Volume 6 Number 1 (February 2014) 31-36

Genotyping of Clostridium perfringens isolated from healthy

and diseased ostriches (Struthio camelus)

Jamshid Razmyar1*,Gholam Ali Kalidari1, Ali Tolooe2, Mehrnaz Rad3 and Ahmad Reza Movassaghi3

1
Department of Clinical Sciences, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad,
Mashhad, Iran. 2Graduated DVM, DVSc in Poultry Health & Diseases, University of Tehran, Tehran,
Iran. 3Department of Pathobiology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad,
Mashhad, Iran.

Received: May 2013, Accepted: December 2013.

ABSTRACT

Background and Objectives: Clostridium perfringens is more prevalent type of clostridia genus isolated from the intestinal
tract of ostrich (Struthio camelus). Necrotic enteritis (NE) is a potentially fatal gastrointestinal (GI) disease of poultry and
other avian species, which produces marked destruction of intestinal lining in digestive tract caused by C. perfringens.
Pathogenicity and lesions are correlated with the toxins produced, thus toxin typing of the bacterium has diagnostic and
epidemiological signiicance. The aims of the present study were to determine the biotypes of C. perfringens among ostrich’s
farms either diseased and healthy ones and to screen the isolates for major toxin genes (cpa, cpb, etx, and iA, cpb2, and cpe).
Materials and Methods: Thirty isolates of C. perfringens were obtained from NE-positive and NE-negative ostrich locks
in Khorasan-e-Razavi porvince and analyzed by multiplex PCR assay.
Results: All isolates were positive for alpha toxin gene (cpa) and ive of those were positive for beta toxin gene (cpb). The
presence of cpb2 gene was detected in a high percentage of isolates originating from both healthy (93.3%) and diseased
locks (80%). None of the isolates carried enterotoxin gene (cpe).
Conclusion: The results suggest that types A and C of C. perfringens are the most prevalent types in ostrich in Iran. Due
to detection of beta2 toxin gene in isolates from both healthy and diseased birds, it appears that the presence of cpb2 is not
considered a risk by itself.

Keywords: Necrotic enteritis; ostrich, Struthio camelus, Clostridium perfringens, toxin genes, Iran

INTRODUCTION intestinal lora, nutrition, environmental factors

and pathogens (1). Some viral agents have been
In recent decades, ostrich (Struthio camelus) farms isolated from outbreaks of enteritis in ostrich chicks
are increased in number in Iran. Enteritis can be a (2). However, in most cases they were not primary
major cause of mortality in intensively reared ostrich pathogens (1, 2). Bacteria play a more important role
chicks, practically never occurring in chicks reared than viruses, and those involved in ostrich enteritis
on pasture, and it is inluenced by a multiple factors. comprise the Gram-negative bacteria, Campylobacter
These can be divided into the following categories: jejuni, Erysipelothrix rusioopathiae and clostridia (1).
Clostridia produce a myriad of potent toxins that are
* Corresponding author: Jamshid Razmyar Ph.D
responsible for severe disease in humans and other
Address: Department of Clinical Sciences, Faculty of animals (3). Clostridial enteritis in ostriches is an
Veterinary Medicine, Ferdowsi University. acute and severe disease able to cause a high mortality
Tel: :+98-511-8803767 among young and adult birds (1). Clostridium
Fax: +98-511-8763852 perfringens was more prevalent type of clostridia
E-mail: [email protected] genus that was isolated from the intestinal tract of

31
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RAzMyAR ET AL .

ostrich (4). Necrotic enteritis (NE) is a potentially beta2 toxin have been proposed to be important for
fatal gastrointestinal (GI) disease of poultry and other the pathogenesis of intestinal disorders (8, 11). The
avian species observed with variable frequency, which C. perfringens enterotoxin (CPE) mediated food
produces a marked destruction of intestinal lining in poisoning ranks among the most common foodborne
the digestive tract caused by C. perfringens (5, 6 ). illnesses worldwide (3, 8). Poultry meat is suspected
NE is characterized by reduced growth performance, of being the likely vehicle causing food poisoning
decreased feed eficiency, and depression in its mild due to C. perfringens, which is widely distributed
form and by anorexia, severe morbidity, and signiicant in the intestinal contents of healthy and diseased
mortality at its worst. C. perfringens is a Gram- poultry that contaminate carcasses and meat during
positive spore-forming anaerobic bacterium that is dressing in abattoirs or poultry processing plants (12).
widespread in the environment, commonly found in Understanding of the potential reservoirs and routes
soil and sewage and in the gastro-intestinal tract of of transmission of C. perfringens strains causing
animals and humans as a member of the normal gut food poisoning are necessary. Beta2 toxin have been
microbiota (5). C. perfringens causes histotoxic and the subject of numerous studies, and the possible
enteric infections in humans and animals by virtue of association of cpb2-harbouring C. perfringens isolates
production of a large number of toxins (3). There are and the occurrence of enteric disease in humans and
at least 17 different toxins known, but each individual domestic and wild animals have been investigated
C. perfringens strain produces a selection of these (11).
toxins (5). The differential production of four major Various epidemiological and experimental studies
toxins, alpha (CPA), beta (CPB), epsilon (ETX), of C. perfringens strains from various species of
and iota (Iota) toxin, is the basis for classifying C. animal and human have been published but very
perfringens isolates into ive types, A to E. Alpha few studies have been published on C. perfringens-
toxin is found in all types, whereas the beta toxin induced NE in ostrich and very little knowledge of
can be detected in types B and C only. Moreover, the the C. perfringens genetic proile in ostrich isolate
epsilon toxin can be found in types B and D; and iota is available. In the present study, for the irst time,
toxin in type E only (5). Pathogenicity and lesions are the collected C. perfringens isolates from healthy
correlated with the major toxins produced, thus typing and diseased ostrich locks were analyzed by using
of the bacterium has diagnostic and epidemiological a multiplex PCR assay in order to determine the
signiicance. Toxin typing of C. perfringens is presence of alpha (cpa), beta (cpb), epsilon (etx), iota
important since particular toxin types are associated (iA), beta2 (cpb2), and enterotoxin (cpe) toxin genes.
with speciic enteric diseases in animals. NE is
primarily caused by C. perfringens type A and to a MATERIALS AND METHODS
lesser extent type C strains (5, 6). Various aspects of
NE have been intensively studied for several decades. Bacterial isolation and characterization. The
Despite considerable research efforts, however, the C. perfringens isolates used in this study were isolated
pathogenesis of this condition in poultry and especially from ostriches displaying clinical signs of necrotic
in ostriches is still poorly understood. A good deal of enteritis and ostriches dead from other diseases with
research effort has been devoted to characterizing a the exception of enteritis. Samples were obtained
deinitive toxin that is responsible for causing NE from 30 ostrich locks in Khorasan-e-Rzavi province.
in poultry. In addition to the so-called major toxins, Fifteen ostrich locks were diagnosed as having acute
there are other minor toxins or enzymes produced by form of NE but ifteen locks were chosen from
some strains of C. perfringens, which may play a role apparently healthy locks. Ostriches from healthy
in pathogenicity (3). These compounds include beta2, locks were subjected to the necropsy and isolates of
NetB, TpeL, delta, theta, kappa, lambda, mu, nu, C. perfringens were obtained aseptically with sterile
gamma, eta, neuraminidase, urease and enterotoxin swabs from gut samples of ostriches. In addition, all
(3, 7). While the roles of beta, iota, and epsilon toxins dead ostriches in diseased locks were subjected to
in enteritis pathogenesis among animals are well the necropsy and sampled by scrubbing the intestinal
documented, the roles of other toxins, such as alpha, wall of affected birds. Gram stain of tissue specimens
NetB, TpeL and beta2 toxin, in NE pathogenesis are from ield cases of NE was done, too. Subsequently,
still unclear (7- 10). Toxins such as enterotoxin and 90 intestinal samples were streaked onto blood agar

32 IRAN. J. MICROBIOL. Vol. 6, No. 1 (February 2014), 31-36 http://ijm.tums.ac.ir

 GENOTypING Of OSTRICH’S C. perfringenS ISOLATES

plates containing 7% deibrinated sheep blood and intestines of ostriches. Fifteen isolates were isolated
incubated anaerobically at 37ºC for 48 hr. Colonies from NE-positive and the remainders from ostriches
which showed characteristic dual hemolytic zones without necrotizing enteritis. Only one isolate from
were picked up and sub-cultured in Tryptose Sulite each bird was considered. All bacterial isolates
Cycloserine agar (TSC) and Tryptose Sulite Neomycin exhibited the characteristic features of C. perfringens.
agar (TSN) for puriication. The identity of the isolates The colonial characters on blood agar showed dew
was conirmed by their colonial and microscopical drops, smooth, grayish, and convex colonies with a
morphology, hemolytic pattern and Gram staining. All double zone of haemolysis. Clostridium perfringens
culture media and additives used in this study were formed black colonies in TSC and TSN agar.
purchased from Merck (Germany). Reference strains Microscopic characters revealed Gram positive non
of Clostridium perfringens ATCC 13124 (cpa); CIP motile rods with boxcar-shaped square cells. The
106157 (cpa, cpe); CIP 60.61 (cpa, cpb, etx, cpb2) Gram stain of tissue specimens from ield cases of NE
were used as positive controls. All treatments of birds demonstrated that rod-shaped bacteria with typical
were conducted according to Animal Care Guidelines C. perfringens morphology formed large clumps
of the Research Committee, Faculty of Veterinary primarily around the necrotic areas, and only seldom,
Medicine, Ferdowsi University of Mashhad. single colonies of C. perfringens were seen in the
vicinity of non-necrotic epithelium.
Multiplex pCR. A single colony of each strain
was suspended in 100 μl distilled water, boiled for Multiplex pCR. All C. perfringens isolates from
10 min and then centrifuged at 10,000 × g for 10 min. NE-positive (No:15) and NE-negative (No:15)
The supernatants were collected carefully and used ostriches were positive for cpa and negative for etx
as template DNA for PCR (10). Six pairs of primers and iota toxin genes. In addition, 4 isolates (26.6%)
were used to determine the presence of cpa, cpb, iA, from NE-positive and only 1 (6.6%) isolate from NE-
etx, cpe (13), and cpb2 (14) genes using a multiplex negative ostriches were positive for cpb. The results
PCR technique for all isolates. The primers and showed that 25 isolates (83.3%) belonged to toxin
other materials used in PCR reaction were provided type A and 5 (16.7%) belonged to toxin type C (Fig.
by Ampliqon (Odense, Denmark). Ampliication 1). Only one of 5 C types isolates belonged to NE-
reactions were carried out in a 50μl reaction volume negative ostriches. All C. perfringens isolates were
containing 5 μl 10 x PCR buffer, 5 mM dNTPs, 25 also negative for cpe gene but 26 out of 30 (86.7%)
mM MgCl2, 5U of Taq DNA polymerase, 0.5 mM of from diseased and healthy ostrich locks were positive
each cpa oligo, 0.36 mM of each cpb oligo, 0.36 mM for cpb2 gene (Fig. 1). Only 1 isolate (3.3%) from
of each cpb2 oligo, 0.52 mM of each iA oligo, 0.44 NE-negative ostriches was negative for cpb2 gene.
mM of each etx oligo, 0.34 mM of each cpe oligo,
and dH2O. Ten μl of template DNA was added to DISCUSSION
the mixture. Several bacterial strains as positive and
negative controls (described above) were included in Traditionally, typing of C. perfringens strains
all PCR reaction sets. Ampliication was programmed involved sero-neutralisation of culture iltrates in
in a thermocycler (Techne TC-3000, England) as vivo. Mice or guinea pigs were injected with culture
follows: 95°C for 3 min followed by 35 cycles of 94°C supernatants of C. perfringens, along with antitoxin,
for 1 min, 55°C for 1 min, 72°C for 1 min, and a inal and death (mice) or dermonecrosis (guinea pigs)
extension at 72°C for 10 min (13). The ampliication was assessed (15). This assay was extremely time-
products were detected by gel electrophoresis in consuming as growth of the organism was required.
1.5% agarose gel in 1 x TAE buffer, stained with 0.5 It was also expensive as two of the toxins, epsilon and
μg/ml EtBr. Ampliied bands were visualized and iota, required trypsin for activation, but a third toxin,
photographed under UV transillumination. beta toxin, was inactivated by trypsin. Therefore each
culture supernatant was assayed numerous times;
RESULTS with and without trypsin, and with and without the
ive different preparations of neutralizing antisera
Characterization of bacterial isolates. A total of (16). Multiplex PCR assay is a useful alternative to
30 isolates of C. perfringens were obtained from the traditional assays and as a replacement for standard

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 RAzMyAR ET AL .

180
109 CFU/g in diseased birds (7). The presence of
C. perfringens in the intestinal tract or inoculation
of the animals with high doses of C. perfringens,
however, does generally not lead to the development
of necrotic enteritis. One or several predisposing
factors may be required to elicit the clinical signs and
lesions (5). Additionally, it was shown that strains
isolated from necrotic enteritis outbreaks did not
produce more alpha toxin compared to isolates from
the gut of clinically healthy broilers (19).
Alpha toxin is a phospholipase C sphingomyelinase
181
fig. 1. Agarose gel (1.5%) electrophoresis of multiplex that hydrolyzes phospholipids and promotes
Agarose gelof
PCR products (1.5%) electrophoresis of
C. perfringens isolates.
multiplexLanes M: size
PCR products of membrane disorganization (3). Until recent years;
isolates.
marker Lanes M: size marker
(GeneRuler 100 bp(GeneRuler
DNA Ladder, 100 bp Fermentas);
DNA Ladder, Fermentas);
lane lane 1:
(CIP 60.61); lane 2: Positive control alpha
for toxin has been proposed as the main virulence
1: Positive control for cpa, cpb, etx and cpb2 (CIP 60.61);
(CIP 106157); lane 3: Negative control; lanes 5, 9 and 13: field isolates of
lane 2: Positive control for cpa and cpe (CIP 106157); lane factor for NE in poultry. Recently, the role of
3: Negative control;
(type C); lanes lanes
7, 11 and 5, isolates
14 field 9 and of 13: ield isolates of C. C. perfringens alpha toxin in NE is disputed (7,
perfringens obtained from healthy and diseased ostriches, (type C). lanes 4: field
10, 11, 27). Keyburn et al. described a novel pore-
that were positive for cpa and cpb (type C); lanes 7, 11 and
(type A); lanes 6, 10 and 12 field isolates of
14 ield isolates of C. perfringens obtained from healthy and forming toxin, NetB, a virulence factor which is
diseased ostriches, that were positive for cpa , cpb and cpb2 involved in the pathogenesis of NE (10). Subsequent
(type C). lanes 4: ield isolates of C. perfringens obtained studies showed that netB being the only virulence
from healthytyping
Traditionally, ostriches,
of that were positive for cpa (type A);
lanes 6, 10 and 12 ield isolates of C. perfringens obtained factor to date shown to be essential in producing
from healthy and diseased ostriches, that were positive for disease (7). Severity of disease may vary with the
assessed
cpa and(15).cpb2This assayA).
(type was extremely time
presence of other virulence determinants, of which
ta toxin, was inactivated by trypsin. Therefore eachthe
culture
best implicated accessory toxin is another novel
in vivo
the typing
five different methodsof (13).
preparations In the
neutralizing present
antisera (16). study, a toxin, TpeL, a member of Large Clostridial Toxins
total of 30 isolates of C. perfringens 8
were cultured (LCT) family (20), which is present in some type A
from the intestines of healthy and diseased ostriches. NE isolates (21). There is evidence that netB-positive
C. perfringens types A (cpa positive) and C (cpa and strains that are also tpeL-positive cause more severe
cpb positive) were identiied in the samples but type disease than strains that lack tpeL (22). The presence
A was the dominant type (83.3%). This inding was of netB and tpeL genes among C. perfringens isolated
in agreement with previous studies done on poultry in this study was investigated in separate studies
(13, 17, 18). From 5 C. perfringens type C isolates (Unpublished data).
in this study, 4 obtained from NE-positive and only 1 Beta toxin is present in C. perfringens type C
isolate from NE-negative ostriches. Very few studies found in poultry and we isolated 5 cpb-positive
have been published on genotyping of C. perfringens C. perfringens in ostrich isolates in the present study.
obtained from ostrich. To the best of our knowledge, Beta toxin is thought to be pore-forming in nature,
the present study is the second investigation on causing increased permeability (3). It is a highly
genotyping of C. perfringens obtained from ostrich. trypsin-sensitive protein (3) which is responsible for
Songer and Meer (1996) conducted the genotyping mucosal necrosis and possibly for central nervous
of 9 C. perfringens isolates from ostrich by multiplex system signs in C. perfringens-induced disease in
PCR. All isolates were type A and one isolate was cpe domestic animals (6). Although this toxin is cytotoxic,
positive (18). its mode of action has not yet been elucidated in
Because C. perfringens type A is highly prevalent pathogenesis of C. perfringens in avian and typical
in the intestines of healthy animals, controversy exists disease cannot be reproduced by this toxin alone (6).
about its real pathogenic role. The intestinal number All isolates tested in this study were cpe-negative.
of C. perfringens in healthy and in NE-affected birds C. perfringens enterotoxin (CPE) is the most
are different. The C. perfringens population is found important virulence factor when type A isolates cause
to be normally less than l02 to 104 colony-forming human GI diseases, although less than 5% of type
units (CFU) per g of the intestinal contents in the A isolates produce this toxin (12, 23). In contrast to
small intestine of healthy chickens compared to 107- the well-established role of this toxin in human GI

34 IRAN. J. MICROBIOL. Vol. 6, No. 1 (February 2014), 31-36 http://ijm.tums.ac.ir

 GENOTypING Of OSTRICH’S C. perfringenS ISOLATES

disease, the data implicating CPE in animal disease the presence of cpb2 in majority of both healthy and
remains more ambiguous (8). Animals with diarrhea diseased ostriches.
are rarely tested for the presence of CPE in their feces
and diagnostic criteria for establishing CPE-mediated ACKNOWLEDGEMENTS
animal disease are lacking (8).
Biological activity of beta2 toxin was similar to This research was funded by a grant (No.
that of the beta toxin, although it may possess weaker 16142) from the Research Council of the Ferdowsi
cytotoxic activity. A possible pore formation or other University of Mashhad. We would like to thank Mr.
mechanisms leading to cell membrane disruption A. Kargar for his assistance in laboratory works and
appear to be its most plausible function (3). CPB2 Dr. Shojadoost from University of Tehran, Iran, for
toxin can be produced by all types of C. perfringens. the gift of reference strains.
In this study, we have reported a high percentage
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