1  

Isolation and identification of Salmonella from the environment of

traditional poultry farms in Khartoum North

By

Hisham Ibrahem MohammedAhmad Saeed

B. V. M

U of K

2004

Supervisor

Dr. Awad Elkarim A. Ibrahem

B. V. Sc, M. Sc. & Ph. D.

A thesis submitted for the partial fulfillment of the requirements

for Master Degree in Microbiology

Department of Microbiology

Faculty of Veterinary Medicine

University of Khartoum

March 2010

i  

DEDICATION

To my parents,

Wonderful Brothers and Sisters

And all my teachers throughout my life

With respect

ii  

ACKNOWLEDGEMENT

To begin with, my gratitude and praise are due to almighty Allah,

the Beneficent and the Merciful for the precious gift of health and the

capability to accomplish this work.

I am deeply indebted and thankfulness to my supervisor Dr.

Awad Elkarim Abdelghaffar Ibrabim for his priceless guidance, close

supervision, helpful, patience and contiuous encouragement.

I am deeply thankful to my wonderful friend Reem Merghani Ali

Nageela for her continuous support to accomplish this work.

I would like to thank all the technicians of the department of

microbiology for their great help.

I am also grateful to my colleagues Ahmed Mohammed saad and

Amani Mahmoud.

Finally my thanks extended to everybody he gave me his time,

support and help.

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ABSTRACT

Avian salmonellosis is a large group of acute or chronic

diseases of fowl caused by different species of the genus Salmonella.

It is a problem of economic concern to all phases of the poultry

industry from production to marketing.

Our study was conducted to investigate the incidence of

Salmonella species in the feed and environment of open system

poultry farms in Khartoum North area during the period from August

to November 2009.

A total of 80 samples were taken from six poultry farms of layers

and broilers located in Al-Halfaya, Shambat, Hillat Kuku and Al-

Zakiab area. The samples include: poultry feed from feeders (27

samples), litter (27 samples) and drinking water from drinkers (26

samples).

Isolation of Salmonella were carriedout in selective classical

medium (DCA) after enrichment in selenite-f-broth. Four salmonella

isolates represent (5%) of total samples were recovered; three isolates

(75%) from litter samples and one isolate (25%) recovered from water

sample, no Salmonellae recovered from feed samples. Three isolates

belong to S. enteritidis while the fourth isolate belongs to S. arizonae.

All four Salmonella isolates were recovered from two farms:

three isolates recovered from a farm located in Al-Halfaya (layers)

and one isolate recovered from a farm located in Shambat, (broilers)

no isolates were recovered from Hillat Kuku or Al-Zakyab area farms.

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Other Enterobacteria were also isolated and included 15 (18.75%)

Serratia spp., 11 (13.75%) Proteus spp., 8 (10.00%) Citrobacter spp.,

1 (1.25%) Kluyvera spp., 1 (1.25%) Enterobacter spp., 1 (1.25%)

Yersinia spp., and 1 (1.25%) Hafnia spp.

All isolates were identified to the species level using cultural

characteristics and biochemical reactions.

Antimicrobial sensitivity test to the four Salmonella isolates was

carried out. Each isolate was tested to 10 different antimicrobial

agents using Mueller and Hinton Agar Medium. All isolates found

sensitive to chloramphenicol, ceftizoxime, amikacin and resistant to

gentamycin, tetracycline, ambicillin\ sulbactam and piperacillin\

tazobactam.

v  

ملخص األطروحة

سالمونيله الطيور مصطلح يطلق على مجموعة آبيرة من األمراض الحادة والمزمنة

مع التوسع الكبير في صناعة الدواجن . التي تسببها أنواع مختلفة تنتمي إلى جنس السالمونيله

أصبحت سالمونيله الطيور من أهم األمراض البكتيرية المنقولة بواسطة البيض وهو مرض

.عاد اقتصادية في جميع مراحل صناعة الدواجن ذو أب

تتلخص الدراسة في تحديد وجود أنواع جنس السالمونيله في علف وبيئة الدواجن في

مزارع تربية الدواجن ألغراض إنتاج البيض واللحم بمنطقة الخرطوم بحري في الفترة من

. 2009أغسطس إلى نوفمبر

مزارع بنظام 6من ) ماء شرب 26فرشة ، 27علف ، 27( عينة 80تم جمع

.التربية المفتوح في مناطق الحلفاية ، حلة آوآو ، شمبات ، ومنطقة الزاآياب

:وُصنفت آاآلتي % ) 5( عزالت من بكتريا السالمونيله 4تم عزل

تم . عزالت من السالمونيله الملهبة لألمعاء وعزلة واحدة من السالمونيله األرزونية 3

عزالت من عينات الفرشة وعزلة واحدة من عينات ماء الشرب ولم يتم 3الحصول على

تم الحصول على األربع عزالت للسالمونيله . التحصل على عزالت السالمونيله من العلف

.مزرعة إلنتاج البيض في الحلفاية وأخرى إلنتاج اللحم في شمبات : من مزرعتين فقط

:تم عزل البكتريا وُصنفت آاآلتي

، %) 10( 8، ستروباآتر %) 13.75( 11، بروتيس %) 18.75( 15ا سريشي

جميع العزالت تم %) . 1.25( 1و هافنيا %) 1.25( 1، انتيروباآتر %) 1.25( 1آلوفيرا

.تصنيفها حتى مرحلة النوع اعتمادًا على الخصائص المزرعية واالختبارات البيوآيميائية

جميع عزالت . مضادات حيوية 10استخدام تم إجراء اختبار الحساسية للسالمونيله ب

السالمونيله أظهرت حساسية للكلورامفينيكول والسفتيزوآزيم و األميكاسين وأظهرت مقاومة

.آاملة للجنتاميسين و التيترسيكلين و األمبيسلين مع السالباآتام والبيبراسيلين مع التازوباآتام

vi  

List of contents

Subject Page

Dedication …………………………………………………... i.

Acknowledgment ………………………………………….... ii.

Abstract……………………………………………………… iii.

Arabic Abstract …………………………………………….. v.

List of Contents …………………………………………….. iv.

List of Tables ……………………………………………….. x.

List of Figure ……………………………………………….. xi.

Introduction ………………………………………………... 1

Chapter One: Literature Review ………………………….. 3

1.1History of Salmonella ………………………………….. 3

1.2 Classification of Salmonella …………………………... 3

1.3 Morphology of Salmonella …………………………… 5

1.4 Antigenic Structure …………………………………….. 5

1.5 Prevalence of Salmonella …………………………….. 7

1.5.1 Prevalence in Poultry …………………………..…… 7

1.5.1.1 Isolation of Salmonella from different poultry

sources ……………………………………………….……... 7

1.5.1.1.1 Chicks …………………………………………... 7

1.5.1.1.2 Poultry flocks …………………………………… 8

1.5.1.1.3 Poultry feed …………………………………….. 9

1.5.1.1.4 Poultry carcasses and other poultry sources .…… 9

1.5.2 Prevalence in animals……………………..………..… 11

1.5.3 Prevalence in man……………………….…………… 12

1.6 Pathogenicity of Salmonella ………………....………… 14

1.7 Laboratory diagnosis………………………....………… 17

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1.7.1 Isolation of Salmonella ……………………………… 17

1.7.1.1 Cultural characteristics …………………………… 17

1.7.1.1.1 Enrichment media ……………………………… 18

1.7.1.1.2 Differential and selective solid media …..……… 19

1.7.1.2 Biochemical reactions ……………………..……… 22

1.7.2 Serological tests ……………………………..……… 23

1.8 Drug susceptibility ………………………………..…… 24

1.9 Control and treatment ………………………..………… 26

1.10 Salmonella vaccine ………………………...………… 28

Chapter Two: Materials and Methods: ………...…………… 30

2.1 Sampling ………………………………….…………… 30

2.1.1 Source of specimens ………………………………… 30

2.1.2 Collection of specimens ………………..…………… 30

2.1.2.1 Feed samples ……………………………………… 31

2.1.2.2 Litter samples ……………………………………… 31

2.1.2.3 Drinking water sample …………………………… 33

2.1.3 Transport and storage of samples …………..……… 33

2.2 Bacteriological investigation ………………………… 33

2.2.1 Culture media ……………………………………… 33

2.2.1.1 Liquid media …………………………………….. 33

2.2.1.2 Semi-solid media ………………………………… 35

2.2.1.3 Solid media ……………………………………… 35

2.2.2 Solutions and Reagents …………………………… 38

2.2.2.1 Normal saline solution …………………………… 38

2.2.2.2 Methyl Red ……………………………………… 38

2.2.2.3 Kovac’s reagent ………………………………… 38

2.2.2.4 Oxidase test reagent ……………………………… 39

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2.2.2.5 Potassium Hydroxide solution ……………………. 39

2.2.2.6 Andrade’s indicator ……………………………… 39

2.2.2.7 Voges- Proskauer (V.P) test reagent …………….. 39

2.2.2.8 Lead acetate paper ……………………………… 39

2.2.2.9 Bromo-thymol blue ………………………………. 40

2.2.2.10 Phenol red ……………………………………….. 40

2.2.3 Sterilization procedures …………………………… 40

2.2.3.1 Hot air oven ……………………………………… 40

2.2.3.2 Autoclaving ………………………………………. 40

2.2.3.3 Disinfectants and antiseptics …………………… 40

2.2.3.4 U. V Light ……………………………………….. 40

2.2.4 Cultivation of samples ……………………………… 41

2.2.4.1 Inoculation of enrichment medium ……………… 41

2.2.4.2 Inoculation of plates ……………………………… 41

2.2.4.3 Purification and storage of isolates ……………… 42

2.2.5 Identification of the isolated bacteria ……………… 42

2.2.5.1 Microscopic examination ………………………… 42

2.2.5.2 Biochemical tests for identification of bacteria … 43

2.2.5.2.1 Primary biochemical tests ……………………. 43

2.2.5.2.2 Secondary biochemical tests …………………… 44

2.2.6 Antimicrobial sensitivity test ……………………… 46

Chapter Three: Results …………………………………… 49

3.1 Isolation of bacteria ………………………………….. 49

3.2 Site of isolation ………………………………………. 49

3.3 Properties of Salmonella …………………………….. 50

3.3.1 Cultural properties …………………………………. 50

3.3.1.1 Growth in liquid media …………………………. 50

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3.3.1.2 Growth on solid media …………………………… 50

3.3.2 Microscopic properties …………………………….. 50

3.3.3 Biochemical reactions ………………………………. 51

3.4 Sensitivity to antimicrobial agents …………………… 51

Chapter four: Discussions ………………………………… 59

Conclusion ……………………………………………….. 63

Recommendations ………………………………………… 63

References ………………………………………………. 64

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List of Tables

Table title Page

Origin, type and number of samples collected in the study…... 32

Antibiotics used in sensitivity testing ………………………... 47

Standard zone of inhibition to different antimicrobial agents... 48

Isolated Enterobacteria from different samples in the study … 52

Isolated Salmonellae from different samples and areas in

Khartoum North………………………………………………. 53

Sensitivity of Salmonella isolates to different Antimicrobial

Agents…………………………………………………………. 54

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List of Figure

Figure Title Page

Growth of Salmonella on Nutrient Agar……………………. 55

Growth of Salmonella on Desoxychocolate Citrate Agar

(DCA)………………………………………………………..

55

Growth of Salmonella on Triple Sugar Iron Agar (TSI)……. 56

The sensitivity of Salmonella to different antibiotics …….. 57

The sensitivity of Salmonella to different antibiotics………. 58

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INTRODUCTION

Members of the family Enterobacteriaceae are gram- negative,

non-spore forming rods. Some of them are human and animal

pathogens producing intestinal infection and food poisoning. The

genera of pathogenic importance in poultry include Salmonella and

Escherichia (Holt et al., 1994).

Avian salmonellosis is an inclusive term designating a large

group of acute or chronic diseases of fowl caused by different species

of the genus Salmonella including S. pullorum (Pullorum disease),

S.gallinarum (Fowl typhoid), S. arizonae (Arizonae infection), S.

enteritidis and others (Paratyphoid infection) (Carter and Wise, 2004).

Paratyphoid infections are economically among the most

important bacterial disease of the hatching industry and result in high

death losses among all types of young poultry (Hofstad et al., 1978).

In addition, the occurrence of this disease in valuable breeding stocks

is extremely costly. Also fertility, hatchability and egg production

may be seriously impaired (Graham and Michael 1936; Pomeroy and

Fenstermacher 1941).

Adult birds infected with paratyphoid organisms generally show

no outward symptoms; however, they may serve as intestinal carriers

of the infection over long periods of time and serve as the chief source

of paratyphoid infections of most species of poultry (Olesuik et al.,

1969; Hofstad et al., 1978).

Fecal contamination of egg shells with paratyphoid organisms

during the process of laying or from contaminated nests, floors, or

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incubators after laying is of foremost importance in the spread of the

disease. Also the disease may be transmitted directly to young birds

from older fowl that are chronic intestinal carriers of the infection but

exhibit no visible symptoms (Hofstad et al., 1978).

Evidence has been presented that poultry feeds may be a

common and very important source of paratyphoid organisms. The

level of Salmonella contamination in poultry feeds is normally low;

however, it has been shown that even one organism per 15 grams of

feed can produce infection (Harry and Brown, 1974).

Salmonellosis in poultry resulted in continuous increase of

public health problems as stated by Corrier et al., (1990).

Contamination of poultry meat with Salmonella was investigated by

many scientists in Sudan as well as in many countries. In Sudan

Mamon et al., (1992) succeeded to isolate 21 Salmonella enteritidis

from embryonated eggs. Yagoub and Mohammed (1987) studied the

occurrence of Salmonella in poultry carcasses in Khartoum state; 23

serotypes were identified and most of them were S. monas and S.

amek.

The objectives of this study were:

1- To investigate the contamination of poultry environment: feed,

drinking water and poultry litter with Salmonella species in

Khartoum North area poultry farms.

2- To determine the antimicrobial sensitivity of Salmonella isolates

to most common used antimicrobial agents in the Sudan.

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CHAPTER ONE

LITERATURE REVIEW 1.1 History of Salmonella:

Salmon and Smith were the first to isolate Salmonella from pigs

in (1885) (cited by Ryan and Ray, 2004). The typhoid bacillus was

first isolated in 1884, when the German microbiologist Gaffkey

obtained Salmonella typhi from human spleen (Scherer and Miller,

2004). Salmonella is an important genus of the family

Enterobacteriaceae. Members of the genus are Gram-negative,

facultative anaerobes and inhabit the intestinal tract of man and

animals. They may be recovered from a wide range of hosts such as

poultry, swine, human, foods and environment. Members of the genus

Salmonella may be pathogenic to wild or domestic animals and human

(Holt et al., 1994).

It's important pathogen to the food industry and has been

frequently identified as the etiological agent of food-borne out breaks

(Siqueira et al., 2003; Zhao et al., 2001). In human they cause

enteritis, enteric fever and systemic infection (Brook et al., 2004).

1.2 Classification of Salmonella:

The scientific classification of salmonella was described by

Hafez (2005) as follow:

Domain: Bacteria, Kingdom: Monera, Phylum: Proteobacteria,

Class: Gamma proteobacteria, Order: Enterobacteriales, Family:

Enterobacteriaceae and Genus: Salmonella.

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The members of the genus Salmonella were originally classified

on the basis epidemiology, host range, biochemical reactions and

structures of the O, H and Vi antigens (Brook et al., 2004).

Recent advances in Salmonella taxonomy divide the genus in to

two species: Salmonella bongori and Salmonella enterica (Le Minor

and Popoff, 1987). S. bongori contains less than 10 serovars while S.

enterica contains more than 2500 serovars and are divided into six

subspecies namely enterica, salamae, arizonae, diarizonae, houtenae

and indica.

All centers of disease control and prevention recommended that

Salmonella species should be named by their genus and serovar e.g.

Salmonella typhi instead of Salmonella enterica subspecies enterica

serovar typhi.

Most commonly, the Salmonella was classified according to

serology. The division is first by the somatic (O) antigen and by the

flagellar (H) antigen. (O) antigen is of lipopolysaccharide nature and

(H) antigen of protein nature (Kauffmann White Scheme 1960).

The genus Salmonella can roughly be classified into three

groups (Hafez and Jodas 2000). First group includes highly host

adapted and invasive serovars such as S. gallinarum, S. pullorum in

poultry and S. typhi in human. Second group includes non-host

adapted and invasive serovars such as S. typhimurium, S. arizonae and

S. enteritidis. Third group contains non-host adapted and non invasive

serovars, most of these serovars are harmless for animals and human.

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1.3Morphology of Salmonella:

Salmonellae are Gram-negative straight rods, (0.7- 1.5 x 2.0 – 5.0),

conferring the general definition of the family Entrobacteriaceae,

(Collee et al., 1996), non-acid- fast, non-capsulated and non- spore

forming. Most serotypes are motile with peritrichous flagella, but

Gallinarum- Pullorum is non- motile; non- motile variants (OH→ O

variation) are occasionally found in other serotypes. Most strains of

most serotypes form type 1 fimbrae (mannose- sensitive,

haemagglutinating); Gallinarum- Pullorum and a few strains in other

serotypes either form type 2 fimbriae (non- haemagglutinating) or are

non- immediate; most strains of S. paratyphy A are non- fimbriated

(Duguid et al., 1966).

1.4 Antigenic structure:

Although the principal varieties of enteric bacilli can be

identified by their reactions in differential media, final identification

of many species, as well as many strains, is based on antigenic

structure. However, strains with the same antigenic pattern may

exhibit different metabolic reactions (fermentative variants or

biotypes), three kinds of surface antigens (H, O and K or Vi),

determine the organism's reaction with the specific antiserum (Davis

et al., 1990).

Salmonella, like other Gram-negative bacteria, has somatic (O)

antigens, which are lipopolysaccharide components of the cell wall,

and flagellar (H) antigens, which are proteins (Mandel et al., 1985;

Collee et al., 1996 and Brook et al., 2004).

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Capsular antigens are called K Ag (German. Kapsel), and a

specific capsular antigen of S. typhi is called Vi due to it's role in

virulence. Fimbrial antigens previously designated as K antigens

before their structure was recognized, and they now bear descriptive

names (CFA i and ii: colonization factors and antigens 1 and 2) (Davis

et al., 1990).

There are about 60 (O) antigens, and many H antigens, each of

which is designated by number and letter (Mandel et al., 1985;

Paniker and Vilma, 1997; Brooks et al., 2004).

The polysacharide Vi Ag in certain species is usually too thin to

be seen as capsule. However, it does inhibit O agglutination unless it

is destroyed (along with H-Ag ) by boiling for two hours .

The (O) Ags in smooth (S) strains cover the underline R Ag

which becomes accessible to antibodies in rough (R) mutants. The

change from S to R may take place without the loss of flagellar or Vi

Ags. Rough strains tend to agglutinate spontaneously unless

suspended in media of proper ionic strength (Davis et al., 1990). The

smooth- to – rough variation is associated with change in colony

morphology and loss of the O-Ag of virulence. The colony becomes

large, rough and irregular. R forms may be common in laboratory

strains maintained by serial sub-cultivation (David et al., 1989).

Mucoid colonies, associated with development of a new mucoid

or M-Ag, have been described with Salmonella paratyphi B and some

other species (Ananthanarayan and Paniker, 1997).

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1.5 Prevalence of Salmonella:

1.5.1 Prevalence in poultry:

1.5.1.1 Isolation of Salmonella from different poultry sources:

1.5.1.1.1 Chicks:

Nicklas (1987) reported that 50 deliveries of one day- old

chicks were examined for Salmonella as a part of routine

microbiological monitoring. Paper floor inserts and faeces from the

transport boxes were immersed in peptone water and then cultured in

two different enrichment media. Salmonella was isolated from 6of the

50 samples, one isolate was identified as S. muenchen and the other 5

as S. serovar siegburg.

Al Obaidi et al., (1987) stated that in a survey over 18 months,

S. neuikerk (8 isolates) and S. montevideo (6 isolates) were recovered

from 30 day- old chicks. S. neuikerk was also recovered from 3 of 30,

7 day- old chicks and from 2 of 20, 30 day- old chicks. S. Virchow

was recovered from one chick in each of the two later groups.

Salmonella was not isolated from chicks more than 45 day- old.

Only S. java was recovered from litters in pens containing

chicks 7 and 30 days old. It was concluded that antibiotics in feed

increase the rate of isolation of Salmonella from these birds.

Choudhery et al., (1993) isolated Salmonella species from 9

(10.57%) infected yolk sacs of 20 chicks and 80 dead chicks on 85

farms.

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1.5.1.1.2 Poultry flocks:

Desoutter (1986) reported that 20 outbreaks of salmonellosis in

poultry have been observed since 1981. S. typhimurium was isolated

in 50% of cases, S. London of 20%, S. muenchen in 20% and S.

Arizona in 10%, while S. pullorum- gallinarum has never been

isolated.

Majid et al., (1991) found that 18 (13.5%) of 133 commercial

poultry flocks were affected by salmonellosis. Prevalence was higher

in flocks with poor management conditions. Of 18 typed isolates, 10

were S. gallinarum or S. pullorum.

Pacini et al., (1993) reported that the incidence of different

serotypes of Salmlonela isolated from faecal samples of poultry

during 1984-1992 showed a decrease in frequency of S. infantis and S.

typhimurium and an increase in S. enteritidis.

Menzies et al., (1994) found that the predominant serotypes in

poultry during 1979- 1994 were S. typhimurium (22%) and S.

entritidis (11.7%). There was no significant annual trend in avian

salmonellosis, although a peak incidence occurred between 1986-

1987 caused by an increased outbreaks involving S. enteritidis and S.

typhimurium.

In Sudan, Ezdihar (1996) examined 610 samples from infected

chickens and reported the isolation of 14 bacterial genera which

included Klebsiella, Citrobacter, Serratia, Morganella, Enterobacter,

E. coli, Salmonella, Proteus, yersinia, Edwardsiella, Hafnia,

Acinetobacter and Shigella.

9  

1.5.1.1.3 Poultry feed:

Salmonella have been isolated from poultry feed stored at 25C

after 16 month of storage (Williams and Benson, 1978). Survival and

heat resistance of Salmonella in meat, bone meat, dry milk and poultry

feed is related to moisture content and relative humidity (Carlson and

Snoeyenbos, 1970). The only feed ingredient that is resistant to

contamination by Salmonella is liquid animal and vegetable fat

(Harris et al., 1997). Fatty acids have been shown to inhibit the

growth of gram- negative bacteria (Khan, 1969).

Jardy and Michard (1992) tested samples of row poultry feed

components for Salmonella. The most commonly isolated serovars

were S. senftenberg, S. rissen, S. tennesee, S. landott, S. mbandaka, S.

agona and S. havana.

1.5.1.1.4 Poultry carcasses and other poultry sources:

Yagoub and Mohammed (1986) determine that 58 Salmonella

strains were isolated from 1488 samples from slaughtered chicks over

18 months in Khartoum North and Omdurman.

Twenty three serotypes were identified, the most common

serotypes were S. monas (25.6%) and S. amek (16.3%) and of

serotypes except S. uganda had previously been isolated in Sudan

(Khan, 1969).

Baumgartner et al., (1992) isolated 132 Salmonella isolates

from 945 broiler carcasses, 47 (36.2%) were S. infantis, 39 (30%) S.

typhimurium and 25 (19.2%) S. enteritidis.

10  

Mrdjen et al., (1992) examined 658 samples (eggs, chicks, and

broilers), and isolated 93 Salmonella strains:

26 (28%) were S. enteritidis, 24 (26%) S. typhimurium,

23(25%) S. verchow, 12 (13%) S. infantis, 4 (5%) S. lindenburg,

2(3%) S. gallinarum- pullorum, 1 each of S. berdeney and S.

manbattan.

Orhan and Gruler (1993) isolated Salmonella from internal

organs, cloacal swabs, feed samples and eggs. These strains were

identified as S. gallinarum (25) and S. enteritidis (13) while 63 strains

of S. pullorum were isolated by Pan et al., (1993).

Choudhury et al., (1993) isolated Salmonella from 9 (10.6%)

infected yolk sacs of 20 sick and 80 dead chicks. Also S. enteritidis

phage type 4 was isolated from the reproductive tract of 10 of 37

laying hens (Hoop and Pospischil).

Pope (1994) reported that S. enteritidis was isolated from

environmental samples of (2.7%) of layer flocks and (3%) of broiler

flocks.

Salmonella may be isolated from most body tissues including

liver, heart, gizzard and cooked chicken products (Jerng Klinchan et

al., 1994), small intestine, caeca (Wieliczko, 1994), eggs shell

fragments, external rinses, intestinal tracts from one day- old chicks

(Bailey et al., 1994).

Enrichment broth was considered by most workers as an

essential part of Salmonella isolation. Delayed secondary enrichment

11  

was required to isolate Salmonella from 36(15%) of the 226

Salmonella positive chickens and to detect 20% of the total isolation.

The optimum incubation time for Salmonella enrichment

cultures were obtained by inoculation of enrichment broth on to

plating media after 23 hrs at 37C, after 48 hrs at 37C, after 3 days

delayed secondary enrichment (DSE) and after a 5- day DSE

procedure. Inoculation of the enrichment broth onto plating media

after 24 hrs incubation followed by 5-day DSE, made the detection of

96-98% of Salmonella positive samples possible and was the best

combination of condition (Waltman et al., 1993).

1.5.2 Prevalence in animals:

Buxton and Fraser (1977) reported that animals were affected

by different serotypes of Salmonella species. The serotypes usually

affecting horses are S. abortus equi and S. typhimurium. While S.

Dublin and S. typhimurium are the most common causes of bovine

salmonellosis which affect cattle of all ages, and the disease may be

acute or chronic. Sheep and goats are affected by S. abortus ovis and

S. typhimurium and other serotypes including S. dublin which have

been recorded less frequent. Pigs constitute one of most important

reservoirs of Salmonellae and are susceptible to the disease caused by

a wide variety of serotypes. Most important of these are S.

choleraesuis and S. typhimurium.

The transmission of Salmonella spp. to animal's feed was noted

by Jones et al., (1982) who detected the same serotypes (S.ser.

mbandaka) in both cattle and unopened bags of vegetable fat on the

same farm site. However, Grimont et al., (2000) noted that the habitat

12  

of Salmonella spp. is limited to the digestive tract of animals and

humans, and that it's presence in other environments may be limited to

faecal contamination.

Marx (1969) noted that S.enteritidis was isolated from field

mice (Apodemus sylvaticus) as early as 1900. Later,Singer et al.,

(1992) isolated S. enteritidis again from mice (Family: muridae).

Other Salmonella serovars such as S. derby and S. typhimurium were

isolated from rats (Schnurrenberger et al., 1968).

In birds, Salmonella was isolated in a study from racing pigeons

(Adesiyun et al., 1998) but not in wild pigeons (Nielsen and Clausen,

1975). Salmonella isolated from wild birds such as crows and gulls

(Kapperud and Rosef 1983; Devis and Murray, 1991). Evidence also

exists that Salmonella may survive in the intestinal tracts of insects

(Everard et al., 1979). Jones et al., (1991) and Kopanic et al., (1994)

suggested that insects may be vectors for the transmission of

Salmonella spp.

1.5.3 Prevalence in man:

The incidence of human salmonellosis has increased greatly

over the past 20 years and this can mostly be attributed to epidemics

of S. enteritidis in poultry in numerous countries (Barrow et al., 2003;

Guard- Petter, 2001). The association between egg consumption and

S. enteritidis outbreaks is a serious international economic and public

health problem (Center of Disease Control, 2000 and 2003; Guard

Petter, 2001; Patrick et al., 2004).

13  

Transmission to hen may originate from contaminated food or

water or by contact with wild animals. But the main concern with this

bacterium is the existence of silence carriers. These animals can, in

turn, transmit the bacterium to their flock- mates through horizontal

transmission or to their off- spring by vertical transmission. However,

they are difficult to be distinguished from healthy animals, thus are

responsible for transmission to human.

Zhao et al., (2001) and Siqueira et al., (2003) reported the

occurrence of 1.4 million cases of human salmonellosis in United

States of America.

The transmission of Salmonella is usually associated with

consumption of contaminated food (Soumt et al., 1999). However, a

great number of outbreaks might be associated with contaminated

water, which is known to be an important transmission route (Fertado

et al., 1998).

Mead et al., (1999) and Murray (2000) stated that 95% of

salmonellosis cases originate from food materials.

In a comparative study in England, Humphrey (2000) reported

that 30% to 80% Salmonella spp. were isolated from alfalfa seeds,

chocolate, cheddar cheese, red meat, salad, milk and vegetables

(Inami and Moler, 1999; Humphrey 2000).

Kotova et al., (1988) conducted study on human developing the

Salmonella carrier state (S. enteritidis and S. dublin) after acute

salmonellosis and as a result of occupational exposure to poultry. The

Salmonella species most frequently isolated from poultry employees

14  

was S. typhimurium, while S. newport, S. enteritidis and S. Dublin

were also isolated.

In Sudan, Seed Ahmed, (2007) conducted study on Salmonella

carrier state among human, he has tested 5493 stool samples taken

from food handlers working in food service in Khartoum, only S.

paratyphi B was isolated from 17 samples.

1.6 Pathogenicity of Salmonella:

The pathogenesis of Salmonella has been extensively studied in

the mouse (Haraga et al., 2008). In susceptible mice, Salmonella

causes an acute systemic disease with limited intestinal manifestations

(Santos et al, 2001). Recently, a model of Salmonella enterocolitis has

been developed in streptomycin-treated mice (Barthel et al., 2003).

Studies using these mice and other animal models of

Salmonella diseases have yielded substantial data about the molecular

players involved at different levels. The Salmonella pathogenicity

islands (SPIs) I and ІI are two major virulence determinants of S.

enterica. They encode type III secretion systems (T3SS) that form

syringe-like organelles on the surface of gram-negative bacteria and

enable the injection of effector proteins directly into the cytosol of

eukaryotic cells (Galan, 2001; Waterman and Holden, 2003). These

effectors ultimately manipulate the cellular functions of the infected

host and facilitate the progression of the infection. SPI (I) and SPI (II)

play several roles in different organs within the host. SPI (I) primarily

promotes the invasion of non-phagocytic intestinal epithelial cells and

the initiation of the inflammatory responses in the intestines

(Coombes et al., 2005; Hapfelmeier et al., 2004). It is also involved in

15  

the survival and persistence of Salmonella in the systemic

compartment of the host (Brawn et al., 2007; Steele-Mortimer et al.,

2002).

The first characterized role of SPI (II) was its ability to promote

Salmonella survival and multiplication in phagocytic cells that

constitute the main reservoirs for dissemination of the bacteria into

systemic organs (Waterman and Holden, 2003). SPI (II) also plays an

important role in the intestinal phase of Salmonella infection in mice

(Coombes et al., 2005; Cobum et al., 2005; Hapfelmeier et al., 2005).

The regulation of SPI (I) and SPI (II) gene expression involves

numerous transcriptional regulators located both inside and outside

these pathogenicity islands. The regulation of SPI (I) is particularly

complex. SPI (I) encodes for the five regulators HilA, HilC, HilD,

InvF, and SprB. The first four of which are involved in regulatory

pathways that lead to the activation of SPI (I) genes and of genes

encoding T3SS effectors located outside SPI (I). In contrast to SPI (I)

the regulation of SPI (II) genes is simpler with the SsrAB two-

component system being the only transcriptional regulator encoded

within SPI (II) that activates the expression of SPI (II) genes and of

genes encoding T3SS effectors located outside SPI (II). Interestingly,

SPI (I) regulators can regulate SPI (II) genes. These include HilA that

binds and represses the promoter of ssaH (Thijs et al., 2007), and

HilD that binds and activates the promoter of the ssrAB operon

(Bustamante et al, 2008). In contrast, SsrAB has never been shown to

act on the expression of SPI (I) genes.

16  

Few studies have investigated the role of SPI (1) and SPI (II)

during the infection of chickens. In studies using Typhimurium, two

approaches have provided data about the roles of SPI (I) and SPI (II).

The first approach compared colonization in chickens by infecting

with single strains and enumerating colonies from internal organs.

Porter and Curtiss (Porter et al., 1997) found that mutations in

structural genes of the SPI (I) T3SS resulted in a reduction of the

colonization of the intestines in day-old chickens. Jones et al., (2007)

generated strains with deletions of spaS and ssaU, genes that encode

structural proteins of the SPI (I) and SPI (II) T3SS respectively, and

compared their ability to colonize the cecum and liver in one-day and

one-week old chickens to that of wild type. They concluded that both

SPI (I) and SPI (II) play major roles in both the intestinal and the

systemic compartments, with SPI (II) contributing more than SPI (I)

in both compartments. The second approach screened random

transposon libraries for reduced recovery from the chicken

gastrointestinal tract through cloacal swabbing. Turner et al., (1998)

analyzed a library of 2,800 mutants for intestinal colonization in

chickens. Among the mutants that showed reduced intestinal

colonization they found one in which the SPI (I) gene sipC was

inactivated. No mutations in SPI (II) genes were identified in this

screen.

Morgan et al., (2004) screened a library of 1,045 mutants in

chickens and found two mutations in SPI (I) genes and one in a SPI

(II) gene that led to a reduction in colonization ability. The SPI (I)

mutants were unable to be recovered from 50% or 100% of the day

old birds tested, while the single SPI (II) gene was unable to be

17  

recovered in only 33%. In this study fourteen strains with mutations in

SPI (I) and fifteen strains with mutations in SPI (II) did not show any

defect in colonization. The authors of these two studies concluded that

SPI (I) and SPI (II) play a marginal role in the colonization of chicken

intestines by S.typhimurium.

Dieye et al., (2009) reported that SPI (I) contributes to the

colonization of both the cecum and spleen of the chicken. In contrast,

SPI (II) contributes to colonization of the spleen but not the cecum

and, in the absence of SPI (1) inhibits cecal colonization.

Additionally, they show that the contribution of SPI (I) in the spleen is

greater than that of SPI (II). These results differ from those observed

during the infection of mice by Typhimurium, where SPI (II) plays a

major role during systemic colonization.

1.7 Laboratory diagnosis:

1.7.1 Isolation of Salmonella:

1.7.1.1 Cultural characteristics:

Salmonella are facultative anaerobic. The optimum growth

temperature is 37ºC, but some growth is observed in a range from

about 5- 45ºC. Salmonella can grow within a pH range of

approximately 4.0 to 9.0, with an optimum pH around 7.0

(Cruickshank 1972).

18  

1.7.1.1.1 Enrichment media:

These are liquid media used to assist isolation of Salmonellae

from feces, sewage, food stuffs and other materials containing a mixed

bacterial flora. A larger amount of the material can be inoculated into

an enrichment medium than on to an agar plate, so facilitating the

isolation of Salmonellae when these are present only in small

numbers. During incubation any Salmonellae multiply rapidly, while

E. coli and most other bacteria are inhibited. After 18- 24 hours the

enriched culture is plated onto a differential agar medium e.g.

Desoxycholate- Citrate Agar (DCA) or Xylose Lysine Desoxycholate

(XLD), on which the production of Salmonella- like colonies may be

observed (Barrow and Feltham, 1993).

a- Selenite broth:

Selenite broth is an enrichment broth medium used for the

isolation of Salmonella and Shigella species. Casein and meat

peptones provide nutrients. Selenite inhibits enterococci and coliform

those are part of the normal flora if they are subcultuered within 12 to

18 hrs. However, reduction of selenite produces an alkaline condition

that may inhibit the recovery of Salmonella. Lactose and phosphate

buffers are added to allow stability of the pH. When fermenting

organisms produce acid, the acid neutralizes the effect of the selenite

reduction and subsequent alkalinization. Cystine added to selenite

broth enhances the recovery of Salmonella (Murray et al., 2005).

19  

1.7.1.1.2 Differential and selective solid media:

These media are valuable for the isolation of Salmonellae from

feces and other materials contaminated with many bacteria of other

kinds. They include:

a- Mc Conkey Bile- Salt lactose Agar:

After 18 to 24 hrs the colonies are pale yellow or nearly

colourless, 1-3 mm in diameter, and easily distinguished from the

pink- red colonies of lactose fermenting commensal coliform bacilli,

e.g. Escherichia coli which also grow well on this unselective

differential (indicator) medium (Koneman et al.,1997).

b- Brilliant Green Mc Conkey Agar:

The addition to Mac Conkey agar of brilliant green 0.004

g\liter, which is inhibitory to E. coli, Proteus species and the other

commensal enterobacteria likely to out number the Salmonellae in

faeces, makes this an excellent selective aswell as differential medium

for Salmonellae except S. typhi which is not grow well on it.

Salmonellae appear as low convex, pale- green translucent

colonies 1-3 mm in diameter. Lactose fermenting bacteria, including

rare strains of Salmonella serotypes, produce blue- purple colonies

(Murry et al., 2005).

20  

c- Leifson,s Deoxycholate- Citrate Agar (DCA):

The colonies of Salmonellae on DCA are similar to or slightly

smaller in size than those on Mac Conkey agar. They are pale, nearly

colorless, smooth, shiny and translucent. Sometimes they have a black

center and sometimes a zone of cleared medium surrounds them, but

these characters may required 48 hrs of incubation for their

development. Salmonellae are easily distinguished from the opaque

pink colonies of lactose- fermenting coliform bacilli, which are largely

inhibited on this selective differential medium (Cheesbrough, 2000).

d- Wilson and Blair's Brilliant- green Bismuth Sulphite Agar

(BBSA):

This medium is particularly valuable for the isolation of S.

typhi. Cultures should be examined after 24 hrs, then after 48 hrs.

Crowded colonies about 1 mm diameter may appear green or pale-

brown. Larger discrete colonies have a black center and clear edge

(Barrow and Feltham, 1993).

e- Taylor's Xylose Lysine Deoxycholate (XLD):

It is a popular medium for the primary plating of faeces from

suspected Salmonella and Shigella infection. It gives colony

appearances that distinguish Salmonellae from Shigellae, and these

pathogens from from the many non- lactose fermenting strains of non-

pathogenic enterobacteria which form pale colonies similar to theirs

on Mac Conkey and DCA. Colonies of Salmonellae and Shigellae are

red (alkaline to phenol red) because Shigellae do not form acid from

xylose, lactose and sucrose in the medium within 24 hrs and because

21  

Salmonellae neutralize the acid they form from the limited amount of

xylose by decarboxylating the lysine. Most Salmonellae (and

Edwardsiellae) are distinguished from the Shigellae because they

produce hydrogen sulphide, which reacts with ferric ammonium

citrate in the medium to produce black centers in their red colonies

(Cheesbrough, 2000).

f- Rambach,s Agar:

This recently described medium (1990), which contains

propylene glycol (PG) and a novel chromogenic substrate (Merk) to

detect β- glactosidase activity, is claimed to allow detection of 98% of

,customary, Salmonellae. Non- typhoidal Salmonellae (β-

glactosidase- negative) form acid from the metabolism of PG and,

with suitable pH indicator, grow as red colonies. (Dusch and Altwegg,

1995).

g- Salmonella- Shigella Agar:

It is a selective and differential medium used for the isolation

and differentiation of Salmonella and Shigella from clinical specimens

and other sources. The nutritive base contains animal and casein

peptones and beef extract. The selective agents are bile salts, citrates,

and brilliant green dye, which inhibit gram- positive organisms.

The high degree of selectivity of the medium results in the

inhibition of some strains of Shigella, and the medium is not

recommended as a primary medium for isolation of this species. The

medium contains only lactose and thus differentiates organisms on the

basis of lactose fermentation. The formation of acid on fermentation

22  

of lactose causes the neutral red indicator to make red colonies. Non-

lactose fermenting organisms are clear on the medium. As with

Hektoen enteric agar, sodium thiosulfate and ammonium citrate allow

the differentiation of organisms that produce hydrogen sulphide.

Lactose fermenters, such as E. coli, have colonies, which are pink

with a precipitate; Shigella appears transparent or amber with black

centers. Some strains of Shigella dysenteriae are inhibited (Murray et

al., 2005).

1.7.1.2 Biochemical reactions:

All serotypes of the genus Salmonella and those of the former

genus 'Arizona' are now considered to belong to one species for which

the name Salmonella enterica has been proposed, it comprises species

which, historically, have been numbered but now are named (Le

Minor, 1985).

Carbohydrates are generally fermented with the production of

acid and gas. S. typhi, S. gallinarum and rare anaerogenic variants in

other serotypes, e.g. S. typhimurium, form only acid. Typically,

glucose, mannitol, arabinose, maltose, dulcitol and sorbitol are

fermented, but not lactose, sucrose, salicin or adonitol; the ONPG test

for β- glactosidase is negative. Among exceptional strains,

choleraesuis and some strains of S. typhi do not ferment arabinose

(Duguid et al., 1975.).

Many strains of Salmonella arizonae ferment lactose rapidly or

slowly as well as having activity to β- glactosidase enzyme (Holt et

al., 1994).

23  

Salmonella decarboxylate the amino acids lysine, ornithine and

arginine, but not glutamic acid. S. typhi is exceptional in lacking

ornithine decarboxylase and S. paratyphi in lacking lysine

decarboxylase (Collee et al., 1996).

Most Salmonellae have the following reactions: indole not

produced. Methyl- red positive. Acetyl- methyl carbinol not produced

(e.g. Voges- proskauer negative). Citrate utilized except by S. typhy

and S. paratyphy A. Malonate not utilized. Gluconate not utilized.

Urease not produced. Phenylalanine deaminase not produced.

Hydrogen sulphide produced in ferrous chloride gelatin medium,

except by S. paratyphi A, S. choleraesuis, S. typhisuis and S. sendai.

No growth in KCN medium. Gelatin not liquefied.

1.7.2 Serological tests:

These are satisfactory for establishing the presence and

estimating the prevalence of the infection within a flock. The tests that

are readily applied include tube agglutination (TA) test, rapid serum

agglutination (RSA) test, stained antigen whole blood (WB) test and

micro-agglutination (MA) test (Gast, 1997). Other serological tests

include micro-antiglobulin (Coombs), immunodifusion,

haemagglutination and enzyme-linked immunosorbent assay (ELISA).

The rapid serum agglutination test can be used under field

condition and the reaction can be identified immediately. Chickens

can be tested at any age, although some authorities specify a minimum

age of 4 months (Wray, 2000).

24  

1.8 Drug susceptibility:

An increase of Salmonella strains showing resistance and

multiple resistances against different antibiotics have been found from

isolates from poultry in recent years. Kheir El-Din et al, (1987)

examined in vitro the sensitivity of 89 isolates of S. gallinarum, S.

pullorum, S. virchow and S. newport against 11 antibiotics. The result

reveal that 70- 80% of the isolates were sensitive to flumaquine and

chloramphenicol, and that 38- 57% were moderately sensitive to

nitrofurantoin, ampicillin and neomycin and only 15- 18% were

weakly sensitive to lincomycin and streptomycin, but completely

resistant to erythromycin, penicillin, tetracycline and trimethoprim.

Bolinski et al., (1994) reported that flumaquin inhibited 86% of

Salmonella strains, followed by apramycin, ampicillin, oxitetracycline

and gentamycin. The minimum inhibitory concentration of flumaquine

varied between 1.0 and 5.0 µg /ml. On the other hand, Ghosh, (1988)

found that 36 strains of S. virchow were highly sensitive to

gentamycin, streptomycin and kanamycin but resistant to bacitracin,

penicillin, sulphaphenazole and tetracycline.

Lee et al., (1993) determined that 57% of 105 Salmonella

isolates were resistant to one or more antimicrobial agents and 45%

were resistant to two or more agents. Highest resistance was to

tetracycline 45%, streptomycin 41%, sulfisoxazole 19% and

gentamycin 10%.

Jacobs et al, (1994) reported that 7.5% of 94 Salmonella

isolates were resistant to nalidixic acid and fumaquine but did not to

ciprofloxacin.

25  

Roliniski et al, (1994) determined that 52.98% of S. enteritidis

and S. typhimurium were resistant to nitrofurans, oxytetracycline,

sulphonamides alone and with trimthoprim. The similar levels of

resistance (49.84%) were shown by S. gallinarum isolates to

oxytetracycline and sulphonamides alone and with trimethoprim and

only 8% were resistant to nitrofurans.

Esaki et al., (2004) isolated 94 Salmonella strains of 10

serotypes from different poultry farms (broiler and lying hens). 39 of

them were resistant to flumaquine, nalidixic acid and oxolinic acid.

All strains were sensitive to ciprofloxacin. The most frequent

serotypes were S. enteritidis and S. heidelberg.

Currently, S. typhi isolates resistant six different antimicrobial

agents prevail in highly endemic typhoid areas, particularly China,

Pakistan and India. These strains of S. typhi carry a 120- kb plasmid

that encodes resistance to ampicillin, chloramphenicol, streptomycin,

sulphonamides, tetracycline and trimethoprim. In addition, S. typhi

strains isolated from recent outbreaks in Tadjikistan and Pakistan have

also acquired resistance to ciprofloxacin (a fluoroquinolone), one of

the preferred antibiotics for treatment of typhoid fever (Hampton et

al., 1998).

Resistance of non- typhoidal Salmonellae is also a growing

health problem. Particularly troubling is the penta- resistant strains of

S. typhimurium known as DT104 (Definitive phage type 104), which

emerged in Great Britain in 1984 and was reported in 1997 to have

been isolated in the United States of America (CDC, 1997). This

strain has been isolated from numerous species of animals and is

26  

resistant to ampicillin, chloramphenicol, streptomycin, sulphonamides

and tetracycline (R type ACSSuT). In addition, there have been

reports of resistance to two other antibiotics, trimethoprim and

fluoroquinolones, in Great Britain (Threlfall et al., 1996).

Fluoroquinolones are the drugs of choice for an invasive

extraintestinal infection in adults, whereas extended-spectrum

cephalosporins (ESC) are preferentially used to treat salmonellosis in

children (Fey et al., 2000). However, the use of these drugs is

becoming compromised by the increasing development of the ESC

and quinolone resistance all over the world.Treatment failures due to

in vivo acquisition of an extended spectrum β-lactamase (ESBL) gene

(Su et al., 2003) or a reduced susceptibility to ciprofloxacin

(Aarestrup et al., 2003) in Salmonella are now well established.

Salmonella strains resistant to ESC have been reported since the

late 1980s, and their numbers have increased ever since. Two major

resistance mechanisms have been identified in Salmonella. In the first,

the isolates express plasmid-mediated AmpC-like β-lactamases that

hydrolyze the cephamycins, extended-spectrum cephalosporins, and

monobactams. In the second mechanism, the Salmonella strains

express an ESBL that is able to hydrolyze monobactams and oxyimino

cephalosporins (such as cefotaxime and ceftazidime) but not the

cephamycins (Miragou et al., 2004).

1.9 Control and treatment:

Reddy et al, (1987) determined synergy between sulphadiazine

and trimethoprim against S. gallinarum. The minimum inhibitory

concentrations (MIC) of the two drugs used separately were 15.2 and

27  

0.8 µg\ml respectively, and when combined they were 1.9 and 0.1

µg\ml. Trimethoprim plus sulphadiazine added to the drinking water

at 60+300 mg\L for 7 days reduced liver and spleen lesions and the

number of bacterial isolates from chicks experimentally infected with

2×109 S. gallinarum.

Humphrey and Laning (1988) evidenced that treatment of feed

given to lying hens with 0.5% formic acid significantly reduced the

isolation rate of Salmonella and was associated with reduction in the

incidence of infection in newly- hatched chicks. These improvements

were not sustained until slaughtered, as growing birds acquired

Salmonella, probably from feed which was not acid treated. These

finding indicate that formic acid treatment of chicken food could have

important benefits for the public health.

The administration of injectable antibiotics such as gentamycin

in the hatchery played a pivotal role in controlling the spread of S.

arizonae in turkey pouts (Shivaprasad et al., 1998). Antibiotics have

been used to control S. enteritidis infection in several experimental

and commercial contexts. Treatment of chicks with polymyxin B

sulphate and trimethoprim both prevent and cleared experimental

infection (Goodnough and Johnson, 1991). Administration of

flavophospholipol or salinomycin sodium as feed additives reduced

faecal shedding (Bolder et al, 1999). Provision of a competitive

exclusion culture to restore a protective normal microflora after

treatment with enrofloxacin reduced the isolation of S. enteritidis from

broiler breeders and their environment (Reynolds et al., 1997).

28  

Mcllory et al., (1989) reported that antibiotics were used

effectively both as therapeutic and prophylactic agents as a part of

control efforts for S. enteritidis in broiler and broiler breeder's flocks

in Northern Ireland.

To control this zoonosis, a number of prophylactic means have

been developed. Vaccination has a general effect and may reduce

animal contamination and rate of excretion of the bacterium through

faeces (Zhang-Barber et al., 1999). Other methods aimed to reduce the

introduction of the bacterium into the gut, which is based on the on the

early implementation of an adult- type intestinal flora which compete

with S. entritidis (Rabsch et al., 2000) or acidification of feed which

deters bacterial growth. Genetic methods may also be successful in

increasing resistance to systemic disease or carrier state (Beaumonal

et al., 1999), thus reducing the need for antibiotic treatments and risk

of antibiotic resistance.

1.10 Salmonella vaccine:

Worldwide, salmonellosis is a serious medical and veterinary

problem and raises great concern in the food industry. Vaccination is

potentially an effective tool for the prevention of salmonellosis.

Whole-cell killed vaccines and subunit vaccines were used with

variable results for the prevention of Salmonella infection in humans

and animals (Mastroeni et al., 2001). Recent advances in the genetics

of Salmonella led to the development of attenuated Salmonella

vaccine strains with single or multiple defined mutations in the

bacterial genome (Mastroeni et al., 2001). Live attenuated Salmonella

vaccines were also used successfully as carriers for the delivery of

29  

heterologous antigens to the immune system. Flagellin, the major

structural protein of flagella, is used for the serotyping Salmonella.

Purified flagellin induces a high systemic, humoral and mucosal

immune response in C3H/HeJ mice (Strindelius et al., 2004).

While mice immunized intraperitoneally with flagellin show a

strong systemic (immunoglobulin G IgG) response against flagellin,

mice immunized mucosally with the strain did not (Strindelius et al.,

2004).

Nevertheless, flagella elicit a strong immune response in

chickens (Mazumoto et al., 2004; Van Zijdirveld et al., 1992) and are

useful serological markers that carry the serotype-specific H-antigenic

determinants (Kauffmann, 1964) in the central variable domain of the

protein (Van Asten et al., 1995). Therefore, deletion of fliC, the gene

that codes for flagellin (FliC) should allow serological differentiation

between animals immunized with the _fliC vaccine strain and animals

infected by wild-type S. enterica serovar Enteritidis. Specific

antibodies against S. enterica serovar Enteritidis FliC were detected in

sera of spray-inoculated young chickens but not in sera of young

chickens inoculated orally with S. enteric serovar Enteritidis

(Mazumoto et al., 2004).

30  

Chapter two

Materials and Methods

2.1 Sampling:

2.1.1. Source of specimens:

The specimens were taken from poultry farms (layers and

broilers) in Khartoum North area. Samples (feed, litter and drinking

water) were selected from six poultry farms (three broiler farms and

three layer farms) during the period between August to November

2009.

Sites in the area from which samples were collected include

(Table 1):

- Two farms in Shambat area.

- Animal production research center, Kuku (broilers and layers).

- A farm in Al-Halfaya area (layers).

- A farm in Al-Zakyab (broilers).

2.1.2. Collection of specimens:

A total of eighty samples were collected. These samples

comprise 27 feed samples, 27 litter samples and 26 samples of

drinking water (table 1):

31  

2.1.2.1. Feed samples:

A mount of 10 grams feed was taken for every sample from

poultry feeders. Every sample taken from 5 different feeders in the

house. Then samples placed in sterile containers. Sterile spoons were

used for sample collection.

2.1.2.2. Litter samples:

Litter samples were collected in a mount of 10 grams for every

sample and then placed in sterile containers. Sterile spoons were used

for sample collection.

32  

Table (1): Origin, type and number of samples collected in the study

No. and type of samples examined source

water

litter

feed

4

4

4

Amr farm, Shambat (broilers)

5

5

5

Dr. Wafa farm, Shambat (layer)

10

10

10

Animal production research center, Kuku (tow farms, broilers and layers)

3

4

3

Dr. Haytham farm, AlHalfaya (layer)

5

4

5

Dr. Jalal farm, AlZakyab (broilers)

33  

2.1.2.3. Drinking water samples:

30 ml of water samples were collected from drinkers. Every

sample taken from 5 different drinkers in the house. Then samples

placed in sterile containers.

2.1.3. Transport and storage of samples:

All samples were placed on ice in a thermos flask immediately

after collection and transported to the laboratory of bacteriology in

Department of Microbiology (Faculty of Vet. Medicine) and kept at 4º

C.

2.2. Bacteriological investigation:

2.2.1. Culture media:

2.2.1.1. Liquid media:

a. Peptone water (Oxoid Ltd, England):

This medium was used as base of carbohydrates utilization tests

and for other purposes. It was composed of 10 grams peptone, 5 grams

sodium chloride. It was prepared by dissolving 15 grams of powder in

1 liter distilled water, the pH was adjusted to 7.4, then mixed well and

distributed into test tubes 5 ml each and sterilized by autoclaving at

121º C for 15 minutes, then stored in the refrigerator at 4º C until

used.

b. Nutrient broth (Oxoid Ltd, England):

It was composed of 1.0 gram of lab-lemco powder, 2 grams

yeast extract, 5.0 grams peptone and 5.0 grams sodium chloride. It

34  

was prepared by adding 13 grams to 1 liter DW, the pH was adjusted

to 7.4, then mixed well and distributed in 3 ml amounts into bijou

bottles and sterilized by autoclaving at 121º C for 15 minutes, then

stored in the refrigerator at 4º C until used.

c. Selenite-F-broth (Oxoid Ltd, England):

According to manufacturer, the medium was prepared by

dissolving 5.0 grams peptone, 4.0 grams mannitol, 10 grams disodium

hydrogen phosphate and 4.0 grams sodium hydrogen Selenite in one

liter of distilled water, the pH was adjusted to 7.0 and sterilized by

steaming for 20 minutes, mixed well and dispensed into sterile

containers.

d. Methyl Red-Voges Proskauer medium (MR-VP) (Oxoid Ltd,

England):

This medium contains (grams per liter) peptone P (Oxoid L49)

5 grams, dextrose 5 grams and phosphate buffer 5 grams.

It was prepared by adding 15 gram of powder to 1 liter of DW,

mixed well, the pH adjusted into 7.5, distributed into test tubes in 5ml

amount and sterilized by autoclaving at 121º C for 15 minutes.

e. Peptone water sugars:

This medium composed of peptone water and different sugars.

The pH of the peptone water (900 ml) was adjusted to 7.1-7.3 before

10 ml of Andrade’s indicator added, then 100 ml of 10% sugar

solution (glucose or sucrose or mannitol) were added to the mixture,

mixed well and distributed in 2 ml amounts into sterile test tubes

35  

containing inverted Durham’s tube, then sterilized by steaming for 30

minutes and stored in the refrigerator at 4º C until used.

2.2.1.2 Semi- solid media:

a. Hugh and Liefson’s (O\F) medium:

This media contain peptone, NaCl, K2Hpo4, agar and

bromothyonol blue as an indicator. It was prepared according to

Cowan and Steel (1995) by adding the solids in 1 liter DW and boiled

to dissolve completely. The pH adjusted to 7.1 and the medium was

filtered then the indicator was added followed by sterilization at 115º

C for 20 minutes. Sterile glucose solution was then added to give final

concentration of 1%, mixed and distributed aseptically in 10 ml

volumes into sterile test tubes of not more than 16mm diameter.

b. Motility media (Oxoid Ltd, England):

This media prepared by adding 13 grams of nutrient broth 7.5

grams of Oxoid agar No. 1 and dissolved in one liter of distilled water

by heating to 100º C. The pH was adjusted to 7.2 and poured into test

tubes (U shape). The tubes were sterilized by autoclaving at 121º C for

15 minutes, then the media were cooled to use for motility test.

2.2.1.3. Solid media:

a. Nutrient agar (Oxoid Ltd, England):

It consists of (grams per liter) lab-lemco powder 1.0 gram, yeast

extract 2 grams, peptone 5 grams, sodium chloride 5 grams and agar

15 grams.

36  

28 grams of medium were added to 1 liter of distilled water and

boiled to dissolve completely, the pH was adjusted to 7.4, and then the

medium was sterilized by autoclaving at 121º C for 15 minutes and

distributed aseptically in 15 ml amounts into sterile Petri dishes.

Nutrient agar slops were also prepared and stored in refrigerator at 4º

C until used.

b. Triple sugar Iron Agar medium (TSI) (Oxoid):

It contains (grams per liter) Lab-Lemco powder (Oxiod L29) 3

grams, yeast extract (Oxoid L20) 3 grams, peptone (Oxoid L37) 20

grams, sodium chloride 5 grams, lactose 10 grams, sucrose 10 grams,

dextrose 1 gram, ferric citrate 0.3, sodium thiosulfate 0.3, phenol red

0.025 gram and agar No. 3 (Oxoid L13) 12 grams.

Triple sugar iron agar was prepared by adding 65 gram of

powder to 1 liter of DW, the pH adjusted into 7.4, then boiled to

dissolve completely, mixed well, distributed in 5 ml amount into

McCarteny bottles and sterilized by autoclaving at 121º C for 15

minutes. The medium was allowed to set in a slope position about one

inch butt and stored at 4º C.

c. Desoxycholate Citrate Agar (DCA) (Oxoid Ltd, England ):

This medium contains (grams per liter) Lab-lemco powder

(Oxoid L29) 5 grams, peptone (Oxoid L37) 5 grams, lactose 10 grams,

sodium citrate 8.5 grams, sodium thiosulfate 5.4 grams, ferric citrate 1

gram, sodium desoxycholate 5 grams, neutral red 0.02 gram and agar

No. 3 (Oxoid L13) 12 grams.

37  

It was prepared by suspending 52 gram of powder in 1 liter of

DW, the pH adjusted into 7.3, then boiled over flame to dissolve

completely, agitated to prevent charring, and dispensed into sterile

petri-dishes in portions of 15ml and stored at 4º C.

d. Christensen’s Urea Agar (Oxoid Ltd, England):

The medium was composed of (grams per liter) peptone 1.0

gram, dextrose 1.0 gram, sodium chloride 5.0 grams, disodium

phosphate 1.2 grams, potassium dihydrogen phosphate 0.8 gram,

phenol red 0.012 gram and agar 15 grams. According to the

manufacturer instructions, 2.4 grams of dehydrated medium were

dissolved in 95 ml of distilled water by boiling, pH was adjusted to

6.8, sterilized by autoclaving at 115º C for 20 minutes, then cooled to

50º C and aseptically 5 ml of sterile 40% urea solution were added.

The medium was poured into sterile screw-capped bottles 10 ml each,

and then allowed to set in the slope position.

e. Simmon’s Citrate Agar (Oxoid Ltd, England):

It consist of (grams per liter) 0.2 gram of magnesium sulphate,

ammonium dihydrogen phosphate 0.2 gram, sodium ammonium

phosphate 1.0 gram, sodium citrate 2.0 grams, sodium chloride 5

grams, bromo-thymol blue 0.08 gram and agar 15 grams. 23 grams of

dehydrated Simmon’s citrate agar were suspended in one liter of

distilled water, boiled to dissolved completely, the pH was adjusted to

7.0 and sterilized by autoclaving at 121ºC for 15 minutes. It was then

poured into sterile screw-capped bottles and allowed to set in the slope

position.

38  

f. Mueller and Hinton Agar (Oxoid Ltd, England):

This medium used for cultivation of Niesseria and antimicrobial

susceptibility testing. It contains of (grams per liter) beef infusion

from 300 grams, casein hydrolysate 17.5 grams and agar No 1 10.0

grams, and pH adjusted into 7.4.

35 grams of powder were suspended in 1 liter of distilled water,

boiled to dissolved completely, then sterilized by autoclaving at 121ºC

for 15 minutes.

2.2.2. Solutions and Reagents:

2.2.2.1. Normal saline solution:

This was prepared by dissolving 8.5 gram of sodium chloride in

1 liter of DW (Cowan and Steel, 1985).

2.2.2.2. Methyle Red solution:

This solution was prepared by dissolving 0.04 gram of methyl

red powder in 10 ml ethanol and diluted with distilled water to 100 ml

(Barrow and Feltham, 1993).

2.2.2.3. Kovac’s reagent:

This reagent was prepared for indol test. 5 gram of p-dimethyl

aminobenzaldehyde was dissolved in 75 ml of amylalcohol by

warming in water bath (50-55c), and then cooled and 25 ml of HCl

was added. It was protected from light and stored at 4º C (Barrow and

Feltham, 1993).

39  

2.2.2.4. Oxidase test reagent:

This reagent was prepared by dissolving 0.1 gram tetramethyl-

p-phenylene diamine dihydrochloride in 10 ml distilled water (Barrow

and Feltham, 1993). It’s prepared immediately before use because it

easily oxidized.

2.2.2.5. Potassium Hydroxide solution:

This was prepared by dissolving 40 gram of pure potassium

hydroxide in 100 ml DW.

2.2.2.6. Andrade’s Indicator:

This was prepared according to Baker and Silverton (1980) by

dissolving 5 grams of acid fuchsin powder in 1 liter of DW, and then

150 ml of NaOH was added to the solution mixed and allowed to

stand at room temperature for 24 hours.

2.2.2.7 Voges- Proskauer (V.P) test reagent:

This reagent was prepared by mixing 40% potassium hydroxide

(KOH) with 5% alph-naphthol in absolute ethanol.

2.2.2.8 Lead acetate paper:

It was prepared from a filter paper cut into strips of 5-10 mm

wide and 50-60 mm long and impregnated with the hot saturated lead

acetate solution, dried at 50-60ºC and stored at screw-capped

containers. It was used for detection of H2S production.

40  

2.2.2.9 Bromothymol blue:

It was used for citrate medium and (OF) medium. A total of 0.2

gram of the powder was dissolved in 100 ml of distilled water.

2.2.2.10 Phenol red:

It was used for urea agar base medium as 0.2%.

2.2.3. Sterilization procedures:

2.2.3.1. Hot air oven:

Glassware (flasks, test tube, pipettes and petri dishes) and metal

instruments (scissors and forceps) were sterilized in hot air oven at

160º C for 2 hours.

2.2.3.2. Autoclaving:

Culture media and discarded cultures were sterilized by

autoclaving at 121ºC for 20 minutes while glassware with plastic

covers was autoclaved at 121ºC for 15 minutes.

2.2.3.3. Disinfectants and antiseptics:

70% alcohol was used to disinfect the surfaces of benches

before and after use.

2.2.3.4. U. V. light:

It was used to sterilize the vacuum of media pouring room and

laminar-flow cabinets.

41  

2.2.4. Cultivation of samples:

2.2.4.1. Inoculation of enrichment medium:

a. Feed samples:

10 grams of feed sample was inoculated into medical bottle

containing 100 ml of selenite-f-broth and then incubated aerobically at

37ºC for 24 hours.

b. Litter samples:

10 grams of litter was inoculated into medical bottle

containing 100 ml of selenite-f-broth and then incubated aerobically at

37ºC for 24 hours.

c. Water samples:

30 ml of water samples were centrifuged (5000 rounds per

minute for 5 minutes), 1 ml of sediment was inoculated into test tube

containing selenite-f- broth and then incubated aerobically at 4º C for

24 hours.

2.2.4.2. Inoculation of plates:

A loop of the inoculated selenite-f-broth was streaked on a plate

of deoxycholate citrate agar and incubated aerobically at 37º C for 24

hours.

42  

2.2.4.3. Purification and storage of isolates:

Non- lactose fermenter colonies were purified by repeated

subculture on nutrient agar. Pure isolates were stored on nutrient agar

slopes in the refrigerator at 4º C.

2.2.5. Identification of the isolated bacteria:

Identification of purified isolates was performed according to

Cowan and Steel (1985).

2.2.5.1. Microscopic Examination:

a. Gram’s stain:

Smears were prepared from the culture by emulsifying a part of

a colony in a drop of normal saline on a glass slide, dried and fixed by

heating. Then the slides were flooded by crystal violet for 1 minute

and then washed with tap water. Iodine solution was applied for 1

minute, and then the slide was washed with tap water. The smear was

then decolorized with few drops of acetone for seconds and washed

immediately with water. Then the smear was flooded with diluted

carbol fuchsin for 30 seconds and washed with tap water. Slides were

then blotted with filter paper and examined under oil immersion lens.

Gram-positive bacterial cells appeared violet in color while that of

gram-negative bacteria appeared red.

43  

2.2.5.2. Biochemical tests for identification of bacteria:

2.2.5.2.1 Primary biochemical tests:

a. Oxidase Test:

The test was carried out according to Cruickshank (1972).

Strips of filter paper were soaked in 10% solution of tetramethyle -p-

phenylene diamine dihydrochloride in a petri dish and then left to dry.

Then a fresh young test culture, on nutrient agar, was picked up with a

sterile glass rod and streaked on that filter paper. A dark purple color

that developed within five to ten seconds was considered positive

reaction.

b. Catalase Test:

Catalase test was carried out according to Cowan and Steel

(1985). A drop of 3% aqueous solution of hydrogen peroxide was

placed on a clean microscope slide. A colony of test culture, on

nutrient agar was then placed on the hydrogen peroxide drop. The test

was considered positive when gas bubbles appeared on the surface of

the culture material.

c. Glucose utilization Test:

The sugar media were inoculated with the test organism and

incubated at 37ºC over night. They were examined daily for 7 days.

Acid production was indicated by the development of pink color in the

medium, Gas production was indicated by air trapped in the Durham’s

tube.

44  

d. Oxidation-Fermentation (O/F) Test:

The test was made by growing the test culture in tow tubes of

Hugh and Lifeson’s medium. A layer of soft paraffin was added to

one tube to a depth of about 1 cm. Both tubes were incubated at 37º C

and examined daily. Oxidizer organisms showed acid production in

the upper part of medium in the paraffin-covered tube and at the

bottom in the open tube.

e. Motility (Oxoid Ltd, England):

Motility medium (Semi-solid medium in U- shape tube) was

inoculated at the top of one end of the tube with tested organism and

incubated at 37ºC for about 4 days. Positive test was indicated by

presence of growth in the other sides of the tube.

2.2.5.2.2 Secondary biochemical tests:

a. Urease Test:

Suspected Salmonella colonies were streaked on urea agar

slope, incubated 37º C for 2 days. A positive reaction was indicated by

a change of color to pink.

b. Indole Test:

The test culture was inoculated into peptone water medium and

incubated at 37º C for 48 hours. 1 ml of Kovacs’s reagent was run

down to the side of the tube. A pink ring which appeared on the

surface within 1 minute indicated positive reaction.

45  

c. Methyl Red (MR) Test:

The test organism was inoculated in glucose phosphate peptone

water, incubated 37º C for 2 days. Five drops of methyl red reagent

were added. A positive reaction was indicated by appearance of a red

color.

d. Voges Proskauer (V.P) Test:

The test organism was inoculated in glucose phosphate peptone

water, and then 3 ml of 5% alcoholic solution of α-naphthol and 1ml

of 40% KOH aqueous solution was added. A positive reaction was

indicated by development of bright pink color within 30 minutes.

e. Citrate utilization:

An isolate colony from nutrient agar was picked up with a

straight wire, then inoculated in Simmon’s citrate agar and incubated

at 37º C and examined daily. A positive test was indicated by change

of color from green to blue.

f. Hydrogen sulphide (H2S) Production:

The test culture was inoculated by stabbing the butt and

streaking the slope of triple sugar iron agar in McCarteny bottles and

incubated at 37ºC for 2 days. A positive reaction was indicated by

development of a black color.

g. Sugar fermentation test:

The sugar media were inoculated with the test organism and

incubated at 37ºC overnight. They were examined daily for seven

46  

days. Acid production was indicated by development of pink color in

the medium, Gas production was indicated by air trapped in the

Durham’s tube. The sugars used in these tests were lactose, salicin,

sucrose, maltose, manitol, rafinose, inositol, xylose and sorbitol.

2.2.6 Antimicrobial sensitivity test:

Sensitivity of Salmonella isolates to a number of antimicrobial

agents (Table 2) was determined by the standard disk diffusion

method (Buxton and Fraser, 1977). Each isolate was tested to 10

different antimicrobial agents used for Gram-negative bacteria.

Colonies from each isolate were emulsified in 2 ml nutrient broth and

shaken thoroughly to obtain a homogenous suspension of the test

culture. The plates were then flooded with the bacterial suspension,

tipped in different directions to cover the whole surface with the

suspension. Excess fluid was aspirated and the plates were left for 15

minutes to dry.

The antimicrobial disks were placed on the agar medium by

using sterile forceps. The plates were then incubated at 37ºC and

examined after 24 hours for zones of inhibition which were measured

in mm. The isolates were described as resistant, intermediate and

sensitive to different antimicrobial agents according to Bauer et al.,

(1966) (Table 3).

47  

Table (2): Antibacterial used in antimicrobial sensitivity test

Antimicrobial

Code

Conc\ disc

Ambicillin\Sulbactam Axiom 20 mcg

Co- Trimoxazole Axiom 25 mcg

Cefotaxime Axiom 30 mcg

Piperacillin\Tazobactam Axiom 100\10 mcg

Chloramphenicol Axiom 30 mcg

Ciprofloxacine Axiom 5 mcg

Ceftizoxime Axiom 30 mcg

Tetracycline Axiom 30 mcg

Gentamycin Axiom 10 mcg

Amikacin Axiom 30 mcg

48  

Table (3): Standard zone of inhibition to different antimicrobial

agents

Antimicrobial agent

Code

Disk

potency

Zone of inhibition (Diameter in mm)

Resistant Intermediate Sensitive

Ampicillin\Sulbactam AS 20 mcg. 11 or less 12- 14 15 or more

Co- Trimoxazole BA 25 mcg. 10 or less 11- 15 16 or more

Cefotaxime CF 30 mcg 14 or less 15- 22 23 or more

Piperacillin\Tazobactam

TZP 100\10 mcg.

17 or less 18- 20 21 or more

Chloramphenicol CH 30 mcg. 12 or less 13- 17 18 or more

Ciprofloxacine CP 5 mcg. 15 or less 16- 20 21 or more

Ceftizoxime CI 30 mcg. 14 or less 15- 19 20 or more

Tetracycline TE 30 mcg. 14 or less 15- 18 19 or more

Gentamycin GM 10 mcg. 13 or less 14- 15 16 or more

Amikacin AK 30 mcg. 14 or less 15- 16 17 or more

49  

CHAPTER THREE

RESULTS

3.1 Isolation of bacteria:

A total of 80 samples were subjected to bacteriological

examinations. Forty-three Gram-negative Enterobacteria were

isolated from 80 samples; 12 samples showed no bacterial growth, 20

samples did not give typical reactions of Enterobacteria with oxidase,

catalase, OF and glucose fermentation so they were not further

identified, 5 samples gave only lactose fermenter colonies (pink

colonies) in DCA. All samples which gave positive results for catalase

and OF test and negative result for oxidase test and ferment glucose

with gas were further identified.

The isolated Enterobacteria belong to eight genera which

included Serratia species (15), Proteus species (11), Citrobacter

species (8), Salmonella species (5), Yersinia species (1), Kluyvera

species (1) Enterobacter species (1) and Hafnia species (1) (Table 4).

3.2 Site of isolation:

Three isolates of Salmonella were recovered from litter from

Al-Halfaya area farm (layers). These three isolates included tow

isolates of S. enteritidis, and one isolate of S. arizonae. One isolate of

S. enteritidis obtained from poultry drinking water taken from drinkers

in Shambat farm (broilers). No Salmonella isolates obtained from

Animal production research center farms or from Al-Zakyab farm

(Table 5).

50  

3.3 Properties of Salmonella:

3.3.1 Cultural properties:

3.3.1.1 Growth in liquid media:

Growth in selenite-f-broth was detected by brown precipitate in

the medium after 24 hours of incubation at 37ºC.

Growth in nutrient broth and peptone water was indicated by

the formation of turbidity and slight white sediment after 24 hours of

incubation at 37ºC.

3.3.1.2 Growth on solid media:

On nutrient agar Salmonella colonies were moderately large (2-

4 mm), circular with smooth surface and grayish- white in color after

24 hours at 37ºC (Figure 1).

Growth on deoxycholate citrate agar showed slight opaque

dome- shaped colonies measured (2-4 mm) with central black spots

(indicated production of hydrogen sulfide) surrounded by a zone of

clearance after 48 hours at 37ºC (Figure 2).

On triple sugar iron agar Salmonella colonies produced

hydrogen sulfide which was indicated by black discoloration, gas

production causes bubbles in the agar, and pH change was indicated

by production of red color in the slant (Figure 3).

3.3.2 Microscopic properties:

All Salmonella isolates were found Gram-negative, short rods

occurred singly or in groups.

51  

3.3.3 Biochemical reactions:

Salmonella isolates were oxidase and urease negative. They

produced gas from glucose and manitol, while sucrose, salicin and

lactose were not fermented. Hydrogen sulfide was produced by the

isolates (Table 5).

3.4 Sensitivity to antimicrobial agents:

Sensitivity test to the four Salmonella isolates against 10

antibacterial agents was carried out. All isolates found sensitive to

chloramphenicol, ceftizoxime, amikacin and resistant to gentamycin,

tetracycline, ambicillin\sulbactam and piperacillin\tazobactam. All

isolates were found sensitive to co- trimoxazole except one isolate of

S. enteritidis found sensitive; two isolates of S. enteritidis were

resistant to cefotaxime while other two isolates were moderately

sensitive to this agent; all isolates found resistant to ciprofloxacin

except one isolate of S. enteritidis (Figure 4).

52  

Table (4): Isolated enterobacteria from different samples in the study.

Rate of isolation

No. of isolates

Bacteria spp.

18.75 %

15

Serratia spp

13.75 % 11

Proteus spp

10.00 % 8

Citrobacter spp.

5.00 %

4

Salmonella spp.

1.25 %

1

Kluyvera spp.

1.25 %

1

Yersinia spp.

1.25 % 1

Enterobacter spp.

1.25 %

1

Hfnia spp.

15.00% 12

No growth

26.25%

21

Other bacteria

53  

Table (5): Isolated Salmonellae from different samples and areas in Khartoum North.

Species of Salmonella

isolates

No. of

Salmonella isolates

No. and types of samples from which salmonellae were isolated

Source of samples

Water

Litter

Feed

S. enteritidis S. arizonae

4

-

3

-

Al-Halfaya farm (layer)

S. enteritidis

1

1

-

-

Shambat farm (broiler)

54  

Table (8): Sensitivity of Salmonella isolates to different antimicrobial agents

AK GM TE CI CP CH TZP CF BA AS

Name of isolate

S R R S R S R IN S R HAR

S R R S R S R IN S R HEN1

S R R S R S R R R R HEN2

S R R S IN S R R S R SHEN

Key:

(S) Sensitive. (R) resistant. (IN) intermedia

(BA) Co-Trimoxazole. (CF) Cefotaxime (AK) Amikacin

(CP) Ciprofloxacin. (CI)Ceftizoxime (TE) Tetracycline.

(GM) Gentamycin. (AS) Ampicillin\Sulbactam.

(CH) Chloramphenicol (TZP) Piperacillin\Tazobactam .

HAR (S. arizonae from Halfaya) HEN1 (S. enteritidis from Halfaya)

HEN2 (S. enteritidis from Halfaya) SHEN (S. enteritidis from Shambat)

55  

Figure (1) Growth of Salmonella on Nutrient Agar

Figure (2) Growth of Salmonella on Desoxychocolate Citrate Agar

56  

Figure (3) Growth of Salmonella on Triple Sugar Iron Agar (TSI)

57  

Figure (4) the sensitivity of Salmonella to different antibiotics

58  

0

5

10

15

20

25

30

AS BA CF TZP CH CP CI TE GM AK

Zone

 (mm) o

f inh

ibition

Antibiotics

Figure (5) Sensitivity of Salmonella  isolates  to different antibiotics

HAR (S.arizonae from Al‐Halfaya) HEN1 (S. enteritidis from Al‐Halfaya)

HEN2 (S.entretidis from Al‐Halfaya) SHEN ( S.entretidis from Shambat)

(AS) Ampicillin\Sulbactam  20mcg                      (BA) Co‐ Trimoxazole  25mcg                             (CF) Cefotaxime  30mcg                (TZP) Piperacillin \ Tazobactam 100\10mcg     (CH) Chloramphenicol   30mcg                          (CP)   Ciprofloxacine   5mcg  (CI)   Ceftizoxime 30mcg                                      (TE)  Tetracycline  30mcg                                   (GM)  Gentamycin  10mcg   (AK)  Amikacin 30mcg

59  

CHAPTER FOUR

DISCUSSION

Salmonellosis is a major public health concern and continues to

have a serious economic importance in the poultry industry in all

countries (Morales and McDowell, 1999). With the great expansion of

the poultry industry, the wide spread occurrence of the avian

salmonellosis has ranked it as one of the most important egg- borne

bacterial diseases of poultry.

The present study was conducted to investigate the

contamination of poultry feed and poultry environment with

Salmonellae in poultry traditional farms in Khartoum North.

Salmonellae were isolated together with other bacterial genera as

Serratia, Proteus, Citrobacter, Enterobacter, Yersinia, Kluyvera and

Hafnia. Although all collected samples in the study were cultured first

in the selenite-f- broth, gram- negative bacteria other than Salmonella

were isolated. This can be explained by the fact that selenite-f-broth

enriches the growth of Salmonella and Shigella but do not kill other

enteric bacteria which under other conditions (subculture in DCA) can

grow.

In this study Serratia represented the most dominant isolate and

counted for (18.75%), followed by Proteus (13.75%), Citrobacter

(10.00%), Salmonella (5.00%), Yersinia (1.25%), Enterobacter

(1.25%), Kluyvera (1.25%) and Hafnia (1.25%).

The Salmonella isolation rate (5%) was comparable to that

reported in other studies.

60  

Yagoab and Mohammed (1987) examined 1488 samples and

isolated 58 Salmonellae which comprise 3.9% of total isolates. In

another study Ezdihar (1996) examined 610 samples from poultry in

the Sudan and isolated 45 Salmonellae which counted for 7.4% of the

total isolates. The later study showed higher isolation rate compared to

the finding of this study and that may be due to the large difference in

the number of samples collected in both studies. Hiba (2007)

examined 102 samples from sick chickens in Khartoum state and

isolated three Salmonella which counted (2.9%).

Salmonella was isolated only from samples obtained from a

farm of layers in Al-Halfaya and from a farm of broilers in Shambat.

It was not isolated however, from animal production research center

farms or from a farm in Al-Zakiab area. This finding did not indicate

that Salmonella was not present in these areas, but might be due to the

small number of collected samples. On the other hand it confirms the

presence of Salmonella contamination in farms from which

Salmonellae were isolated.

The higher isolation rate was obtained from a farm of layers in Al-

Halfaya, with the fact that all samples were collected from open

system farms; this can be due to poor hygiene in this farm.

Among the examined samples, the highest rate of isolation was

obtained from litter samples (three isolates) then water samples (one

isolate).

This finding indicates a high shedding of Salmonella from the

intestinal tracts of birds in this farm. S. enteritidis is the most

important serovar in poultry flocks and recently it was of high

61  

occurrence worldwide (Pitol et al, 1991). Phillips and Optiz (1995)

showed that S.enteritidis could attach to granulose cells in the

preovulatory membrane and subsequently infect the ovum during the

ovulation. On the other hand, S. enteritidis had the ability to penetrate

eggs through the shell pores and causes egg contamination.

In the present study three isolates of S. enteritidis were

recovered, our finding confirmed previous records (Mamon et al.,

1992; Hiba., 2007) that S.enteritidis was detected in Khartoum state.

As long as the Sudan depends on importation of chickens it could

have been come with infected imported flocks.

From the view point of public health, human salmonellosis was

reported to increase recently in France and United States of America

due to S. enteritidis (Barrow et al., 2003). It was reported to cause

food poisoning due to consumption of under cooked egg dishes

(Quinn, 2002). Isolation of this bacterium from some farms in

Khartoum state represents a real threat to the public health.

S. arizonae was widely distributed in nature in a variety of

avian, mammalian and reptile species (Cambre et al., 1980). The

variety of infection sources in the nature will expose hen flocks to

infection. S. arizonae was reported to cause arizonae infection in

chickens (Carter and Wise, 2004).

The antimicrobial sensitivity test was carried out for salmonella

isolates. All strains of Salmonella were found sensitive to

chloramphenicol, ceftizoxime, amikacin and resistant to

ambicillin\sulbactam, piperacillin\tazobactam, tetracycline and

gentamycin. Also all isolates were found sensitive to co-trimoxazole

62  

except one isolate of S. enteritidis; two isolates of S. enteritidis were

found sensitive to cefotaxime while S. arizonae and the other isolate

of S. enteritidis were moderately sensitive; S. arizonae and two

isolates of S. enteritidis were showed resistance to ciprofloxacin while

the other isolate of S. enteritidis was moderately sensitive.

Resistance to gentamycin has been reported by Lee et al.,

(1993) which determined 10% resistance to this agent from 105

Salmonella isolates. Also there was an increasing development of

quinolones resistance all over the world (Fey et al., 2004). Treatment

failure due to a reduced susceptibility to ciprofloxacin in Salmonella is

now well established (Aarestrup et al., 2003).

In general, Salmonella is the most important agent implicated in

outbreaks in food-borne diseases around the world (Lacey, 1993).

Effective control or eradication programs for salmonellosis depend on

good management system, identification of carrier birds and accurate

medication.

63  

Conclusion

In search for Salmonellae in poultry environment and feed in

open system farms for layers and broilers in Khartoum North, only

four isolates of Salmonella were recovered from litter samples and

drinking water sample while no Salmonella isolates were recovered

from feed samples.

Three isolates belong to S. enteritidis while the other was S.

arizonae.

Isolation of three S. enteritidis isolates out of 80 samples is

consider a high isolation rate and may represent a real threat for public

health for this organism implicated in serious health problems.

All isolates were found sensitive to chloramphenicol, amikacin

and ceftizoxime while resistant to tetracycline, gentamycin,

ambicillin\sulbactam and piperacillin\tazobactam.

Recommendation:

1- Further studies are needed to investigate the relation between

contamination of poultry environment with Salmonellae and

Salmonella infections in poultry and threat to public health.

2- Application of quick procedures (e.g. PCR) is needed to trace

sources of contamination.

3- Drug resistance among Salmonella bacilli has emerged

worldwide, there for we strongly recommended for more prudent uses

of antimicrobial agents in both medical and veterinary fields.

64  

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