HARAMAYA UNIVERSITY

COLLEGE OF VETERINARY MEDICINE

Topic: SALMONELLA AS ENTERIC ZONOOTIC AGENT ALONG CAMEL MEAT AT

THREE SELECTED DISTRICTS OF EASTERN ETHIOPIA

Adem Hiko (DVM, MS.c., PhD, Vet. Public Health and Biomedical sciences/food safety)
(Principal applicant)

Email: [email protected]
Mobil: 0940240080
1. Fekadu Mosisa (BS.c, Food Technology and process Engineering) (Co-applicant)
Email: [email protected]; [email protected]
Mobil: 0910189039

Thematic Research Code: HURG—2014—03—04

Total cost of the project: 100,000.00 ETB

Thematic Research Proposal Submitted to: Haramaya University office of research affairs
Haramaya, Ethiopia
July, 2015

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EXECUTIVE SUMMARY

Background: Functional non-alcoholic soft drinks including bottled water industry and
technology products currently constitute the fastest growing sectors. These drinking items are
handled at different locations and hygienic levels under public health risk. New microbial arising
from the increasing suitable ingredient within the products worldwide. Problems of statement:
Different types of either imported and/or locally manufactured soft drinks are transported from a
distance to consumer location. Such products have risk of contamination with spoilage and
public health pathogenic microbial. The same is true in Ethiopia. Although fragments of
information are available, mainly on microbiological quality of traditional beverages of Ethiopia,
none of them included multiple of commercial soft drink examination for spoilage and foodborne
infectious microbiological quality yet. These are the question of this project. Objectives: The aim
of this research is to determine the microbial load of commercial available soft drinks; to isolate
possible zoonotic agents; and to perform antimicrobial susceptibility test on isolates of zoonotic
agents. Materials and methods: The study will be carried out in Chiro and Dire Dawa towns, in
the eastern part of Ethiopia from July, 2015-June, 2016. Simple random sampling will be applied
on all types of commercial soft drinks at different supply station in the towns with collection of
384 samples from each towns with total of 768 samples. All possible commercially packaged,
non-alcoholic, ready-to-drink (RTD) beverages comprise a diverse group of products and brands
used by consumers links bottled waters, sports and energy drinks, fruit or vegetable juices, dairy
and yogurt, and others will be included. For those products manufactured in Ethiopia, samples
will be collected both from producing companies and supply stations. However, those imported
products will be sampled from supply stations in the study areas. The sample will be purchased
and will be analyzed for microbiology quality and zoonotic agent isolation. Drug susceptibility
test will be done on the isolates using disc diffusion techniques. Beneficiaries: In fact, the
beneficiaries of the project are community in the study area at local level, national population at
country level and the world at global level. The estimated total costs required for this project is
100,000.00 ETB.

Key words: Commercial; soft drinks; microbiology; quality; public Health; Ethiopia

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TABLE OF CONTENT

LIST OF TABLE III

LIST OF FIGURE IV
ABBREVIATION V
INTRODUCTION 1
1.1.Back ground 1
1.2 Statement of the problem 4
1.3 Research question 5
1.4. Scope and significance of the research project 5
1.5. Beneficiaries of the project 5
1.6 Aim and objectives of the project 6
2. REVIEW OF LITERATURE 6

2.1. Overview of food hygiene and food safety 6

2.1.1. Food hygiene and food safety practices in Ethiopia 7
2.2. Over view of salmonella 7
2.2.1 Evolution of Salmonella 7
2.2.2 Characteristics of Salmonella 8
2.2.3. Classification and nomenclature 9
2.2.4. Epidemiology 11
2.2.5. Clinical signs 14
2.2.6. Immunity 15
2.2.7 Diagnosis 15
2.2.8. Clinical feature 19
2.2.9. Treatment and Control 20
2.2.10. Antimicrobial resistance 21
2.2.11. Public Health Significance 22
2.2.12. Economic importance 22
3-METHODS AND MATERIALS 22

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 3.1 Study area 22
3.2. Study population 23
3.3. Study design 23
3.4. Sampling 23
3.5. Sampling procedure 24
3.6. Sample processing 25
3.7. Isolation and identification of Salmonella 26
3.7.1. Pre-enrichment in non selective liquid medium 26
3.7.2. Enrichment in selective liquid media 26
3.7.3. Plating out and identification 26
3.7.4. Confirmation 27
3.8. Antimicrobial Susceptibility Testing 28
3.8.1 Disk diffusion testing 28

3.9. Data management and analysis 30

4. Expected out put 30

5- Work plan 30
6. BUDGET BREAKDOWN 31
5.1 Material expense (chemical and reagent) 31
5.2 Antimicrobial drug sensitivity test expense 31
5.3 Stationary expense 32
5.4- Miscellaneous expense 32
5.5- Personal expense 32
5.5 Perdium 33
5.6 Transport expense 33
5.6 Budget summary 34
7. REFERENCE 35

8. APPROVAL SHEET 43

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 LIST OF TABLE

Table Page
1. Type of sample and study area 24

2 Project work plan 31

3 Material expense 31

4-Stationary expense 32

5-Miscellaneous expense 32

6- Personal expense 33

7- Perdium 33

8 -Pransport expense 34

9 -Budget summery 34

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 LIST OF FIGURE

Figure Page
1: Classification of the genus Salmonella 11

2–Salmonella isolation procedure 28

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 ABBREVIATION
AWACAB Awoday Administrative Council Agricultural Bureau
BGA Brilliant Green Agar
BPW Buffered Peptone Water
BSA Bismuth Sulfite Agar
CDC Center for disease control and prevention
DDACAB Dire Dawa Administrative Council Agricultural Bureau
DIASALM Diagnostic Semi-solid Salmonella Medium
FAO Food and Agricultural Organization
H2S Hydrogen Sulphide
HACCP Hazard Analysis Critical Control Point
HeK Hektoen enteric
HRAO Harari Regional Agricultural Office
IFT Institute of Food Technology
ISO International Organization for Standardization
LIA Lysine Iron agar
m.a.s.l Meter above sea level
MKTTn Muller-Kauffmann Tetrathionete with novobiocinnin
MSRV Modified Semisolid Rappaport Vassilidis
OIE Office International des Epizooties
PCR Polymerase Chain Reaction
RV Rappaport Vassiliadis broth
SC Selenite Cysteine
SSA Salmonella Shigella Agar
TBG Tetrathionate Brilliant Green
TSI Triple Sugar Iron agar
VP Voges-Proskauer
WHO World Health Organization
XLD Xylose lysine desoxycholate
XLD Xylose Lysine Desoxycholate
μg Microgram

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1. INTRODUCTION

1.1. Back ground

Salmonella infections continue to be a major public health problem in both developed and
developing countries. Salmonellosis typically contracted through the consumption of
contaminated food, water, or through contact with an infected host (Alcaineet al., 2006).
Although approximately 2500 Salmonella serotypes have been identified, most Salmonella
infections are caused by a single species (Mulveyet al., 2006).

The genus of Salmonella is a group of bacteria that have long had substantial effect on humans,
domesticated animals, and food. These bacteria are responsible for millions of deaths worldwide
every year, as well as billions of probable infections, and are easily spread intra-andinter-
species. With incidence rates increasing, as well as the evolution of antibiotic-resistant strains,
Salmonella poses a substantial risk to the public health and livelihood. (Pui et al., 2011).

The one-humped camel (Camelus dromedarius) plays an important role as a primary source of
subsistence in the lowlands of Ethiopia. It lives in arid and semi-arid areas which are not suitable
for crop production and where other livestock species hardly thrive. Pastoralists in the eastern
lowlands of Ethiopia rely mainly on camels for their livelihood. Ethiopia possesses over 1
million dromedary camels (FAO, 2002) and the majority of these camels are found in eastern
part of the country. In spite of the large number of camels in Ethiopia, the productivity of camels
is generally low and the camel has been given little research and development attention.
Camel is a potential source of meat protein in sub-Sahara Africa; the meat is consumed to meet
the nutritional requirements in the form of animal protein. Members of Salmonella enterica
subspecies enterica are widely distributed in the environment and the gastrointestinal tracts of
animals (FAO, 2002).

Foodborne diseases are among the most widespread global public health problems of recent
times, and their implication for health and economy is increasingly recognized (Van der Venter,
1999; Gomez et al.1997). These diseases are attributable to a wide range of pathogens and
toxins. Salmonella is a leading cause of foodborne illnesses (White et al., 2001; D’Aoust, 1997;

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D’Aoust, 1991a; WHO, 1988). According to the World Health Organization reports of 1995,
88% of all foodborne diseases were caused by Salmonella (Gabertet al., 1999). Salmonellosis,
the disease caused by infection with Salmonella, is a common intestinal illness caused by
numerous Salmonella serovars and manifested clinically in animals (Radostitset al., 1994) and
humans (Hohmann, 2001) as an acute enteritis and chronic enteritis, an acute septicaemic disease
or as subclinical infections (Acha and Szyfres, 2001).

Salmonella is a common foodborne pathogen that often affects humans who consume
contaminated meat and other contaminated food products. The Centers for Disease Control and
Prevention (CDC) found that Salmonella was the most common infection (1.2 million U.S.
illnesses annually) and the most common cause of hospitalization and death of all foodborne
illnesses (2011). Meat and other common food products have been under constant scrutiny over
the years after several Salmonella outbreaks occurred. Contamination of these products can often
be traced back to the slaughtering and processing of these meats (Hjartardóttir, Gunnarsson, and
Sigvaldadóttir, 2002), but prevention mechanisms can come into play before the animal arrives
at the slaughtering plant.

The epidemiology of foodborne problems like salmonellosis is complex and expected to vary
with change in the pathogens themselves, industrialization, urbanization and change of lifestyles,
knowledge, belief and practices of food handlers and consumers, demographic changes
(increased susceptible population), international travel and migration, international trade in food,
animal feed and in animals, and poverty and lack of safe food preparation facilities (Van der
Venter, 1999; Altekruseet al., 1998; WHO, 1988).

In industrialized countries the incidence of salmonellosis is on the rise due to the emergence and
increase of S. Enteritidis and S. Typhimurium DT 104 (Wray and Davies, 2003; Gomez et al.,
1999; Van der Venter, 1999). Hundreds of millions of people worldwide suffer from
communicable and non-communicable diseases caused by contaminated food. These diseases
take a heavy toll in human life and sufferings, particularly among infants and children, the
elderly and other susceptible persons. They also create an enormous social, cultural and

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economic burden on communities and their health system (Van der Venter, 1999). Interest in
Salmonella has heightened in recent years due to the increased susceptibility of AIDS patients to
salmonellosis, the devastating effects of S. Enteritidis in the poultry industry, and the
globalization of agricultural trade (Clarke and Gyles, 1993). Persistent and severe salmonellosis
has also been recognized as a problem among patients with AIDS (Gerald, 1991).

In developing countries a rapidly growing industry of intensive animal production is

accompanying the process of urbanization with all its environmental and behavioral changes
favorable for Salmonella to prevail (WHO, 1988). Most food industries in developing countries
are not well aware of food safety issues, and knowledge of modern technologies, Good
Manufacturing Practices (GMP), hygiene, Hazard Analysis Critical Control Point (HACCP)
system, and quality control is often limited or absent. Cold storage facilities are inadequate and
quality of water used for food processing may not be suitable. The vast numbers of laborers that
handle food in factories, as well as on farms, are illiterate and untrained. In developing countries
including Ethiopia, foodborne illnesses are perceived as mild and self-limiting diseases. Their
severe and chronic health consequence is often overlooked, as are their consequences on trade
and the economy. In such countries lack of information leads to lack of appreciation of the health
significance of unsafe food and this, in turn, leads to low priority and sometimes no resources
assigned to food safety (Van der Venter, 1999).

Outbreaks of salmonellosis have been reported for decades, but within the past 25 years the
disease has increased in incidence in many continents. The disease appears to be most prevalent
in areas of intensive animal husbandry, especially of poultry or pigs and dairy cattle reared in
confinement (OIÉ, 2000; D’Aoust, 1989). There are pandemics of S. Enteritidis and
S.Typhimurium DT 104 which resulted in enacting regulations in many countries to control the
prevalence of salmonellosis in farm animals in order to prevent foodborne infection (Wray and
Davies, 2000).

The clinically normal carrier animal is a serious problem in all host species. Foods of animal
origin, particularly meat, poultry, and, in some instances, unpasteurized egg products are
considered to be the primary sources of human salmonellosis (Acha and Szyfres, 2001; White et
al., 2001; Wray and Davies, 2000; Gomez et al., 1997; D’Aoust, 1997; Nielsen et al., 1995;

3
Tauxe, 1991; WHO, 1988; Koulikovskii, 1982). Most of these food products, e.g. beef, mutton
and poultry, become contaminated during slaughter and processing, from the gut contents of
healthy excreting animals. In the same way, all food that is produced or processed in a
contaminated environment may become contaminated with salmonellae and be responsible for
outbreaks or separate cases of disease as a result of faults in transport, storage, or preparation
(D’Aoust, 1997; Koulikovskii, 1982). Food producers, processors, and distributors need to be
reminded that even low levels of salmonellae in a finished food product can lead to serious
public health consequences and undermine the reputation and economic viability of the
incriminated food manufacturer (D’Aoust, 1989).

Studies show that antimicrobial-resistant salmonellae are increasing due to the use of
antimicrobial agents in food animals, which are subsequently transmitted to humans usually
through the food supply (White et al., 2001; Angulo et al., 2000; Fey et al., 2000; Mølbaket al.,
1999; Tollefsonet al., 1998; D’Aoust 1989). If the frequency of drug resistance increases, the
choice of antimicrobials for treatment of systemic salmonellosis in humans becomes more
limited.

The isolation of drug-resistant strains of Salmonella first started in the 1960s, with the first
examples being resistant to only one antibiotic, while the discovery of MDR (multi-drug
resistance) has been observed with increasing frequency recently (Azia Elizabeth R. (2015).
Globally, the three main causes of antimicrobial resistance have been identified as use of
antimicrobial agents in agriculture, over-prescribing by physicians, and misuse by patients (IFT,
2003). Salmonella Typhimurium DT 104 has a broad host reservoir and is usually resistant to
five antibiotics (ampicillin, chloramphinicol, streptomycin, sulphonamides, and tetracycline) and
can be resistant to others (fluoroquinolones) (IFT, 2003; Glynn et al., 1998). Routine assessment
of patterns of emerging antibiotic resistant Salmonella strains is of paramount importance
because such information channeled to physicians and veterinarians help to timely redirect drug
use so as to diminish the development and spread of resistance. It also helps to know the extent
and temporal trends of antimicrobial susceptibility. The ultimate outcome will be to prolong the
efficacy of existing and new antimicrobial agents which are desperately needed to control both

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human and animal diseases and to minimize the spread of resistant zoonotic pathogens to
humans (Tollefsonet al., 1998).
In Ethiopia there have been several studies conducted on salmonellosis which suggest an
increase in the antibiotic resistance of Salmonella to commonly used antimicrobials in both the
public health and veterinary sectors (Getenet et al., 2008).

1.2 Statement of the problem

Detection of Salmonella before contaminated foods can be consumed is therefore an essential

feature of safeguarding public health and incidentally preserving the reputations and fortunes of
food manufacturers and processors.

Surveillance of Salmonella in all the different stages of feed-food chain constitutes an important
element in the exploration of epidemiology of foodborne salmonellosis, and in the development
and implementation of efficient Salmonella control strategies.

Efficient laboratory methods for isolation, identification and typing of Salmonella are essential
elements in Salmonella monitoring and control programmes. This protocol describes methods
that have been extensively documented in the scientific literature, are accepted by international
standardization bodies and can be applied under most conditions and circumstances in
laboratories worldwide.

In Ethiopia, as in other developing countries, it is difficult to evaluate the burden of

salmonellosis because of the limited scope of studies and lack of coordinated epidemiological
surveillance systems. In addition, under-reporting of cases and the presence of other diseases
considered to be of high priority may have overshadowed the problem of salmonellosis. The real
situation of antibiotic resistance is also not clear since Salmonella are not routinely cultured and
their resistance to antibiotics cannot be tested. As in a developed country, however, to control the
spread of salmonellosis, surveillance for Salmonella serovars and the assessment of antimicrobial
susceptibility is essential. As a result of this food borne illness is the major constraint and causes
an important public health problem in Ethiopia.

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1.3 Research question
The project will answer the following questions;
 The different camel meat contamination points/location by Salmonella during pre and
post slaughter operation to point out critical control locations.
 Public health risk of salmonellosis associated with the consumption of contaminated
camel meat in Eastern Ethiopia for awareness creation and control action.

1.4. Scope and significance of the research project

The ultimate goal of the current research project is to partly contribute towards the development
of national food safety strategies, which aim to protect the consumer from enteric zoonotic
foodborne diseases. This is through provision of basic data on the situation of Salmonella in
camel meat slaughter operation in Dire Dawa, Awoday and Harari municipal abattoirs.

1.5. Beneficiaries of the project

The beneficiaries of this project include research scientists, government agencies involved in
surveillance activities, the food industry and the general public.

1. Research scientists will benefit from a greater understanding of the diversity of genotype and
phenotype of the Salmonella pathogen. Salmonella is used as a model pathogen organism in
thousands of research labs around the world. These labs tend to use a single or a limited number
of strains in their studies. However, it is not known how generally applicable the conclusions are
from experiments from a single strain. Our study will provide a baseline data for the diversity of
genotype and phenotypes of closely related isolates of S. Typhimurium.

2. Government agencies involved in surveillance of bacterial pathogens will benefit from the
data and data analysis generated in this project

3. The food industry and in particular the camel rearing industry is interested in decreasing the
incidence of Salmonella in the food chain. The output from this project will provide the
knowledge required to discriminate between pathogens of high and low risk to human disease.

4. In the longer term the general public specifically Eastern Ethiopian people will benefit from
potential advances in diagnostic and surveillance methodologies that will become available with
the knowledge generated from the proposed work. The network of scientists involved as
collaborators on the research project have the expertise to deliver these improvements in parallel
and as a direct result of the proposed work.

1.6 Aim and objectives of the project

1. To determine the prevalence and distribution of Salmonella serotypes in camel meat
slaughtered at Dire Dawa, Harar and Awoday Municipal Abattoirs

2. To determine the possible sources of Salmonella serotypes along camel slaughter abattoir
lines.
3. To test the antimicrobial susceptibility of the Salmonella isolates.

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2. REVIEW OF LITERATURE
2.1. Overview of food hygiene and food safety
Foodborne diseases remain a real and formidable problem in both developed and developing
countries, causing great human suffering and significant economic losses. Up to one third of the
population of developed countries may be affected by food borne diseases each year, and the
problem is likely to be even more widespread in developing countries, where food and water-
borne diarrhoeal diseases kill an estimated 2.2 million people each year, most of them Children
(FAO/WHO, 2006). The problem is severe in developing countries due to difficulties in securing
optimal hygienic food handling practices. In developing countries, up to an estimated 70% of
cases of diarrheal disease are associated with the consumption of contaminated food (Knife and
Abera, 2007).
Food safety is therefore a fundamental public health concern, and achieving a safe food supply
poses major challenges for national food safety officials. Changing global patterns of food
production, international trade, technology, public expectations for health protection and many
other factors have created an increasingly demanding environment in which food safety systems
operate. An array of foodborne hazards both familiar and new, pose risks to health and obstacles
to international trade in foods. These risks must be assessed and managed to meet growing and
increasingly complex sets of national objectives (CAC, 2007).

2.1.1. Food hygiene and food safety practices in Ethiopia

Foodborne diseases are common in developing countries including Ethiopia because of the
prevailing poor food handling and sanitation practices, inadequate food safety laws, weak
regulatory systems, lack of financial resources to invest in safer equipment, and lack of education
for food handlers (WHO, 2004). National Hygiene and Sanitation Strategy program (MoH,
2005) reported that in Ethiopia more than 250,000 children die every year from sanitation and
hygiene related diseases and about 60% of the disease burden was related to poor hygiene and
sanitation in Ethiopia. Unsafe sources, contaminated raw food items, improper food storage, poor
personal hygiene during food preparation, inadequate cooling and reheating of food items and a
prolonged time lapse between preparing and consuming food items were mentioned as
contributing factors for outbreak of foodborne diseases (Linda du and Irma, 2005).

Studies conducted in different parts of the country showed the poor sanitary conditions of
catering establishments and presence of pathogenic organisms like campylobacter, Salmonella,
Staphylococcus aureus, Bacillus cereus and Escherichia coli, (Bayleyegn et al., 2003; Abera et
al., 2006; Knife and Abera, 2007; Tefera et al., 2009 and Mekonnen et al., 2013).
Of the foods intended for humans, those of animal origin tend to be most hazardous unless the
principles of food hygiene are employed. Animal products such as meats, fish and their products
are generally regarded as high-risk commodity in respect of pathogen contents, natural toxins
and other possible contaminants and adulterants (Yousuf et al., 2008). Bacterial contamination of
meat products is an unavoidable consequence of meat processing (Jones et al., 2008). Even if
data regarding meat borne diseases in Ethiopia are extremely scarce, a few studies conducted in

7
different parts of the country have shown the public health importance of several bacterial
pathogens associated with foods of animal origin (Bayleyegn et al., 2003; Ejeta et al., 2004:
Adem et al., 2008; Kumar et al., 2009 and Tefera et al., 2009). Salmonella remains a leading
etiological agent in bacterial foodborne diseases (D’Aoust, 1991).
2.2. Over view of salmonella

2.2.1 Evolution of Salmonella

The genus Salmonella was named after Daniel E. Salmon who first reported the isolation of
Salmonella from a pig in 1885 and named the organism Bacterium choleraesuis (currently
known as Salmonella enterica serovar Choleraesuis). A non-human Salmonella spp., S.
Choleraesius, was isolated from a swine’s intestine by Theobald Smith (1859-1934) under the
direction of Daniel E. Salmon (1850-1914) in 1885 (Ellermeier and Slauch, 2006). Dr Salmon
was the administrator of the USDA research program, and thus the organism was named after
him (Rao, 2004; FDA/CFSAN, 2008). It is conjectured that Salmonella and E. coli, which
evolved from a common ancestor emerged about the time of the emergence of mammals, and
emerges as mammalian and avian pathogens through the acquisition of pathogenicity islands and
of a virulence plasmid plus through variation in lipopolysaccharide antigens (Wray, C. and
Wray, A., 2001). It is hypothesized that accumulation of single mutations, insertions or deletions
with the genome of modern-time Salmonella appears to have generated many pseudo genes,
suggesting its recent evolutionary origin (Papagrigorakis et al., 2007).

2.2.2 Characteristics of Salmonella

The family Enterobacteriaceae consists of Gram-negative, facultatively anaerobic non-spore-
forming rods. Salmonella conforms to the general definition of the family (Quinn et al., 1999;
Intorre et al., 2005; Forshell and Wierup, 2006). Members of the genus are motile by
peritrichous flagellation except S. Pullorum and S. Gallinarum, which lack flagella. Nonmotile
variants can also arise as a result of a faulty assembly of flagellar subunits or deficiencies in the
motor functions of these appendages. Salmonellae are chemoorganotrophic, with an ability to
metabolize nutrients by the respiratory and fermentative pathways (D’Aoust, 1997; 2000).

2.2.2.1. Biochemical characteristics

Salmonellae are generally unable to ferment lactose, sucrose or salicin, although glucose and
certain other monosaccharides are fermented with the production of gas. They are usually
catalase positive, oxidase-negative and reduce nitrates to nitrites. The microorganisms use citrate
as the sole carbon source, decarboxylate lysine, arginine and ornithine and produce hydrogen
sulphide. The methyl-red reaction is positive, the Voges-Proskauer test is negative and indole is
negative. Phenylalanine is not delaminated, urea is not hydrolysed, gelatin is not liquified rapidly
in nutrient media and neither DNAase nor lipase are produced. Salmonellae may harbor
temperature phages or plasmids that code for metabolic characters used in identification (e.g.
H2S, lactose or sucrose fermentations) (Coetzer et al., 1994; D’Aoust, 2000; Jay, 2000; Mølbak
et al., 2006).

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2.2.2.2. Growth and destruction
Salmonellae are able to grow on a large number of culture media and produce visible colonies
well within 24 h at about 37oC. The parameters of pH, water activity (aw), nutrient content and
temperature are all interrelated for salmonellae, as they are for most other bacteria. The pH for
optimum growth is around neutrality (between 6.6 and 8.2), with values above 9.0 and below 4.0
being bactericidal. A minimum growth pH of 4.05 has been recorded for some (with
hydrochloric and citric acids), but depending on the acid used to lower the pH, the minimum may
be as high as 5.5. Aeration was found to favor growth at lower pH values (Jay, 2000; D’Aoust,
2001).

Salmonella grows in the temperature range of 2 - 47oC with rapid growth between 25 and 43oC
(D’Aoust, 2000; 2001). The lowest temperatures at which growth has been reported are 5.3oC
for S. Heidelberg and 6.2oC for S. Typhimurium. Temperatures of around 45oC have been
reported by several investigators to be the upper limit for growth. Regarding available moisture,
growth inhibition has been reported for aw values below 0.94 in media with neutral pH, with
higher aw values being required as the pH is decreased toward growth minima (Coetzer et al.,
1994; Jay, 2000; D’Aoust, 2001).

Salmonellae are unable to tolerate high salt concentration. Brine above 9% is reported to be
bactericidal. Nitrite is effective, with the effect being greatest at the lower pH values. This
suggests that the inhibitory effect of this compound is preferable to the undissociated HNO2
molecule (Jay, 2000).

With respect to heat destruction, all salmonellae are readily destroyed at milk pasteurization
temperatures. Some investigators also found that cells grown at 44oC were more heat resistant
than those grown at either 15ºC or 35ºC (Jay, 2000).

Some food products are packaged under vacuum or gaseous mixtures of carbondioxide, nitrogen
and oxygen to increase product quality and extend shelf life. The benefits of modified
atmosphere packaging (MAP) stem are from the inhibition of endogenous spoilage micro flora
and enhanced organoleptic stability of the product. The high concentrations of carbondioxide
(>50%v/v) commonly used in MAP inhibit the growth of Salmonella spp. but exert little or no
effect on survival (D’Aoust, 2001).

2.2.3. Classification and nomenclature

Historically Salmonella had been named based on the original places of isolation such as
Salmonella London and Salmonella Indiana. This nomenclature system was replaced by the
classification based on the susceptibility of isolates to different selected bacteriophages which is
also known as phage typing. Phage typing is generally employed when the origin and
characteristic of an outbreak must be determined by differentiating the isolates of the same
serotype. It is very reproducible when international standard sets of typing phages are used More
than 200 definitive phage types (DT) have been reported so far. For example, S. Typhimurium
DT104 designates a particular phage type for Typhimurium isolates (Hanes, 2003; Andrews and
Baumler, 2005 and Pui et al., 2011).

9
Epidemiologic classification of Salmonella is based on the host preferences. The first group
includes host-restricted serotypes that infect only humans such as S. Typhi. The second group
includes host-adapted serotypes which are associated with one host species but can cause disease
in other hosts serotypes such as S. Pullorum in avian. The third group includes the remaining
serotypes. Typically, Salmonella Enteritidis, Salmonella Typhimurium and Salmonella
Heidelberg are the three most frequent serotypes recovered from humans each year (Gray and
Fedorka-Cray, 2002 and Boyen et al., 2008).
The genus consists of two species: the first is S. enterica which is divided into six
subspecies(Figure 1): S. enterica subsp. enterica, S. enterica subsp. salamae, S. enterica subsp.
arizonae, S. enterica subsp. diarizonae, S. enterica subsp. houtenae and S. enterica subsp.
indica; and the second is S. bongori (formerly called S. enterica subsp. bongori) (WHO, 2003c).
Salmonella enterica subspecies is mainly isolated from warm-blooded animals and accounts for
more than 99% of clinical isolates whereas remaining subspecies and S. bongori are mainly
isolated from cold-blooded animals and account for less than 1% of clinical isolates. As an
example, the Kauffmann species Salmonella Typhimurium is now designated as Salmonella
enterica subspecies I serotype Typhimurium. Under the modern nomenclature system, the
subspecies information is often omitted and culture is called S. enterica serotype Typhimurium
and in subsequent appearance, it is written as S. Typhimurium. This system of nomenclature is
used nowadays to bring uniformity in reporting (Andrews and Baumler, 2005 and Parry, 2006).
Kauffmann-White scheme classifies Salmonella according to three major antigenic determinants
composed of flagellar H antigens, somatic O antigens and virulence (Vi) capsular K antigens.
This was adopted by the International Association of Microbiologists in 1934. Agglutination by
antibodies specific for the various O antigens is employed to group Salmonellae into the 6
serogroups: A, B, C1, C2, D and E. For instance, S. Paratyphi A, B, C and S. Typhi express O
antigens of serogroups A, B, C1 and D, respectively. More than 99% of Salmonella strains
causing human infections belong to Salmonella enterica subspecies enterica. Although not
common, cross-reactivity between O antigens of Salmonella and other genera of
Enterobacteriaceae do occur (Pui et al., 2011).
Therefore, further classification of serotypes is based on the antigenicity of the flagellar H
antigens which are highly specific for Salmonella (Scherer and Miller, 2001). In brief, O
antigens are lipopolysaccharide (LPS) of the outer bacterial membrane. They are heat stable,
resistant to alcohol and dilute acids. H antigens are heat-labile proteins associated with the
peritrichous flagella and can be expressed in one of two phases. The phase 1 H antigens are
specific and associated with the immunological identity of the particular serovars whereas phase
2 antigens are non-specific antigens containing different antigenic subunit proteins which can be
shared by many serovars. K antigens which are heat- sensitive carbohydrates are produced by
Salmonella serovars that express a surface-bound polysaccharide capsular antigen (Hu and
Kopecko, 2003; Yousef and Carlstrom, 2003). Therefore, further classification of serotypes is
based on the antigenicity of the flagellar H antigens which are highly specific for Salmonella
(Scherer and Miller, 2001). In brief, O antigens are lipopolysaccharide (LPS) of the outer
bacterial membrane. They are heat stable, resistant to alcohol and dilute acids. H antigens are
heat-labile proteins associated with the peritrichous flagella and can be expressed in one of two
phases. The phase 1 H antigens are specific and associated with the immunological identity of
the particular serovars whereas phase 2 antigens are non-specific antigens containing different
antigenic subunit proteins which can be shared by many serovars. K antigens which are heat-

10
sensitive carbohydrates are produced by Salmonella serovars that express a surface-bound
polysaccharide capsular antigen (Hu and Kopecko, 2003; Yousef and Carlstrom, 2003).

Figure 1: Classification of the genus Salmonella

Source: Langridge et al., (2008).
Note: Numbers in brackets indicate the total number of serotypes included in each subspecies.
* Common serotypes are listed but other serotypes may cause bacteraemia or focal infection;
subsp = subspecies.

2.2.4. Epidemiology
Salmonella is one of the leading causes of bacterial foodborne disease in industrialized as well as
developing countries even though the incidence seems to vary between countries (Radostits et
al., 1994; D’Aoust, 1997; 2000; Giovannacci et al., 2001; Molla et al., 2003; Chiu et al., 2004;
Vieira-Pinto et al., 2005). The wide variations in the national prevalence of salmonellosis likely
arise from limited scope of studies and lack of coordinated epidemiological surveillance systems,
under-reporting of cases and the presence of other diseases considered to be of high priority
(Radostits et al., 1994; D’Aoust, 2000; 2001; Molla et al., 2003).
The last few decades have witnessed marked increases in the incidence of human Salmonella
infections. Several factors, including tourist travel, international movement of foods and food
ingredients, animal feed trade and the importation of infected animal replacement stocks
contribute to the national succession of Salmonella serotypes in human populations and in the
food chain. The prominence of agricultural products as major reservoirs of Salmonella spp.
arises from the ubiquity of the microorganism in the natural environment, the intense on-farm

11
husbandry practices that favor the spread of salmonellae among reared animals, the use of
untreated sludge to fertilize agricultural land and rendering of animal offals into frequently
contaminated feed proteins (D’Aoust, 1997; 2001; Mølbak et al., 2006).
According to the WHO Global Salm-Surv, during 2000-2002, S. Enteritidis was by far the most
common serotype reported from humans globally. In 2002, it accounted for 65% of all isolates,
followed by S. Typhimurium at 12% and S. Newport at 4%. Among nonhuman isolates, S.
Typhimurium was the most commonly reported serotype in all the 3 years, accounting for 17%
of isolates in 2002 followed by S. Heidelberg (11%) and S. Enteritidis (9%). Salmonella
Enteritidis, S. Typhimurium and S. Typhi were ranked among the 15 most common human
serotypes in all regions of the world throughout the 3-year study period. Salmonella Agona, S.
Infantis, S. Montevideo, S. Saintpaul, S. Hadar, S. Mbandaka, S. Newport, S. Thompson, S.
Heidelberg, and S. Virchow were also widespread. In Africa in 2002, S. Enteritidis and S.
Typhimurium were each reported from approximately one fourth of isolates from humans.
Among nonhuman sources, S. Anatum and S. Enteritidis constituted the largest proportion of
isolates (Galanis et al., 2006; Swaminathan et al., 2006).
2.2.4.1. Distribution
The primary habitat of Salmonella is the intestinal tract of farm animals, humans, birds, reptiles,
and occasionally insects. Although their primary habitat is the intestinal tract, they have also
been found in spleen, liver, bile, mesenteric and portal lymph nodes, diaphragm, and pillar as
well. As intestinal forms, the organisms are excreted in feces from which they may be
transmitted by insects and other living creatures to a large number of places. Moreover, they may
also be found in polluted water (Radostits et al., 1994; Quinn et al., 1999; Jay, 2000; Vieira-
Pinto et al., 2005).

Salmonellae can survive for 9 months or more in the environment in sites such as moist soil,
water, fecal particles and animal feeds, especially in blood-and-bone and fish meals (Quinn et
al., 1999; D’Aoust, 2001; Mølbak et al., 2006). The ubiquity of salmonellae in the natural
environment, coupled with the intensive husbandry practices used in the meat, fish and shellfish
industries and the recycling of offal and inedible raw materials into animal feeds, has favored the
continued prominence of this human bacterial pathogen in the global food chain (D’Aoust,
1997).
European Union scientific committee concluded that the food categories that possibly pose the
greatest hazard to public health include: raw meat and some meat products intended to be eaten
raw, raw or undercooked poultry meat products, eggs and products containing raw eggs and
unpasteurised milk and some milk products. Sprouted seeds, and unpasteurised fruit juices are
also of concern (D’Aoust, 1997; Jay, 2000; Chiu et al., 2004; Forshell and Wierup, 2006).
Contamination of milk usually occurs after the milk leaves the cow, even though the organism
can be excreted into the milk during the acute phase of the disease and occasionally by carrier
animals (Radostits et al., 1994). Moreover, salmonellae have been found in commercially
prepared and packaged foods. Among the contaminated foods were cake mixes, cookie dough,
cornbread mixes, coconut meal, salad dressing, mayonnaise, milk and other foods (Jay, 2000;
D’Aoust, 2001; Mølbak et al., 2006).

12
2.2.4.2. Host range
Salmonellae have a wide variety of domestic and wild animal hosts. All members of the genus
are considered to be potentially pathogenic, although serotypes may differ widely in their host
range and the pathogenic syndromes that they produce. From the epidemiological point of view,
salmonellae can be classified in to three main groups. The first group (human-adapted)
comprises S. Typhi, S. Paratyphi A and S. Paratyphi C, which infect humans only and are spread
directly or indirectly from person to person. The second group includes serotypes that are host-
adapted for particular species of vertebrates. Included are S. Gallinarum (poultry), S. Dublin
(cattle), S. Abortus-equi (horses), S. Abortus-ovis (sheep) and S. Choleraesuis (swine). Some of
these are also pathogenic for human (especially S. Dublin and S. Choleraesuis). The third group
(nonhost-adapted) contains the majority of the other Salmonella serotypes with no particular host
preference that infect both humans and other animals (WHO, 1988; Jay, 2000; Forshell and
Wierup, 2006).

2.2.4.3. Source of infection and transmission

Salmonellae are mainly transmitted by the fecal-oral route. They are carried asymptomatically in
the intestines or gall bladder of many animals, and are continuously or intermittently shed in the
feces (Radostits et al., 1994; OIE, 2005; Forshell and Wierup, 2006). They can also be carried
latently in the mesenteric lymph nodes or tonsils; these bacteria are not shed, but can become
reactivated after stress or immunosuppression. Fomites and mechanical vectors (insects) can
spread Salmonella (OIE, 2005).
Vertical transmission occurs in birds, with contamination of the vitelline membrane, albumen
and possibly the yolk of eggs. Salmonella spp. can also be transmitted in utero in mammals
(OIE, 2005).
Animals can become infected from contaminated feed (including pastures), drinking water or
close contact with an infected animal (including humans). Birds and rodents can spread
Salmonella to livestock. Carnivores are also infected through meat, eggs and other animal
products that are not thoroughly cooked. Cats sometimes acquire Salmonella Typhimurium after
feeding on infected birds or spending time near bird feeders (OIE, 2005; Forshell and Wierup,
2006).
People are often infected when they eat contaminated foods of animal origin such as meat or
eggs (OIE, 2005). Ingesting organisms in animal feces can also infect them, either directly or in
contaminated food or water. Directly transmitted human infections are most often acquired from
the feces of reptiles, chicks and ducklings. Livestock, dogs, cats, adult poultry and cage birds can
also be involved (OIE, 2005).
The trade of live animals within and between countries spreads Salmonella. Moreover, trade in
contaminated animal feed products has also significantly contributed to the spread of Salmonella
and several large outbreaks in humans have been traced back to contaminated animal feed.
Salmonella is additionally spread between countries by humans as a result of foodborne
infections acquired abroad. The overall importance of these routes of transmission may reflect
the prevalence of Salmonella contamination of food (including food of animal origin) in a
particular country (Forshell and Wierup, 2006).

13
2.2.4.4. Pathogenesis
After entering the small bowel, salmonellae must traverse the intestinal mucus layer before
encountering and adhering to cells of the intestinal epithelium. Salmonellae express several
fimbriae that contribute to their ability to adhere to intestinal epithelial cells (Radostits et al.,
1994; Quinn et al., 1999; Ohl and Miller, 2001). Once across the intestinal epithelium,
salmonellae encounter another obstacle of innate immunity, the submucosal macrophage.
Salmonella serotypes that cause systemic infection enter macrophages, again apparently by
induced macropinocytosis, and subsequently activate virulence mechanisms that allow evasion
of the microbiocidal functions of the phagocyte, permitting survival and replication in the
intracellular environment. Migration of infected phagocytes to other organs of the
reticuloendothelial system probably facilitates dissemination of bacteria in the host (Ohl and
Miller, 2001).
The invasive strains that produce septicemia are able to escape destruction by the host and to
multiply within the macrophages of the liver and spleen as well as intravascularly. The invasive
abilities of some strains of S. Typhimurium are increased by the presence of genes carried on a
plasmid. O-repeat units of the lipopolysaccharide prevent destruction within the blood stream. It
is thought that they may mask determinants on the bacterial cell surface that would normally
bind complement and activate it by means of the alternate pathway. This would reduce chances
of chemotaxis, opsonization and phagocytosis. As a result there will be multiplication of the
organisms in the body that subsequently leads to a severe endotoxaemia. Furthermore, these
invasive salmonellae secrete siderophores, which remove iron from iron-binding proteins of the
host (Radostits et al., 1994; Quinn et al., 1999).

2.2.4.5. Carrier states

Because salmonellae are facultatively intracellular organisms that survive in the phagolysome of
macrophages, they can evade the bactericidal effects of antibody and complement. Thus,
persistence of infection in animals and in the environment is an important epidemiological
feature of salmonellosis. When an animal is infected with S. Dublin it may become a clinical
case or an active carrier, passing organisms constantly or intermittently in the feces. It may also
become a latent carrier with infection persisting in lymph nodes or tonsils but no salmonellae in
the feces, or even a passive carrier, which is constantly picking up infection from pasture, but is
not invaded so that when is removed from the environment the infection disappears. The
importance of the latent carriers is that they become active carriers or even clinical cases under
stressful conditions. Although all infected adults become carriers it is rarely for any length of
time, and calves rarely become carriers. In sheep and cattle the carrier state may persist for as
long as 10 weeks and in horses up to 4 months (Radostits et al., 1994).
The intermittent fecal shedding that may follow the acute phase of human salmonellosis may be
of short duration (convalescent carrier) or may persist for one or more years (chronic carrier) if
the condition is not effectively treated. Carrier states are of concern to the food manufacturing
and food service industries because of the perceived risk of cross contamination of foods by
infected food handlers and potentiation of foodborne disease outbreaks. Although temporary
exclusion of food handlers with non-typhoid diarrheal illness from work in sensitive areas is
common practice, there is lack of unanimity on the need to exclude asymptomatic workers from
their food-related duties and need to subject them to follow-up stool testing (D’Aoust, 2001).

14
2.2.5. Clinical signs
The disease manifestations depend on the virulence of different Salmonella serovars; the number
of Salmonella ingested and host immunity. Many Salmonella infections are opportunistic
infection in compromised hosts. The majority of Salmonella infections in a herd over time are
subclinical; the clinical infections are only the tip of the iceberg, even during outbreaks of
clinical disease (Gay, 2003). A sudden change in herd resistance caused by concurrent disease,
for example, fascioliasis, bovine virus diarrhea virus, nutritional stress, or severe weather can
result in clinical disease (Wray and Davies, 2003). Salmonellosis is manifested in animals in
three major forms: enteritis, septicemia, and abortion. However, in an outbreak, or even in a
single animal, any combination of the three may be observed (Wray and Sojka, 1977). Fever,
inappetence, and depression are commonly observed in acutely ill animals. Enteritis caused by
Salmonella results in the passage of foul-smelling, watery feces, which may contain fibrin,
mucus, and sometimes blood (Wray and Sojka, 1977). When the enteric disease is severe, death
may result from dehydration, electrolyte loss, and acid-base imbalance (Clarke and Gyles, 1993).
Septicaemic form often leads to abortion. Salmonella Dublin has been associated with outbreaks
of abortion in cattle, and several other serovars adapted to animal hosts have a particular
association with abortion (Wray and Sojka, 1977). The signs and lesions are not pathognomonic
and in many cases, especially in poultry and pigs, Salmonella infections may be inapparent (OIÉ,
2000).

2.2.6. Immunity
According to Wray and Davies (2000) the relative importance of humoral and cell-mediated
immunity has been the subject of debate, but it is now generally accepted that both play a part in
producing protection. Nevertheless, there is widespread agreement that cell-mediated immunity
is more important than humoral antibodies in the resistance to salmonellosis. Cell-mediated
immunity has its basis in enhanced microbicidal activity of the host’s macrophages for the
organism and is not serotype specific. The bactericidal actions of phagocytes supported by the
lytic actions of T-killer lymphocytes and activated serum complement generally circumscribe the
non-typhoid infection to a local intestinal manifestation with no systemic involvement (D’Aoust,
1991a). Humoral immunity contributes to bacterial clearance, and is serotype-specific (Gillepsie
and Timoney, 1981). Both live and inactivated vaccines have been used to prevent salmonellosis
by immunization (Wray and Davies, 2000) but not the infection or carrier status (Acha and
Szyfres, 2001). Although many publications attest to the efficacy of live vaccine in experimental
trials, few are commercially available (Wray and Davies, 2000). Live vaccine stimulates a
greater cell-mediated immune response than bacterins, which primarily promote a humoral
response with little or no association with protection. Oral administration (whether bacterins or
live vaccine) has the advantage of producing local immunity in the intestine and reducing
elimination of salmonellae in feces (Acha and Szyfres, 2001).

2.2.7 Diagnosis
Salmonella can be isolated either from tissues collected aseptically at necropsy or from feces,
rectal swabs, environmental samples, food products and feedstuffs. When infection of the
reproductive organs, abortion or conceptus occurs, it is necessary to culture fetal stomach
contents, placenta and vaginal swabs and, in the case of poultry, embryonated eggs. Individual
samples for bacteriological tests should be collected as aseptically as possible by following the
15
respective standards. Moreover, precautions should be taken to avoid cross contamination of
samples during transit and at the laboratory. Packages should also be kept cool and accompanied
by adequate information (OIE, 2005).
Conventional cultural methods for the detection of foodborne Salmonella spp. generally consist
of five distinct and successive steps: pre-enrichment in nonselective media, selective enrichment
in broth media, plating on differential agar, biochemical screening and serological confirmation
(D’Aoust, 2000; 2001).

2.2.7.1. Pre-enrichment in nonselective liquid medium

The number of Salmonella in feces from asymptomatic animals, environmental samples, animal
feed and food is usually low, and are often accompanied by considerably larger numbers of other
Enterobacteriaceae or other families. Therefore, it is necessary to use pre-enrichment media to
assist the isolation. Furthermore, pre-enrichment is necessary to permit the detection of sub
lethally injured Salmonella (ISO 6579, 2002).
Pre-enrichment in a nonselective broth medium for 18-24h at 35-37oC allows the small numbers
of salmonellae, which may otherwise be killed by the toxic effect of enrichment media, to
multiply, or it may help to resuscitate salmonellae that have been sub lethally damaged, e.g. by
freezing, heating, exposure to biocides or desiccation. Pre-enrichment may not be the best
method for isolating less vigorous Salmonella strains, such as the host-adapted strains, from
feces because of overgrowth by competing organisms during nonselective pre-enrichment
(D’Aoust, 2000; 2001). The use of short (6-8h) pre-enrichment for greater method brevity is not
recommended because it fails to provide salmonellae with sufficient time to adapt to its new
environment, repair cellular damage and actively grow to high numbers (D’Aoust, 2001).
The traditional pre-enrichment media include nonfat dry milk with added brilliant green dye for
the pre-enrichment of cocoa and chocolate products, brilliant green water for milk powder,
tripticase soy broth supplemented with potassium sulfite to neutralize spice-dependent
bacteriostasis and buffered peptone water, nutrient or lactose broths for other foods and
agricultural products, however, buffered peptone water is the most commonly used pre-
enrichment medium in the recovery of Salmonella in foods (D’Aoust, 2001).

2.2.7.2. Enrichment in selective liquid media

Enrichment media are liquid or semi-solid agar media that contain additives that selectively
permit salmonellae to grow while inhibiting the growth of other bacteria (D’Aoust, 2000; 2001).
Some, however, are also relatively toxic to certain serotypes of Salmonella, e.g. selenite inhibits
S. Choleraesuis, and brilliant green is toxic to many strains of S. Dublin. Elevated temperatures
have also been used to increase the selectivity of enrichment medium, and a temperature of 43°C
is used in some laboratories, although this may be inhibitory with some media, e.g. tetrathionate
and Rappaport-Vassiliadis at 43°C inhibit temperature-sensitive strains, especially S. Dublin and
41.5°C is now recommended for incubation of Rappaport-Vassiliadis broth. Selective motility
enrichment may also be used to increase the sensitivity of Salmonella isolation and semi-solid
enrichment media, e.g. modified semi-solid Rappaport-Vassiliadis or Diagnostic Semi-Solid
Salmonella medium (DIASALM), may provide greater sensitivity. The formulation of the
medium, temperature and duration of incubation, and the volume of the samples used to
inoculate the medium, may all serve to improve the isolation rate, and these variables should
always be taken into account. Examples of selective enrichment medium are sodium

16
tetrathionate, as in Muller-Kauffman broth, selenite cysteine (SC), tetrathionate brilliant green
(TBG) broth and Rappaport-Vassiliadis (RV) broths, or semi-solid Rappaport-Vassiliadis
medium. Additions such as Ferrioxamine E may be added to selective media to enhance isolation
of Salmonella from iron or nutrient-limited samples such as eggs, water or soil (D’Aoust, 2001;
ISO 6579, 2002).
The concurrent use of two enrichment media where one medium is incubated at 41-43oC and the
other at a more permissive temperature (35-37oC) maximizes detection of the target
microorganism. These conditions accommodate fastidious Salmonella spp. that grow poorly or
are inhibited at elevated temperatures and whose growth is hampered in highly selective
enrichment media such as TBG and RV. Numerous studies on the reliability of enrichment
conditions support the following decreasing order of effectiveness: TBG43 ≥ RV43 > TBG35 >
SC35 (D’Aoust, 2000; 2001).

2.2.7.3. Plating out and identification

Enrichment cultures are plated onto selective agar media for the presumptive identification of
Salmonella colonies on the basis of discriminating biochemical reactions. Standard plating media
include brilliant green agar (BGA), brilliant green sulfa (BGS), Bismuth sulfite agar (BSA),
xylose-lysine-desoxycholate (XLD) and Hektoen enteric (Hek) agars that report on acid
production from lactose and/or sucrose utilization through determinant colour changes in the
media. Salmonella spp that typically produce hydrogen sulfide appear as charcoal black colonies
with or without a black halo that produce a metallic sheen under reflected light. The Rambach
and SM-ID plating media produce discriminating colour reaction in the presence of isolates that
are galactosidase positive and that produce acid from propylene glycol (Rambach) and
glucuronate (SM-ID). Plating media generally yield suspect colonies within 18-24 h incubation
at 35-37oC, except BSA, which may require 48 h incubation for the development of presumptive
Salmonella colonies. Comparative studies support the following ranking in decreasing order of
effectiveness: BSA > BGS > BGA  Rambach = SM-ID > XLD > Hek (D’Aoust, 2000; 2001;
ISO 6579, 2002).
2.2.7.4. Confirmation
For confirmation, at least five presumptive (typical or suspect) Salmonella colonies will be
selected from every selective plating media. If the suspected colonies on each plate are fewer
than five, all the colonies will be selected. The selected colonies will be streaked onto the surface
of pre-dried nutrient agar plates, in a manner that will allow well-isolated colonies to develop.
Then the inoculated plates will be incubated at 37ºC ± 1ºC for 24h ± 3h. The pure cultures on
nutrient agar will be used for biochemical and serological confirmation (ISO 6579, 2002).

Biochemical confirmation
The use of lactose and/or sucrose, the production of H2S and presence of lysine decarboxylase
and urease are key determinants in the biochemical screening of presumptive Salmonella isolates
(D’Aoust, 2000; 2001; ISO 6579, 2002).
Salmonellae are facultatively anaerobic and, with few exceptions, all produce gas from
fermentable sugars. In culture, nitrates are reduced to nitrites, and hydrogen sulfide is produced.
Glucose and maltose are fermented, dulcitol and inositol are variably used, and sucrose is not
fermented (Coetzer et al., 1994).

17
Serological confirmation
The three kinds of surface antigens, O antigens, H antigens, and Vi antigens, determine the
reactions of the organisms to specific antisera. The detection of the presence of Salmonella O, Vi
and H-antigens is done by the slide agglutination with the appropriate sera, from pure colonies
after auto-agglutinable stains have been eliminated. This method relies on the antibody/antigen
reaction between a test culture and commercially prepared antiserum (Popoff and Le Minor,
2001; ISO 6579, 2002).

2.2.7.5. Typing
Because of the importance of Salmonella in foodborne disease, numerous typing methodologies
have been developed and have been used to trace salmonellosis outbreaks to the contaminated
source and to delineate the epidemiology of Salmonella infections (Kotetishvili et al., 2002;
Botteldoorn et al., 2004). Some of the typing techniques include serotyping and phage typing
(Kotetishvili et al., 2002; Demczuk et al., 2003; Botteldoorn et al., 2004). These techniques are
useful for defining relationships between strains (Botteldoorn et al., 2004).

Serotyping
Serotyping is based on the O and H antigens using a slide agglutination test (Coetzer et al., 1994;
Quinn et al., 1999; Mortimer et al., 2004). Most serotypes exhibit diphasic flagellar antigen
expression by alternately expressing two genes, fliC (phase 1) and fljB (phase 2) which encode
flagellins of different antigenicity. Salmonella serotyping methods recognise 63 distinct phase 1
flagellar antigenic factors and 37 phase 2 flagellar antigenic factors although the latter are not
always present (Mortimer et al., 2004).
Bacterial growth for serotyping should be taken from a triple sugar iron (TSI) agar slant or from
nutrient agar as culture from selective media is often unsuitable for typing. Then a loopfull of
culture of the Salmonella to be serotyped should be suspended in a drop of saline on a
microscope slide and examined for autoagglutination. This can occur with rough strains and will
invalidate the serotyping. Smooth-rough dissociation occurs after subculture and most frequently
from media containing carbohydrates (Coetzer et al., 1994; Quinn et al., 1999). Smooth
Salmonella to be serotyped is emulsified in a drop of 0.85% saline on a clean microscope slide.
A drop of antiserum is added to and mixed well with the Salmonella suspension. The slide is
rocked gently for about 30 seconds and the antigen-antibody mixture examined for agglutination.
The Salmonella is first tested against antisera to the O antigens and then the H antigen (Quinn et
al., 1999).

Phage typing
Phage typing is based on the specificity of a given phage for its host bacterium, and this
relationship allows one to use known phages to identify their specific hosts (Jay, 2000).
Therefore, phage typing of Salmonella isolates is based on the sensitivity of a particular isolates
to a series of bacteriophages at appropriate dilutions. This can be useful to determine whether
isolates, which come from different places at different times, are similar or different in their
reactions with specific sets of phages used for typing (Quinn et al., 1999).
In more detail, phage typing is based on specific bacteriophage reacting with specific receptors
on bacterial cell walls or pili so that they can then invade and lyse the bacteria. The test isolate is

18
exposed to a series of different phage and the pattern of lysis is observed and compared with
other patterns recorded previously. Banks of phage have been developed for a number of
serotypes including S. Typhimurium and S. Enteritidis. Very dry agar plates containing a rich
nutrient source are flooded with a liquid culture of the bacterial isolate. The liquid culture is
removed; the culture film allowed to dry and a set of phage suspensions are inoculated onto the
surface of the plate by means of a multipoint inoculator. Phage spots are left to dry, and plates
are incubated inverted overnight at 37oC. The next day, phage lysis reactions are recorded, and
compared to a set of standards developed by the Public Health Laboratory Service, Colindale,
England guidelines (Schmieger, 1999).

2.2.8. Clinical feature

2.2.8.1. Salmonella infections in humans

Infections with non-typhoid Salmonella serotypes most often result in self-limited acute
gastroenteritis that does not require antimicrobial therapy (D’Aoust, 1991; Chiu et al., 2004).
Nevertheless, approximately 5% of individuals with gastrointestinal illness caused by non-
typhoid Salmonella serotypes develop bacteremia (Chiu et al., 2004). Non-typhoid salmonellosis
in humans is usually manifested as a localized enterocolitis. The incubation period ranges from
5h to seven days, but clinical signs usually begin 12 h to 36 h after ingestion of a contaminated
food. Shorter incubation periods are generally associated with either higher doses of the
pathogen or highly susceptible people (Forshell and Wierup, 2006).
Clinical signs include abdominal pain, nausea, watery diarrhoea with occasional mucus and
traces of blood, mild fever and chills. The diarrhoea varies from a few thin vegetable-soups like
stools to massive evacuations with accompanying dehydration. Vomiting, prostration, anorexia,
headache and malaise may also occur. The syndrome usually lasts for two to seven days.
Susceptibility to infection is highest in infants, elderly people and in immuno-compromised
hosts. A fatal outcome is rare. With the exception of S. Choleraesuis and S. Dublin, which show
a definite predilection for systemic spread, non-typhoid salmonellosis is generally restricted to
the intestinal tract. The excreta of infected patients contain large numbers of Salmonella spp. at
the onset of illness. Those numbers decrease with the passing of time. Some patients become
carriers, and some are still excreting Salmonella spp. after three months (D’Aoust, 1991; Chiu et
al., 2004; Forshell and Wierup, 2006).
In contrast to non-typhoid salmonellosis, human infections of S. Typhi (enteric fever) and
paratyphoid strains are more severe and characteristically involve a systemic spread of the
pathogen and invasion of extra intestinal tissues. Following a period of incubation ranging from
8 to 28 days, non-specific symptoms of fever, chills and abdominal pain with underlying
bacteremia gradually build up in intensity within the first few week of illness. Appearance of
watery diarrhea (or constipation) with persistent abdominal pain, prostration and cutaneous rose
spots that rarely affect the extremities are commonly encountered in the second week of disease.
The course of the disease in the next 2 weeks may involve gradual resolution of symptoms with
possible relapses 7 to14 days after completion of antibiotic therapy or more serious conditions
such as intestinal perforation, osteomyelities and meningitis. The fatality case ratio of S. Typhi
infection ranges from 1% in developed countries to more than 10% in third world countries
(D’Aoust, 1991).

19
2.2.8.2. Salmonella infections in animals
Salmonellae are often carried asymptomatically (OIE, 2005; Forshell and Wierup, 2006).
Clinical disease usually appears when animals are stressed by factors such as transportation,
crowding, food deprivation, weaning, parturition, a concurrent viral or parasitic disease, sudden
change of feed, or overfeeding following a fast. Salmonellosis is common in horses after major
surgery. In some cases, oral antibiotics may also precipitate disease. Although salmonellosis can
be seen in all domestic animals, pregnant, lactating or young mammals and birds are the most
susceptible (OIE, 2005).
The clinical signs vary with the infecting dose, health of the host, Salmonella serotypes and other
factors (OIE, 2005). Host adapted serotypes primarily cause abortions or severe gastroenteritis in
their animal hosts. In food animals, a group of more frequently isolated serotypes, such as S.
Typhimurium, S. Enteritidis, S. Hadar and S. Infantis manifest themselves clinically through per-
acute septicemia, acute enteritis or chronic enteritis. In the subclinical form of the disease, the
animal may either have a latent infection or become a temporary or persistent carrier. The
remaining, less frequently isolated serotypes can colonize animals, usually without significant
clinical signs, but they are all considered capable of causing gastrointestinal infection of varying
severity in humans (Forshell and Wierup, 2006).

2.2.8.3. Salmonella in camel

Numerous serotypes of Salmonella have been isolated from camels, some of them associated
with enteritis (Salmonella typhimurium, S. enteritidis, S. dublin) or with abortion (S. dublin, S.
bovis morbifleans). Most of the serotypes are ubiquitous. Certain serotypes particularly
pathogenic for human beings (S. typhi, S. paratyphi C) have been isolated occasionally from
camels (Ambwani V. & Jatkar P. (1973).

2.2.9. Treatment and Control

2.2.9.1. Treatment
Nursing care is the principal treatment for the enteric form of salmonellosis. Treatment of the
systemic form includes nursing care and appropriate antimicrobial therapy as determined by
retrospectively acquired susceptibility data. Treatment options may be compromised due to
acquisition of resistance (R) plasmids encoding resistance to multiple antibiotics (Hirsh, 1999;
D’Aoust, 2000). In man, antibiotics are not indicated in salmonellae gastroenteritis, except in
very young and those over 60 (Davis et al., 1980) and to individuals with severe invasive
infection (Lesser and Miller, 2001; Fey et al., 2000; Lee et al., 1993), since the disease is brief
and limited to the gastrointestinal tract. In addition, the unnecessary use of antibiotics prolongs
salmonella excretion, promotes the incidence of the carrier state, and favors the acquisition of
resistance by the infecting strain (Hohmann, 2001; D’Aoust, 1991a; Baird-Parker, 1990).

2.2.9.2. Control
The control of Salmonella in meat animals and derived products is a most challenging task
because of the complexity and interdependence of various aspects of animal husbandry,
slaughtering, and food processing (D’Aoust, 2001). Because of the complexity of Salmonella
virulence factors, little progress has been made in converting the available knowledge into

20
therapeutics. Good Agricultural Practices (GAP), Good Manufacturing Practices (GMP), Hazard
Analysis Critical Control Point (HACCP), system appropriate food handling, and adequate water
treatment remain the best preventive measures for most Salmonella infection, although the
typhoid vaccines are effective against S. Typhi in humans, and vaccines for several other
serovars have shown promise in food animals (IFT, 2003; Seifert, 1996). In order to record
marked reductions in the prevalence of Salmonella in food coordinated control efforts at various
critical points from the farm to the “table” is essential. Additionally, comprehensive educational
programs for the consumer and food handler, both in commercial establishments and in the
home, about correct cooking and refrigeration practices for foods of animal origin, and about
personal and environmental hygiene is of paramount importance (IFT, 2003; Acha and Szyfres,
2001; D’Aoust, 1989; WHO, 1988). Veterinary meat and poultry inspection and supervision of
milk pasteurization and egg production are important for consumer protection (Acha and
Szyfres, 2001). The detection of personnel who are carriers of Salmonella, through regular stool
culture is important to the food industry because it may prevent future contamination of products
(Guthrie, 1992). Adequate nutrition and hygiene prior to and during transport and within the
abattoir could be an important intervention to prevent spread of salmonellosis (Wray and Davies,
2003).
2.2.10. Antimicrobial resistance
Salmonella display high natural susceptibility levels to the most commonly used antibacterial
agents (Cabrera et al., 2004). Conventional antimicrobial agents, such as ampicillin,
chloramphenicol, and trimethoprim-sulfamethoxazole, had been the drugs of choice in the
treatment of salmonellosis before the 1980s (Chiu et al., 2004). However, the occurrences of
isolated Salmonella strains showing resistance to one or more antibacterial agent have steadily
increased worldwide, probably due to continuous antibiotic pressure (Cabrera et al., 2004; Chiu
et al., 2004; Schroeter et al., 2004; Agustı´n et al., 2005). This is an important public health
problem that may be related to therapeutic failure. Moreover, the problem is especially relevant
in developing areas, where the lack of economic resources does not allow a wide antibacterial
armamentarium (Cabrera et al., 2004). Extended-spectrum cephalosporins and fluoroquinolones
have been suggested as appropriate alternative agents in the treatment of infections caused by
such multidrug-resistant Salmonella serotypes; however, since 1991, outbreaks or cases of
infections caused by Salmonella serotypes resistant to extended-spectrum cephalosporins or
fluoroquinolones have been increasingly reported (Chiu et al., 2004).

The emergence of antimicrobial resistance in Salmonella is complicated because the use of

antibiotics for therapeutic purposes in veterinary medicine and as growth promoters in animal
feed, thus presenting a potential risk to public health from zoonotic infections (Chiu et al., 2004;
Schroeter et al., 2004; Agustı´n et al., 2005). In addition, pet animals such as frogs and turtles
and their water environment were shown to carry multidrug-resistant Salmonella strains, which
could subsequently cause infections in humans. Therefore, to curb the resistance problem in
Salmonella, it has been suggested that inappropriate use of antimicrobial agents in food animals
should be prohibited (Chiu et al., 2004).

21
2.2.11. Public Health Significance
All strains of Salmonella are known to infect man and many other animal species (Guthrie,
1992). National and global epidemiological registries continue to highlight the importance of
Salmonella spp. as one of the leading cause of foodborne bacterial zoonotic disease in humans
(WHO, 1995; D’Aoust, 2000; David et al., 2001; Giovannacci et al., 2001; Hjartardóttir et al.,
2002; Molla et al., 2003; Mølbak et al., 2006). It is noteworthy that the problem of human
salmonellosis from the consumption of contaminated foods generally remains on the increase
worldwide (D’Aoust, 2000). The cumulative losses of salmonellosis are due to productivity
losses and absenteeism, pain and suffering, lost leisure time and medical costs. The costs of food
safety regulatory programs and costs to the food industry for product recalls and plant closures
due to foodborne salmonellosis outbreaks would increase the size of the estimated lose.
Moreover, the most serious risk is that the transmitted bacteria will have acquired resistance to
specific antibiotics because the animals from which they originate have been treated with the
particular antibiotics repeatedly over long period (Lazarus et al., 2007).
Various clinical forms of Salmonella such as gastroenteritis, bacteremia and other systemic
abnormalities can occur in veterinarians working with Salmonella-infected animals (Radostits et
al., 1994). Moreover, cutaneous salmonellosis has been reported in veterinarians attending to
infected cattle at the time of parturition. The disease was characterized by pustular dermatitis
from which S. Virchow and S. Dublin were isolated (Visser, 1991). Similarly, pustular dermatitis
caused by S. Stanley developed on the arm of a veterinary surgeon after the delivery of a dead
bovine calf. Unlike the previous reports, the veterinarian did not develop any systemic symptoms
and made a full recovery (Lazarus et al., 2007).

2.2.12. Economic importance

Salmonellosis is a significant cause of economic loss in farm animals because of the costs of
clinical disease, which include deaths, diagnosis and treatment of clinical cases, diagnostic
laboratory costs, the cost of cleaning and disinfections, and the cost of control and prevention. In
addition, when the disease is diagnosed in a herd it can create considerable apprehension in the
producer because of its difficulty in identifying infected animals. An estimation of the economic
impact of an outbreak of S. Dublin infection in a calf-rearing unit indicated that the cost of the
disease represented a substantial proportion of the gross margin of rearing calves. The losses
incurred by livestock producers include reduced feed efficiency and reduced weight gains or
deaths because of salmonellosis (Radostits et al., 1994).

22
3-Methods and Materials

3.1 Study area

The study will be carried out in three selected districts of Eastern Ethiopia; Awoday, Dire Dawa
and Harar Towns. Awoday is located at approximately 500 km Far from Addis Ababa.
Geographically this area lies between 9026’N latitude and 4203’E longitude, at an altitude of
2000 meters above sea level. The area has a mean temperature ranging from 10 to18 OC with
relative humidity of 65%. It receives an average annual rainfall of 800mm with bimodal
distribution of the seasonal pattern peaking in mid-April and mid-August of the year; however
there is a variation from year to year (AWAO, 2015).

Dire Dawa Administration Council (DDAC) is situated in the eastern part of Ethiopia at about
515 km of Addis Ababa. The area is located between 9° 27’ N and 9°49’ E latitudes and 41°38’
and 42° 19’E longitude. The rain fall pattern of the area is characterized by small rainy season
from February to May and big rainy season from July to September. The dry season extends
from October to January. The mean annual rain fall in the study area varies from 550 mm in the
lowland northern part to above 850 mm in the southern mountain. The monthly mean maximum
and minimum temperature ranges from 34.6°C to 14.5°C respectively. The entire territory of
DDAC rests on an elevation ranging between 950 meters above sea level in the north east, to
2260 meters above sea level in south west (DDACAB, 2015).

Harari is found at 525 Km East of Addis Ababa the capital city of Ethiopia. The town is located
at 42.04 - 42.22 °E Longitude and 100-250 °N Latitude. It has an average altitude of 1780 m.a.s.l
and average temperature of 22.65 °c. The annual rain fall on average is 700 mm (HRAO, 2015).

3.2. Study population

The study will be carried out on apparently healthy slaughtered camel and apparently healthy
abattoir personnel and the abattoir environment (Table 1).

23
3.3. Study design
The cross sectional study will be undertaken on apparently healthy slaughtered camel and
apparently healthy abattoir personnel and the abattoir environment in Eastern Ethiopia (Dire
Dawa, Awoday and Harari town) from Sep, 2017 to April 2018. The explanatory variables
considered will be Salmonella status of animal sample (feacal, skin swab, carcass swab, hepatic
lymph node, mesenteric lymph node) and environmental sample (eviscerating knives and axle,
eviscerator's hands, water used to wash the carcass, apron and the slaughtering room swab)
(table 1).

3.4. Sampling

The sample size required for this study was determined depending on the expected prevalence of
Salmonella and the desired absolute precision according to Thrusfield (2005) as follows:

N = (Zα)2 P (1-Pexp)
d2
Where:
N = number of sample size.
P = expected prevalence
d = marginal error between the samples and population (0.05)
Zα = the standard normal deviation corresponding 95% of confidence level = 1.96

The sample size required for this study was determined based on the Salmonella prevalence and
distribution of serotypes in apparently healthy slaughtered camels (Camelus dromedarius) in
Eastern Ethiopia with prevalence of 16.2% done by Molla et al. (2004). The estimate was desired
to be with 5 % sampling error and 95 % confidence. Using this information, the number of
sample will be 208 which is about 70 camels from each study towns.

Accordingly, it will be sampled from 3 study area (Diredawa, Harari and Awoday towns) that is
(210*3) =630 and distributed to study towns using simple random sampling. Thus, a total of
seven camel samples will be taken at three locations from the selected camels. This are before
slaughtery (fecal sample and skin swabs); immediately after slaughter (samples of meat, hepatic
lymph node and mesenteric lymph node), and post quality inspection (meat sample at abattoir
and meat sample at the public supply (at butchery) will take. Hence, a total of 90*7 =630
samples will be collected in parallel, 6 environmental samples constricting one sample each swab
samples from slaughter room, personnel hand, cutting knife, cutting axle, apron, and sample of
water will take. Hence, a total of 6*12(trip) =72 environmental sample will be collected prior to
slaughter. So, a total of 630 +72=702 samples will be examined during study period.

3.5. Sampling procedure

Samples will be collected twice a month interval. A minimum of 8 animals sample per visit
implying (8×7= 56 sample) and 6 environmental samples leads to minimum of 62 samples will
be collected.

24
Table 1. Sample animal distribution by study area, type and sources of laboratory sample.

Study variables Possible number of

samples
Study municipal Dire Dawa 70
Abattoirs camels in Awoday 70
number Harar 70
Over all animal sample 210
Animal sample Carcass swab post slaughter 70
Hepatic lymph node 70
Carcass swab post quality inspection 70
Carcass swab at public location (butcheries ) 70
Sub Total Laboratory sample 280
Environmental Personnel Hands 12
sample Apron 12
Knife 12
Axle 12
Room 12
Water sample 12
Meat transporting facility swabs 12
Sub Total Laboratory Sample 84
Over all laboratory sample 364

From each slaughtering camel; animal sample (feces, skin swabs, mesenteric lymph nodes,
hepatic lymph nodes and carcass swabs (during post quality inspection at abattoir and public
location at butchery)) will be collected in a separate sterile containers. Samples will also
collecting from environmental sample (swab samples from eviscerating knives, eviscerator’s
hand, cutting axle, apron and slaughtering room, and sample of water used to wash the carcass).

25
Skin swabs will take before bleeding was done. A swab moistened with 10ml buffered peptone
water (BPW) was used to rub the external skin where slaughtering incision was made and over
the skin where incisions were made for flaying.

Knives and axle used for evisceration will be sampling prior to slaughter. The knife(from the tip
to the base ) and axle blade was twice swabbed using sterile cotton wool moistened with 10ml
BPW (Botteldoornet al., 2003).
About 25 gm of mesenteric and hepatic lymph node samples will be collected in sterile universal
bottles by opening of abdomen and liver respectively (Vieira-Pinto et al., 2005) and feaces will
be collected from rectum of animal prior to slaughter and put into sterile flask.

Carcass swabs will be collecting immediately at the end of slaughtering process according to
Botteldoornet al. (2003). The swab sampling was performed according to the protocol described
by Bhandareet al. (2007) and ISO 17604 (2003). Briefly, carcass swabs is collecting from the
brisket and rib medial, brisket and rib lateral, loin and flank region by the swab technique for
which sterile cotton wool swabs (3 cm long and 1 cm in diameter) held by wooden sticks
moistened with 10ml BPW were used. An area of about 100 cm 2 was marked with a sterile
template of 10 cm × 10 cm on each site on the carcass. Then the swabs were rubbed over the
whole area with pressure continuously for 30seconds, moving first horizontally and turning the
swab so that all sides were used, and the swabs were transferred to a screw-capped test tubes
containing 10 ml of BPW, breaking off the wooden shaft against the inside of the tube.
Subsequently, the same procedure was repeated to sample the same area with dry swab that was
placed into the same container having 10ml BPW.

Swabs from eviscerator’s hands will be collected immediately prior to slaughter. Both hands
were swabbed using a swab moistened with 10ml BPW. Swabs from slaughter room and apron
will also immediately before slaughtering operation is held. In addition, water sample of 25ml
will also collecting aseptically on every visit to the abattoir prior to slaughter.
During sample collection, each sample will legibly and clearly labeled with identifying
information, which included date of sampling, type of sample and species of the animal from
which the sample was obtained. The samples will be transported on ice to the Haramaya
University, Faculty of Veterinary Medicine microbiology laboratory for processing and analysis
upon arrival.

3.6. Sample processing

The mesenteric and lymph node samples aseptically freed from the surrounding tissue, and 25gm
was weighed each, immersed briefly in boiling water approximately for 10 seconds separately to
decontaminate the surface following the method by Vieira-Pinto et al. (2005). Each lymph node
was then cut into smaller pieces on sterile cutting board by using sterile scalpel blade. The
minced lymph nodes were then put into sterile stomacher bags and 225 ml of BPW was added
and homogenized for two minutes with stomacher at high speed. Whenever samples were less
than 25 gm, BPW was added to the samples in 1: 9 ratios.
Approximately ten grams of feaces will be collected from rectum of animal prior to slaughter
and were put in sterile flasks. About 90 ml BPW was added and the resulting mixture was
agitated to disperse the content as described in McDowell et al. (2007).

26
Test tubes containing swab samples from the skin, carcasses, eviscerator’s hands, apron and
knife blades were shaken on a vortex mixer for 30seconds for uniform distribution of
microorganisms and then the samples were homogenized in 90 ml BPW and pre-enriched
(Swanenburget al., 2001; Botteldoornet al., 2003; Bhandareet al., 2007).
Twenty-five ml of the water sample was transferred into a sterile flask, and 225 ml BPW was
added. Then the sample was stirred gently (Swanenburget al., 2001).

3.7. Isolation and identification of Salmonella

Salmonella was isolated and identified according to the techniques outlined in the International
Organization for Standardization (ISO 6579, 2002; ISO 6579:2002/FDAM 1, 2007). The
bacteriological media used in different stages of the isolation and identification of Salmonella
were prepared according to the manufacturer’s recommendations. All samples will be processed
separately. In general, the detection of Salmonella necessitates four successive stages: pre-
enrichment in nonselective liquid medium, enrichment in selective liquid media, plating out and
identification and confirmation of identity (Figure 1).

3.7.1. Pre-enrichment in non selective liquid medium

All the samples will be processed separately as in section 3.6. Then, the processed samples in
appropriate amount of BPW (1:9) will be incubated for 18h ± 2h at 37°C ±1°C.

3.7.2. Enrichment in selective liquid media

Rappaport-Vassiliadis with Soya (RVS) broth and Muller-Kauffmann tetrathionate with
novobiocin (MKTTn) broth will be used for selective enrichment of all the samples except the
feaces (ISO 6579, 2002). In the case of feacal samples modified semi-solid Rappaport-
Vassiliadis (MSRV) medium and MKTTn broths will be used (ISO 6579:2002/FDAM 1, 2007).
A 0.1 ml pre-enriched sample was transferred aseptically into a tube containing 10 ml of MSRV
medium for feacal samples or 10 ml of RVS broth for the remaining sample types and incubated
at 41.5°C ±1°C for 24h ± 3h. Another 1 ml of the culture obtained in pre-enrichment broth was
transferred aseptically into a tube containing 10 ml of MKTTn broth and incubated at 37°C ±1°C
for 24h ± 3h.

3.7.3. Plating out and identification

Xylose lysine desoxycholate (XLD) agar and Salmonella - Shigella (SS) agar plates will be used
for plating out and identification purpose. Loopfull of inoculum from each RVS, MSRV and
MKTTn broth cultures will be streaked onto XLD and SS agar plates and the inoculated plates
will be incubated at 37°C for 24 ± 3h. After proper incubation, the plates will be examined for
the presence of typical Salmonella colonies. Typical colonies of Salmonella grown on XLD
medium produce hydrogen sulphide and have a black (H2S) center and a lightly transparent zone
of reddish colour due to the colour change of the indicator (ISO 6579, 2002) while on SS
medium they become colorless colonies with black center. Salmonella H2S negative variants
(e.g. S. Paratyphi A) grown on XLD agar are pink with a darker pink center whereas lactose-
positive salmonellae are yellow with or without blackening.

27
3.7.4. Confirmation
For confirmation, at least five presumptive Salmonella colonies will be selected from every
selective plating media. Whenever the suspected colonies on each plate are fewer than five, all
the colonies will be selected. The selected colonies were streaked onto the surface of pre-dried
nutrient agar plates, in a manner that allow well-isolated colonies to develop and incubated at
37°C ± 1°C for 24h ± 3h. Then, the pure cultures on nutrient agar will be used for biochemical
and serological confirmation (ISO 6579, 2002).

Biochemical confirmation

Colonies suspected to contain Salmonella will be tested biochemically according to the

recommendations of ISO 6579 (2002). The biochemical tests included glucose, lactose and
sucrose fermentation and gas and H2S production in TSI agar, growth in L-lysine
decarboxylation medium, urease, Voges-Proskauer (VP) and Indole production tests.
TSI and lysine iron agar (LIA) slants will be inoculated by stabbing the butt and streaking the
slant. In addition, pure colonies were inoculated onto urea broth, methyl red - Voges-Proskauer
medium and SIM medium for further characterization. For comparison purposes and ease of
identification on plates, presumptive Salmonella colonies were also inoculated onto RambachTM
agar plates. The inoculated media will be then incubated at 37°C for 24 ± 2 h according to the
recommendations of ISO 6579 (2002).

Figure 2–salmonella isolation procedure

PRE-ENRICHMENT
Test portion, 25g + buffered Peptone water, 225ml*
o
16-20 h, 37 C

SELECTIVE ENRICHMENT
Culture, 0-1ml + Culture, 10ml +
Rappaport (RV) broth 10ml Tetrathionate Broth (Müller Kauffman) 10 ml
o o
18-24 h, 42 C 18-24 h, 42 C
(2 periods) (2 periods)

SELECTIVE DIAGNOSTIC ISOLATION

28
 Plate on Brilliant Green Agar (Edel and Kampelmacher)
and any other solid selective medium
o o
24 h, 35 C or 37 C
(48 h, if necessary)
Pick five presumptive Salmonella colonies from each agar plate
and inoculate on nutrient agar
o
18-24 h, 35 C or
o
37 C

BIOCHEMICAL CONFIRMATION
(TSI, UREA BROTH, VP, INDOL, LDC)
o
24 h, 37 C

SEROLOGICAL CONFIRMATION
Slide agglutinations - O, Vi, H antisera

3.8. Antimicrobial Susceptibility Testing

3.8.1 Disk diffusion testing

The antimicrobial susceptibility testing will be done by the agar disk diffusion method as
described by the National Committee for Clinical Laboratory Standards (NCCLS, 2005), now
known as the Clinical and Laboratory Standards Institute (CLSI). The pure Salmonella isolates
confirmed by the biochemical testing procedure as described in ISO 6579: 2002 will be tested for
antimicrobial susceptibility. The antibiotics to be used will be selected among the currently
available and commonly used chemotherapeutic agents for treatment of Salmonella infection in
human and animals.
3.8.1.1 Preparation of Mueller-Hinton Agar
The Mueller-Hinton agar will be prepared as per the instructions provided by the manufacturer.
After autoclaving the media at 121°C for 15 minutes, it will be cooled to 50°C and
approximately 30 ml to 50 ml will be poured into the 15 x 150 mm Petri dishes. The depth of the
agar in the Petri dishes was maintained approximately at 4 mm. The freshly prepared plates will
be used on the same day.

3.8.1.2 Turbidity standards (McFarland) for inoculum preparation

McFarland 0.5 turbidity standards were prepared as per the standard guidelines described by the
Clinical and Laboratory Standards Institute (CLSI). A volume of 0.5 ml of a 1.175 % (wt/vol)
barium chloride dihydrate (BaCl2.2H2O) solution will be added to 99.5 ml of 0.18 mol/L (1 %
vol/vol) sulphuric acids with constant stirring to maintain the suspension. The turbidity standard
was then aliquoted into test tubes of 4 ml each, identical to those used to prepare the inoculum
suspension. McFarland standards then will be stored in the dark at room temperature (22 °C to
25 °C). Before each use, the standards will be shaken well, mixing the fine white precipitate of
barium sulphate in the tube.

29
3.8.1.3 Inoculum preparation
At least 3-5 well isolated colonies of the same morphological type will be selected from the agar
plate culture. The top of each colony was touched with a loop, and the growth was transferred
into a tube containing 4 ml tryptic soy broth. The broth culture was either directly adjusted to the
McFarland standards or by incubation at 35ºC until it achieved or exceeded the turbidity of the
0.5 McFarland standards (usually 2-6 hours). The turbidity of the actively growing broth culture
was adjusted with sterile broth to obtain turbidity optically comparable to the point of the 0.5
McFarland standards.
3.8.1.4 Inoculation of test plates
Optimally within 15 minutes after adjusting the turbidity of the inoculum suspension, a sterile
cotton swab will dipped into the adjusted suspension. The swab then rotated several times
pressed firmly on the inside wall of the tube above the fluid level. This removed excess inoculum
from the swab. The dried surface of a Mueller-Hinton agar plate will be inoculated by streaking
the swab over the entire sterile agar surface. This procedure was repeated by streaking two more
times, rotating the plate approximately 60° each time to ensure an even distribution of inoculum.
As a final step the rim of the agar was swabbed. The procedure will be done under laminar flow
to avoid contamination. The lid will be left ajar for 3-5 minutes but no more than 15 minutes, to
allow for any access surface moisture to be absorbed before applying the drug impregnated
disks.
3.8.1.5 Application of disks to inoculated agar plates
The drugs used for disk diffusion testing will be: amoxicillin 25 μg (AMX), ampicillin 10 μg
(AMP), chloramphenicol 30 μg (CHL), ciprofloxacin 5 μg (CIP), clindamycin 2 μg (CLN),
erythromycin 15 μg (ERY), gentamycin 10 μg (GEN), kanamycin 30 μg (KAN), nitrofurantoin
300 μg (NIT), spectinomycin 100 μg (SPC), tetracycline 10 μg (TET) and trimethoprim 5 μg
(TMP). The predetermined battery of antimicrobial disks was dispensed onto the surface of the
inoculated agar plate. Each disk will be pressed down individually to ensure complete contact
with the agar surface. The disk placed in the agar surface was not closer than 24 mm from center
to center. The plates will be inverted and placed in an incubator set to 35°C within 15 minutes
after the disks were applied.

3.8.1.6 Reading plates and interpreting results

After 16-18 hours of incubation, each plate will be examined. The resulting zone of inhibition
was uniformly circular with a confluent lawn of growth. The diameters of the zones of complete
inhibition (judged by the unaided eye) will be measured by sliding caliper, which will held on
the back of the inverted Petri plate. The petri plates were held a few inches above a black, non-
reflecting background.
Transmitted light from the colony counter was used to examine the zones for light growth wher
ever indicated, within apparent zones of inhibition. The zone margin was taken as the area
showing no obvious, visible growth that can be detected with the unaided eye. Faint growth of
tiny colonies, which can be detected only with a magnifying lens at the edge of the zone of
inhibited growth, was ignored. The sizes of zones of inhibition were interpreted by referring to

30
zone diameter interpretive standards from NCCLS (2005) and the isolates will be reported as
susceptible, intermediate or resistant to the agents that will be tested.

3.9. Data management and analysis

All data will entering into a Microsoft Excel spreadsheet and checked for accuracy. After
validation, data will be transferred to STATA software version 12.1 for analyses. The
explanatory variables considered (carcass swab, skin swab, eviscerating knife and axle swab,
eviscerator’s hand swab, feacal sample, mesenteric and hepatic lymph node, room swab, apron
swab and water sample Salmonella status and total slaughter volume) will be separately analysed
to see their associations with the outcome of the bacteriological status of the carcass.

4. Expected out put

Expected outputs of this project are vital information on the camel as asymptomatic carriers of
Salmonella in the study region; it equally revealed the trends in the prevalence of newly
emerging or rarely reported Salmonella serotypes, and the antimicrobial resistance profile of
Salmonella from camel in eastern Ethiopia, this project can be considered as a base for
Salmonella control, prevention and therapeutic strategies and addresses gaps in the fundamental
knowledge impacting the ability of the Eastern Ethiopian camel meat to deliver safe meat with a
reduced risk of contamination with food borne pathogens and in particular Salmonella.

5- Work plan
The study is anticipated to be completed in definite one year period. The activities within the
study are out lined as follows.

Table 2 . Project work plan

Major Activities to be done Date

Collection of literature review July -August, 2017

1
Proposal development and defense September 20- 25, 2017
2
3 Data Collection and laboratory work October 2017 – March, 2018
4 Data analysis April 1-April 15, 2018
5 Revision through research draft April 16- 19, 2018

31
 6 Thesis submission April 20-30 , 2018
7 Research defense May , 2018

6. BUDGET BREAKDOWN
5.1 Material expense (chemical and reagent)
Table 3. Material expense

Titl SALMONELLA AS ENTERIC ZONOOTIC AGENT ALONG CAMEL MEAT AT THREE

e SELECTED DISTRICTS OF EASTERN ETHIOPIA

Theme 2-Human Health, nutrition and welfare

Sub theme 2.1- Health promotion and disease prevention
Investigator Melkamu Melese
Research code HURG-2017-02-01
No. Items Unit Quantity Unit price Total price
1 Buffered peptone water quantity 1 720 710
2 Tetrathionate broth quantity 1 720 700
(Müller-Kauffmann)
3 Rappaport Vassiliadis quantity 1 750 750
soy peptone broth

4 XLD- agar plate quantity 1 750 750

5 MKTTn quantity 1 650 650
6 TSI agar quantity 1 600 650
7 Nutrient agar plate quantity 1 550 650
8 Salmonella- shigella quantity 1 500 500
agar
9 Glove box 2 300 300
10 Ice box carrier pcs 1 500 500
11 Muller Hinton Agar quantity 1 650 650
12 Brilliant green ( BGA) quantity 1 500 500
agar
13 Apron pcs 1 200 200
14 Detergent litre 2 50 100
15 L-lycine quantity 1 450 450
decarboxylation
medium
16 Urea broth quantity 1 600 600
17 Methyl red quantity 1 650 650
18 Rambach TM agar plate quantity
19 Antimicrobial disk stick 12 470 5640
Sub total 14300

32
MKTTn*- muller-kauffmantetrathinate with novobiocin TSI*- Tripe Sugar Iron XLD*-
Xyllose lysine desoxycholate

5.3 Stationary expense

Table 4-stationary expense

No Items Unit Quantity Unit Price Total Price

1 Note books Pcs 2 55 110
2 Bag ( leather) No. 1 500 500
3 Flash 32GB No. 1 280 280
4 CD-RW No. 3 32 96
5 Permanent marker pkt 5 30 150
6 Printing paper pkt 5 110 550
Sub total 1686

5.4- Miscellaneous expense

Table 5-miscellaneous expense

No. Item descripition Estimated cost

1 Thesis binding 500
2 Printing 2000
3 For mobile 1000
4 For internet service 2100
Sub total 5600

5.5- Personal expense

Table 6- personal expense

No Personnel Number Purpose Number of Rate per Total

of days day Expense
personnel

Researcher 1 Data Collection 32 193 6176

1
Laboratory 3 Laboratory 12*3=36 149 5364
2 Technician work
HU

33
 3 Guiding 15 206 3090
Major Advisor 1 research work

1 Guiding
4 Co- advisor research work 15 206 3090

5 Assistance data 12 for data 12*12=14 30 4320

recordor collection 4
6 Laboratory cleaner 1 For cleaning 20 30 600

7 Secretary 1 For writing 1500 1500

8 Local language 6 Translation 6*20=120 30 3600

translator
Sub total 27740

5.5 Perdium
Table 7- Perdium

No Descripition Expense Remark

.
1 Researcher 6000 Expense
during trip
2 Major advisor 2000 Expense
during trip
Sub total 8000

5.6 Transport expense

Table 8 -transport expense

No Person Departure Destination Purpose No. of Cost Per Total

trips Trip
Researcher HU To AA Lab, 1*2=2 330 660
1 equipment
purchase
2 Researcher HU Diredawa(st 8*2=16 20 320
udy area) Data collection

HU Harar Data collection 8*2=16 15 240

(Study area)
HU Awoday Data collection 8*2=16 10 160
(study area)

34
 Major HU Study Supervision 3*2=6 45 270
3 Advisor area( 3
abattoirs)
Subtotal 1650

5.6 Budget summary

Table 9 - budget summery

No Budget title Total cost

1 Stationery expense 1686

2 Personal expense 27740

3 Transport expense 1650

4 Chemical and reagent expense 14300

5 Perdium 8000

6 Miscellaneous expense 5600

Sub total 58976

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8. RESEARCHERS CURRICULUM VITAE (CV)

8.1. Adem Hiko (CV-1), 2016 (Principal investigator)

1. Personal information

Full Name: Adem Hiko Woshie (Associate Professor)

Sex: Male
Nationality : Ethiopian
Address contact: College of Veterinary Medicine (CVM) and School Food Technology and
Process Engineering (FTPE); Haramaya University (HU)
P.O.BOX: 289, Haramaya University Mobile phone: +251 940 24 00 80
Email: [email protected]
2. Educational background and qualification

Year Date of Degree/Certificate Institute

Graduation
April 2011- April, 2014 April 25, 2014 PhD Biomedical Sci./ Food safety FU Berlin, Germany
Oct. 2005-July 2007 Aug. 23, 2017 MS.c in Tropic. Vet. Public Health AAU, Ethiopia

43
 Oct. 1999-July 2005 July 23, 2005 DVM AAU, Ethiopia
3. Certificates

Different national and international certificates on One Health aspect, biomedical disciplines and
others.

4. Work experiences:

 Asst. Professor and participant in different HU Affairs, Community engagement services,

researches, teaching course at different sections of the University (2014-present). (HU, Ethiopia)
 PhD researcher in general Biomedical Sciences in relation to food safety, quality, production &
processing technology, animal and human health concerns (Jan. 2011-April, 2014) (FU Berlin,
Germany)
 Asst. Professor and research coordinator participant in different HU Affairs, Community
engagem.ent services, researches, teaching course at different sections of the university (2007-
2010) (HU, Ethiopia)
5. Publications (some):

 Hiko A., Ejeta A. G.: First-time detection of mycobacterium species from goats in Ethiopia.
Trop. Anim. Health Prod. 43:133 –139. (2011).
 Hiko A., Asrat D., Zewde G.: Occurrence of Escherichia coli O157:H7 in retail raw meat
products in Ethiopia. J Infect Develo. Count. 2(5): 389-393. (2008).
 Esayas F., Hiko A., Ali A, Kedir A. Kokorevica L.: Infectiousness of brain and salivary gland
suspension from rabies suspected dogs. Intr. J. Public Hlth. Epidemiolo. 1 (3), 031-034. (2012)
 Bedada B. A., Hiko A.: Mastitis and antimicrobial susceptibility test at Asella, Oromia Regional
state, Ethiopia. Journal of Microbiology and Antimicrobials. 3(9): 228-232. (2011).
6. References:

 Solomon Abera (BSc, MS.c, PhD, Assoc. Prof.): Head, Departments of Food Process
Engineering , Haramaya University, E-Mail: [email protected]
 Gobena Ameni (DVD, PhD, Assoc. Prof.): Aklilu Lemma Institute of Pathobiology, AAU,
Mobile phone: (+251-) 911-413-073, E-mail: [email protected]

8.2. Melkemu MaleseCV-3

1. Personal information

Full Name: Fekadu Mosisa Wako (MSc Student at CVM)

Sex: Male
Nationality : Ethiopian
Address contact: Department of Food Technology and Process Engineering (FTPE) Institute of
Technology, Haramaya University (HU)
P.O.BOX: 272, Haramaya University
Mobile phone: +251 910189039
Email: [email protected]
2. Educational background and qualification

Year Date of Degree/Certificate Institute

Graduation
Sept, 2008/9- July, July 06, 2013 BSc in Food Technology and Haramaya University
2013 Process Engineering

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3. Teaching Skill

4. References

1. Solomon Abera (Dr.Eng); E-mail [email protected]

2. Negussie Bussa ( Associate prof.); E-mail [email protected]
3. Dr. Asfaw Kebede; E-mail: [email protected]
4. Prof. B. K. Kumbhar; E-mail: [email protected]

8- Approval sheet

TO Office of the Vice-President for Research Affairs

HARAMAYA UNIVERSITY
From-Melkamu Melese (Msc student in Veterinary Public Health)
Haramaya University
Date-

S/r-Requesting for project work approval

45
Here by I was assigned to conduct my research project study in three selected district of Eastern
Ethiopia; Diredawa, Harari and Awoday town municipality abattoirs on the Title”
SALMONELLA AS ENTERIC ZONOOTIC AGENT ALONG CAMEL MEAT AT
THREE SELECTED DISTRICTS OF EASTERN ETHIOPIA” .So, I request you politely to
approve my research project term paper attached 42 copies of page.
Best Regard!

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