic acid.
The peptidorhamnomannans can react with
the carbohydrate-binding protein concanavalin A,
4.14.6 Genome but the rhamnomannan is nonreactive. The reac-
tive fraction is extracted and is known as
S. schenckii possesses six to eight chromosomes S. schenckii conA-binding fraction (SsCBF).
of 460–6,200 kb, with a total genome size of It is used in serodiagnosis for the detection of
approximately 28 Mbp. The average guanine IgG in human and feline patients. The SsCBF
cytosine (GC) content of the genome is is composed of three glycoproteins, i.e. gp70,
54.7 mol%. The genome is diploid containing gp84 and gp58. Among them, gp70 is the major
approximately50fg DNA per cell in both the antigen which is present in both yeast and myce-
filamentous and yeast phases. The mitochondrial lial phases. The protein (gp70) acts as adhesin to
DNA (mtDNA) analysis helps to establish the bind with the extracellular matrix protein such
fungal types in epidemiological study. So far, as fibronectin and mice dermis.
32 mtDNA types of S. schenckii are detected
which is based on restriction enzyme analysis
of mtDNA with HaeIII. 4.14.9 Virulence Factors
The virulence factors explored by different

4.14.7 Isolation, Growth and Colony investigations till date are enlisted in Table 4.34.
Characteristics
S. schenckii can be isolated in Sabouraud 4.14.10 Transmission

dextrose agar with chloramphenicol and in the
media containing cycloheximide (mycobiotic The transmission of S. schenckii occurs through
agar). The plates are incubated at room tempera- the traumatic inoculation of the fungi into the
ture (25–28 C) for 2–7 days. The primary mould skin and subcutaneous fat associated with
colonies are creamy, wrinkled and leathery scratches, small injuries and pricks with thorns
which become dark coloured at the centre with of plants (roses). Certain job-related and free-
advancement of age. time activities, such as gardening, fishing, hunt-
The dimorphic conversion into the yeast ing, farming and mining, are associated with the
phase can be detected in enriched media such transmission. Human-to-human spread and
as blood agar, chocolate agar and brain–heart occurrence of epidemics are rare.
infusion agar. The conversion requires incuba- The zoonotic transmission takes place follow-
tion at 37 C for 3–5 days with 5–7 % CO2 ing the bite or scratches by animals such as cats,
tension. The yeast colonies are creamy and dogs, squirrels, armadillos and insects [fire ants
yellow to tan in colour. (Solenopsis)]. The largest outbreak of human
Table 4.34 Virulence mechanisms and factors possessed by S. schenckii

Thermotolerance The strains of S. schenckii which can tolerate and grow at 37 C are able to produce
lymphatic sporotrichosis, whereas the strains devoid of thermotolerance property are
capable of producing cutaneous lesions only
Melanin Both the yeast and mycelial phases can synthesise melanin. The conidia utilise
1,8-dihydroxynaphthalene (DHN) pentaketide pathway, whereas 3,4-dihydroxy-L-
phenylalanine (L-DOPA) is utilised by hyphae for synthesis of melanin. Following
roles of melanin in pathogenesis are established
(a) Melanin produced in the conidia can resist the host cell phagocytosis and
diminishes the respiratory burst mediated by human monocytes and murine
macrophages. The melanin acts as scavenger for free radicals
(b) The melanin-producing strains are more invasive in experimental animals than
the albino strains
(c) The melanin pigment may hamper the treatment of sporotrichosis
Fibronectin, laminin and type II They act as adhesin, and they are required for attachment of the yeast or mycelium
collagen-binding proteins, gp70 with the host cells and dissemination of infection throughout the body. Laminin-
binding protein is expressed by both the yeast and mycelial phases, whereas
fibronectin binding protein is expressed by yeast cells only and it is associated with
virulence. The gp70 protein prefers to bind with the dermis
Capsular material The capsular material helps in adhesion of fungi with the host cells
Ergosterol peroxide It is synthesised by yeast cells to protect themselves from reactive oxygen species
which is generated by the phagocytes for killing of the yeast cells
Peptidorhamnomannans It is present in the cell wall, and it causes suppression of immune response until the
sixth week of infection and may act as adhesin to bind the fungi with the host cells
Acid phosphatases This enzyme is produced by both the yeast and mycelial phases and the conidia of
S. schenckii. The enzyme helps in intracellular survival of the fungi after
phagocytosis
Proteinase I and II They can hydrolyse the stratum corneum cells in vitro
sporotrichosis due to cat bite or scratches immune status. After transmission, the mycelial
occurred in Brazil (1998–2001), in which phase is converted into the yeast cells due
156 cases (out of 178) had direct contact with to exposure to the higher temperature or uni-
the cats infected with S. schenckii. The cats are dentified signal. The yeast cells are either
considered as the most potent animals capable of localised in the subcutaneous tissues or spread
zoonotic transmission probably due to the pro- via lymphatics or blood producing either fixed or
duction of large amount of yeast cells. The yeast lymphocutaneous or disseminated clinical forms,
cells can directly infect the human even in the respectively. The disseminated form occurs
absence of cat scratches or biting history. either due to immunosuppression or due to inoc-
Rarely, S. schenckii is transmitted through ulation of huge quantity of yeast cells from the
inhalation route producing extracutaneous form infected cats at the multiple locations of the host.
of sporotrichosis, i.e. granulomatous pneu- The fixed form is characterised by ulcerated
monitis. The laboratory-acquired infection is lesion with erythematous edges. Most of the
also reported during culturing of the fungi with- clinical cases (75 %) manifest the lympho-
out proper precautions. cutaneous form which starts with the formation
of a papule at the site of trauma. The papule is
converted into a subcutaneous nodule with ulcer-
4.14.11 Pathogenesis ation and pus formation. Secondary lesions arise
along the course of lymphatics. However, the
Clinical manifestation varies with the load and involvement of lymph node and other vital
pathogenicity of the fungi along with the host organs is rare in human. The disseminated form
4.14 Sporothrix 117
Table 4.35 Major diseases of animal and human caused by S. schenckii

Fungi Host Disease
Sporothrix Dogs The dogs are most commonly affected among the animals. Lymphocutaneous
schenckii form is most predominant. Other forms such as cutaneous and disseminated
sporotrichosis are also detected. The primary lesion develops as subcutaneous
nodule (1–3 cm in diameter) at the site of trauma which ulcerates and discharges
pus. The dissemination may occur along the course of lymphatics which produces
cording and new nodules. Occasional involvement of lymph node is noticed.
The lesions are neither painful nor producing itching
Cats The primary lesions are small, draining, puncturing type of wounds which appear
in the head, base of the tail or the legs (Fig. 4.33). With the progression of the
infection, the lesions ulcerate and become nodular. Occurrence of multiple skin
lesions is more than the lymphocutaneous form. Systemic involvement is common
and is not associated with immunosuppression. The systemic involvement may
occur in the bone, lung, liver, spleen, testes, gastrointestinal tract and central
nervous system
Horses Cutaneous nodules develop along the lymphatics usually on the legs. Systemic
involvement may occur like other animals such as cats
Donkey, mule, pigs, Naturally occurring sporotrichosis is detected
fowl, goat, cattle
Human It is also known as ‘Rose gardeners’ disease’ and is characterised by the formation
of subcutaneous nodule. Four clinical forms are detected, i.e. fixed cutaneous,
lymphocutaneous, disseminated and extra cutaneous. In adults and children, the
lesions appear in arm and face, respectively. Lymphocutaneous form is the most
predominant
shows multiple skin lesions at the noncontiguous 4.14.13 Immunity

sites, and it involves the bones, joints, lungs,
meninges and osteoarticular system. The inhala- The innante immune response of the host largely
tion of spores rarely produces the extracutaneous depends on the interaction between the host
form which is characterised by haematogenous pattern recognition receptors (PRR) and the
dissemination and formation of osteoarticular fungal pathogen-associated molecular pattern
lesion in most of the cases. (PAMP). This interaction is not very much
In cats, occurrence of multiple skin lesions explored in S. schenckii. The experiments
is more (39 %) than the lymphocutaneous form suggest the possible interaction between the
(19 %). Systemic involvement is common and is host TLR4 and fungal cell wall lipid which
not associated with immunosuppression. The fre- can activate the polymorphonuclear cells to
quency of granuloma formation is low (12 %), phagocytose the yeast cells of S. schenckii.
and huge amount of fungal elements are detected The fungi also activate the alternative comple-
in the skin which make them more susceptible to ment pathway. The complement product (C3b)
S. schenckii. deposits on the fungal cell wall and acts as
opsonin to help in the phagocytosis. After
phagocytosis, the superoxide anions, produced
4.14.12 Disease Produced by the macrophages, can act as fungistatic or
fungicidal.
The major animal and human diseases produced The cell wall components of S. schenckii
by S. schenckii are enlisted in Table 4.35. induce granuloma formation in the host which
is a T cell-dependent inflammatory and acid–Schiff (PAS) stain. The cells appear as

protective response. The CD4+ T cells produce spherical with PAS-positive capsule.
IFNγ which stimulates the macrophages. The The granuloma produced by the fungi
pro-inflammatory cytokines such as IL1 and consists of three zones. The central or chronic
TNFα are produced by the peritoneal exudate zone contains neutrophils, histiocyte and
cells (PEC) after 8–10 weeks of the infection. lymphocytes along with ‘asteroid bodies’.
The TNFα further acts on activated macrophages Surrounding the central zone, there is a sec-
to release nitric oxide. The nitric oxide is the ond zone which contains epithelioid cells and
key lethal factor for the yeast and conidia of giant cells (strange body and Langerhans
S. schenckii. The nitric oxide also acts as a medi- type). The third and outer zone is composed
ator which can prime the macrophages for potent of lymphocytes, plasmocytes and fibroblasts.
oxidative burst. The Th1-mediated immunity However, the zones are not a constant diag-
thus acts as key factor to prevent the dis- nostic feature and the cells can mingle with
semination of infection. In contrast, the Th2 each other. In dogs, the granuloma is
response participates in the advanced stages of characterised by increased neutrophil infil-
sporotrichosis only. tration and absence of lymphocytes and
macrophages which can differentiate it with
the leishmaniasis lesion.
4.14.14 Diagnosis The ‘asteroid bodies’ or ‘asteroid
corpuscles’ (found in 20–66 % cases) originate
4.14.14.1 Clinical Specimens from the Splendore–Hoeppli reaction in sporo-
The clinical specimens for sporotrichosis include trichosis. The Splendore–Hoeppli reaction is a
pus or exudates from the deeper part of the localised immunological response in fungal,
lesion, saliva, urine, blood, cerebrospinal fluid, bacterial or parasitic infection, and it is
synovial fluid and tissue biopsy specimens. characterised by the formation of radiating
homogenous, refractile, eosinophilic club-like
4.14.14.2 Laboratory Examination material surrounding a central eosinophilic
1. Direct examination: The wet mounts with focus (Fig. 4.32). The asteroid bodies in Spo-
10 % KOH are employed on the clinical spec- rotrichosis consists the viable yeast cells in the
imen for detection of the yeast cells which is
diagnostic of sporotrichosis. However, detec-
tion of yeasts depends on the fungal burden
in the body. So detection is easier in cat
specimens than human clinical samples due
to high fungal burden in cats. Staining can be
performed with Gram stain or Giemsa stain
(for diluted pus sample). They are not so much
confirmatory because ‘cigar-shaped buds’
are present only in 1–2 % cases. Instead,
fluorescent staining offers confirmatory diag-
nosis of the yeasts.
2. Isolation and identification: Media and
incubation condition as described earlier will
serve the purpose.
3. Histopathology: The yeast cells of Fig. 4.32 Splendore–Hoeppli reaction in sporotrichosis.
Arrow indicates the radiating club-like material
S. schenckii can be demonstrated in the tissues surrounding a central eosinophilic focus (Photograph
with haematoxylin and eosin (H&E), Gomori courtesy: Prof. P.P. Gupta, Ex-Director of Veterinary
methenamine silver (GMS) or periodic Research, Punjab Agricultural University, Punjab, India)
4.14 Sporothrix 119
centre along with the host IgG and IgM on the immunosuppressed patients and false-positive
spikes of the radiated crown. Thus, the aster- reaction is produced in healthy patients previ-
oid bodies use the host immune molecules to ously suffered from the infection. Experimen-
provide a safe shelter to the yeasts. tally the sporotrichin test with SsCBF antigen
4. Serological tests: The serological tests such in guinea pig was found reactive. In Brazil,
as agarose gel precipitation test (AGPT), the sporotrichin test was carried out among
tube agglutination (96 % sensitivity), latex the captive mammals present in a zoo for
agglutination (94 % sensitivity) and immuno- epidemiological investigation.
electrophoresis can be employed for detection 6. Molecular biology: The chitin synthase gene
of antibodies against S. schenckii. The AGPT (ChS1)-based PCR was first developed which
does not cross-react with the sera from leish- can detect up to 10 picogram of genomic
maniasis. However, these tests are not so S. schenckii DNA. Further PCR-based assays
much sensitive for the diagnosis of cutaneous are also developed, detecting DNA topoisom-
form of the infection. Enzymatic immune erase II and internal transcriber space in the
assay such as ELISA is a preferred choice of rRNA gene of S. schenckii.
serodiagnosis. The test was initially devel-
oped against the S. schenckii conA-binding
fraction (SsCBF) antigen. However, in sero- 4.14.15 Treatment
diagnosis of cutaneous form, this antigen-
based ELISA produces cross-reaction with Potassium iodide (KI) or sodium iodide (NaI)
other infection such as paracoccidioi- is commonly used for the treatment of sporotri-
domycosis. Later, the exoantigens produced chosis especially for the lymphocutaneous and
by a mycelial phase of S. schenckii strain, fixed forms of the infection in cats, dogs and
isolated from Brazil, were used in ELISA horses. The compounds have high efficacy with
which produced no cross-reaction with minimal side effects. It is easy to administer and
antigens and serum samples from patients is relatively low in cost in comparison to the
with coccidioidomycosis, histoplasmosis or standard antifungals. KI has immunostimulation
paracoccidioidomycosis. property which can activate the neutrophils and
5. Skin test for detection of delayed-type hyper- monocytes to destroy the fungi. Some authors
sensitivity (DTH): The ‘sporotrichin test’ is a claim its proteolytic nature which helps to
tuberculin-like skin test for the detection of resolve the granulomas. However, exact mecha-
DTH against sporotrichosis. The antigen used nism of action is not known. The KI therapy
for the test varies with the country and accord- should be continued for 2–3 months for complete
ingly the result also varies. The 5 McFarland cure. However, in human, some side effects such
standard suspension of heat-killed yeast cells, as nausea, vomition, diarrhoea, abdominal pain
extracted mycelial antigens at 1:2,000 dilution and metallic taste are noted. Some cats are
(approximately 5 107 cells/mL) or diluted also susceptible to iodide toxicosis.
yeast phase antigens at 1:4,000 were used as In human, itraconazole is the first choice treat-
antigen in Brazil and Mexico, respectively. ment with the dose rate of 100–200 mg/day
The antigen is inoculated at 0.1 mL into the orally (for lymphocutaneous and fixed forms).
forearm or back of the suspected patient. It has minimal toxicity and good tolerance even
A positive result involves an indurated, ery- in long-term treatment except during pregnancy.
thematous and painful area of more than 5 mm In cats and dogs, also itraconazole is preferred
in diameter at the site of the injection 48 h nowadays. For disseminated form of sporotricho-
after inoculation. The test is sensitive for sis, amphotericin B is preferred even in pregnant
detection of lymphocutaneous and fixed women (after 12 weeks of pregnancy) and
cutaneous forms. Rarely, the false-negative immunosuppressed patients. Fluconazole is less
reaction is produced in anergic or effective and is preferred only for those patients
(Chennai), India, and named them as ‘morbus

tuberculosis pedis’. Due to its widespread occur-
rence among barefooted natives in Madurai
(India), it was popularly known as ‘Madura
foot’. In 1860, Carter established the term
‘mycetoma’ and its etiological correlation with
the fungi. In 1874, Carter wrote a monograph
entitled ‘On mycetoma or the fungus disease of
India’ where he established firmly the correlation
between the fungus and the menace.
Laveran (1902) first isolated Streptothrix
mycetomi as the fungal agent from the mycetoma
(black grain). Brumpt (1905) reclassified
Streptothrix mycetomatis as Madurella myce-
Fig. 4.33 Sporotrichosis in a cat (Photograph courtesy – tomi. British Medical Research Council (1977)
Prof. Alexandro Bonifaz, Head, Department of Mycology further changed the name from Madurella
and Dermatology service, General Hospital of Mexico,
Mexico City) mycetomi to Madurella mycetomatis which is
accepted worldwide.
Radaeli (1911) first isolated a fungus
who are allergic to itraconazole. The ketocona-
(Monosporium apiospermum) from the white-
zole is not used due to poor efficacy and high
grain mycetoma in Sardinia. Currently this
level of toxicity. The terbinafine and
fungus is known as Pseudallescheria boydii
posaconazole showed good in vitro activity
(teleomorph).
against S. schenckii. The thermotherapy is
Scedosporium as a fungal causative agent of
recommended for fixed cutaneous form in preg-
human otitis was first described in 1889.
nant or drug-allergic patients because
Saccardo (1911) first illustrated its etiological
S. schenckii cannot grow at more than 40 C.
role in human mycetoma and suggested the
The hot baths (45 C) for 15–20 min three
name as Scedosporium.
times a day and topical thermal compression are
advised (Fig. 4.33).
4.15.1 Aetiology
4.15 Mycetoma (Madurella, Mycetoma (maduromycosis) is chronic

Pseudallescheria, pyogranulomatous infection of the skin and
Scedosporium) subcutaneous tissues caused by either fungi
(eumycetoma) or bacteria (actinomycetoma).
In ancient Sanskrit writing (Atharva Veda), the A wide variety of fungi such as Madurella
first description of mycetoma (Pada Valmikan) mycetomatis, Pseudallescheria boydii (previ-
was documented. In the seventeenth century, ously Allescheria boydii), Trematosphaeria
a German physician (Engelbert Kaempfer) work- grisea (previously Madurella grisea), Asper-
ing in India first reported clinical human cases of gillus, Fusarium, Curvularia, Acremonium,
mycetoma which is followed by case reports Paecilomyces and others cause mycetoma in
of French missionaries in Pondicherri, India human and animals (Table 4.36). Among them,
(eighteenth century). In 1842, John Gill, a British Madurella mycetomatis is the major causative
Army physician working at South India, also agent of black-grain mycetoma in human which
reported a case of human mycetoma. Godfrey is usually found in tropical region (India, Sudan
(1846) first described an authentic clinical report and Madagascar). In temperate and subtropical
of four human mycetoma cases from Madras region (United States, Brazil), Pseudallescheria
4.15 Mycetoma (Madurella, Pseudallescheria, Scedosporium) 121
Table 4.36 Fungal species associated with mycetoma and properties of grains produced
Fungi Grain colour Size and texture
Madurella mycetomatis Black 0.5–4 mm, hard
Pseudallescheria boydii White 0.5–1 mm, soft
Trematosphaeria grisea (Madurella grisea) Black 0.3–0.6 mm, hard
Aspergillus flavus Green 0.5–3 mm, soft
Aspergillus nidulans White 0.5–4 mm, soft
Fusarium oxysoprum White 0.5–1 mm, soft
Curvularia geniculata Black 0.5–1 mm, hard
Acremonium falciforme White 0.5–1 mm, soft
Falciformispora senegalensis (Leptosphaeria senegalensis) Black 0.5–2 mm, hard
Neotestudina rosati White 0.5–1 mm, hard
Exophiala jeanselmei Black 0.2–0.3 mm, soft
Cylindrocarpon cyanescens White –
Polycytella hominis White –
Plenodomus avramii Black –
Corynespora cassiicola Black –
Pyrenochaeta mackinnonii Black –
Phialophora verrucosa Black –
boydii is the most common etiological agent of produces two types of conidia. Round conidia
eumycetoma in human. are detected in flask-shaped phialides, and
In addition, Microsporum canis, Cladophialo- pyriform conidia are observed in branched or
phora bantiana and Curvularia lunata are also unbranched conidiophores without phialides.
detected as aetiology of mycetoma in cat, dog, In Trematosphaeria grisea (Madurella grisea),
and dog and parrot, respectively. two types of hyphae are observed, i.e. thin
(1–3 μm width) and broad (3–5 μm width).
Pseudallescheria boydii (teleomorph) has
4.15.2 Morphology more than one anamorph, i.e. Scedosporium
apiospermum (anamorph) and Graphium
The mycetomal lesion consists of hyphal eumorphum (synanamorph). P. boydii is a homo-
aggregates, known as ‘grain’. The morphology thallic fungus. The reproductive structure
and colour of grain help in the identification of (cleistothecium/ascocarp) is produced in nutri-
fungal species (Table 4.36). Most of the grains tionally poor media. It initiates with the forma-
are round or oval and trilobed. Two types of tion of coiled ascogonia which is converted into
grains are detected, i.e. filamentous and vesicular matured cleistothecia (100–300 μm) within
(rare). The filamentous grain is composed of 10 days. The cleistothecial wall is composed of
branched, septate and brown hyphae. The vesic- thin, flat, polygonal brown cells, and it contains
ular grains consist vesicles and light-coloured globose or subglobose ascus which bears eight
hyphae at the centre and cementing substance at ascospores inside. The ascospores (4–5 μm
the periphery (melanin, heavy metal, protein and 7–9 μm) are unicellular, oval or ellipsoidal
lipid). The cementing substances interfere the and yellow-brown. The ascospores contain an
penetration of host immune effector molecules oil droplet inside (Fig. 4.34).
and antifungals. Scedosporium (anamorph) contains hyaline,
Microscopic study revealed that the thallus of septate and cylindrical hyphae (2–4 μm in diam-
Madurella mycetomatis is composed of regularly eter). The conidiogenous cells emerge from
septate hyphae. Most of the cultures are sterile. the hyphae which produce oval, brown, sticky
Sometimes conidiation is observed which conidia (4–9 μm 6–10 μm) which do not
Fig. 4.34 Cleistothecium

and ascospores of
Pseudallescheria boydii
(Photograph courtesy:
Professor Ali Zarei
Mahmoudabadi,
Jundishapur University of
Medical Sciences, Ahvaz,
Iran)
Fig. 4.35 Conidial

arrangement of
Scedosporium (Photograph
courtesy: Professor Ali
Zarei Mahmoudabadi,
Iran)
contain any oil droplet inside. These conidia 4.15.3 Classification

are produced as single from one conidiophore
(solitary annelloconidia) and are truncated at Classification of Madurella was possible with the
the base (Fig. 4.35). help of molecular tools (sequencing of internal-
The Graphium synanamorph mycelium is transcribed spacer (ITS) and beta-tubulin genes)
composed of erect, stiff, olive-brown bundles because the genus lacks sexual reproduction
of hyphae, terminating in a brush of slender or specialised morphological features and
conidiogenous cells. The conidiophores are detection of asexual conidia is also extremely
cemented together to form synnemata (united difficult. Based on nuclear sequence data,
conidiophores) to produce clusters of conidia. Madurella is placed under two different orders,
The conidia are extra slender and less pigmented i.e. Sordariales and Pleosporales. The type
than Scedosporium conidia, however, truncated species M. mycetomatis belongs to the order
at the base-like Scedosporium conidia Sordariales along with M. pseudomycetomatis,
(Fig. 4.36). M. fahalii and M. tropicana, whereas
Fig. 4.36 Synnemata and

conidia of Graphium
(Photograph courtesy:
Professor Ali Zarei
Mahmoudabadi,
Iran)
Trematosphaeria grisea (previously Madurella polluted environmental niches having poor air
grisea) belongs to the order Pleosporales. circulation and high osmotic pressure.
Pseudallescheria boydii (teleomorph) and Mycetoma may occur throughout the world
Scedosporium apiospermum (anamorph) belonged but is common in tropical and subtropical zone
to phylum Ascomycota, class Euascomycetes, (between the latitudes of 15S and 30N) with
order Microascales and family Microascaceae. major foci in Asia (southern India), Africa
The genus Pseudallescheria is composed of (Somalia, Senegal, Sudan, Mauritania, Niger,
seven species such as P. boydii, P. africana, Ethiopia, Chad, Cameron) and South and Central
P. angusta, P. desertorum, P. ellipsoidea, America (Mexico, Argentina, Brazil, Venezuela)
P. fimeti and P. fusoidea. Among them, P. boydii (‘mycetoma belt’). Sudan has the highest inci-
is the only pathogenic species. Molecular phyloge- dence of mycetoma in human which is followed
netic analysis revealed that P. boydii is a species by Mexico. The endemic zone should have arid
complex consisting 44 haplotypes. and hot climate with limited rainfall. The
cases are also reported from the United States
(Texas, California), Germany, Egypt, Turkey,
4.15.4 Susceptibility to Disinfectants Philippines, Japan, Lebanon, Thailand, Iran,
The Netherlands, Ceylon and Saudi Arabia.
M. mycetomatis is susceptible to 70 % ethanol
and moist heat (60 C for 30 min).
4.15.6 Genome
The mitochondrial genome of M. mycetomatis is
Madurella resides in the soil and sometimes a compact, circular DNA molecule (45,590 bp).
in thorns of the plants (Acacia) which also Most part of genome is AT rich and the overall
helps in the transmission of the infection. GC content is only 26.8 %. The genome encodes
Pseudallescheria boydii/S. apiospermum is for the rRNAs, tRNAs (27), proteins of respira-
detected in brackish water, saltwater, sewage, tory chain complexes (11), ATP synthase
soil, swamps, coastal tidelands, manure of poul- subunits and 6 intronic proteins including the
try, cattle and bat guano. They can tolerate high ribosomal protein (rps3).
temperature, low-oxygen tension and high salt The genomic organisation of Pseudalles-
concentration (5 %). So they are common in cheria boydii is still unknown.
4.15.7 Isolation, Growth and Colony agar, soil extract agar and water agar. The
Characteristics colonies of M. grisea are leathery, folded with
radial grooves, light brown to greyish in colour
The grains are collected from the mycetomal which later become dark brown to reddish-brown
lesion for isolation of etiological fungi or bacte- and have a brownish-black reverse.
ria. The grains are washed with sterile saline and The colonies of P. boydii in Sabouraud glu-
antibiotics, crushed with sterile glass rod and are cose agar look different from the upper surface
spread over the plate. The medium commonly (obverse) and from the reverse. The colour is
used is Sabouraud dextrose agar with antibiotics initially white and later becomes dark grey or
such as gentamicin sulphate (400 mcg/mL), pen- smoky brown (obverse), while from the reverse,
icillin G (20 U/mL), streptomycin (40 mcg/mL) the colonies appear pale with brownish-black
and chloramphenicol (50mcg/mL) for isolation zones. During preservation of the culture for
of Madurella. Some species of Madurella are many years, the colonies become dirty white
inhibited by antibiotics, so one plate without coloured and have fur-like surface without any
antibiotic should also be kept. Recently, the use conidia.
of trypticase soy agar with 5 % horse serum
has been proposed to enhance the growth of
M. mycetomatis. M. mycetomatis grows at 4.15.8 Antigenic Characteristics
37 C, while M. grisea grows at 30 C.
Sabouraud glucose agar is preferred for isola- M. mycetomatis secretes enzymes such as
tion of P.boydii/Scedosporium apiospermum fructose-bisphosphate aldolase (FBA) and pyru-
which requires 25 C as optimum growth tem- vate kinase (PK) which act as immunodominant
perature. This fungus can tolerate up to antigens. The antibodies against them are
37–42 C, low-oxygen tension or anaerobic con- detected in the sera of patients.
dition, high concentration of magnesium chloride P. boydii has a peptidopolysaccharide
(5 %) or sodium chloride (5 %). Addition of antigen (peptidorhamnomannan) which has a
benomyl, amphotericin B or dichloran makes similarity with Sporothrix schenckii pepti-
the medium selective for isolation of P. boydii/ dorhamnomannan, but they do not cross-react
Scedosporium apiospermum. during serological reactions.
The colonies of M. mycetomatis are white,
flat, woolly or leathery at the beginning which
later produce a diffusible brownish pigment and 4.15.9 Virulence Factors
become folded and heaped due to production of
aerial mycelium. The sporulation is better The virulence factors of M. mycetomatis and
observed in straw extract agar, wheat extract P. boydii are enlisted in Table 4.37.
Table 4.37 Virulence mechanisms and factors possessed by M. mycetomatis and P. boydii
Virulence mechanisms/
factors Functions
Ability to grow at 37 C It helps in better adaptation of M. mycetomatis to the host body temperature
Grain formation Within the host tissues, both M. mycetomatis and P. boydii produce grain (sclerotia or
granules) to evade the host immune response. Grains are fungal aggregates along with a
matrix or cementing substance
Peptidase (28 KDa, The extracellular peptidases are released by P. boydii. The peptidases split host fibronectin
34 KDa) and laminin which may help to escape the fungi from host effector cells such as fibronectin-
activated macrophage and monocyte
Serine protease It is released by S. apiospermum which splits host fibrinogen and helps in tissue invasion
(33 KDa)
4.15.10 Transmission cementing substance for their survival. This

fungal aggregate is known as ‘grain’ (sclerotia
In the endemic zone, M. mycetomatis, or granules). Sometimes the grains do not pos-
Scedosporium and other fungal and bacterial sess any cementing substances, and the hyphal
agents can grow saprobically in the environ- cell walls become thickened near the periphery
ment (thorns, metal piece, wire, wood splinter, of the grain which can protect the fungi from host
nails, speculated seeds, etc.). During penetrating immune system. The host innate immune system
trauma, puncture or lacerated wound of the host, attempts to clear the infection through activation
these etiological agents can enter the subcutane- of complement cascade and complement
ous tissue through the contaminated objects. product-mediated chemotaxis of granulocytes
Sometimes the objects (thorn) are detected towards the fungal grains. In natural infection,
within the mycetoma lesion. There is no evi- granulomas develop which contain the fungal
dence of transmission from animal to human or grains at the centre, surrounded by a zone of
human to human. polymorphonuclear neutrophils (PMN). Some-
In human, there is no racial or ethnic predom- times the PMNs are detected within the grains
inance. However, males aged between 16 and also. With the progress of the infection, the
30 years are more susceptible than females. granulomas mature and contain lymphocytes,
In a study in West Bengal (India), occurrence of macrophages, plasma cells, giant cells and
mycetoma between male–female was 183: 81. mononuclear cells.
4.15.11 Pathogenesis 4.15.12 Disease Characteristics
Following entry of the organism through the The clinical features of eumycetoma in human
wound, the fungi produce an aggregate with a and animals are described in Table 4.38.
Table 4.38 Clinical features of mycetoma in human and animals

Species Clinical features
Human The incubation period is unknown. Mean duration of eumycetoma is 9.8 years. The
infection starts with a small, firm, painless tumour-like mass in subcutaneous tissue which
further spreads into the subcutaneous fat, muscle, ligaments and bones. With the progress,
multiple nodules develop, ulcerate and drain through the sinuses. The sinus tracts appear
within 3 months–1 year. The discharge is purulent or serosanguinous and it contains the
grains. The draining sinuses may heal and a new one opens simultaneously. The lesion is
painless due to nerve damage by fibrous tissue reaction. Intense pain is felt only when there
is bone involvement and secondary bacterial infection. Most of the mycetoma cases (70 %)
occur in the feet. It may occur at other sites such as the hand, knee, arm, leg, head, neck and
face, thigh, perineum and rarely at chest and abdominal wall, eyelid, testis, vulva, middle
ear, paranasal sinus, mandible and lymph nodes
Horses Mycetoma in horses is characterised by the subcutaneous nodules which may suppurate and
drain a serosanguineous or purulent discharge through multiple sinus tracts. The discharges
contain the fungal grains
Cats Subcutaneous nodules are detected in dorsal trunk which drains purulent exudate
containing yellow-coloured granules
Dogs Nodules are observed in subcutaneous tissues and sometimes in the ribs and thorax with
visible granules. Rarely disseminated mycetoma is found which involves the lymph nodes,
heart, kidneys, adrenal glands, bone, pancreas, brain and eyes
Goats Mycetomal lesions are observed in hind legs and scapula
Cattle Lesions are found in the skin, nasal cavity and lymph node
Grand Eclectus parrot Black nodules were detected in thoracic cavity and lung causing central nervous system
(Eclectus roratus roratus) disorder and wing paralysis
4.15.13 Immunity bacterial etiological agent, if any. For the

detection of fungal aetiology, the slides
Macrophages are the first line of defence against are immersed in 20 % KOH followed by
Scedosporium apiospermum which are able fluorescence microscopy using the Uvitec,
to phagocytose both the hyphae and conidia. Blankophor or calcofluor white. However,
The hyphae are opsonised with the serum, and the fluorescence microscopy cannot differen-
the macrophages can produce oxidative burst/ tiate between the fungal genera.
superoxide anion (O2) to control the opsonised 2. Isolation and identification: Media and incu-
hyphae. The polymorphonuclear neutrophils bation condition as described earlier will
(PMN) can also participate in phagocytosis serve the purpose.
and oxidative burst-mediated killing of Scedo- 3. Histopathology: The grains can be identified
sporium. However, the cytokine (IL15) can in haematoxylin and eosin (H&E)-stained tis-
enhance this oxidative burst of PMNs against sue sections. Gram–Weigert stain can be used
Scedosporium prolificans but not against to detect bacterial mycetoma and periodic
Scedosporium apiospermum. acid–Schiff (PAS) and Gomori methenamine
The cell wall α-glucan of P. boydii is silver, and Gridley stains are used to detect
recognised by toll-like receptor (TLR2) and hyphae and fungal spores in eumycetoma.
CD14 of host macrophages and dendritic cells. The grains have differentiating characteris-
The interaction can transduce the signal to tics in the tissue section which creates an
secrete pro-inflammatory cytokines. idea regarding the aetiology. For example,
the black grains of Trematosphaeria grisea
(M. grisea) have a non-pigmented centre
and dark-coloured periphery with brown
4.15.14 Diagnosis
cementing substances. Further, the tissue
reaction is also suggestive for the fungal
aetiology. The grains are surrounded by
The diagnosis of mycetoma depends on three
neutrophils in M. mycetomatis infections and
major clinical symptoms (triad of clinical
by eosinophils in P. boydii infections. How-
symptoms) such as edematous subcutaneous tis-
ever, most of the fungi-causing eumycetoma
sue, multiple draining sinuses and excreation of
cannot be differentiated by histopathology.
grains. The clinical specimens include pus, exu-
For example, the fungal species such as
date, bandage gauze and biopsy tissues which are
Scedosporium, Aspergillus and Fusarium pro-
used for naked eye examination to detect the
duce hyaline hyphae with septation at regular
grains. The physical properties of the grains are
intervals and dichotomous branching. Some-
indicative of the etiological agent (Table 4.36).
times Scedosporium produce terminal or
Fine needle technique is also used for the
intercalary chlamydospores which are con-
collocation of grains from the lesions. A fine
fused with the yeasts.
needle attached to a syringe is inserted within
4. Serological tests: Indirect haemagglutination
the lesion, and it is moved up and down in 3–4
assay (IHA) and immunodiffusion test
different locations to collect the exudates. The
(ID) are developed for the detection of
bloody material is kept for some time to clot.
Pseudallescheria boydii. For the detection of
M. mycetomatis antibodies in human, immu-
4.15.14.2 Laboratory Examination nodiffusion and counter immune electropho-
1. Direct examination: For the detection of etio- resis (CIE) are widely used. Sometimes, ID
logical agent (fungi or bacteria), the grains are tests produce false-negative reaction espe-
crushed and fixed with 10 % formal saline on cially during the early phase of the infection
a microscopic slide. The slide can be stained and when the treatment is ongoing. The CIE is
with modified acid-fast staining to detect the found to be superior than ID for better
4.16 Pythium 127
sensitivity. ELISA is also used for the granulomatous disease in horses known as
detection of antibodies. Previously the ‘bursattee’ in local language. Later the aetiology
ultrasonicated mycelial extract (cytoplasmic of the disease was correlated with the fungi based
protein) was used as antigen in serological on histology. In 1901, Dutch investigators
tests which produce variable results and (de Haan and Hoogkamer) working in Indonesia
cross-reaction with other fungi. The use of isolated the fungi and identified them as
recombinant antigens [fructose-bisphosphate Zygomycetes (due to lack of sporulation) from
aldolase (FBA) and pyruvate kinase (PK)] in the horses having similar kind of infection
ELISA produces promising result for detec- (Hyphomycosis destruens equi). Witkamp
tion of M. mycetomatis antibodies. (1924), another Dutch scientist, also isolated the
5. Molecular biology: The PCR is developed for fungi from the horses showing a similar kind of
the detection of M. mycetomatis and syndrome. In 1974, Austwick and Copland
Pseudallescheria boydii using the primer for recognised them as a member of the genus
internal-transcribed spacer (ITS) located Pythium under oomycete family. Based on this
between 18S and 28S genes and beta-tubulin finding, Chandler et al. (1980) proposed the term
(BT2) genes, respectively. Rolling circle pythiosis for the infection. In 1987, de Cock first
amplification (RCA) technique is used to detect identified them as a new species under the genus
P. boydii which is based on the amplification Pythium and proposed the nomenclature of
of the ITS region with pan-fungal primers. Pythium insidiosum.
The loop-mediated isothermal amplification
(LAMP) method is also developed for the con-
firmation of P. boydii which do not require
4.16.1 Morphology
sophisticated instruments and is suitable for
endemic zones of developing countries.
Pythium can produce wide (4–10 μm), hyaline,
coenocytic or sparsely septate hyphae which pro-
duce branches at right angle (lateral pegs). In
4.15.15 Treatment older cultures, sporangia-like swellings
(12–30 μm) are observed. However, it is not
In human, ketoconazole or itraconazole with sur- considered as a true fungus due to lack of chitin
gical excision is recommended for eumycetoma. and sterol in cell wall and cytoplasmic mem-
Further, treatment with cotrimoxazole- brane, respectively. The cell wall is composed
streptomycin, trimethoprim and sulphame- of cellulose and β-glucans.
toxazole, and tetracyclins or rifampicine also
produces promising result. The study with exper-
imental Scedosporium infection in laboratory
4.16.2 Life Cycle
mice revealed that posaconazole, fluconazole
and voriconazole are effective in reduction of
The motile zoospores are considered as an infec-
fungal burden.
tious form of P. insidiosum. They are released in
In animals especially in horses, surgical exci-
the aquatic environment and infect the damaged
sion with thiabendazole powder applied topically
skin or gastrointestinal mucosa of animals and
is found effective against M. mycetomatis
human. The formation and release of zoospores
infection.
take place from the zoosporangium. The
zoosporangia are produced from the hyphae
which are in contact with certain plants. The
4.16 Pythium long, narrow and thin-walled tubular structure
with widen tip is developed from zoosporangia
Smith (1884), a veterinarian working in India, (discharge tube). The sporangial protoplasm
first described a chronic cutaneous enters the discharge tube to produce a terminal
vesicle. The vesicle expands in size and it is was detected, and the new genus Phytopythium
released from the discharge tube as a zoospore. has been proposed. Pythium is necrotroph
The zoospores are motile with two flagella pres- and zoospores are produced from a vesicle in
ent in anterior (tinsel) and posterior (whiplash) the zoosporangia, whereas Phytophthora is
site. The tinsel flagellum produces the thrust and hemibiotroph and the zoospores are produced
the whiplash flagellum acts as rudder during directly within the sporangia.
the swimming of the zoospores.
The swimming of the zoospores is directed
by chemotaxis (calcium, sugars and amino 4.16.4 Reproduction
acids released from the damaged plant root,
animal skin or tissues), electrotaxis and auto- Some strains of P. insidiosum can undergo sexual
aggregation. The aggregation of zoospores in a reproduction to produce oospores. The genera-
site attracts more zoospores to adhere which tion of oospores occurs in the oogonium (female
is known as auto-aggregation. The zoospores structure) which is intercalary, smooth and
adhere with the damaged skin surface of the subglobose, whereas the antheridium (male
animals and human by formation of a cyst-like structure) is diclinous (unisexual). The antherid-
structure. The gelatinous sticky glycoprotein ium produces a rigid fertilisation tube from
covering of the cyst helps in adherence. Germ their tip during conjugation. The oospores are
tubes are produced from the cyst which generates aplerotic which cannot fill the oogonium
hyphae. The hyphae penetrate the host tissues completely and often pressed to one side of the
to establish the infection. oogonium.
The asexual reproduction generates zoospores
which are described earlier.
Pythium belonged to the kingdom Stramenopila, 4.16.5 Susceptibility to Disinfectants

class Oomycetes, order Pythiales and family
Pythiaceae. Currently there are 250 numbers of Pythium are susceptible to 0.5 % solution of
species under the genus Pythium. Most of them sodium hypochlorite, quaternary ammonium
are nonpathogenic, even some are beneficial compounds.
except the P. insidiosum which causes diseases
in mammals. Molecular phylogenetic studies
also revealed its distant relationship with the 4.16.6 Natural Habitat and Distribution
fungal kingdom. All the Pythium species are
divided into 11 different phylogenetic groups P. insidiosum resides in the freshwater sources.
(A–K) and P. insidiosum belonged to group Other species under the genus Pythium may
C. Further molecular studies based on heteroge- inhabit in the soil, marine water (P. grandio-
neity of rRNA genes separated P. insidiosum into sporangium) or freshwater. Some species are
three different clades based on geographical parasitic to insect (mosquito), algae, fish and
locations. The clade I isolates belonged to other fungi. They are used as a biological control
North, Central and South America, clade II measure against the mosquitoes such as Aedes
isolates are from Asia and Australia, whereas aegypti, Culex quinquefasciatus and treehole
clade III isolates are from Thailand and the mosquito (Ochlerotatus sierrensis).
United State. The clade I and II isolates are Pythiosis is common in animals and human of
closely related, but clade III is significantly Southeast Asia including India, Indonesia, Japan,
different from the others. Korea, New Guinea and Thailand; coastal
Recently similarity between one of the phylo- Australia and New Zealand; and South America
genetic clade (K) of Pythium and Phytophthora such as Argentina, Brazil, Colombia, Venezuela,
4.16 Pythium 129
Costa Rica, Guatemala, Haiti, Panama and 4.16.9 Antigenic Characteristics

Nicaragua, North America, Mexico and the
United States. The antigens of P. insidiosum can induce
humoral immunity in the host, although it cannot
clear the infection effectively. The
immunodominant antigens of P. insidiosum are
4.16.7 Genome
28 KDa, 30 and 32 KDa (twin antigen), 34 KDa,
38 KDa and 74 KDa proteins. Among them,
The pyrosequencing study of plant pathogenic
recent study indicated the 74KDa protein as
Pythium genome (P. aphanidermatum, P. arrhe-
major immunodominant antigen. Further, there
nomanes, P. irregulare, P. iwayamai, P. ultimum
are two diffuse proteins (49 KDa, 51 KDa)
var. sporangiiferum, P. vexans, P. ultimum var.
detected only in the animal strains of
ultimum) revealed the smaller genome size than
P. insidiosum but not in human strains.
other members of the family sequenced so far
(Hyaloperonospora, Phytophthora). The annota-
tion study showed the presence of 11,957–15,291
numbers of predicted genes in the Pythium 4.16.10 Virulence Factors
genome. Pythium species have 1.5–1.7 introns
per gene and average exon length is The virulence factors of P. insidiosum are
312–630 bp. All the sequenced genome has com- enlisted in Table 4.39.
mon 76 clusters containing 782 secreted proteins
(core Pythium secretome).
4.16.11 Transmission
4.16.8 Isolation, Growth and Colony Direct contact with P. insidiosum zoospores-
Characteristics infested water is the major route of transmission
in animals and human. The damaged skin or
P. insidiosum can be isolated in common fungal gastrointestinal mucosa helps in the adherence
media such as Sabouraud dextrose agar, vegeta- of the zoospores and dissemination of infection.
ble extract agar, peptone yeast glucose agar, In horses, most of the pythiosis lesions occur in
cornmeal agar, potato flakes agar and V8 the legs and ventral abdomen (Fig. 4.37).
agar with streptomycin (200 mcg/mL) and ampi- Recently, P. insidiosum is also isolated from the
cillin (100 mcg/mL). As an alternative, Campy larvae of Culex quinquefasciatus (tropical mos-
blood agar with trimethoprim, vancomycin, quito) in India which indicates the possible role
polymyxin B, cephalothin and amphotericin B of mosquito in propagation of infection.
can be used for isolation. The small pieces of The large, male, young (1–3 years) and out-
fresh, non-macerated tissues are directly placed door working dog breeds (e.g. Labrador
on the surface of plates. The plates can be
incubated both at 25 or 37 C along with a beaker Table 4.39 Virulence mechanisms and factors pos-
sessed by P. insidiosum
filled with distilled water to provide the moisture.
The typical colonies are observed within Virulence
factors Functions
12 –24 h. The colonies are submerged, white
Protease P. insidiosum produces three or more
to colourless, and have an irregular radiate proteases of different molecular weight.
pattern. In the laboratory, zoospore production They are considered as serine proteases.
can be induced by keeping boiled grass blades The proteases help in host tissue invasion
on the surface of a 1–2-day-old colony growing Mechanical The mechanical force is exerted by the tips
on 2 % water agar and incubating at 37 C for force of the elongating hyphae which also help
in host tissue invasion
18–24 h.
4.16.13 Disease Produced
The major human and animal diseases produced

by P. insidiosum are enlisted in Table 4.40.
4.16.14 Immunity
The antigens of P. insidiosum can induce

humoural immunity (Th2 mediated) in the host
which generates precipitate and agglutinate anti-
P. insidiosum antibodies. These antibodies are
nonprotective and can be only used for the diag-
nosis of the infection, whereas the cell-mediated
immunity (Th1 mediated) produced by activated
macrophages, mast cells, eosinophils and other
inflammatory cells causes extensive tissue dam-
age, but it can effectively clear the infection.
During natural infection, P. insidiosum always
presents the antigens in such a manner that
the antigen-presenting cells (APC) secrete
Fig. 4.37 Ulcerative lesion of pythiosis in a horse
(Photograph courtesy: Prof. P.P. Gupta, Ex-Director of IL4 which converts the Th0 cells into Th2
Veterinary Research, Punjab Agricultural University, cells and produces Th2-mediated immunity. In
Punjab, India) vaccinated or cured patients, higher amounts of
IFNγ and IL2 are detected, indicating the gene-
ration of Th1 response which has played a
retrievers) are more susceptible to the infection. protective role.
In human, agriculture or water leisure activities
are the major predisposing factors. However,
no zoonotic transmission is detected so far. 4.16.15 Diagnosis

4.16.12 Pathogenesis The ‘kunkers’ from the horses can be collected
and transported into the laboratory in saline
After adherence of the P. insidiosum zoospores water with antibiotics (streptomycin and ampi-
with the damaged skin or gastrointestinal cillin, tetracycline, chloramphenicol). Other
mucosa, the cysts are produced. The encysted specimens include skin scrapings, tissue biopsies
zoospores produce a germ tube stimulated with (deep wedge biopsies), exudates and fine needle
the host body temperature. The germ tube aspirates from lymph node. Transport of the clin-
develops into hypha which invades the host ical samples in ice is not recommended because
tissues with the help of the virulence factors the low temperature (4 C) can inhibit the growth
(Table 4.39). In human, the hyphae also pene- of P. insidiosum.
trate the blood vessels and disseminate through-
out the body. Sometimes the blood vessel 4.16.15.2 Laboratory Examination
penetration causes thrombosis. The infection is 1. Direct examination: The wet mount with
life-threatening in human and animals if not 10 % KOH can be prepared directly from
treated early. the clinical specimens. The presence of
4.16 Pythium 131
Table 4.40 Major diseases of animal and human caused by P. insidiosum

Host Disease
Horse Cutaneous and intestinal forms are detected. In cutaneous form, large, rounded,
granulomatous, nodular and ulcerative lesions are observed, composed of eosinophils
and the fungal hyphae. The tissues contain ‘kunkers’ (necrotic yellow-grey materials)
which is a specific symptom of pythiosis in horses. The kunkers are produced due to
degranulation of eosinophils by the invading hyphae. New eosinophils replace the older
ones which accumulate in the tissues as a necrotic mass. The clinical signs associated
with cutaneous form are itching, biting of the wound area and auto-mutilation,
lameness, enlargement of regional lymph nodes, anaemia and hypoproteinaemia. The
cutaneous form is known as ‘swamp cancer’ or ‘Florida horse leeches’. In intestinal
form (rare), stenotic fibrous and GI tract lesions are observed
Dogs Intestinal form is more frequent than the cutaneous form. The symptoms are vomition,
weight loss and intermittent diarrhoea. There is segmental transmural thickening of the
stomach, small intestine, colon, rectum, oesophagus or pharynx. Enlargement of
mesenteric lymph node is observed which becomes palpable in the mid-abdomen. In
cutaneous form, the lesions are found in the face, leg, ventrum, perineum and tail
Cattle Pythiosis in cattle is a sporadic infection which is common in subtropical area during
rainy season. Granulomatous lesions are observed in the limbs. The animals cannot
stand and in untreated cases death may occur due to dehydration. Secondary bacterial
infection especially with anaerobes may occur producing ‘infectious pododermatitis’
Sheep Granulomatous lesions are observed in limbs and rhino-pharyngeal area. Clinical
symptoms include nasal discharge, swelling of nostrils and face
Cats Pythiosis is rare in occurrence. Both cutaneous and intestinal forms are reported. The
cutaneous lesions are detected in legs, digits or footpads
Camel, Bear, Jaguar, Bengal The pythiosis lesions are observed in face and vulva of camel, preputial gland and
Tiger, Californian nestling gastrointestinal tract of bear, gastrointestinal tract of tiger, and wings, neck, head and
white-faced ibis bird limbs of Californian nestling white-faced ibis birds
Human The infection is noticed in apparently healthy persons having contact with the
contaminated water. There are four clinical forms of human pythiosis which are
cutaneous (infecting the face or limbs as a granulomatous and ulcerating lesion),
vascular (affects arteries and causes arterial occlusion and aneurysm), ocular and orbital
(corneal ulcers) and disseminated pythiosis (vital organs)
coenocytic or sparsely septate hyphae in the The granulomas are composed of

slide indicates pythiosis. macrophages, plasma cells, multinucleate
2. Isolation and identification: Media and incu- giant cells and fewer neutrophils and
bation condition as described earlier will eosinophils. The fungi are found within
serve the purpose. The ideal clinical necrotic area or at the centre of granulomas.
specimens for culture are kunkers, tissue 4. Serological tests: In the laboratory, different
biopsies. The samples are washed thrice with serological tests such as immunodiffusion,
the sterile saline solution before culture and ELISA, immunochromatographic assay and
are cut into 5–10 mm blocks. The tissue Western blot are developed for the detection
blocks are impregnated directly into the agar of anti-P. insidiosum antibodies. Immunodif-
plates. fusion test is however unable to detect the
3. Histopathology: The histological sections are antibodies in dogs, whereas ELISA shows
stained with H&E, Gomori methenamine potential in early detection of postoperative
sliver (GMS) and periodic acid–Schiff recurrence. A simple and reliable haemagglu-
(PAS). However, the hyphae are clearly visi- tination test is also developed in which agglu-
ble only with GMS stain. Affected tissues tination of sheep red blood cells coated with a
contain multiple foci of necrosis surrounded P. insidiosum extract is possible using the
by neutrophils, eosinophils and macrophages. serum samples of the patients.
The immunohistochemical techniques Immunotherapy with killed ultrasonicated

include immunofluorescence and immunoper- mycelium or secreted antigens from broth culture
oxidase staining which can be used in after precipitation produced rapid cure of the
paraffin-embedded fixed tissues. lesions in horses and dogs, when combined with
5. Molecular biology: The P. insidiosum- surgery or antifungals.
specific PCR is developed using the internal-
transcribed spacer (ITS) of rRNA gene as a
target. The PCR is successfully applicable for 4.17 Emerging and Uncommon
the confirmation of DNA extracted from the Pathogenic Fungi
culture, fixed tissues and clinical specimens if
preserved properly either at 70 C or at 4.17.1 Phaeohyphomycosis
ambient temperature with 95 % ethanol.
Recently a nested PCR is also successfully Phaeohyphomycosis is used to describe the fun-
employed for the detection of P. insidiosum gal menace ranging from cutaneous colonisation
from paraffin-embedded tissues. to abscess formation and dissemination, caused
by a group of melanised fungi which reproduce
in culture by unicellular budding. The fungi
4.16.16 Treatment belong to phylum Ascomycota, subphylum
Pezizomycotina and class Dothideomycetes.
Surgical intervention is the choice of treatment in There are two orders, i.e. Capnodiales and
animals and human. In cutaneous pythiosis, Pleosporales, under the class Dothideomycetes.
amputation of legs is recommended when the The order Capnodiales consists of Cladosporium
lesions appear in the extremities. However, cladosporioides and the order Pleosporales
chances of recurrence are also higher (45 %) consists of Alternaria alternata, A. infectoria
after surgical removal of cutaneous lesions in and Bipolaris spicifera as major aetiological
horses and dogs. In animals with gastrointestinal agent. The other aetiological fungi such as
pythiosis, segmental lesions should be resected Cladophialophora bantiana, Exophiala attenuata,
with 3–4 cm margins. Due to anatomical loca- Fonsecaea multimorphosa and Phialophora
tion, certain area such as oesophagus, gastric verrucosa belong to the order Chaetothyriales
outflow tract, rectum and mesenteric root cannot under the class Eurotiomycetes. The poultry
be removed completely. black yeast (Ochroconis gallopava) belongs
Due to the absence of ergosterol in the cyto- to order Ochroconiales under the class
plasmic membrane, P. insidiosum is resistant Eurotiomycetes.
against most of the antifungals such as ergosterol The black fungi mycelia are composed of
synthesis inhibitors (azole group). However, the cylindrical hyphae with thick cell wall in which
azole group of antifungals (itraconazole, ketoco- melanin is deposited. Some of them (Exophiala)
nazole, miconazole and fluconazole), terbinafine also have yeast-like phases and they are known
and amphotericin B can also alter the permeabil- as ‘black yeast’. The conidiogenous cells emerge
ity of fungal cytoplasmic membrane and thus from the hyphae and produce conidia. The
may have little antifungal effect against Pythium. method of conidiogenesis (blastic or thallic) and
So, the antifungal therapy with itraconazole physical properties of conidia help in the identi-
(10 mg/kg by oral route in every 24 h) and fication of fungal species (Table 4.41; Figs. 4.38,
terbinafine (5–10 mg/kg by oral route in every 4.39, 4.40, and 4.41).
24 h) is recommended for 2–3 months after sur- The melanised fungi (Alternaria, Bipolaris)
gery to reduce the chances of recurrence. are ubiquitous saprobe inhabiting soil and plants.
Currently combination of azole group and The black yeasts (Chaetothyriales) can survive in
caspofungin (inhibitor of β-glucan synthesis) polluted environment also rich in aromatic
produces promising result against P. insidiosum. hydrocarbons. Their ability to use hydrocarbons
4.17 Emerging and Uncommon Pathogenic Fungi 133
Table 4.41 Physical properties of conidia and colony characteristics produced by different melanised fungi
Fungi Conidia Colony characteristics
Cladosporium The conidiophores are branched or unbranched. During Colonies are olivaceous to black and
conidiogenesis, ramoconidia (shield cells) are produced velvety
first which give rise to branching chains of dark-coloured,
single- or double-celled conidia with prominent attachment
scar (hila)
Alternaria Chains of conidia are found which are large, dark, euseptate Colonies are woolly, pale to olivaceous to
alternata and muriform black. The growth of the colony is rapid
A. infectoria The conidia have long apical beaks which serve as
secondary conidiogenous cells
Bipolaris There is bipolar germination of conidia. The conidiophores Colonies are woolly, grey to black, with
spicifera are geniculate (bent) with flattened hilum. B. spicifera has rapid growth
three distosepta (pseudosepta where inner walls are
involved) and four cells
Exophiala The conidiogenous cells produce conidia by short Colonies are primarily mucoid which
percurrent proliferations (annellations). The tip of an later become more filamentous
annellide increases in length and becomes narrower as each
subsequent conidium is formed. Sometimes phialides are
also present
Fonsecaea The conidia are produced from swollen denticles (small Colonies are olivaceous to black and
projection). The secondary and tertiary conidia in chains velvety
(four conidia) are also produced on sympodial
conidiophores or on phialides
Phialophora It has dark, funnel-shaped collarettes Colonies are olivaceous to black and
verrucosa velvety
Ochroconis The conidia are clavate and borne from denticles Colonies are brown, velvety with a red
gallopava diffusing pigment
Fig. 4.39 Conidial arrangement of Fonsecaea

Fig. 4.38 Conidial arrangement of Fonsecaea multimorphosa (Photograph courtesy: Prof. Sybren de
multimorphosa (Photograph courtesy: Prof. Sybren de Hoog, CBS Fungal Biodiversity Centre, The Netherlands)
Hoog, CBS Fungal Biodiversity Centre, The Netherlands)
explains about their affinity for central nervous bantiana, Ochroconis gallopava) in Sabouraud
system due to the structural similarity between dextrose agar, brain–heart infusion agar, potato
the hydrocarbon and neurotransmitter. dextrose agar, malt extract agar, V8 juice agar,
Most of the species are able to grow at 37 C cereal agar, carnation leaf agar, cornmeal dextrose
or at higher temperature (Cladophialophora agar (nonselective) or cycloheximide-containing
Table 4.42 Virulence mechanisms and factors possessed

by melanised fungi
Melanin The fungi-obtained
melanin from
dihydroxynaphthalene,
a cell wall constituent.
The pigment prevents the
lytic effects of hydrolytic
enzymes on the fungal cell
wall and scavenging free
radicals released by
phagocytic cells during the
oxidative burst. Thus,
Fig. 4.40 Conidia of Ochroconis gallopava (Photograph melanin helps in evasion of
courtesy: Prof. Sybren de Hoog, CBS Fungal Biodiversity immune response. It also
Centre, The Netherlands) provides resistance against
physicochemical agents,
toxic metals, desiccation,
ionising radiation and
antifungals
Cell wall The cell wall is unusually
thick where the melanin can
be deposited
Yeast-like phase It helps in evasion from
phagocytosis
Aspartic protease It degranulates eosinophils
(Alternaria)
Additional Transmission of infection
conidiogenesis
Thermotolerance These fungal properties
(Ochroconis gallopava, help in the establishment of
Exophiala), osmotolerance, infection
adhesion, hydrophobicity,
production of extracellular
polysaccharides,
siderophores and acidic or
alkaline secondary
Fig. 4.41 Phialophora verrucosa (Photograph courtesy: metabolites
Prof. Sybren de Hoog, CBS Fungal Biodiversity Centre, Assimilation of aromatic It helps in the survival of the
The Netherlands) hydrocarbons fungi in polluted
environment
media (act as selective media except for

Ochroconis gallopava). Antibiotics are also Cladosporium cladosporioides is transmitted
added to suppress the bacterial contamination. through inhalation in a humid and overcrowded
All of the fungal species produce velvety or pen. Minor skin abrasions and trauma also play a
woolly colonies with pigmentation in the media role in the transmission of melanised fungi in
(Table 4.41). human and animals.
There are several virulence factors and The Chaetothyriales group of black yeasts
mechanisms used by the melanised fungi to prefer to infect invertebrates and cold-blooded
establish an infection (Table 4.42). vertebrates with a moist skin and having water-
The transmission of Ochroconis in poultry associated life. Black yeast infection of
occurs through the inhalation of fungal elements mammals is unusual due to the presence of
from the contaminated litter. In sheep, water repellent skin and the capacity to produce
granuloma with lymphocytes, granulocytes and sections of the nodules in cats reveal granu-
giant cells (highly developed immune system). lomatous dermatitis containing neutrophils,
In debilitated mammals such as immunocompro- macrophages and giant cells. The pigmented,
mised animals or wild animals kept in captivity, yeast-like cells and hyphal elements are identified
chances of infection are more. in the tissue sections of nodules in cutaneous or
Ochroconis gallopava was reported to subcutaneous form and black granular necrotic
cause epidemic encephalitis in poultry, turkey, areas throughout the liver in systemic form.
captive owl and cats. It causes cerebral Other histopathological stains such as melanin
phaeohyphomycosis in quail chicks and grey- Fontana–Masson, periodic acid–Schiff (PAS)
winged trumpeters (Psophia crepitans). The and Gomori methenamine silver can be used for
birds showed sudden ataxia, intermittent torticol- the detection of light-melanised fungi. Isolation of
lis and rigidity of legs. the melanised fungi in artificial culture is required
In cats, cutaneous, subcutaneous and systemic to confirm the infection. However, the specimens
phaeohyphomycoses were reported which from the patients receiving antifungals may not
were caused by Cladophialophora bantiana, produce any growth, and in such cases histopa-
Phialophora verrucosa, Fonsecaea pedrosoi, thology helps in identification. The nucleic acid
Exophiala spinifera, Alternaria alternate and sequencing of the internal-transcribed spacer
A. infectoria. The infection showed nasal, ocular, (ITS1 of rRNA gene) is developed for the detec-
renal and cerebellar involvement. tion of black yeast (Chaetothyriales) infection in
In dogs, Ochroconis gallopava and Bipolaris animals.
spicifera were detected to cause systemic Treatment of melanised fungi in human and
phaeohyphomycosis which involved the brain, animals is difficult due to the presence of mela-
bone and kidney. Cladosporium cladosporioides nin in their cell wall which make them refractory
was detected to cause granulomatous encephali- to the most of the antifungals. In animals, the
tis and nephritis. Onychorrhexis (breakage of systemic antifungals such as amphotericin B,
nails) on the digits and blackened corium, black itraconazole, voriconazole, posaconazole, caspo-
plaque-like lesion in mouth was observed in fungin and anidulafungin are used for the treat-
dogs suffering from Alternaria infection. ment of black yeast infection. Clean and hygienic
In sheep (merino breed), Cladosporium pen and poultry houses can prevent the transmis-
cladosporioides causes systemic phaeohypho- sion of black yeast.
mycoses which was characterised by anorexia,
fever and respiratory distress followed by death.
In horse, firm brown-coloured skin nodule, alo- 4.17.2 Pneumocystis
pecia and scaling in head and neck were detected
due to Alternaria infection. Carlos Chagas (1909) first observed the cystic
The clinical specimens include tissue forms of Pneumocystis in the lungs of rats and
biopsies, aspirates and body fluids. The postmor- guinea pigs. Vanek and Jirovec (1952) first
tem samples such as the brain sections can also described Pneumocystis as an aetiological agent
be collected from the animals died due to of endemic pneumonia in children. Later (1960
suspected encephalitis. The blood from the onwards) it was recognised as an opportunistic
suspected animals is collected for culture if the pathogen in immunocompromised children.
lesions are not accessible. Gram stain, KOH wet In 1980s, Pneumocystis was recorded as most
mount and fluorescent staining (calcofluor white) widespread opportunistic pathogen in individuals
are the most commonly used methods for the suffering from AIDS. With the advancement
direct examination of specimens. The histo- of antiretroviral therapy, the incidence of
pathological staining of brain tissues with Pneumocystis infection in human subsided.
haematoxylin and eosin shows the invasion of Earlier Pneumocystis was considered as
septate, melanised hyphae. The histological protozoa. The molecular phylogenetic study
based on 16S rRNA and mitochondrial gene endoplasmic reticulum-like structure. A nucleus
established its correlation with the kingdom is present in the variable position of the cell with
Fungi. Recent classification included a nucleolus either centrally or peripherally. The
Pneumocystis under the phylum Ascomycota, vacuoles, microtubules and a budding Golgi
class Archiascomycetes, order Pneumocystidales complex are also sometimes observed. Some
and family Pneumocystidaceae. Pneumocystis trophozoites contain filopodium-like structure
infects mammals only and is highly host specific. which helps in making contact with host cells.
Previously all the forms of Pneumocystis was Both the developmental stages lack sterol (ergos-
designated as Pneumocystis carinii as an honour terol) in their cell wall.
to Antonio Carini, an Italian biologist who Pneumocystis is ubiquitous in nature which is
described them in rats. At present P. carinii is commonly detected in soil, pond water and
used to describe the rat or other animal-related indoor or outdoor air. The antibodies are detected
forms of Pneumocystis. It is detected in dogs, in children (7 months–4 years) and adults (both
cats, sheep, goat, monkey, rat and mice. Human in HIV and non-HIV healthy patients) through-
form of Pneumocystis is designated as out the world without any clinical syndrome,
Pneumocystis jirovecii in honour of Jirovec. indicating their exposure to the organism. The
In the life cycle of P. carinii, two develop- fungi cause a major opportunistic infection
mental stages exist, i.e. the mature cyst and the (pneumonia) throughout the world, and the prev-
trophozoite. The matured cysts are spherical alence rate is rising in developing countries of
(5–8 μm in diameter) containing up to eight Asia (Thailand, Vietnam, Cambodia), Africa
numbers of intracystic bodies (1.2 μm mean (Tanzania, Congo, Ivory Coast, Zimbabwe,
diameter). The cysts are surrounded by two cell Kenya, Ethiopia, South Africa) and North and
layers, i.e. outer layer (15 nm thick) and inner South America (Mexico, Panama, Guatemala,
layer (35 nm thick). The cyst protoplasm consists Venezuela, Brazil, Chile, Argentina and Peru).
of a matrix and intracystic bodies. The matrix In India, the prevalence rate of pneumonia
contains mitochondria, ribosome, empty caused by Pneumocystis is low (5–6.1 %).
vacuoles and membrane debris. The intracystic The genome of P. carinii is haploid and
body is spherical to oval containing a centrally consists of 13–15 linear chromosomes (300–700
located nucleus, endoplasmic reticulum, free Kbp each). The genes present in the genome
ribosome, mitochondria and glycogen particles. contain 60–65 % AT sequences and introns
After release of intracystic bodies, the cyst (less than 50 bp, nine per gene in number).
becomes banana shaped which is known as Pneumocystis isolation in artificial culture is
remnants of the ruptured cysts. The intracystic not possible probably due to its stringent require-
bodies originate trophozoites. ment which cannot be reproduced outside
The trophozoites are 0.3 μm in diameter, vary the host.
in shape and produce clusters, and they are The major antigen of Pneumocystis is a sur-
associated with pneumocytes in the lung tissue. face glycoprotein (major surface glycoprotein/
They are haploid in nature and they can undergo glycoprotein A, 90–120 KDa), encoded by msg
binary fission (endogeny). Later two trophozoites gene. The relatively conserved carboxy-terminal
conjugate to produce a diploid cell which region of the protein (MsgC) is immunodominant
undergoes meiosis (three times) to produce a in nature, and anti-MsgC antibody titre is often
spherical cell (cyst) with eight intracystic bodies. detected in infected patients. The Msg protein
The trophozoites are surrounded with a helps in interaction with a number of host cell
20–30 nm thick dense coat which contains proteins (fibronectin, vitronectin, surfactants)
small electron translucid areas. The coat helps and attachment with the host epithelial cells.
in anchoring and nutrient uptake. The trophozo- Pneumocystis can secrete proteases [kexin
ite cytoplasm is rich in free ribosome, glycogen (Kex1, Prt1)] having similarity with serine
particles, mitochondria with cristae and proteases (Kex2) produced by Saccharomyces
cerevisiae. The kexin acts as virulence factor by Pneumocystis. The natural antibodies help in rec-
proteolytic processing and activation of Msg ognition of fungi by dendritic cells and enhance
protein. dendritic cell maturation and migration into the
Transmission of P. jirovecii occurs from a draining lymph nodes. The natural antibodies
common environmental source or through also help in the development of anti-
person-to-person contact. The animal-to-human Pneumocystis-adaptive immunity containing
transmission of Pneumocystis is not observed. both humoural and cellular responses. The anti-
The infection can also reactivate from latent Pneumocystis humoural immunity plays a major
childhood contamination during immunocom- role in protection. The passive transfer of hyper-
promised condition. The childhood contamina- immune antiserum or monoclonal antibodies
tion may remain latent in the host for a conferred protection in experimentally infected
prolonged period due to changing capability of mice. As a component of cellular immunity, opti-
Pneumocystis Msg structure to evade immune mum level of CD4+ T cells (Th1, Th2, Th17)
response. The seroconversion rate of along with functional co-stimulatory molecules
Pneumocystis in children was detected as 5 % (CD2, CD28) is associated with the control of
per month. The children may act as reservoir and Pneumocystis infection. The level of Th2 was
transmit the infection to other persons who are detected to be maximum during experimental
healthy or immunocompromised. In healthy infection. The natural IgM is required for the
persons again it maintains the latency, whereas development of Th2 response, Th17 cell differen-
in immunocompromised patients, it produces tiation and immunoglobulin class switching,
fatal pneumonia. In animals, inhalation is the whereas CD8+ T cells offer a partial protection
major route of transmission. only. Further, γδ T cells may play a role in
After transmission, the organisms proliferate controlling inflammation and pulmonary injury
extracellularly in the lung alveoli. The virulence during diminished αβ-T cell concentration.
factor (Msg) of Pneumocystis interacts with alve- The diagnosis depends on direct examination
olar epithelial cells for attachment. Diffuse alve- of clinical specimens such as sputum, saliva,
olar lesions are observed which suggest the bronchoalveolar lavage (BAL) and lung tissues.
involvement of host immune response with T The commonly used stains are Giemsa, methena-
cell-mediated inflammation. mine silver, toluidine blue and Diff-Quik. How-
P. jirovecii causes pneumonia (PcP) in immu- ever, direct detection method especially in
nocompromised human patients due to histological sections requires expertise. Instead,
malignancies, immunosuppressive therapy, con- detection of Pneumocystis antigen (β-D glucan)
genital immunodeficiency or other infection in BAL and antibodies in serum is an alternative
(HIV) and malnourished children. P. carinii can diagnostic strategy. The PCR is a better choice
infect Arabian foals with or without severe com- for diagnosis targeting the genes encoding Msg
bined immunodeficiency. The clinical signs in antigen or mitochondrial large subunit rRNA
horses include dyspnoea, cough and nasal dis- (mtLSU) gene. The real-time PCR is also devel-
charge. The lungs become firm, meaty and mot- oped for the detection of Pneumocystis heat
tled. The lung parenchyma resists of being cut shock protein 70 gene (hsp70). Currently
and bulges on cut section during in vitro test. attempts are in progress with cdc2 as target
Naturally occurring P. carinii infection is also gene to increase the sensitivity and specificity
reported from piglets, goats, dogs, cats and non- of the molecular assay.
human primates. In goats, P. carinii infection Trimethoprim–sulphamethoxazole is the drug
was associated with Mycobacterium avium of choice for treatment and prophylaxis of
subsp. paratuberculosis. Pneumocystis-associated pneumonia in human.
The natural antibodies (IgM) generated with- Some patients with HIV infection (25 %) are
out any antigenic stimulation as a component of unable to tolerate a full course of trimethoprim–
innate immunity offered protection against sulphamethoxazole. In such cases, intravenous
pentamidine and intravenous clindamycin with

oral primaquine or atovaquone are effective
alternatives. In animals, echinocandin (prevent
synthesis of β-D glucan) was detected to be
active against matured cyst but not against tro-
phozoite forms. Corticosteroid as adjunct therapy
was successful in curing of the infection.
Amphotericin B or triazoles are not very effec-
tive due to lack of sterol in the cell wall.
4.17.3 Prototheca
Zopf and Kühn (1880) isolated an unknown organ-

ism from the slime flux of a linden tree which was
Fig. 4.42 Prototheca sporangium with a central rounded
later characterised and identified as a fungi due to
endospore (1,000x) in haematoxylin–eosin-stained tissue
cultural similarity with yeasts (Krüger 1894). In smear (Photograph courtesy: Prof. Cornelia Lass-Flörl,
1913, similarity of the organisms with algae was Medizinische Universität Innsbruck, Austria)
established due to its internal sporulation (Chodat
1913). Later it was reclassified as algae under the sporangia of P. wickerhamii (3–10 μm in diame-
genus Prototheca and family Chlorelaceae (West ter) are generally smaller than the sporangia of
1916). Davies et al. (1964) first described human P. zopfii (7–30 μm in diameter). Initially the
infection due to Prototheca which was detected spores are irregular in shape. The spores increase
from cutaneous lesion of a farmer at Sierra Leone. in size and disrupt the sporangial membrane and
However, the etiological correlation between thus they are released from the sporangia. The
Prototheca and bovine mastitis was established spore release requires 5–6 h time and sufficient
earlier (Lerch 1952). nutrients. The newly released spores again
Prototheca is an achlorophyllous mutant of the increase in size and repeat the cycle.
green algae with nonfunctional chloroplast. It was Prototheca is ubiquitous in nature and widely
originated from Chlorella during evolution, and distributed throughout the world. The organism
later it acquired different cell wall composition, was isolated from slime flux of trees, soil, sew-
physiology and better ability to survive environ- age, fresh and salt water, urban water supply
mental stress. They are unicellular, oval or spheri- system, pasture mud, dairy barn drainage and
cal (3–30 μm in diameter) and surrounded by fruits (banana). Prototheca prefers to inhabit in
bilayered cell wall. The cell wall does not contain the water bodies containing plant-decomposed
glucosamine, muramic acids, chitin and cellulose. materials. The organism was also isolated from
The nonpathogenic species can produce capsule. skin, sputum and faecal samples of healthy
There are five major species under the genus human and animals such as cattle, pig, horses
Prototheca. Among them, P. wickerhamii and and sheep.
P. zopfii are pathogenic, whereas P. stagnora, The mitochondrial genome of P. wickerhamii
P. ulmea and P. moriformis are considered as is 55,328 base pairs in size with more AT content
nonpathogenic. (74.2 %). The genes present in the genome can
Asexual reproduction takes place in which the encode cytochrome oxidase, apocytochrome b,
cytoplasmic cleavage produces endospores/ NADH dehydrogenase (nad1-7, nad4L, nad9),
aplanospores/sporangiospores (2–20 in number). ATPase (atp6, atp9, atp1), ribosomal RNAs
The endospores are generated within a sporan- (5SrRNA, small subunit (srn), large subunit
gium, a thick-walled, oval or spherical body (lrn) RNA), tRNAs and ribosomal proteins. The
(10–30 μm in diameter) (Fig. 4.42). The plant-specific genes such as orf25, orf244, orfB
and rrn5 are detected in the mitochondrial Prototheca (P. zopfii genotype 2) causes sub-
genome of P. wickerhamii which indicates their clinical or chronic mastitis in cattle without sys-
relationship with algae, the simplest plant. temic involvement. The clinical syndrome is pain
Prototheca is heterotrophic in nature. They in udder, watery milk secretion with clots and
require external sources of organic carbon (glu- decreased milk production. The somatic cell
cose, fructose and galactose), nitrogen (protein or count varies between 6 and 9 million cells/mL.
inorganic nitrogen), oxygen and thiamine for The infection is limited within the udder and
their growth. They can be isolated in blood regional lymph node. However, sporangia and
agar, brain–heart infusion agar and Sabouraud sporangiospores are detected within the macro-
dextrose agar (SDA) without cycloheximide. phage and neutrophils in the alveolar lumen and
The specific medium for Prototheca should con- interstitium, indicating the intracellular nature of
tain folate (for inhibition of bacteria) and the infection. So it is difficult to eradicate and it
5-fluorocytosine (for inhibition of yeast). The becomes chronic mastitis which persists for sev-
plates are incubated at 25–37 C for 48–72 h. eral lactation periods. The chronic mastitis
P. wickerhamii colonies are smooth, moist, becomes progressive interstitial mastitis with
cream-coloured and yeast-like. P. zopfii colonies consequent alveolar atrophy and severe
in SDA are large, dull white, irregularly mar- decreased milk production.
gined and granular with a central protrusion. P. zopfii (sometimes P. wickerhamii) causes
P. zopfii has three biotypes (I, II, III) which systemic protothecosis in dogs. The transmission
are based on carbohydrate assimilation. The bio- of infection occurs by ingestion route. The algae
type II (associated with bovine mastitis) can fer- pass through the intestinal mucosa and are
ment glucose and glycerol but not galactose disseminated throughout the body via blood or
(delayed fermentation may occur in some lymph. Gastrointestinal disorders producing
isolates), whereas biotype III (swine isolates) vomition, tenesmus and intermittent diarrhoea
cannot ferment glycerol. The antigenic structure (with blood and slime) are most commonly
differs between the three biotypes. Based on 18S manifested. Diffuse hyperaemia, haemorrhages,
rRNA gene sequence and cellular fatty acids, the ulcerations and multiple granulomas are
biotype III is recently proposed as a novel species observed in the intestinal mucosa (especially in
(P. blaschkeae sp. nov.). Further, all the three the colon) which may result intestinal stricture
biotypes are currently considered as three and obstipation. The infection can spread to the
genotypes (1–3) of P. zopfii. central nervous system, cardiovascular system,
The transmission of Prototheca occurs by urinary tract, liver, skeletal muscle, lymph
direct contact or traumatic inoculation in nodes, thyroid gland, pancreas, peritoneum and
human, dogs and cats. In cattle, the organism diaphragm. The eye infection leading to blind-
enters from contaminated environment (faeces, ness due to glaucoma and retinal ablation is
wet litter) through direct contact with teat end, another common clinical sign.
especially 25–30 min after milking when the teat P. wickerhamii causes cutaneous protothecosis
canal sphincter is relaxed to prevent the ascend- in cats and dogs. It is characterised by ulcerative
ing infection. Alternatively, Prototheca is also lesions, scabs and pyogranulomatous dermatitis in
transmitted through intramammary infusion the limbs, trunk and mucosal surfaces.
material. The cow-to-cow transmission during Human infection is primarily caused by
milking is also possible. The predisposing factors P. wickerhamii (rarely by P. zopfii). Three clini-
include poor milking hygiene, prolonged antimi- cal forms such as cutaneous, olecranon bursitis
crobial therapy and warm and humid weather. In and systemic form are commonly detected. The
dogs, transmission may occur via ingestion also cutaneous form occurs in immunocompromised
which results systemic infection. Man-to-man or individuals, and it is characterised by the forma-
man-to-animal and vice versa transmission of tion of vesiculobullous and ulcerative lesion with
Prototheca is not detected. purulent discharge, erythematous plaques,
pustules, papules, nodules, pyodermic and PAS staining with diastase can differentiate
herpetiform, and hypopigmented or atrophic Prototheca and green algae. The green algae
lesions in exposed areas such as extremities and contain cytoplasmic starch granules that are
face. The olecranon bursitis takes place as a PAS negative following diastase treatment. Sero-
result of injuries or grazing of the elbow. The logical tests for the detection of anti-Prototheca
signs include mild induration of the bursa along antibodies can be performed in cattle to confirm
with tenderness, erythema and production of the subclinical infection. The PCR based on 18S
serosanguineous fluid. The systemic form occurs rDNA sequence is developed for differentiation
in immunocompromised patients. The tissues of three P. zopfii genotypes. Taqman probe-based
and organs such as the skin, subcutaneous tissue, quantitative PCR is recently developed for the
gut, peritoneum, blood and spleen are affected. differentiation of P. zopfii genotype which is
The clinical specimens for diagnosis of based on small subunit ribosomal DNA
Prototheca infection in animals include faeces, sequences.
urine, mastitic milk, skin scrapings and lesion In human, amphotericin B is used in systemic
exudates, whereas from human, skin scarificates protothecosis which prevents the ergosterol syn-
(cutaneous protothecosis) or joint punctuate thesis of Prototheca. The immidazoles have also
(olecranon bursitis) can be collected. Direct attempted without any conclusive findings. Sur-
examination of the wet mount slides prepared gical excision is preferred in some cases. In
from the clinical specimen or culture and stained immunocompromised individuals, the prognosis
with lactophenol cotton blue or calcofluor white is grave.
can be done (Fig. 4.43). The tissues can be In dogs with systemic protothecosis,
stained with Gridley fungus stain, Grocott’s amphotericin B and immidazoles are used,
modification of Gomori methenamine silver, although the effect of such antifungals is not
PAS with or without diastase and haematoxy- concluded. Surgical excision is performed in
lin–eosin (less sensitive). Prototheca appears as cutaneous form.
large, spherical, oval or elliptical, non-budding The effective treatment for protothecal bovine
cells both in wet mount and tissues. The size of mastitis is yet not developed, although the
Prototheca sporangia is usually smaller in vitro antifungal sensitivity of P. zopfii was
(10–30 μm) than the other fungi, and it contains observed against amphotericin B, nystatin, poly-
2–20 numbers of sporangiospores. In Gram- myxin, gentamicin and neomycin. The control of
stained smears, spores stain as gram positive mastitis can be achieved by detection and exclu-
and the empty sporangia are gram negative. The sion of infected animals from the herd and by
adaptation of common hygienic measures such as
teat dipping before and after milking, etc.
4.17.4 Lobomycosis
Lobomycosis is a chronic granulomatous fungal

infection of the skin and subcutaneous tissues in
human, bottlenose dolphin (Tursiops truncatus)
and Guiana dolphin (Sotalia guianensis). The
first human case was described by Jorge Lobo
(1931) in Brazil. Lobo designated the infection
as ‘keloidal blastomycosis’ due to the presence
of organisms resembling Paracoccidioides
Fig. 4.43 Prototheca (1,000x) in PAS stained smear
(Photograph courtesy: Prof. Cornelia Lass-Flörl, brasiliensis. Later the disease was named Jorge
Medizinische Universität Innsbruck, Austria) Lobo’s disease.
It is caused by Lacazia loboi, a yeast-like transformed into squamous cell carcinoma. In

organism which is noncultivable in artificial cul- dolphins, lobomycosis is characterised by white
ture medium. Lacazia loboi is taxonomically to pink, verrucous lesions which may ulcerate
related (sister taxon) with Paracoccidioides and form large plaques.
brasiliensis and belongs to the order Onygenales. Diagnosis depends on direct examination of
Both of the fungi share antigenic proteins (gp43, the smears prepared from the clinical specimens
chitin synthase). The sequencing of L. loboi gp43 (exudate) which show yeast-like rounded, thick-
revealed its higher degree of nucleotide similar- walled Lacazia loboi cells, occurring in chains of
ity (85 %) with P. brasiliensis gp43 and compar- 2–10. The confirmation depends on
atively lower similarity (75 %) in amino acid histopathological examination showing atrophic
level. Probably the high degree of nucleotide epidermis and development of fibrous granuloma
similarity occurs due to lack of known sexual in the dermis. The granuloma is composed of
reproduction and contact with its natural habitat histiocytes and giant cells containing the typical
for prolonged period (40–50 years) which helps thick-walled L. loboi cells. The tissue sections
to retain the original nucleotide motif in L. loboi are stained with periodic acid–Schiff, Gomori
genome. Further genomic study revealed about methenamine silver (GMS) and Gridley’s silver
the loss of certain nucleotides (reductive evolu- stain to detect the yeast-like cells.
tion) which was earlier observed in other uncul- The localised lesions in the extremities are
tivated, host-restricted pathogens such as treated with cryosurgery or surgical excision in
Mycobacterium leprae, Rickettsia prowazekii such a manner so that the margins of the lesions
and Buchnera. The reductive evolution occurs are free of infection to avoid recurrence. The
due to lack of known sexual cycle to acquire disseminated lesions are treated with chemother-
new DNA during recombination, and as a conse- apy, such as clofazimine and itraconazole or a
quence decreased evolutionary fitness with every combination of both antifungals.
possibility of early extinction takes place.
Lobomycosis is endemic in South and Central
Americas. The endemic zone should be located 4.17.5 Lagenidium
200–250 m above sea level and should have
dense vegetation, annual rainfall < 2,000 mm, Lagenidium giganteum was first described by
mean temperature of 24 C and mean relative Couch (1935) from copepods and mosquito larvae
humidity >75 %. However, the infection is also in North Carolina, USA. Grooters et al. (2000) first
reported from Europe, Canada, the United States isolated and identified Lagenidium from a dog
and South Africa. In this non-endemic zone, the with severe multifocal cutaneous lesions and
persons who had travelled to Central or South regional lymphadenomegaly.
America or had contact with an infected dolphin The genus Lagenidium belongs to the class
were detected with lobomycosis. Oomycetes, and it contains more than 50 species.
Transmission of Lacazia loboi in human Lagenidium species are parasites of algae, fungi,
occurs through traumatic inoculation such as rotifers, nematodes, crustaceans and mosquito
scratches obtained while working in agriculture larvae. Lagenidium giganteum is a facultative
field and during insect and animal bites, etc. pathogen of mosquito larvae and is used as a
Lobomycosis in human is characterised by for- biological control agent of mosquito (Culex,
mation of cutaneous nodules, papules or plaques Anopheles, Mansonia). Canine pathogenic
of various sizes having smooth, verrucous or Lagenidium species are antigenically related
ulcerated surfaces after a prolonged incubation with L. giganteum, although their life cycle and
period (more than 40 years). The lesions com- biology are unexplored.
monly appear in the extremities and ears which Lagenidium possesses broad (7–25 μm in
are sometimes disseminated into other body parts diameter), poorly septate or coenocytic, hyaline
such as lymph nodes. The chronic lesions may be and branched hyphae. L. giganteum produces
both asexual (zoospores) and sexual spores The diagnosis of lagenidiosis is based on
(oospores). The zoospores are biflagellate and histological observation, isolation of fungi and
motile which can infect the mosquito larvae. serological and molecular tests. In contrast to
The zoospores do not have a cell wall and are Pythium, the hyphae of Lagenidium can be
fragile in nature (48 h survival period). The observed in haematoxylin–eosin (H&E)-stained
oospores are resistant to desiccation and mechan- smear and the smears stained with Gomori
ical abrasion and remain viable for several years methenamine silver (GMS). The Lagenidium
in the environment. hyphae are broader (7–25 μm) than Pythium
L. giganteum was isolated from different parts hyphae (4–10 μm). The ELISA can detect the
of the world including North America, Europe, anti-Lagenidium antibodies in canine serum.
Africa, Asia and even Antarctica. Canine However, these antibodies may produce cross-
pathogenic Lagenidium is so far reported from reaction with Pythium infection. So,
young- to middle-aged dogs residing at south- Lagenidium-ELISA should always be performed
eastern United States. They reside in the aquatic along with Pythium-ELISA to get a confirmative
environment as a saprophyte. The dogs suffering diagnosis. The PCR is developed using the
from Lagenidium infection had previous internal-transcribed spacer (ITS) of rRNA gene
exposures to ponds or lakes. Lagenidium are as a target. The PCR is successfully applicable
sterol auxotrophs and incorporate sterols from for the confirmation of DNA extracted from the
the environment rather than producing them. culture and clinical specimens if preserved prop-
Lagenidium can be isolated in peptone yeast erly. The sequencing of the PCR product and its
glucose (PYG) agar with ampicillin alignment study can definitively diagnose the
(100 mcg/mL) and streptomycin (200 mcg/mL) fungi.
and 2 % Sabouraud dextrose agar. The small In animals, surgical resection of infected
pieces of tissues are directly placed on the sur- tissues with wide margin is the treatment of
face of plates. The plates are incubated at 37 C. choice for lagenidiosis. Amputation is suggested
The typical colonies are observed within if the infection occurs in limbs. Assessment of
24–48 h. The colonies are submerged, glabrous internal organs is recommended before surgical
and white to colourless. The zoospores can be resection to determine the stage of dissemination,
artificially produced in Sabouraud dextrose agar if any. Treatment with antifungals is not so much
(pH 7.0) and corn meal agar. successful. However, use of itraconazole and
The infection produced by Lagenidium is terbinafine combined with surgical resection
known as lagenidiosis. The lagenidiosis in was found effective in dogs. In human suffering
dogs is characterised by progressive cutaneous from keratitis, repeat penetrating keratoplasty
or subcutaneous multifocal lesions which successfully eradicated the infection and
involve the extremities, mammary gland, peri- prevented the recurrence.
neum and trunk. The lesions are nodular or
ulcerated with draining tracts. The dissemina-
tion of infection occurs into the regional lymph 4.17.6 Basidiobolus and Conidiobolus
nodes and abdominal great vessels. Sometimes
the infection in dogs is associated with Basidiobolus and Conidiobolus belong to the
hyperglobulinaemia, hypoalbuminaemia and order Entomophthorales (entemon ¼ insect)
hypercalcaemia. Cats, ruminants and other under the class Zygomycetes and phylum
domestic animals are not reported to be infected Zygomycota. Recently a new subphylum
with Lagenidium. However, lagenidiosis is a (incertae sedis), namely, Entomophthoro-
common problem in shrimp culture, causing mycotina, is proposed to include the order
lethargy and mortality of larvae. In human, Entomophthorales. Major species under the
severe keratitis is reported with Lagenidium genera are Basidiobolus ranarum, Conidiobolus
infection. coronatus, C. incongruus and C. lamprauges.
The hyphae are broad, thin walled, coenocytic (Basidiobolus). The typical colonies are flat,
with little branches. The hyphal diameter of waxy in consistency and grey in colour. With
Basidiobolus is 5–20 μm (mean 9 μm) and the maturity, the colonies become heaped and
Conidiobolus is 5–13 μm (mean 8 μm). Both of radially folded. The aerial hyphae are generated
the fungi produce asexual (conidia) and sexual and the different conidia are discharged. The
spores (zygospores). The conidiophores are discharged spores (primary, secondary and
unbranched. Primary conidia are produced singly capilliconidia) cover the colony surface and lid
at the swollen tip of the conidiophores which are of the Petri dish.
discharged forcibly. The primary conidia are The transmission of the infection occurs by
spherical, 12–40 μm in diameter, and have a percutaneous entrance of the spores through the
prominent basal papilla or hair-like projections trauma, insect bite, etc. Mites and other insects
(villose conidia). Secondary conidia are can carry the fungal spores (capilliconidia of
generated from the primary conidia which are Basidiobolus ranarum) on their surface. Some-
smaller. Sometimes, oval to elongate spores times ingestion and inhalation of the spores can
with a terminal knob are produced which are also transmit the infection. Dogs get infected
known as ‘capilliconidia’. The zygospores are while chewing of the wood which is old and
thick-walled, smooth, undulant and contain a decaying. The reptiles and amphibians excrete
characteristic ‘copulatory beak’ (remnant of the the spores in the environment, although the sta-
copulation tube). bility of the spores in the environment is low.
Most of them are saprophytes and are com- The virulence factors include thermotolerance,
monly found in soil, decaying plant materials. serine protease, lipase and collagenase.
They are also present in insects and faeces of Thermotolerance (37 C) helps in adaptation of
amphibians and reptiles. Both of the fungi are the fungi in mammalian host. The serine protease
worldwide in distribution but are more prevalent is involved in discharge of fungal spores. Other
in tropical countries or countries with warm and proteases, lipase and collagenase cause tissue deg-
moist climate such as India, Australia, Africa, radation and help in invasion of the fungi. Cellular
Central America, Brazil, Colombia and south- degradation products provide nutrition to the fungi.
eastern United States. The major diseases produced by Conidiobolus
Both of the fungi can be isolated in common and Basidiobolus are enlisted in Table 4.43.
media such as Sabouraud dextrose agar and The clinical specimens for the diagnosis of
potato dextrose agar. The plates are incubated Conidiobolus and Basidiobolus infection in
at 25 C for 2days (Conidiobolus) to 5 days animals and human include polyp, scrapings,
Table 4.43 Major diseases of animal and human caused by Conidiobolus and Basidiobolus
Fungi Host Disease
Conidiobolus Horses Equine conidiobolomycosis (rhinophycomycosis): It is characterised by
pyrogranulomatous lesion in nasal mucosa which causes epistaxis, nasal discharge and
dyspnoea
Sheep, deer, Nasopharyngeal infection with or without local dissemination into face,
human retropharyngeal region and retrobulbar space. Nasal polyp may develop which can
block the passage. The human infection was previously described as ‘subcutaneous
phycomycosis’
Dogs Nasopharyngeal conidiobolomycosis: It is characterised by the formation of severe
chronic ulcerative dermatitis in the nasal planum, ulcer in hard palate, regional
lymphadenopathy, mutifocal ulcerative subcutaneous lesions and pneumonia
Cats Lesion in the gastrointestinal tract, lungs, hard palate
Basidiobolus Dogs Gastrointestinal tract lesion, focal preputial/vulvar lesion, ulcerative draining skin
lesions (rare)
Horses Pruritic granulomatous lesions in the trunk, neck, thorax, ventral abdomen and head
exudates, nasal discharge and bronchoalveolar adiaconidia by the fungi, the infection is known
lavage. The direct examination of the specimens as adiaspiromycosis.
can be done with KOH mount which reveals the Emmonsia belongs to the family
presence of broad, thin-walled, coenocytic Onygenaceae. The major species under the
hyphae with little branches in positive cases. genus are Emmonsia crescens (formerly
The branches are emerged at right angles from Chrysosporium parvum var. crescens) and
the hyphae (‘lateral peg’). The histological E. parva. The hyphae are hyaline, septate and
sections are characterised by granulomatous branched. The conidiophores come up at right
inflammation infiltrated with neutrophils, plasma angle from the vegetative hyphae and produce
cells, eosinophils and multinucleate giant cells. small aleurioconidia (2–4 μm). These spores are
In haematoxylin–eosin-stained smear, the granu- transmitted into the host by inhalation. In the
loma contains hyphae at the centre as clear or lung tissue (or in artificial culture at 40 C), the
basophilic mass. It is surrounded by a wide spores are converted into a spherical structure
(2.5–25 μm) eosinophilic sleeve (‘eosinophilic (adiaconidia). The adiaconidium matures into a
cuff’) which differentiates Conidiobolus and thick-walled spherule (200–400 μm in diameter).
Basidiobolus infection (Entomophthorales infec- No further replication of spherule is observed.
tion) from others such as pythiosis and The spherule wall is refractile and bilayered.
lagenidiosis. This sleeve is an antigen–antibody The outer layer is narrow and eosinophilic,
complex and the incident is called ‘Splendor- whereas the inner layer is wide, hyaline and
e–Hoeppli phenomenon’. The serological test chiefly made of chitin. The spherules in the tissue
such as ELISA for the detection of antibodies is appear as empty or may contain small eosino-
also developed. philic globules. When the spherule concentration
The most effective treatment in animals is increases in the body, they may collapse the lung
aggressive surgical resection of infected tissues alveoli and produce respiratory distress and
which is followed by itraconazole for 2–3 failure.
months. If surgery is not preferred by the owners, The adiaspiromycosis is detected in human,
the combination of itraconazole or amphotericin dogs, goat, horse and soil burrowing mammals
B lipid complex is recommended. The subcuta- such as armadillos, ground squirrels, mink, mice,
neous infection can be treated with itraconazole, skunks, etc. In human, three clinical forms are
amphotericin B lipid complex and potassium reported depending on the concentration of
iodide. The equine conidiobolomycosis is also spores transmitted into the body. The forms are
treated with direct injection of amphotericin B solitary granuloma, localised granulomatous
within the lesion along with systemic sodium and form and disseminated form. In low concentra-
potassium iodide therapy. tion of spores, pulmonary nodules develop and
the infection remains asymptomatic. In
disseminated form, fever, cough and dyspnoea
4.17.7 Adiaspiromycosis (Adiaspirosis, are observed due to compression and displace-
Haplomycosis) ment of alveolar parenchyma by the expanding
granulomas. Rarely the infection may spread into
Adiaspiromycosis is a non-contagious, self- the other body parts such as the skin, peritoneum
limiting pulmonary infection of mammals and bone. In animals, the infection is mostly
(rarely human) caused by dimorphic fungi of asymptomatic and light grey to yellowish lesions
the genus Emmonsia (previously known as are observed in the lungs.
Chrysosporium). The fungus was first isolated The diagnosis depends on the histopathological
by Emmons and Ashburn (1942) from wild detection of characteristic spherule in the lung
rodents in Arizona. The fungus is currently tissue. In the tissues, the spherule is detected in
worldwide in distribution and it is present as the centre of a granuloma produced by giant cells,
saprophyte in the soil. Due to production of epithelioid cells and fibrous tissues. All the
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Collection and Transport of Clinical
Material for Isolation of Fungi 5
The collection of proper clinical specimens with Ear and nose: The ear and nasal swabs are
sterile instruments and their timely shipment collected by gently mopping the ear or nasal
with appropriate arrangements into the labora- canal with the help of sterile cotton swabs. The
tory is a crucial matter for isolation and identifi- swabs should be moistened with transport
cation of fungi. The correct type of specimen medium before collection. The collected swabs
with sufficient quantity is required for proper are put into Stuart transport medium or sterile
identification. Table 5.1 describes the types of Sabouraud broth or BHI broth for transport. The
clinical specimens that can be collected from fine-needle aspirates from nasal polyp can be
different body systems and types of fungal collected with a long needle. In rhinosporidiosis,
infection that can be identified from those nasal scrape is preferred over the fine-needle
specimens. The collection methods of clinical aspirates because the lesions bleed easily.
materials and their despatch are mentioned Gastrointestinal tract: The faeces should be
below. collected in sterile, wide-mouth and screw-
Cerebrospinal fluid (CSF): The CSF is col- capped short jars. Rubber caps should be avoided
lected slowly by lumbar puncture between third because the gas generated in the collected faeces
and fourth vertebrae with a needle (18–20 s.w.g.) may blow the cap. The faeces and urine should
and a stylet (10 cm). However, in animals, the not be mixed together. Rectal or cloacal swabs
collection of CSF is not always safe. The animals can be collected as described above. Gastric
have increased CSF pressure and the collection lavages are collected by flushing the empty stom-
of CSF rapidly decreases the pressure with fatal ach with sterile water. The washings are taken
consequences. into sterile screw-capped glass containers.
Eye: The corneal swabs are collected by hold- Respiratory system: In human the sputum is
ing the palpebra apart and gently swabbing the collected in empty stomach in a screw-capped
surface with the help of sterile cotton swabs. The wide-mouth sterile container. In full stomach
corneal scrapings are taken from the base and the sample may be contaminated with bacteria
margin of corneal ulcers with a sterile spatula and other saprophytic fungi. The fresh single
under local anaesthesia. The swabs and scrapings cough specimen expectorated in the early morn-
are put into Stuart transport medium or sterile ing is the most ideal for fungal investigation. In
Sabouraud broth for transport. The anterior animals, the sputum is collected with small throat
chamber fluid of eye can be collected immedi- swabs as described above. The sputum should be
ately after death of the animal. It is done by transported and processed rapidly to reduce the
inserting a small gauge needle through the cornea contamination level. The bronchoalveolar lavage
into the anterior chamber and aspirating the fluid (BAL) is preferably collected with a broncho-
into a 3 or 5 ml syringe. scope from human patients who cannot produce

156 5 Collection and Transport of Clinical Material for Isolation of Fungi
Table 5.1 Types of clinical samples collected for fungal identification

Body parts/
system Clinical specimens Fungal infection
Central nervous Cerebrospinal fluid Cryptococcus, Coccidioides, Histoplasma, Blastomyces,
system Histoplasma capsulatum var. capsulatum, Penicillium
marneffei, Sporothrix
Eye Swabs, corneal scrapings, anterior Blastomyces
chamber fluid
Ear Swab Aspergillus
Nose Swabs, exudates, nasal scrape, fine- Rhinosporidium, Aspergillus fumigatus (horses),
needle aspirates biopsy from nasal Cryptococcus
polyp
Oral cavity Swabs, oropharyngeal washing, palatal Penicillium marneffei
papule scrapings
Gastrointestinal Faeces, gastric lavage Histoplasma capsulatum var. capsulatum, Penicillium
tract marneffei, Rhizopus
Respiratory Sputum, Bronchoalveolar lavage, Aspergillus, Blastomyces, Coccidioides, Histoplasma
system tracheal aspirate capsulatum var. capsulatum, Mucor, Penicillium marneffei,
Rhizopus, Sporothrix
Urinary tract Urine Blastomyces, Cryptococcus, Histoplasma capsulatum var.
capsulatum, Penicillium marneffei, Sporothrix
Blood Candida, Cryptococcus, Histoplasma capsulatum var.
capsulatum, Mucor, Penicillium marneffei, Rhizopus,
Sporothrix
Udder/ Milk Aspergillus, Candida, Cryptococcus, Mucor
mammary gland
Aborted foetus Abomasal content, liver, lung Aspergillus, Rhizopus
Vagina Swabs/discharge Rhizopus
Lymph node Aspirates, biopsy Blastomyces, Coccidioides, Cryptococcus, Penicillium
marneffei, Pythium insidiosum
Joint Synovial fluid Blastomyces, Sporothrix
Nail Clippings Epidermophyton, Trichophyton, Mucor
Skin Scrapings, crust Trichophyton, Microsporum, Mucor, Aspergillus (horse),
Candida, Coccidioides, Cryptococcus, Penicillium
marneffei, Pythium insidiosum, Rhizopus
Pus/exudates/fine-needle aspirates Blastomyces, Coccidioides, Histoplasma capsulatum var.
from lesion/nodule farciminosum, Madurella mycetomi, Sporothrix
Hair Microsporum, Trichophyton
Viscera Tissues Blastomyces, Candida, Cryptococcus, Histoplasma
capsulatum var. capsulatum, Madurella mycetomi,
Penicillium marneffei, Pythium insidiosum,
Rhinosporidium, Rhizopus, Sporothrix
sputum. However, flexible fiberoptic bronchos- growth of microbes. So chances of bacterial

copy is not a sterile procedure. Contamination contamination are maximum.
can take place from upper airways and some Blood: The blood sample should be collected
microbes colonise in the tracheobronchial tree from animals by venipuncture. In large animals
which is also detected in BAL samples. jugular vein or caudal vein is preferred. In pigs
Urinary tract: ‘Mid-stream’ urine collected and birds, vena cava and wing vein (brachial
during early morning is the best specimen for vein) are selected, respectively. The skin of the
mycological investigation and should be sent to collection site should be shaved and cleaned with
the laboratory immediately. It is a good media for 70 % ethanol and dried before collection. Whole
5.1 Transport of Clinical Materials 157
blood (with anticoagulant) in triplicate is used for swabs are placed into glass tubes with Sabouraud
isolation of fungi. The serum is used for serolog- broth. The pus can be also collected by fine-
ical diagnosis of fungi and testing with paired needle aspiration technique. After collection of
sera (sera collected during acute phase and con- pus or exudate in sterile syringe, the needle
valescent phase) can confirm the infection. After should be discarded and the syringe should be
collection the serum is preserved with merthio- capped before transport to reduce the environ-
late (1:1,000) or carbolic acid (0.5 %). mental contamination. The skin biopsies should
Mammary gland: Milk from the mastitic be collected both from periphery and centre of
animals is collected for fungal investigations. the lesion. The punch biopsy technique can be
The udder is washed with antiseptic solution and used under local anaesthesia.
the teats are cleaned with ethanol (70 %) swabs Hair: The affected hairs should be plucked
before collection. The primary stripping is from the lesion without breakage. The use of
discarded and later 10–15 ml of mid-stream milk Mackenzie’s hair brush, a hard-bristled hand
is collected in a sterile and capped container. brush (2.5 in. 1.5 in.), produces better isola-
Aborted foetus: The abomasal content is col- tion of dermatophytes than the hair plucking. In
lected in sterile, wide-mouth glass bottle and the human, tweezer (forcep) is used to pluck the
organs are taken in sterile petri dishes. hairs. About 10–15 hairs are collected and placed
Vagina: The area should be disinfected with into brown paper or sealed envelope for trans-
antiseptic and dried before collection of the vagi- port. The hair soiled with pus or exudates should
nal discharge. The discharge is collected in a ster- not be collected.
ile screw-capped glass container with a pipette.
Joint: The synovial fluid in sufficient amount
is collected from the joint with sterile needle and 5.1 Transport of Clinical Materials
syringe and is transported in a screw-capped
bottle. All the clinical materials should be packed prop-
Nail: The nails are swabbed with 70 % ethanol erly according to the guidelines of dangerous
and dried before collection. The affected nail bed goods regulations (DGR, available in http://
is exposed by removing the onycholytic nail www.iata.org/publications/dgr). The packaging
plate with a nail clipper. They are wrapped in should be strong enough so that no sample is
brown paper (or coloured paper) and are kept in a leaked from the container. Three-tier packaging
tight container preferably without moisture for which consists of a primary container (leak proof),
transport into the laboratory. The powdery nail secondary packaging and a rigid outer packaging
shavings are sent in glass plates. is recommended. The dry ice or pre-frozen pack
Skin: The lesion area should be cleaned with can be used surrounding the secondary packaging.
70 % ethanol to remove the bacterial contamina- The fungal agents causing animal or human
tion. The scales from the active lesions should be diseases are not included in the DGR list of infec-
collected with a blunt scalpel (or toothbrush). tious agents affecting animals (UN 2900) or
The scrapings are packed in envelope or brown human (UN 2814) except Coccidioides immitis
paper and put into airtight container for transport. (culture only). So the packaged clinical materials
To collect the exudates or pus from the skin can be labelled as ‘Diagnostic specimen’/‘Clinical
lesion, the moist cotton swabs are gently rubbed specimen’/‘Biological substance category B
over the lesion surface. After collection the (UN 3373)’ which should be clearly visible.
Diagnostic Techniques for Fungi
6
The conventional and modern diagnostic cell debris and leaves the sugar-
techniques which are used for identification of containing fungal cell wall. The diges-
fungi are discussed below. tion of keratin with the KOH can be
(a) Direct examination: It provides rapid infor- speed up with heat. An alternative prep-
mation about the presence or absence of aration is 20 % KOH with 36 % DMSO
fungi. Accordingly the treatment of the that provides rapid diagnosis without
affected animal or human may be started. heating and the specimens can be pre-
The direct examination depends on the qual- served for a long time. Addition of
ity of the sample. Parker ink with 10 % KOH (1:10) or
(i) Smears are prepared from the clinical chlorazol azole black (0.1 %) further
samples such as sputum, blood, exudates, improves the visualisation. Currently
urine deposit, tissue impression, etc. Parker ink is not available and chlorazol
in triplicate. The first two smears is mostly preferred. Chlorazol helps in
are stained with Giemsa, Gram’s or identification of hyphae by staining the
Wright’s stain (Penicillium). The third carbohydrate-rich wall without staining
smear is kept as reserve. Gram stain effec- the contaminants such as cotton or elas-
tively stains the yeast cells (Candida, tic fibres.
Histoplasma yeast phase). The yeast cells (iii) Fluorescent staining: Treatment of
are also demonstrated by the negative specimens with calcofluor white or
staining with Diff-Quik (DQ) which blankophor or acridinium orange can
demonstrates the unstained yeast cells also produce early diagnosis with better
present in the stained background. The sensitivity than wet mount. These fluo-
fine-needle aspirates can be stained with rochrome stains bind with chitin of fun-
the May –Grunwald–Giemsa method. gal cell wall and produce fluorescence
(ii) Wet mount: The clinical materials such under ultraviolet light. The arthrospores
as skin scrapings, hair including the hair appear as brightening structures.
bulb, nail fragments, sputum and pus can However, it can also non-specifically
be observed under microscope by wet stain the plant and fatty substances
mount with 10 % KOH preparation. (Fig. 6.1).
The wet mount preserves fungal mor- (iv) Capsule staining: Capsule of Cryptococ-
phology without any distortion and cus can be demonstrated by negative
helps in identification but lacks sensitiv- staining with India ink, nigrosin and
ity. The KOH acts as a clearing agent Romanowski (Fig. 6.2). Romanowski
which digests the proteinaceous host stain produces clearer capsule against

160 6 Diagnostic Techniques for Fungi
inoculated at the four corners of the plug and

a sterile coverslip is placed over the plug. A
filter paper soaked with sterile distilled water
is placed in the plate to prevent the drying of
the agar. The lid is closed and the plate is
kept at 25 C for 5–7 days. When the sporu-
lation occurs, the coverslip is taken out care-
fully and placed over a drop of mounting
fluid (Lactophenol cotton blue/Narayan
stain) in a clean glass slide keeping the fun-
gal growth side downwards. Alternatively
the mounting fluid is added over the used
slide and a clean coverslip is placed over
it. The slide is observed under low and high
Fig. 6.1 Prototheca zapfiri in calcofluor white dry magnification.
staining (1,000) (Photograph courtesy: Prof. Cornelia (c) Isolation of pathogenic fungi: Isolation of
Lass-Flörl, Medizinische Universität Innsbruck, Austria) fungi is still considered as gold standard
method for diagnosis in spite of the fact that
it is time consuming and it requires expertise.
The conventional solid medium includes
Sabouraud dextrose agar with cycloheximide
(actidione, 400 mg/L) and chloramphenicol
(50 mg/L). The cycloheximide inhibits the
growth of bacteria, yeasts and certain
moulds. So it is not incorporated in the
media intended for isolation of Aspergillus,
Cryptococcus, etc. The incubation time and
temperature varies with the fungal species.
However, the complete mould growth
Fig. 6.2 Encapsulated Cryptococcus in India ink prepa-
requires 2–5 weeks time. The yeasts are
ration (400) (Photograph courtesy: Prof. P. P. Gupta,
Ex-Director Veterinary Research, Punjab Agricultural isolated in blood agar, corn meal agar,
University, Punjab) Sabouraud’s dextrose agar (with antibiotics),
honey agar, brain–heart infusion agar and
the lightly stained background. The cap- malt agar. The plates are incubated at
sule itself can be stained with Mayer’s 28–37 C for 2 days–2 weeks. Dimorphic
mucicarmine stain. conversion (mycelium to yeast phase) can
take place when the subculture of mycelium
(b) Slide culture technique: This technique helps can be performed in brain–heart infusion
in detection of detailed fungal morphology agar with blood at 35–37 C. Identification
such as arrangement of hyphae, pattern of of fungi depends on front and reverse pig-
conidia formation, etc. A clean glass slide is mentation of the colonies, characteristics of
placed over a V-shaped, sterile glass rod in a the colonies and their microscopic appear-
petri dish. A piece of agar block (plug/1 cm2) ance (Fig. 6.3).
is cut from the already prepared Sabouraud (d) Germ tube test: The test is performed to
dextrose agar plate. The agar plug is placed confirm the presence of Candida albicans
over the slide. The fungus to be studied is or C. tropicalis. A single candidal colony is
6 Diagnostic Techniques for Fungi 161
(f) Hair perforation test: This test can differentiate

between Microsporum canis–M. equinum and
Trichophyton mentagrophytes–T. rubrum. The
child hairs are collected which should be pref-
erably free from dust, oil and dandruff. The
hairs are cut into small pieces (1–2 cm) and
are placed into a glass petri dish. The petri dish
along with hair is sterilised by autoclaving. The
plate is filled with sterile distilled water with
yeast extract (10 %) and small pieces of test
fungus culture. The positive control plates are
also prepared in similar way with M. canis or
T. mentagrophytes culture. All the plates are
incubated at 25 C and are examined weekly
for 4 weeks. Individual hair is placed on the
mounting fluid and is observed under micro-
Fig. 6.3 Fungal colony (Photograph courtesy: Prof. scope to detect whether any hair perforation
Sybren de Hoog, CBS Fungal Biodiversity Centre, The takes place like the positive control. If there is
Netherlands) no hair perforation up to 30 days, it is consid-
ered as negative.
picked up and mixed with 0.5 ml of rabbit or (g) Wood’s lamp examination: The ectothrix
sheep or bovine or human serum and dermatophyte infection of hair can be
incubated at 37 C for 1–2 h. After the incu- detected by ultraviolet rays (Wood’s lamp).
bation, a drop of preparation is observed The ectothrix spores (majority of
under the phase contrast or dry objective of Microsporum) attached with the hair shaft
the microscope. In positive case, small tubes produce a greenish-yellow fluorescence
projecting from the yeast cells without any when exposed to Wood’s lamp in a dark
constriction at the point of attachment are room. However, majority of Trichophyton
observed. This is a diagnostic feature of species (except T. schoenleinii) are negative
C. albicans and C. tropicalis (prolonged under Wood’s lamp. This test is useful in
incubation for 3 h). long-haired animals where the dermatophyte
(e) Hair bait test: The test is performed to detect lesions are not prominent in the skin. If the
keratinophilic dermatophytes from the soil. hairs are contaminated with oil-based lotions
The collected surface soil is kept in sterile used for treatment, it may produce false-
petri dish up to half of the depth. The top positive fluorescence under Wood’s lamp.
layer of the soil is made wet by sprinkling (h) Animal inoculation technique: Animal inocu-
sterile distilled water. The sterile human or lation technique is performed to isolate the
horse hairs (20–30) are spread over the sur- fungi from clinical materials and to assess
face. The petri dish is incubated at 30 C for a the pathogenicity of the fungal isolates sub-
week. After 7 days, the plate is opened to ject to permission of animal ethics committee.
detect any white/brown growth surrounding Isolation of fungi in laboratory animals helps
the hair. If the growth is detected, the hairs to obtain pure culture in artificial media.
are observed under the microscope with Table 6.1 describes the species of laboratory
mounting fluid and are transferred into animals used for isolation of different patho-
Sabouraud dextrose agar (SDA) plates. The genic fungi. White mice and guinea pigs (for
SDA plates with hairs are incubated again at dermatophytes) are preferred for pathogenic-
30 C for a week to confirm the dermatophyte. ity testing of the fungi. The animals are
162 6 Diagnostic Techniques for Fungi
Table 6.1 Use of laboratory animals for isolation of fungi

Laboratory animal Fungi
Mice Aspergillus, Blastomyces, Coccidioides, Histoplasma, Sporothrix, Candida
Guinea pigs Blastomyces, Coccidioides, Cryptococcus, Dermatophytes, Histoplasma
Hamsters Blastomyces
Rabbit Aspergillus, Coccidioides, Candida
Rat Coccidioides, Sporothrix
inoculated by intraperitoneal, intravenous, (i) Polymerase chain reaction (PCR): PCR is

intratesticular and intracerebral routes. the process by which in vitro amplification
Intratesticular and intracerebral routes are of target DNA takes place, making their
used for pathogenicity assessment of millions of copies. The amplified DNA
Sporothrix and Cryptococcus neoformans, becomes visible under ultraviolet light.
respectively. The process is highly sensitive, specific
(i) Antigen detection test: The antigen detection and requires less time and little expertise.
tests are useful in immunocompromised However, liberation of DNA form fungal
patients or in early stage of the infection cell is challenging due to the presence of a
when detectable antibodies are not produced. highly rigid cell wall which often produces
Galactomannan (GM) is the predominant false-negative result. Conventional PCR is
antigen released by Aspergillus fumigatus developed for detection of dermatophytes
which can be detected by latex agglutination (T. rubrum, T. interdigitale, M. gypseum,
test and sandwich ELISA. However, the GM M. canis, T. tonsurans, T. violaceum,
detection assay is not specific for Aspergillus E. floccosum) using nuclear ribosomal
as it is cross-reacting with other fungi such as DNA (rDNA), mitochondrial DNA
Penicillium, Fusarium, Alternaria and (mtDNA), chitin synthase-1 (chs-1), topo-
Histoplasma. Similarly, detection of D glu- isomerase II (TOP2) genes, small (18S
can (BDG) can give a presumptive diagnosis rRNA) and large-subunit (28S rRNA) of
of Aspergillus, Candida, Fusarium, ribosomal RNA as major target genes.
Pneumocystis, etc. Currently Blastomyces Identification of Candida is possible by
adhesin (BAD1) is the target antigen for spe- PCR using rRNA (5.8S, 18S, 28S), internal
cific detection of Blastomyces which do not transcribed spacer (ITS) and intergenic
cross-react with Histoplasma. spacer (IGS) region genes as target.
(j) Serological tests: ELISA-based serological (ii) Molecular characterisation: PCR may
tests can detect the specific fungal antibodies. produce false-positive result when
However, serological tests are not so much conserved rRNA or other genes are
reliable. The false-positive result occurs due targeted and airborne fungal contamina-
to cross-reaction, superficial colonisation and tion occurs. So, further confirmation of
persistence of the infection, and false-negative PCR amplicon is required by molecular
result occurs in immunocompromised patients characterisation. Randomly amplified
producing low or undetectable level of polymorphic DNA (RAPD), PCR finger-
antibodies. printing, amplified fragment length poly-
(k) Molecular biological techniques: morphism (AFLP) and nucleotide
Conventional methods used in diagnostic sequencing have been successfully
mycology usually are less specific and applied to species identification in
more time consuming. Further, a trained dermatophytes but were mostly unable
mycologist is needed to identify the fungi to discriminate between the strains. The
morphologically. So molecular biological sequencing of the internal transcribed
techniques are better alternative approach. spacer (ITS) and non-transcribed spacer
6 Diagnostic Techniques for Fungi 163
(NTS) region of the rRNA genes are and it has revolutionised the diagnostic
used for phylogenetic analysis and identi- microbiology. The method is specific,
fication of dermatophyte strains. Confir- sensitive and quick to process, and it has
mation of Aspergillus fumigatus isolates relatively low cost than the nucleic acid
by PCR-RFLP can be performed using sequencing. The technique can identify
BccI, MspI and Sau3AI restriction the fungi both from culture grown in SDA,
enzymes. blood agar and chrom agar and from the
(iii) PCR-ELISA: Recent progress includes clinical specimens such as urine. The
the use of PCR-ELISA which can spe- intact cells of the target fungi are directly
cifically identify the PCR amplified streaked onto the MALDI target plate and
product with the help of enzyme overlaid with a matrix solution lysing the
labelled probe producing colour reac- cells. The cell components after lysis are
tion in positive cases. The uniplex- embedded into the matrix. Matrices act as
PCR-ELISA can identify Trichophyton ionising agent and it also helps in energy
rubrum, T. interdigitale, T. tonsurans transfer from the laser to the analyte
and T. violaceum individually. (cell components). The laser fire desorbs
(iv) Real-time PCR: Real-time PCR is devel- the analytes from the target plate. The
oped using Taqman probe or light cycler resulting ions are accelerated in an electric
system for identification of Candida, field and focused to fly along the flight tube
Coccidioides, Histoplasma capsulatum attached with a detector. Small ions move
var. capsulatum, Mucor and Penicillium faster than the larger ones to reach the
marneffei. detector. The differences in the time
(v) DNA microarray: DNA microarray is required for the ions to reach the detector
developed for simultaneous detection of show differences in analysed peak (spec-
several pathogenic fungi such as Mucor, trum) which is specific for a species and
Aspergillus, Candida, Cryptococcus, subspecies. The species is identified by
dermatophytes, etc. comparing the test spectra with references
(vi) Loop-mediated isothermal amplification available in central database. The MALDI-
(LAMP): It is developed for the confir- TOF-MS is successfully used in differentia-
mation of mycetoma caused by tion between Trichophyton species of
Pseudallescheria boydii which do not Arthroderma benhamiae and Microsporum
require any sophisticated instrument. canis, differentiation of Candida species,
(l) Matrix-Assisted Laser Desorption Ioniza- identification of different phenotypic
tion Time-of-Flight Mass Spectrometry variations of Aspergillus species and differ-
(MALDI-TOF-MS): MALDI-TOF-MS is entiation of filamentous fungal species
used in identification of bacteria and fungi, including Rhizopus.
Appendix
KOH solution (10 %)

Composition of Commonly Used
Mounting Fluids/Stains Potassium hydroxide (KOH) 10.0 g
Distilled water 100 ml
Use: Demonstration of dermatophytes from skin
Andre and Hoyer’s fluid (mounting fluid) scrapings, hair including the hair bulb, nail fragments,
Arabic gum 15.0 g sputum and pus
Chloral hydrate 100.0 g
Glycerol 10.0 g Lactophenol cotton blue (mounting fluid)
Distilled water 25.0 ml
Phenol 20.0 g
Use: Primary study of fungal morphology Lactic acid 20 ml
Glycerol 40 ml
Giemsa stain Distilled water 20 ml
Giemsa 1.0 g After dissolving by gentle heat add 0.05 g cotton blue
Glycerol 66.0 ml Use: It is used for primary study of fungal morphology
isolated from animals and human
Absolute methyl alcohol 66.0 ml
Commercially the above composition is available which
is mixed with supplied buffer (1:9) Narayan stain (mounting fluid)
Use: Demonstration of yeast cells and moulds Methylene blue 0.5 ml
Dimethyl sulphoxide 7.0 ml
Glycerine jelly (mounting fluid) Glycerine 4.0 ml
Gelatin 1.0 g Use: It is used for primary study of fungi and algae
Glycerol 7.0 g morphology isolated from animals and human
Distilled water (with 1 % phenol) 6.0 ml
Use: Primary study of fungal morphology New methylene blue stain
Potassium oxalate 1.6 g
New methylene blue powder 0.5 g
India ink
Distilled water 100 ml
India ink 1.0 ml
Formalin (40 %) 9.0 ml Use: Differentiation of viable and dead yeast cells. Viable
cells will be unstained and dead cells will be stained as
Use: Demonstration of capsule in Cryptococcus blue colour
I. Samanta, Veterinary Mycology, DOI 10.1007/978-81-322-2280-4, 165

166 Appendix
Nigrosin stain Czapek’s Dox Agar

Nigrosin (granular) 10.0 g Potassium chloride (KCl) 0.5 g
Formalin (10 %) 100.0 ml Sodium nitrate (NaNO3) 3.0 g
The solution is placed in hot water bath for 30 min and is Di-potassium hydrogen phosphate (K2HPO4) 1.0 g
filtered twice through double filter paper (Whatman Magnesium sulphate 0.5 g
No. 1) (MgSO4, 7 H2O)
Use: Demonstration of capsule in Cryptococcus Ferrous sulphate 0.01 g
(FeSO4, 7 H2O)
Sucrose/glucose 30.0 g
Phol stain
Agar 15.0 g
Formalin (4 %) 5.0 ml Distilled water 1,000 ml
Methylene blue (3 %) 0.3 ml
Use: Isolation of yeast and mould
Glycerol 3.0 ml
Use: Morphology study of dermatophytes, yeasts and
algae
Dermatophyte test medium
Wright’s stain Papaic digest of soybean meal 10.0 g
Glucose 10.0 g
Wright’s stain powder 0.3 g
Phenol red 0.2 g
Glycerol 3.0 ml
Agar 20.0 g
Methyl alcohol 97.0 ml
Distilled water 1,000 ml
Use: Demonstration of yeast cells and moulds
pH (at 25 C) 5.5 0.2
Use: Selective isolation of dermatophytes
Composition of Commonly Used
Media in Diagnostic Mycology
Mycosel agar
Blood agar Papaic digest of soybean meal 10.0 g

Dextrose 10.0 g
Peptone 15.0 g
Agar 15.5 g
Liver extract 2.5 g
Cycloheximide 0.4 g
Yeast extract 5.0 g
Chloramphenicol 0.05 g
Sodium chloride 5.0 g
Agar 15.0 g
Distilled water 1,000 ml Use: Isolation of pathogenic fungi from contaminated
pH (at 25 C) 7.4 0.2 clinical materials
Use: Isolation of yeast
Potato dextrose agar

Corn meal agar
Potatoes (peeled) 200.0 g
Corn meal 2.0 g
Dextrose 20.0 g
Tween 80 (1 %) 10 ml
Agar 15.0 g
Agar 15.0 g
pH (at 25 C) 5.6 0.2
Use: Detection of chlamydospore-producing Candida and
isolation of fungi Use: Isolation of fungi from clinical materials
Appendix 167
Sabouraud dextrose agar (Emmon’s)

Dextrose 20.0 g Chloramphenicol 50.0 mg
Peptone 10.0 g Actidione 400.0 mg
Agar 20.0 g Distilled water 1,000 ml
Use: Isolation of dermatophytes and dimorphic fungi
pH 7.0 0.2
from clinical materials
Use: Maintenance of fungal culture
Sabouraud dextrose agar (Emmon’s) with antibiotic Sunflower seed agar (Pal’s medium)
Dextrose 20.0 g Pulverised sunflower seed 45.0 g
Peptone 10.0 g Chloramphenicol 100 mg
Agar 20.0 g Agar 20.0 g
Chloramphenicol (dissolved in 95 % ethanol) 50.0 mg Distilled water 1,000 ml
Use: Isolation of Cryptococcus neoformans
Use: Isolation of fungi from clinical materials
Sabouraud dextrose agar (Emmon’s) with antibiotic and

cycloheximide Water agar
Dextrose 20.0 g Agar 20.0 g

Peptone 10.0 g Distilled water 1,000 ml
Agar 20.0 g Use: Cultivation and enumeration of some fungi
(continued)
Glossary
Acropetal A chain of conidia in which the Autopsy Post-mortem examination of human or

youngest conidium is at the tip and the oldest animal
is at the base Basidiospore A haploid spore produced on a
Adiaconidia A large, globose, thick-walled basidium following karyogamy and meiosis
conidium, usually produced by Emmonsia Basidium (plural, basidia) A cell that gives
parvum in the lungs of human and animals rise to a basidiospore
Aerial mycelium Hyphal elements growing Basipetal The youngest conidium is found at
above the agar surface the base of a chain and the oldest is found at
Aleuroconidium (plural, aleuroconidia) A the tip
conidium that develops as an expanded end of Biopsy Collection of tissue from live animal or
an undifferentiated hypha or on a short pedicel human
and is released by rupture of the supporting cell Bipolar budding Blastoconidia developing at
Anamorph Asexual form of the fungus the opposite poles of a parent cell
Annellation Formation of ring-like structures at Blastospores Asexual spores produced by bud-
the conidiogenous end of a conidiophore ding of yeast
Annellide A conidiogenous cell that produces Budding Asexual reproduction in which a bud
conidia in a basipetal way is formed from the parent cell
Anthropophilic An organism that primarily Chlamydospore A conidium with a thickened
infects human cell wall that may be terminal or intercalary
Apophysis Funnel-shaped swelling of a sporan- and serves the function of survival
giophore which is present immediately below Clavate Club shaped
the columella Cleistothecium (plural, cleistothecia) An
Appressorium A swelling on a germ tube or enclosed fruiting body that contains asci
hypha which is used for attachment in an Coenocytic (aseptate) hyphae Hyphae without
early stage of infection septa
Arthrospores Asexual spores produced by Collarette A remnant of a cell wall present at
fragmentation of hyphae the tip of a phialide or around a
Ascocarp A fruiting body containing asci and sporangiophore
ascospores Columella A sterile, dome-shaped expansion at
Ascospore A haploid sexual spore formed in an the apex of the sporangiophore
ascus following meiosis Conidiogenous cell A cell that produces conidia
Ascus (plural, asci) A sac-like structure in Conidiophore A specialised hypha on which
which ascospores are produced. Asci are char- conidia are generated singly or in clusters
acteristic of Ascomycetes Conidium (plural, conidia) A nonmotile asex-
Aspergilloma A fungus ball composed of ual spore which is produced from fungal mito-
Aspergillus hyphae and is located within the sis. It is either unicellular (microconidium) or
lung cavity multicellular (macroconidium)

170 Glossary
Dematiaceous Pigmented fungi Hyaline Translucent or colourless

Denticle A small projection on which conidia Hypha (plural, hyphae) Tube-like structure
are produced which makes the mycelium of a fungus
Dichotomous Fungi having equal branching of Intercalary Occurring within a hypha
hyphae, e.g. Aspergillus Lanose Woolly
Dimorphic Fungi having two different morpho- Merosporangium (plural, merosporangia) A
logical forms, i.e. yeast and mould small cylindrical sporangium with the
Double septum A two-layered septum that sporangiospores aligned in a row
undergoes centripetal separation (schizolysis) Metula (plural, Metulae) A sterile cell below
to release a conidium phialides (e.g. Aspergillus, Penicillium)
Echinulate Covered with spines Mould Mycelial fungus
Ectothrix Dermatophyte infection in which Multipolar budding Blastoconidia developing
arthrospores are produced outside the hair at different sites on the surface of a parent cell
shaft, e.g. Trichophyton verrucosum Muriform Conidia with longitudinal or trans-
Endothrix Dermatophyte infection in which verse wall (e.g. Alternaria)
arthrospores are produced inside the hair Mycelium (plural, mycelia) A mat of
shaft, e.g. Trichophyton schoenleinii intertwined hyphae
Floccose Loose, cottony texture Mycetism Infection of human or animals due to
Foot cell A basal cell of a conidiophore in vivo toxin production by the fungi
(Aspergillus and Fusarium) Mycosis (plural, mycoses) Fungal disease of
Fungus (plural, fungi) Eukaryotic, heteroge- human or animal
nous, unicellular to filamentous, spore bear- Mycotoxicosis Intoxication of human or
ing, chemoorganotrophic organisms which animals due to consumption of mycotoxin-
lack chlorophyll contaminated food or feed
Geniculate Bent like a knee Obclavate Club shaped in reverse
Geophilic Soil-inhabiting organisms Ostiole An opening through which spores or
Germ tube Small tubes projecting from the conidia can escape
yeast cells without any constriction at the Pectinate Like the teeth of a comb
point of attachment, e.g. Candida albicans Pedicel A slender stalk
Gloiospora Conidia aggregated in slimy heads Penicillus (plural, penicillin) Brush-like
at the tip of an annellide or phialide conidiophore of Penicillium
Gymnothecium (plural, gymnothecia) A Perfect stage Sexual state of the fungi
fruiting body composed of loosely interwoven Phialide Sac-like structure that produces
hyphae and asci conidia (e.g. Aspergillus)
Habitat Natural environment of an organism Phialoconidium A conidium produced from a
for multiplication phialide
Haustorium A special hyphal branch which is Phragmoconidium A conidium having two or
used for absorption of food more transverse septa
Heterothallic During sexual reproduction two Poroconidium A conidium produced through a
fusion gametes are produced by different thallus small pore in a conidiogenous cell
Hilum A scar at the base of a conidium Propagule A reproductive unit that gives rise to
Holomorph Whole fungus which includes both an organism
the anamorph and the teleomorph state Pseudohyphae Sometimes the buds from the
Holothallic A mode of conidia formation when parent cell fail to detach and they produce a
all the cell wall layers of the conidiogenous or chain of elongated hyphae like filament called
sporogenous cells are involved ‘pseudohyphae’
Homothallic During sexual reproduction two Pseudomycelia Mass of pseudohyphae
fusion gametes are produced by the same thallus Pyriform Pear shaped
Glossary 171
Rhizoid Short branching hypha resembling a Sporodochium A cushion-shaped mass of

root (e.g. Rhizopus) hyphae bearing conidiophores
Saprobe (or saprotroph) An organism that Sterigmata Specialised hypha bearing a
obtains its nutrients by absorption of soluble conidium (e.g. Aspergillus)
organic compounds from nonliving or Stolon A running hypha from which rhizoids
decaying organic matter, plant or animal and sporangiospores arise
Sclerotium (plural, sclerotia) An organised Sympodial Conidiophores which produce conidia
mass of hyphae that remains dormant on geniculate or zig-zag basis (e.g. Fonsecaea)
during unfavourable conditions (also called Synnema (plural, synnemata) Erect macro-
‘grain’) scopic structure formed by fused
Septate Hyphae having septum conidiophores that bear conidia terminally,
Septum (plural, septa) Cross wall in a myce- laterally or in both ways
lium or conidia Teleomorph A sexual state of fungi
Sessile Direct hyphal attachment Thallus (plural, thalli) Vegetative body of a
Spherule Circular, thick-walled, closed struc- fungus
ture containing endospores Tuberculate Small spine-like projections/
Spinulose Covered with small spines wart-like structures from a conidium
Spitzenkorper A structure present in fungal Uniserate Phialides arising directly from a
hyphae consists of small vesicles and it vesicle (e.g. Aspergillus)
organises the fungal growth Verrucose Having many warts
Sporangiolum A small sporangium producing a Vesicle Swollen cell produced at the terminal
small number of sporangiospores end of conidiophores
Sporangium (plural, sporangia) Closed spher- Zoospores Motile asexual spores
ical structure containing sporangiospores Zygospores Thick-walled sexual spores
Index
A Apophysis, 73, 74, 79, 169

Abattoir, 16 Apothecia, 8
Abortion, 41, 55, 77, 79, 90 Armadillo, 114, 115, 144
Acacia, 123 Arthroderma, 13, 14, 23, 24, 163
Acervulus, 8 Arthrospore, 7, 16, 20, 24–26, 30, 31, 52, 54, 79, 159, 169
Achorion, 11, 29 Ascospore, 8, 33, 45, 121, 122, 169
Acid-fast, 49, 126 Aspergillosis, 2, 32, 35, 37, 41, 43
Acremonium, 120, 121 Aspergillus, 1, 2, 6, 10, 14, 18, 24, 32–44, 61, 77, 78,
Acridinium orange, 159 83, 87, 88, 121, 126, 156, 162, 163
Adiaconidia, 144 Asteroid body, 118
Adiaspiromycosis, 144–145 AT island, 15
Aerial, 4, 7, 15, 25, 46, 59, 73, 84, 86, 124, 143 Atria, 37
Aflatoxin, 32, 34–36 Auto-inoculation, 11, 71
African glanders, 55 Auxotroph, 142
African histoplasmosis, 65, 90
Agroclavin, 76 B
Ajellomyces, 45 BAD1. See Blastomyces adhesin 1 (BAD1)
Alcian blue, 49, 92 BAL. See Bronchoalveolar lavage (BAL)
Algae, 128, 138–141 Bamboo rat, 83, 87
Allescheria, 120 Barcode sequence, 20
Alopecia, 18, 26, 27, 110 Basic helix loop-helix (bHLH), 87
Alternaria, 10, 35, 43, 88, 132–135, 162 Basidiobolus, 10, 74, 142–144
Amphotericin B, 44, 50, 58, 61, 67, 72, 78, 79, 83, Basidiospore, 8, 96, 169
91, 102, 112, 119, 124, 129, 132, 135, 138, Basidium, 8, 93, 169
140, 144, 145 Basipetal, 85, 169
Amplified fragment length polymorphism (AFLP), Bat, 44, 45, 61, 62, 123
20, 162 BCP milk solid glucose agar, 15
Anamorph, 13, 14, 23, 24, 29, 85, 121, 123, 169 β-D glucan (BDG), 35, 43, 49, 111, 112, 137, 138, 162
Aneuploid, 105 Behcet’s syndrome, 31
Angioinvasion, 40, 43, 76–78 Benzalkonium chloride, 30, 75
Anidulafungin, 135 Berenil, 72
Annelloconidia, 122 Biopsy, 20, 48, 56, 66, 72, 78, 90, 102, 110, 118, 126,
Annotation, 129 156, 157, 169
Antheridium, 7, 8, 128 Bipolar, 102, 133, 169
Anthropophilic, 13, 14, 17, 23, 24, 29–31, 169 Bipolaris, 10, 132, 133, 135
Antibiotic, 6, 15, 32, 37, 41, 43, 55, 61, 66, 75, 84, Black yeast, 132, 134, 135
95, 105, 110, 124, 130, 134, 145, 160, 167 Blankophor, 20, 126, 159
Antifungal, 5, 6, 15, 21, 29, 32, 33, 38–40, 44, 47, 58, Blastoconidia, 7
61, 63, 76, 78, 79, 84, 97, 102, 105–107, 109, Blastomyces, 3, 5, 10, 44–51, 57, 60, 101, 156, 162
110, 112, 119, 121, 132, 134, 135, 140–142, 145 Blastomyces adhesin 1 (BAD1), 45–49, 162
Antigen presenting cells (APC), 96, 130 Blastomycin, 49
Antler hyphae, 4 Blastospores, 3, 7, 111, 169
Aphthae albae, 1, 102 Bread mould, 75
Apical extension, 3 Broad-based bud, 44, 48
Aplanospore, 7, 138 Bronchoalveolar lavage (BAL), 42, 48, 78, 83, 137,
Aplerotic, 128 155, 156

174 Index
Budding, 3, 6, 7, 39, 45, 47, 59, 66, 84, 91, 101–103, 109, Coiling phagocytosis, 42
110, 114, 132, 136, 169, 170 Collarettes, 133, 169
Bursattee, 2, 127 Columella, 74, 79, 80, 169
Complement fixation test (CFT), 49, 57, 66, 67
C Conidia, 7, 11, 12, 14–17, 21, 22, 32–35, 37–40, 42–47,
Cadherin, 108 53, 56, 59, 63, 84–87, 89, 113–116, 118, 121–124,
Calcofluor, 20, 48, 56, 78, 82, 126, 135, 140, 159, 160 126, 132–134, 143, 160, 169
California disease, 55 Conidiobolus, 10, 74, 142–144
Campy blood agar, 129 Conidiogenol, 86
Canary, 83, 93 Conidiogenone, 86
Candida, 3, 6, 10, 30, 35, 43, 83, 95, 102–112, 156, Conidiophore, 7, 32–34, 43, 44, 84, 85, 87, 113, 121,
159, 160, 162, 163, 166 122, 133, 143, 144, 169
Capilliconidia, 143 Copper sulphate, 112
Capsule, 5, 47, 49, 58, 92, 94–101, 113, 118, 138, Copulatory beak, 143
159, 160, 165, 166 Corneal, 41, 48, 131, 155, 156
Carcinogen, 36, 88 Cornmeal tween-80 agar, 111
Carnation leaf agar, 133 Counter immune electrophoresis (CIE), 126
Caspofungin, 78, 112, 132 Crop mycosis, 110, 112
Cat, 16, 17, 23–29, 48, 55, 57, 65, 67, 70, 77, 83, 91, 94, Cross pathway control, 39
100, 110, 114–121, 131, 135–137, 139, 142, 143 Cryptococcus, 3, 5, 6, 10, 35, 49, 74, 88, 91–102, 109,
Cathepsin, 28 156, 160, 162, 163, 165–167
Cave, 24, 61, 62 Crystal violet, 105
Cell wall proteins (CWP), 103 CSF. See Cerebrospinal fluid (CSF)
Ceramide, 59 Curvularia, 120, 121
Cerebrospinal fluid (CSF), 48, 49, 66, 79, 89, 95, 101, Cycloheximide, 6, 15, 25, 30, 35, 46, 52, 61, 75, 88, 95,
118, 155, 156 106, 115, 133, 139, 160, 166, 167
CFT. See Complement fixation test (CFT) Cyclophilin, 64
Chemotaxis, 96, 125, 128 Cyst, 128, 130, 136, 138
Chitin, 4, 5, 7, 19, 20, 26, 33, 35, 37, 38, 42, 48, 50, 56, 59, Cytokine, 19, 28, 38, 42, 47, 56, 63–65, 78, 89, 96, 101,
63, 68, 78, 103, 119, 127, 138, 141, 144, 159, 162 108–110, 118, 126
Chlamydopsore, 7, 24, 62, 103, 111, 126, 166, 169 Czapek dox agar, 87
Chlamydospore agar, 111
Chloramphenicol, 6, 15, 25, 30, 61, 106, 115, 124, 130, D
160, 166, 167 Daisy flower, 113
Chlorazol black, 20 Damage associated molecular pattern (DAMP), 42
Chlorella, 138 Dangerous goods regulations (DGR), 157
Chlorhexidine gluconate, 105 Dapsone, 72
Chlorine, 14, 51 Dectin, 19, 28, 39, 40, 55, 56, 63, 109, 111
Chloroxylenol, 70, 80 Deep sequencing, 34
Christae, 68, 136 Defensin, 40, 110
CHROMagar, 106 Deferoxamine, 76
Chromosome, 5, 15, 34, 52, 61, 70, 81, 87, 94, 105, Delayed type of hypersensitivity, 49
115, 136 Dendritic cells, 38, 42, 54, 55, 64, 96, 99, 109, 111,
Cigar shaped bud, 113, 118 126, 137
Citrinin, 36, 88 Denticle, 113, 133, 170
Cladophialophora, 10, 132, 133, 135 Derjaguin–Landau–Verwey–Overbeek (DLVO)
Cladosporium, 10, 132–135 theory, 108
Classification, 9–11, 13–14, 22–23, 29–30, 34, 45, 51, Dermatitis, 18, 19, 27, 55, 65, 83, 90, 110, 135,
60, 69–70, 74, 80, 85, 92–93, 104, 112, 114, 139, 143
122–123, 128, 136 Dermatophyte test medium, 166
Clavate, 22, 29, 113, 133, 169 Dermatophytosis, 1, 11, 17, 24, 27, 29
Cleistothecia, 8, 45, 121, 169 Desert fever, 55
Clofazimine, 141 Diabetes mellitus, 76, 77, 110
Coccidioides, 1, 3, 10, 49–58, 60, 68, 72, 145, 156, Diabetic ketoacidosis (DKA), 77
157, 162, 163 Diarrhoea, 29, 57, 65, 77, 83, 119, 131, 139
Coccidioides-specific antigen (CS-Ag), 53 Diclinous, 128
Coccidioidin, 53, 57 Diff-Quik (DQ), 49, 137, 159
Coccidioidomycosis, 50, 52, 54–57, 119 Dihydrofarnesol, 30
Coenocytic, 4, 73, 78, 79, 83, 127, 131, 141, 143, 144, 169 Dikaryotic hypha, 93
Index 175
Dimorphic, 3, 5, 14, 50, 58, 79, 80, 82, 84, 85, 87, 91, 102, Fomite, 16, 31, 45
113, 115, 144, 160, 167, 170 Fonsecaea, 10, 132, 133, 135
Diploid, 7, 105, 115, 136 Foot cell, 32, 84, 85, 170
Discharge tube, 127, 128 Formaldehyde, 30, 51, 93, 114
Distosepta, 133 Fruiting body, 8, 43
DMSO, 19, 159 Fumagillin, 37
Dog, 11, 14, 16, 17, 22–28, 30, 31, 41, 44, 46–48, Fumitremorgin, 37
50, 55, 57, 58, 65–67, 70–72, 77, 80, 86, 91, Fungaemia, 55, 99
100, 104, 110, 114, 115, 117–119, 121, 125, Fungi, 1–10, 14–18, 23, 24, 26, 27, 32–43, 45–48,
129, 131, 132, 135–137, 139–144 51–53, 55–57, 61–65, 70, 73–78, 82–94, 96,
Dolphin, 91, 140, 141 100, 107–110, 112, 114–121, 124–128, 131–145,
Domain, 15, 36, 46, 53, 63, 111 155–157, 159–163, 165–167
Downs strain, 61 Fungi imperfecti, 33
DRIP group, 70 Fusarium, 35, 43, 88, 120, 121, 126, 162
E G
Echinulate, 33, 170 Galactomannan, 5, 33, 35, 37, 38, 43, 62, 88, 90, 162
Ecoconazole, 21 Gametangium, 7, 8, 74
Electrotaxis, 128 GC. See Guanine-cytosine (GC)
ELISA. See Enzyme-linked immunossorbent Geniculate, 133, 170
assay (ELISA) Genome, 5, 15, 24–25, 30, 34–36, 38, 46, 52, 57, 61, 70,
Emmonsia, 10, 144, 145 75, 80, 81, 85, 87, 94–95, 105–106, 115, 123, 129,
Encephalitis, 135 136, 138, 139, 141
Endogenous antisense transcript, 94 Genome sequencing and genealogical concordance
Endogeny, 136 phylogenetic species recognition (GCPSR), 85
Endosporulation antigen (EA), 54, 55 Genomic island, 52
Enilconazole, 24, 30, 44 Genomic plasticity, 87
Entomophthoromycosis, 73 Gentamicin, 15, 25, 124, 140
Enzyme-linked immunossorbent assay (ELISA), Geophilic, 13, 14, 23–25, 28, 170
43, 49, 57, 66, 67, 90, 101, 111, 112, 119, Germosporangium, 74
127, 131, 142, 144, 162 Germ tube, 39, 40, 82, 84, 103, 110, 114, 130, 160, 170
Eosinophilic sleeve, 144 Giant cell, 83, 118, 125, 131, 135, 141, 144
Epidermophyton, 4, 10, 13, 22, 23, 29–32, 156 Giemsa, 20, 57–59, 92, 118, 137, 159, 165
Epizootic lymphangitis, 65 Gliotoxin, 36, 37, 40
Ergosterol peroxide, 116 Globose, 33, 73, 121
Erythema, 18, 27, 82, 140 Glucan, 4–6, 33, 35, 38, 50, 59, 80, 106, 113
Eucalyptus, 91, 94 Glutaraldehyde, 30, 45, 93
Exon, 25, 52, 94, 129 GM-CSF. See Granulocyte macrophage-colony
Exophiala, 10, 121, 132–135 stimulating factor (GM-CSF)
Exudate, 11, 41, 48, 56, 66, 83, 110, 118, 125, 126, GMS. See Grocott methenamine silver (GMS)
130, 140, 141, 144, 156, 157, 159 Gomori methenamine silver, 126, 131, 135, 140
G protein-coupled receptor (GPCR), 91
F Grain, 34, 76, 81, 82, 120, 121, 124–126
FAT. See Fluorescent antibody technique (FAT) Granulocyte macrophage-colony stimulating factor
Favic chandeliers, 4, 5 (GM-CSF), 19, 65, 89
Favus, 11, 24, 27 Granuloma, 17, 18, 41, 42, 44, 55, 77, 83, 100, 117, 118,
Fibronectin, 37, 38, 64, 108, 115, 116, 124, 136 135, 141, 144
Filobasidiella, 91, 93 Graphium, 121–123
Filopodium, 136 Griseofulvin, 11, 21, 24, 29
Fine-needle aspirates, 20, 49, 72, 155, 156 Grocott methenamine silver (GMS), 20, 49, 57, 90, 103,
Fission, 6, 84, 85, 90, 136 118, 131, 141, 142
Fluconazole, 21, 44, 50, 57, 58, 61, 67, 102, 119, Grocott’s silver, 43
127, 132, 145 Growth, 5–7, 15, 17, 25, 30–31, 33, 35, 38–40, 42, 46, 47,
Flucytosine, 90 52–54, 61–63, 65, 70–73, 75–77, 79–82, 84,
Fluorescent antibody technique (FAT), 45, 66, 90, 101 87–88, 91, 94–96, 98, 100, 103–107, 115, 124,
Fluorescent in-situ hybridization (FISH), 111 129, 130, 133, 135, 139, 156, 160, 161
Flux, 138 Guanine-cytosine (GC), 15, 25, 52, 87, 115, 123
Folate, 40, 139 Guano, 61, 123
176 Index
H Inhibitory mould agar, 46

Haemagglutination, 66, 126 Insect, 34, 61, 74, 76, 81, 83, 114, 115, 128, 140–143
Haematoxylin and eosin, 49, 57, 59, 72, 78, 83, 90, Integrin, 64, 108
118, 126, 135, 138, 142, 144 Intergenic spacer (IGS), 112, 162
Haemoglobin response gene (HBR), 105 Internal transcribed spacer (ITS), 57, 78, 127,
Haemolysin, 36, 40, 107 135, 142, 163
Hair bait test, 161 Intracystic, 136
Hair perforation test, 161 Introgression, 52
Haploid, 7, 45, 80, 91, 93, 105, 136 Intron, 15, 25, 52, 94, 95, 123, 129, 136
Haploid fruiting, 93 Invasin, 107, 108
Hartley digest agar, 61 Iodide toxicosis, 119
Heat shock protein, 35, 38, 63, 98, 107 Iodophor, 30
Helvolic acid, 37 Isorophyl alcohol, 80
Heterokaryon, 12 Isotropic growth, 40, 54
Heterothallic, 7, 14, 24, 45, 74, 80, 86, 170 Itraconazole, 21, 29, 44, 50, 57, 61, 67, 90, 102,
High efficiency particulate air (HEPA), 56 119, 120, 127, 132, 135, 141, 142, 144
High mobility group (HMG), 46, 74, 80
High resolution melt analysis (HRMA), 83 K
Hila, 133 Karyogamy, 7, 74, 105, 169
Histidine kinase, 39, 44, 60 Keratin, 5, 11, 14, 16, 17, 19, 26, 159
Histiocyte, 58, 72, 83, 89, 90, 118, 141 Keratitis, 41, 64, 65, 90, 142
Histofarcin, 66, 67 Kerion of Celsus, 1, 11
Histo-hazard, 62 Ketoconazole, 21, 29, 44, 58, 61, 67, 72, 90, 102, 127, 145
Histoplasma, 2, 3, 5, 7, 10, 35, 43, 46, 49, 51, 57–68, Kexin, 136, 137
88, 101, 156, 159, 162, 163 KOH, 19, 20, 31, 42, 48, 56, 72, 118, 126, 130, 135,
Histoplasmin, 62, 67 144, 159, 165
Hodgkin’s disease, 54, 84 Kunker, 130, 131
Holomorph, 85, 170
Homothallic, 7, 45, 74, 79, 80, 105, 106, 170 L
Honey agar, 95, 160 Lacazia, 141
Human immunodeficiency virus (HIV), 67, 82, 84, 90, Lactoferrin, 40
94, 100, 136, 137 Lactophenol cotton blue, 43, 140, 160, 165
Hyaline, 12, 79, 84, 113, 121, 126, 127, 141, 144, 170 Lagenidium, 10, 141–142
Hydrogen peroxide, 63, 70 Laminin, 38, 108, 116, 124
Hydrophobic interaction, 108 Larvae, 129, 141, 142
Hyphae, 2–5, 7–9, 11, 12, 19, 20, 22, 26, 27, 29, 31–33, Lateral peg, 127, 144
35, 38–40, 42–47, 50–52, 59, 62, 73, 74, 77–80, Lectin, 19, 37, 40, 96, 97, 108, 111
82–87, 93, 102–104, 107–109, 113, 114, 116, 121, Lid-lifter, 81
122, 126–132, 135, 141–144, 159, 160, 169–171 Linear, 59, 136
Litter, 37, 134, 137
I Lobomycosis, 140–141
Identity island, 94 Long mosaic, 15
Idiomorph, 14, 24, 33, 46, 94 Loop-mediated isothermal amplification (LAMP),
Imizol, 72 109, 127, 163
Immunochromatographic, 131 Lymphocutaneous, 113, 116, 117, 119
Immunocompetent, 27, 31, 50, 82, 83, 89, 100, 145 Lymphocyte, 19, 48, 72, 83, 96, 101, 118, 125, 135
Immunodiffusion (ID), 49, 57, 66, 67, 90, 126, 131 Lymphokine activated macrophages, 48
Immunodominant, 15, 31, 35, 46, 48, 53, 62, 71, 88, Lysozyme, 40
90, 124, 129, 136
Immunofluorescence, 132 M
Immunoperoxidase, 90, 132 Mackenzie’s hair brush, 157
Immunoreceptor tyrosine-based activation (ITAM), 42 Macroconidium, 7, 169
Immunosuppression, 17, 31, 36, 48, 67, 81, 99, 107, Madurella, 120–127, 156
108, 110, 116, 117 Majocchi’s granuloma, 17, 18
Incidental host, 62 Major histocompatibility complex (MHC), 19
India ink, 92, 101, 156, 160, 165 Major repeat sequence (MRS), 105
Indirect haemagglutination assay (IHA), 126 Major surface glycoprotein, 136
Induced endocytosis, 108 Mannan, 5, 16, 19, 33, 37, 56, 59, 96, 106, 108, 111, 112
Infundibulum, 37 Mannan binding lectin (MBL), 37, 40
Index 177
M antigen, 62, 63, 66, 67 O

Mastitis, 37, 41, 42, 55, 73, 82, 83, 90, 91, 94, 100–102, Obstipation, 139
110, 112, 138–140, 157 Ochratoxin, 36, 88
MAT, 14, 23, 24, 46, 74, 87, 94 Ochroconis, 10, 132–135
Matrix Assisted Laser Desorption Ionization Oidia, 7, 114
Time-of-Flight Mass Spectrometry Olecranon, 139, 140
(MALDI-TOF-MS), 20, 78, 163 Onychomycosis, 14, 18, 19, 21, 27, 31
May–Grunwald–Giemsa, 20 Onychorrhexis, 135
Mercuric chloride, 83 Oogonium, 7, 8, 128
Mesomycetozoa, 70 Oosphere, 8
Methenamine silver, 20, 43, 57, 59, 72, 90, 110, 118, Oospores, 8, 128, 142
126, 131, 135, 140–142 Opsonin, 37, 42, 54, 117
Metulae, 43, 84, 85, 87, 170 Osmotrophic, 5
Micafungin, 112 Osteoarticular, 90, 117
Miconazole, 21, 44, 83, 90, 132 Ostiole, 8, 170
Microarray, 83, 163 Oxidative burst, 63, 118, 126, 134
Microconidium, 7, 169 Oxidative stress, 34, 38, 39, 63, 88, 96, 98, 104, 107, 109
Microcystis, 69 Oxoid chromogenic Candida agar (OCCA), 106
Microfocus, 45
Micro RNA, 94 P
Microsporum, 4, 10, 14, 21–29, 32, 121, 156, 161, 163 Paecilomyces, 85, 120
Mitochondria, 5, 20, 25, 30, 38, 68, 69, 75, 81, 87, 88, 92, Panleukopenia, 77, 110
94, 95, 97, 98, 115, 123, 136–139, 162 Papule, 17, 18, 26, 27, 64, 72, 82, 89, 90, 116,
Mitogen activated protein (MAP), 34, 97, 104, 109 140, 141, 156
Monilia, 102 Parrot, 93, 100, 114, 121, 125
Monokaryotic fruiting, 93 Pathogen associated molecular pattern (PAMP), 19, 42,
Mosquito, 128, 129, 141, 142 55, 56, 78, 108, 109, 111, 117
Mould, 3, 5, 9, 46, 49, 50, 52, 58, 59, 75, 76, 87, 88, Pathogenicity, 34, 62, 116, 161, 162
114, 115, 160, 165, 166, 170 Pattern recognition receptor (PRR), 28, 37, 42, 55,
Mould to yeast, 114 56, 78, 96, 109, 111, 117
Mucor, 1, 10, 73–75, 78–83, 101, 156, 163 PCR-ELISA, 20, 28, 32, 163
Mucormycosis, 73, 75–77, 83 Peach flower, 113
Multidrug resistance, 87 Pectinate body, 4, 5
Mycelium, 3, 5–7, 15, 20, 25, 31, 32, 34, 44, 59, 61, Pedunculated, 71, 72
63, 66, 74, 84, 113, 116, 122, 124, 132, 160, 170 Penicillium, 4, 10, 34–36, 43, 61, 83–91, 101, 156,
Mycetism, 1, 170 159, 162, 163, 170
Mycetoma, 1, 120–127, 163 Penicillus, 83, 86, 170
Mycetoma-belt, 123 Peptidoglycan, 104
Mycobiotic agar, 61 Peptidorhamnomannan, 115, 116, 124
Mycology, 1–2, 11–145, 162, 166–167 Peptone yeast glucose (PYG) agar, 129, 142
Mycophagous, 61 Periodic acid Schiff (PAS), 2, 20, 43, 49, 57, 59, 68,
Mycosis, 1, 18, 27, 32, 41, 48, 52, 57, 73, 77, 90, 110, 90, 101, 110, 118, 126, 131, 135, 140, 141
112, 114, 170 Perithecia, 8
Mycosis mucorina, 73 Peritoneal exudate cell (PEC), 108
Mycotoxicosis, 1, 170 Peroxisome, 84
Phaeohyphomycosis, 132–135
N Phagocytosis, 16, 36, 39, 42, 53, 54, 59, 64, 82, 95–97,
Nail, 18, 19, 21, 27, 31, 32, 81–83, 125, 135, 156, 99, 107, 116, 117, 126, 134
157, 165 Phenol, 14, 15, 165, 166
Narrow-based bud, 91 Phenotypic switch, 59, 60, 95
Nasal scrape, 72, 155, 156 Pheromone, 80, 87, 94, 105
Neem, 21, 29 Phialide, 32, 33, 43, 84–87, 121, 133, 169–171
Neosartorya, 33, 34 Phialophora, 10, 121, 132–135
Nephritis, 135 Photophobia, 41, 72
Neutrophil entrapment trap (NET), 42 Phytopythium, 128
Nitric oxide, 53, 64, 65, 89, 98, 118 Pigeon, 45, 91, 93, 94, 100
NOD like receptor, 40, 111 Plaque, 17, 18, 27, 40–42, 82, 83, 110, 135, 139, 141
Nodule, 4, 18, 42, 66, 77, 82–84, 90, 116, 117, 125, Plasma cell, 72, 83, 125, 131
135, 140, 141, 144, 156 Plasmalemma, 3
Non-transcribed spacer (NTS), 20, 163 Plasmodesmata, 74
178 Index
Plasmogamy, 7, 74, 80 Ripening, 88

Pleomorphism, 15, 25 Rodlet, 38, 40
Pleuropneumonia-like organism (PPLO) agar, 61, 67 Rolling circle amplification (RCA), 127
Pneumocystis, 10, 35, 135–138, 162 Romanowski, 101, 159
Pneumonia, 41, 48, 55, 65, 100, 102, 135–137, 143 Rose gardeners’ disease, 114, 117
Polar growth, 40 Rose thorn, 114
Polyketide, 34, 47, 76, 84 Rotifer, 141
Polymerase chain reaction (PCR), 20, 28, 32, 49, 57, 67,
78, 83, 90, 95, 102, 112, 119, 127, 132, 137, 140, S
142, 145, 162, 163 Sabouraud dextrose agar, 15, 25, 30, 46, 52, 95, 124,
Polymorphonuclear, 55, 117, 125, 126 129, 142, 143, 160, 161, 167
Polyp, 68, 71, 72, 143, 155, 156 San Joaquin fever, 55
Polypoidal, 71 Saprophyte, 5, 6, 13, 34, 80, 87, 114, 142–144
Porrigo, 1, 11 Sarcin–ricin loop, 88
Posadas-Wernicker’s disease, 55 Scedosporium, 120–127
Potassium Iodide (KI), 119, 144 Sclerotia, 34, 36, 124, 125, 171
Potato dextrose agar, 15, 46, 87, 106, 115, 143, 166 Scutulum, 18, 20
Potato flake agar, 46 Secreated aspartyl proteinase (SAP), 104
Pottery tree, 93 Secretome, 129
Prearthroconidial cell, 85 Septa, 3, 9, 12, 22, 29, 75, 85, 102, 103, 169–171
Pro-inflammatory, 19, 28, 42, 47, 63, 64, 78, 96, 109, Septate, 4, 12, 22, 29, 32, 43, 50, 59, 73, 84, 113,
118, 126 121, 127, 131, 133, 135, 141, 144, 169, 171
Propolis, 32 Seroconversion, 137
Protein with internal repeats (Pir), 103 Serological, 5, 49, 57, 66, 83, 90, 101, 112, 119, 124,
Protoplasmic streaming, 3 126, 127, 131, 142, 144, 145, 162
Prototheca, 138–140, 160 Sessile, 22, 71, 72, 171
Psedofarcy, 55 Sewage, 24, 75, 123
Pseudallescheria, 120–127, 163 Shield cell, 133
Pseudo-glanders, 65 Sialic acid, 33, 37, 82, 89
Pseudohyphae, 3, 91, 102, 103, 110, 111, 170 Siderophore, 39, 40, 63, 64, 75, 76, 87, 98, 107, 134
Pseudothecia, 8 Signal transduction, 34, 39, 98
Pycnidium, 8 Single nucleotide polymorphism (SNP), 51, 105
Pyriform, 121, 170 Skin scrapings, 19, 31, 48, 56, 75, 78, 82, 89, 110,
Pyrosequencing, 112, 129 130, 140, 156, 159
Pythium, 2, 10, 127–132, 142, 156 Small interfering RNA (siRNA), 94
Sodium dodecyl sulphate, 14
Q Sodium hypochlorite, 21, 30, 61, 80, 93, 128
Quail, 135 Sodium iodide (NaI), 102, 109
Quantitative PCR (qPCR), 83, 140 Spermogonia, 7
Quaternary ammonium, 14, 105, 108 Sphagnum, 114
Quorum sensing, 104 Spherule, 50–57, 72, 144
Spherulin, 57
R Sphincter, 139
Racquet hyphae, 4, 5, 12 Spicule, 46
Radio immune assay (RIA), 49, 57, 66, 67, 124 Spiral, 4, 5, 12
Raduloconidia, 113 Spiraling hyphae, 4
Random sequence tag (RST), 87 Spitzenkorper, 103, 171
Reactive oxygen species (ROS), 38, 39, 42, 54 Splendore–Hoeppli reaction, 118
Real-time PCR, 32, 57, 67, 78, 83, 90, 112, 137, 163 Sporangiospore, 7, 73, 76–80, 82, 138–140, 145,
Regulator gene, 36 149, 161, 170
Reproduction, 6–8, 14, 23–24, 45, 51, 74, 80, 84–87, 92, Sporangium, 7, 68–74, 79, 128, 138, 170, 171
93, 96, 104–105, 114, 122–123, 128, 138, 141, Spore, 3, 4, 6, 7, 12, 24, 30, 32, 37, 45, 54, 69, 76, 80,
169, 170 82, 93, 98, 111, 138, 169, 170
Rhinosporidium, 68–73, 145, 156 Spore-morula, 69
Rhizoid, 3, 73, 74, 79, 171 Sporocarp, 8
Rhizonin A, 76 Sporogen, 86, 170
Rhizopus, 2, 10, 73–81, 156, 163, 171 Sporothrix, 3, 10, 112–120, 124, 156, 162
Rhizoxin, 76 Sporothrix schenckii conA binding fraction, 112–114,
Ribotoxin, 36, 40, 88 117, 124
Index 179
Sporotrichin test, 119 Tube precipitin, 57

Sporulation, 15, 25, 34, 36, 39, 54, 61, 87, 124, 127, Tuberculate, 7, 59, 60, 171
138, 160 Tubular mitochondria, 92, 98
Sputum, 48, 49, 56, 78, 82, 89, 103, 137, 138, 155, 156, Turkey-X disease, 32
159, 165 Tweezer, 157
Stolon, 73, 74, 171 Twin antigen, 129
Strange body, 118 Tzanck cytodiagnosis, 66
Stratum corneum, 17, 19, 26, 31, 116
Stuart transport medium, 155 U
Sugary texture, 35 Ubiquitous, 75, 80, 132, 136, 138
Sunflower seed agar, 95, 106, 167 Udder, 41, 82, 100, 103, 106, 110, 139, 156, 157
Superoxide dismutase, 38, 40, 97, 98, 107, 109 Umbel, 73
Swamp, 123, 131 Unannotated genes, 34
Sympodial, 133, 171 Unfolded protein response (UPR), 39, 40
Synanamorph, 121, 122 Uninucleate, 4, 7, 80, 84, 85
Synnemata, 122, 123, 171 Unisexual mating, 93
Synovial fluid, 48, 118, 156, 157
V
T V8 agar, 129, 133
Talaromyces, 85 Valley fever, 55
Tannery, 16 Van der Waals, 108
Tapering, 79 Vegetable extract agar, 34, 75
TaqMan, 90, 112, 140, 163 Vegetative, 3, 4, 11, 32, 74, 80, 84, 85, 91, 144
Tazobactam, 43 Verrucous, 48, 71, 141
Teleomorph, 13, 14, 23, 24, 33, 45, 51, 85, 91, 93, 120, Very late antigen, 64
121, 123, 170, 171 Veterinarian, 2, 16, 46, 127
Tenesmus, 139 Villose, 143
Tennis racquet, 12 Vitronectin, 108, 136
Terbinafine, 21, 29, 32, 44, 120, 132, 142 Vomition, 29, 41, 57, 77, 119, 131, 139
Terpene, 84 Voriconazole, 44, 50, 61, 67, 76, 79, 102, 112,
Tetraploid, 105 127, 135
Thallus, 3, 80, 121, 170, 171
Thiabendazole, 21, 127 W
Thigmotropism, 107 Wart, 44, 71, 72, 171
Thorn, 114, 115, 123, 125 Whiplash, 128
Thrombosis, 40, 77, 78, 130 White-opaque transition, 104, 106
Thrush, 110 Wire, 125
Tinea, 1, 11, 14, 18, 21, 22, 24, 27, 29, 31, 32 Wood’s lamp, 20, 28, 32, 161
Tinsel, 128 Wood splinter, 125
Titan cell, 91 Wright’s stain, 90, 159, 166
T-lymphocytes, 19, 48, 72, 96
Toll like receptor (TLR), 39, 40, 55, 56, 78, 99, 109, Y
111, 117, 126 Yeast, 2–7, 9, 35, 44–49, 52, 58–67, 69, 79, 80, 82,
Toluidine blue, 137 84, 85, 87–97, 99–104, 107–111, 113–119,
Torticollis, 135 126, 129, 132, 134, 135, 138, 139, 141, 142,
Transtracheal washes, 56 159–161, 165, 166, 169, 170
Trematosphaeria, 120, 121, 123, 126 Yeast to mould, 114
Trichogyne, 7
Trichophyton, 1, 2, 4, 10–23, 28–30, 32, 156, 161, Z
163, 170 Zipper phagocytosis, 42
Trimethoprim–sulphadiazine, 72, 137 Zoophilic, 13, 14, 16, 18, 23, 24
Trisporic acid, 80 Zoosporangium, 127
Trophocyte, 68 Zygomycetes, 3, 10, 73–75, 78, 79, 127, 142
Trophozoite, 136 Zygomycosis, 73, 74, 78, 79
Trumpeter, 135 Zygophore, 74, 80
Tryptoquivaline A, 37 Zygospore, 8, 74, 79, 80, 143, 171

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Indranil Samanta

ISBN 978-81-322-2279-8 ISBN 978-81-322-2280-4 (eBook)

Library of Congress Control Number: 2015930190

Springer New Delhi Heidelberg New York Dordrecht London

Printed on acid-free paper

Springer (India) Pvt. Ltd. is part of Springer Science+Business Media (www.springer.com)

Buenos Aires University School of Medicine Ricardo Negroni

Kolkata, West Bengal, India Dr. Indranil Samanta

4.5.5 Natural Habitat and Distribution . . . . . . . . . . . . 45

4.8.3 Susceptibility to Disinfectants . . . . . . . . . . . . . . 70

4.13.10 Virulence Factors . . . . . . . . . . . . . . . . . . . . . . . 106

4.16.5 Susceptibility to Disinfectants . . . . . . . . . . . . . . 128

Dr. Indranil Samanta obtained his Bachelor of Veterinary Sciences and

I. Samanta, Veterinary Mycology, DOI 10.1007/978-81-322-2280-4_1, 1

2.1 Morphology like filament called ‘pseudohyphae’. The examples

I. Samanta, Veterinary Mycology, DOI 10.1007/978-81-322-2280-4_2, 3

2.1.1 Types of Hyphae

The following hyphal types are observed:

2.1.2 Fungal Cell Structure

Fig. 2.3 Fungal cell wall

Table 2.1 Comparative growth requirements of bacteria and fungi

Fig. 2.5 Types of fungal

Fig. 2.6 Types of fungal

I. Samanta, Veterinary Mycology, DOI 10.1007/978-81-322-2280-4_3, 9

Fungi Zygomycota Zygomycetes Entomophthorales Basidiobolaceae Basidiobolus Basidiobolus ranarum

(Chromista) Lagenidium L. giganteum

4.1 Trichophyton named Achorion schoenleinii (Trichophyton

I. Samanta, Veterinary Mycology, DOI 10.1007/978-81-322-2280-4_4, 11

4.1.1 Morphology The hyphae produce asexual spore known as

Fig. 4.1 Coiled hyphae

Fig. 4.2 Microconidia

4.1.2 Classification The molecular study revealed that none of the

Table 4.2 Teleomorphs of Trichophyton 4.1.4 Susceptibility to Disinfectants

4.1.6 Genome The conventional solid medium includes

4.1.7 Isolation, Growth and Colony 4.1.8 Antigenic Characteristics

Table 4.3 Virulence mechanisms and factors possessed by Trichophyton

4.1.9 Virulence Factors spread the infection by leaving infected hairs

Table 4.4 Major diseases of animal and human caused by Trichophyton

4.1.13 Immunity The activation of Th1 cells produces a cell-

4.1.15 Treatment extracts showed antidermatophytic activity

Fig. 4.3 Macroconidia of

Fig. 4.4 Macroconidia of

Table 4.6 Ecological niche-based classification and host specificity of Microsporum

Onygenales and family Arthrodermataceae. (human associated), zoophilic (animal associated)

4.2.5 Natural Habitat and Distribution

Table 4.8 Genome statistics 4.2.8 Antigenic Characteristics

Table 4.9 Virulence mechanisms and factors possessed by Microsporum

Table 4.10 Major diseases of animal and human caused by Microsporum

4.2.13 Immunity wrapped in brown paper (or coloured paper)

Fig. 4.5 Macroconidia of

4.3.3 Susceptibility to Disinfectants 4.3.5 Genome

4.3.4 Natural Habitat and Distribution

30 C as an ideal temperature for growth of 4.3.10 Pathogenesis

The study indicated the presence of 31 proteins

E. floccosum are able to grow at 37 C and

Fig. 4.6 Conidial

4.4.2 Classification A. flavus follows a life cycle consisting of a

Fig. 4.7 Growth of

4.4.8.2 Gliotoxin 4.4.8.6 Haemolysin

4.4.8.7 Helvolic Acid and Fumagillin 4.4.11 Pathogenesis