Upload
others
View
1
Download
0
Embed Size (px)
Citation preview
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.
iii
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.
iv
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
vii
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
viii
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
ix
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
x
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
xi
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
1
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
2
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.
3
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.
4
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.
5
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).
6
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).
7
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.
8
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
REFERENCES
Aarestrup, F. M., C. Wiuff, K. Molback, and E. J. Threlfall. (2003). Is
it time to change fluoroquinolone breakpoints for
Salmonella spp. Antimicrob. Agents Chemother. 47:827-
829.
Adesiyun, A.A.; Seepersadsingh, N.; Inder, I. and Caesar, K. (1998).
Some bacterial enterobathogens in wildlife and racing
pigeons from Trinidad. J. Wildlife. Dis. 34: 73-80.
AlObaidi, A. S.; AlAni, I. A.; AlSoudi, K. A. and Abdul-Gani, Z. G.
(1987). Incidence of Salmonella in a flock of chicken
native to Iraq. J. Agric. Water Re., Res. Anim. Prod. 6(2):
51-61.
Ananthanaryan, R. and Paniker, C. K. J. (1997). Textbook of
Microbiology 5th ed. Orient Longman Ltd. New Delhi,
India.
Bailey, J. S.; Cox, N. A.; Berrange, M. E. (1994). USDA Agricultural
research services. Poultry Sci. 73(7): 1153-1157.
Barrow, G. I. and Feltham, R. K. A. (1993). Cowan and Steel’s
Manual for the Identification of Medical Bacteria. 3rd ed,
Cambridge University Press, Cambridge.
Barrow, P.R.; Barrow, G.C.; Mead, c.; Wray, and Duchet-Suchaux.
M. (2003). Control of food poisoning in poultry. Worlds
Poult. Sci. J. 59: 373-383.
65
Barthel M, Hapfelmeier S, Quintanilla-Martinez L, Kremer M, Rohde
M, Hogardt M, Pfeffer K, Russmann H, Hardt WD.
(2003). Pretreatment of mice with streptomycin
provides a Salmonella enterica serovar Typhimurium
colitis model that allows analysis of both pathogen and
host. Infect Immun. 71:2839–2858.
Bauer, A. W.; Kirby, W. M.; Sherris, J. C. and Turck, M. (1966).
Antibiotic susceptibility testing by standardized single
disc method. Ameri. J. clin. Pathol. 45: 493-496.
Baumgartenerm A.; Heimann, P.; Schmid, H.; Liniger, N. and
Simmen, A. (1992). Salmonella contamination of poultry
carcasses and human salmonellosis. Archivfur-Leben
Smittelhygiene, 43: 123-124.
Beaumonal, C.; Beaumont, J.; Protais, J. F.; Guillot, P.; Colin, K.;
Proux, N. and Pardon, P. (1999). Genetic resistance to
mortality of day-old chicks and carrier-state of hens after
inoculation with Salmonella enteritidis. Av. pathol. 28:
131-135.
Bolder, N. M.; Wagenaat, F. F.; Pultrulan, K. T. and Sommer, M.
(1990). The effect of flavophospholipol (Flavomycin7)
and salinomycin sodium (Sacox7) on the excretion of
Clostridium perfringen, Salmonella enteritidis and
Campylobacter jejuni in broilers after experimental
infection. Poult. Sci. 78: 1681-1689.
66
Bolinski, Z.; Wlaz, P.; Kowalski, C. and Pacholczyk, N. (1994).
Computer analysis of antibiotics sensitivity of bacteria
isolates from fowls. Medycyna-Weterynaryjna, 50: 65-
70.
Brawn LC, Hayward RD, Koronakis V. (2007). Salmonella SPI (1)
effector SipA persists after entry and cooperates with a
SPI (2) effector to regulate phagosome maturation and
intracellular replication. Cell Host Micro. 1: 63–75.
Brook, G. F.; Butel, J. S. and Morse, S. A. (2004). Jawetz, Melnick
and adelbergs Medical Microbiology 23rd ed. Mc Graw
Hill Companies, Inc.
Bustamante VH, Martinez LC, Santana FJ, Knodler LA, Steele-
Mortimer O, Puente JL. (2008). HilD-mediated
transcriptional cross-talk between SPI-1 and SPI-2. Proc.
Nat. Acad. Sci. USA. 105:14591–14596.
Buxton, A. and Fraser, G. (1977). Immunology, Bacteriology,
Mycology and diseases of fish and laboratory methods,
Animal Microbiology, 1st ed. 1: 103-105.
Cambre, R.C.; Green, D.E.; Nontail, R.J. and Bush, M. (1980).
Salmonellosis and arizonosis in the reptile collection at
the national zoological park. J. Ameri. Vet. Med. Assoc.
77: 717-725.
Carlison, V.L. and Soneyenbos, G.H. (1970). Effect of moisture on
Salmonella populations in animal feeds. Poult. Sci. 49:
717-725.
67
Carter, G.R. and Wise, J. (2004). Essentials of Veterinary
Bacteriology and Microbiology. 6th ed. Blackwell,
publishing.
Center for Disease Control, (2000). Outbreaks of Salmonella
enteritidis infection associated with eating raw or
undercooked shell eggs-United States, 49:1149-1152.
Center for Disease Control, (2004). Outbreaks of Salmonella
enteritidis infection associated with eating raw or shell
eggs-United States, 49:1149-1152.
Chessbrough, M. (2000). District Laboratory practice In Tropical
Countries. Part 2; Cambridge University Press,
Cambridge.
Choudhury, B.; Chanda, A.; Dasgupta, P.; Dutta, R. K.; Lila, Saha;
Santan, Bhuin; Saha, L. and Bhuin, S. (1993). Studies on
yolk sac infection in poultry, Antibiogram of isolates and
correlation between in vitro and in vivo drug action. Ind.
j. Anim. Health. 32(1) 21-32.
Coburn B, Li Y, Owen D, Vallance BA, Finlay BB. (2005).
Salmonella enterica serovar Typhimurium pathogenicity
island (2) is necessary for complete virulence in a mouse
model of infectious enterocolitis. Infect. Immun. 73:
3219–3227.
Collee, J. G.; Fraser, A. G.; Marmiio, B. P.; Simon, A. and Old, D. C.
(1996). Mackie and McCarteny Practical Medical
68
Microbiology. 14th ed. Longman Singapore publishers
(Pte). Ltd. Singapore.
Coombes BK, Coburn BA, Potter AA, Gomis S, Mirakhur K, Li Y,
Finlay BB. (2005). Analysis of the contribution of
Salmonella pathogenicity islands (1) and (2) to enteric
disease progression using a novel bovine ileal loop model
and a murine model of infectious enterocolitis. Infect.
Immun. 73: 7161–7169.
Corrier, D.E.; Purdy, C.W. and Deloach, J.R. (1990). Effect of
marketing stress on faecal excretion of Salmonella spp. In
feeder calves. Ameri. J. Vet. Res. 51: 866-869.
Cowan, S.T. and Steel, K.J. (1985). Cowan and Steel’s manual for
identification of medical bacteria, 2nd ed. Cambridge
University Press, London.
Cruickshank, A. (1972). Medical Microbiology, A Guide to Lab
Diagnosis and control of infection. Edinburgh and
London, 71: 221-236.
David, B. G.; Bishop, M. L. and Mass, D. (1989). Clinical Laboratory
Science: Strategies for practice. JB Lippincott Company,
Philadelphia.
Davis, B. D.; Dulbecco, R.; Eisen, H. N. and Ginsberg, H. S. (1990).
Microbiology 4th ed. J. B. Lippincott Company.
69
Desoutter, D. (1986). Salmonella in poultry farming. Revue-D′-
Elevage-et-de-Medicine-Veterinaire-de-Vouvelle-
Caledonie, 8, 5-7.
Devi, S. J. and Murray, C. J. (1991). Cockroaches (Blatta and
Periplaneta species) as reservoirs of drug resistant
Salmonella. Epidemiology and infection, 107: 357-361.
Duguid, j. P.; Anderson, E. S.; Alfredsson, G. A.; Barker, R. and Old
DC (1975). A new biotyping scheme for Salmonella
typhimurium and its phylogenetic significance. J. Med.
Microbiol. 8: 149-166.
Duguid, S. P.; Anderson, E. S.; Campbell I. (1966). Fimbriae and
adhesive properties in Salmonella. J. pathol. Bacteriol.
92: 107-138.
Dusch, H. and Altwegg, M. (1995). Evaluation of five new plating
media for isolation of Salmonella from human feces. J.
Clin. Microbiol. 33: 802-804.
Eberth, G.J. (1880). Cited by Topley, A.A. and Wilson, G.S. (1955).
Principles of Bacteriology and Immunology, 4th ed. 1:
82-85.
Esaki, H.; Morioka, K.; Ishihara, A.; Kojima, S.; Shiroki, Y. and
Takahashi, T. (2004). Antimicrobial susceptibility of
Salmonella isolated from cattle, swine and poultry (2001-
2002): report from the Japanese Veterinary Antimicrobial
resistance Monitoring Program. J. Microb. 16: 105-116.
70
Everard, C.O.R.; Tota, B.; Bassett, D. and Cameillf, A. (1979).
Salmonella in wildlife from Trinidad and Grenada. J.
Wildlife Dis. 15: 213-219.
Ezdihar, A.A. (1996). Isolation and characterization of Salmonella
from domestic fowl and its environment in the state of
Kordofan. MVSc. Thesis. U of K. Sudan.
Fey, P. D., T. J. Safranek, M. E. Rupp, E. F. Dunne, M. E. Ribot, P. C.
Iwen, P. A. Bradford, F. J. Angulo, and S. H. Hinrichs.
(2000). Ceftriaxone-resistant Salmonella infection
acquired by a child from cattle. N. Engl. J. Med.
342:1242-1249.
Furtado, G.K.; Wall, H.S. and Casemore, D.P. (1998). Outbreaks of
waterborne infectious intestinal disease in England and
Wales (1992-1995). Epidemiology and infection, 21:
109-119.
Galan, J. E. (2001). Salmonella interactions with host cells: type III
secretion at work. Annu. Rev. Cell Dev. Biol. 17:53–86.
Gast, R.K. (1997). Detecting infection of chickens with recent
Salmonella pullorum isolates using standard serological
methods. Poult. Sci. 76: 17-23.
Ghosh, S.S. (1988). Drug resistance pattern of Salmonella virchow
isolated from poultry in North Eastern region in India.
Ind. Vet. J. 65: 1151-1153.
71
Goodnough, M.C. and Johnson, E.A. (1991). Control of Salmonella
enteritidis infections in poultry by polymyxin B and
trimethoprim. Appl. Envi. Microbiol. 57: 785-788.
Graham, R. and Michael, V. M. (1936). Poult. Sci. 15: 83-87.
Grimont, P.A.D.; Grimont, F. and Bouvet, P. (2000). Taxonomy of the
genus Salmonella. In: Salmonella in domestic animals,
Wary, C. and Wray, A. CAB international. Wallingford.
UK, 1: 1-17.
Guard-Petter, J. (2001). The egg and Salmonella enteritidis. Appl.
Envi. Microbiol. 3: 21-430.
Hafez H.M. (2005). Government regulation and control of some
important poultry diseases. World's Poult. Sci. J. 61:
574-575.
Hafez, H.M. and Jodas, S. (2000). Salmonella infection in turkeys. In:
Salmonella in domestic animals. Wary, C. and Wray, A.
CAB international. Wallingford. UK, 1: 133-155.
Hampton,M. D.; Ward, L. R; Rowe, B. and Threlfal, E. J. (1998).
Molecular fingerprinting of multidrug-resistant
Salmonella enterica serotype typhi. Emerg. Infect. Dis. 4:
317-320.
Hapfelmeier S, Ehrbar K, Stecher B, Barthel M, Kremer M, Hardt
WD. (2004). Role of the Salmonella Pathogenicity
Island (1) Effector Proteins SipA, SopB, SopE, and
SopE2 in Salmonella enterica Subspecies 1 Serovar
72
Typhimurium Colitis in Streptomycin-Pretreated Mice.
Infect. Immun. 72:795–809.
Hapfelmeier S, Stecher B, Barthel M, Kremer M, Muller AJ,
Heikenwalder M, Stallmach T, Hensel M, Pfeffer K,
Akira S, Hardt WD. (2005). The Salmonella
pathogenicity island (SPI-2) and (SPI-1) type III
secretion systems allow Salmonella serovar typhimurium
to trigger colitis via MyD88-dependent and MyD88-
independent mechanisms. J. Immunol. 174:1675–1685.
Haraga A, Ohlson MB, Miller SI. (2008). Salmonellae interplay with
host cells. Nat. Rev. Microbiol. 6:53–66.
Harris, I.T.; Fedorka, P.J.; Gray, J.T.; Thomas, L.A. and Ferris, K.
(1997). Prevalence of Salmonella in swine feed. J. Appl.
Vet. Med. Assoc. 210: 382-385.
Harry, E. G. and Brown, W. B. (1974). Fumigation with Methyl
Bromide-Applications in the Poultry Industry. World′s
Poult. Sci. J. 30: 193-216.
Hofstad, M. S.; Calnek, B. W.; Helmboldt, C. F.; Reid, W. M. and
Yoder,Jr. H. W. 1978. Diseases of Poultry, 7th ed. Iowa
State University. USA.
Holt, N.R.; Krieg, P.H.A.; Sneath and Williams, S.T. (1994). Berger's
Manual of Determinative Bacteriology. 9th ed. Williams
and Wilkins Company. Baltimore, 50: 787-791.
73
Hoop, R. K. and Pospischil, A. (1993). Bacteriological, serological,
histochemical and immunohistochemical finding in
laying hens with naturally acquired Salmonella enteritidis
phage type 4 infection. Vet. Rec. 133(16) 391-393.
Humphrey, T. (2000). Public-health aspects of Salmonella infection.
In: Salmonella in domestic animals. Wary, C. and Wray,
A. CAB international. Wallingford. UK, 3: 245-264.
Humphrey, T. J. and Lamming, D. G. (1988). The vertical
transmission of Salmonella and formic acid treatment of
chicken feed. A possible strategy for control.
Epidemiology and infection. 100(1) 43-49.
Inami, G.B. and Moler, S.E. (1999). Detection and isolation of
Salmonella from naturally contaminated alfalfa seeds
following an outbreak investigation. J. Food Prot. 62:
662-664.
Jacobs-Relisma, W.F.;Koneraad, P.M.; Bolder, N. and Mulder, R.
W.(1994). In vitro susceptibility of Campylobacter and
Salmonella isolates from broiler to quinolones,
ampicillin, tetracycline and erythromycin. Poult. Sci. 16:
206-208.
Jardy, N. and Michard, J. (1992). Salmonella contamination in raw
feed components. Microbiol. Alimen. Nutr. 10: 233-
240.
Jerngklinchan, J.; Koowatananukul, C.; Dengprom, K. and Saitanul
(1994). Occurrence of Salmonella in raw broilers and
74
their products in Thailand. J. Food Protect. 57(9) 808-
810.
Jones MA, Hulme SD, Barrow PA, Wigley P. (2007). The Salmonella
pathogenicity island (1) and Salmonella pathogenicity
island (2) type III secretion systems play a major role in
pathogenesis of systemic disease and gastrointestinal
tract colonization of Salmonella enterica serovar
Typhimurium in the chicken. Av. Pathol. (36) 199–203
Jones, F.T.; Axteli, R.C.; Rives, D.V.; Scheideler, S.F. and Wineland,
M.J. (1991). A survey of Salmonella contamination in
modern broiler production. J. Food Prot. 54: 502-507.
Jones, P.W.; Collins, P.; Brown, G.T.H. and Aitken, M. (1982).
Transmission of Salmonella mbandaka to cattle from
contaminated feed. J. Hyg. 88: 255-263. Junk Publishers,
The Hague, the Netherlands.
Kapperud, G. and Rosef, O. (1983). Avian wildlife reservoir of
Campylobacter Jejuni, Yersino spp. and Salmonella spp.
In Norway. Appl. Envi. Microbiol. 45: 375-380.
Kauffman, F. (1960). Tow biochemical subdivision of the genus
Salmonella. Acta Pathology Scand. 49: 393-396.
Kauffmann F. 1964. The world problem of salmonellosis, p. 21–66.
Dr. W.
Khan, A.Q. (1969). Animal Salmonellosis in the Sudan. A PhD thesis,
U of K, Sudan.
75
KheirEl-Din, A.M.W.; Hamed, O.M. and Mashhoor, M.M. (1987).
Chemotherapeutic sensitivity testing and pathogenicity of
Salmonella isolated from poultry farms in Egypt. Vet.
Med. 32: 315-323.
Koneman, W. G; Allen, D.S; Janda, M. W; Sclreckenberger, C. P and
Win, C. W. (1997). Color Atlas and Text Book of
Diagnostic Microbiology 5th ed.
Kopanic, R.J.; Sheldon, B.W. and Wright, C, G. (1994). Cockroaches
as a vector of Salmonella: laboratory and field trail. J.
Food Prot. 57: 125-132.
Kotova, A. L.; Kondrataskaya, S. A. and Yasutis, I. M. (1988).
Salmonella carrier state and biological characteristics of
the infectious agents. J. hyg-Epid.-Microbiol. Immun.
32(1) 71-78.
Le Minor, L. and Popoff, M.Y. (1987). Designation of Salmonella
enterica. International J. Sys. Bacteriol. 37: 465-468.
Le Minor, L.; Le Minor, S.; Grimont, P. A. D. (1985). Rapport
quardrie nnal du centre national des Salmonella sur
I’origine et la repartition en serotypes souches isolees en
france continentale au cours des annees 1980 a 1983.
Revued’Epidemidologic et de santé publique. 33: 13-21.
Lee, L.A.; Threatt, V.L.; Puhr, N.D.; Levine, P. and Tauxe, R.V.
(1993). Anti-microbial resistant Salmonella species
isolated from healthy broiler chickens after slaughter.
Ameri. Vet. Assoc. 202: 752-755.
76
Machado, J. and Bernado, F. (1990). Prevalence of Salmonella in
chicken's carcasses in Portugal. J. Appl. Bacteriol. 69:
477-880.
Majid, A.; Siddque, M. and Khan, M. Z. (1991). Prevalence of
salmonellosis in commercial chicken layers in and around
Faisalabad. Pakist. Vet. J. 11(1) 129-135.
Mamon, I.E.; Khalfalla, A.I.; Bakhiet, M.R.; Agab, H.A.; Sabiel, Y.A.
and Ahmed, el J. (1992). Salmonella enteritidis infection
in the Sudan. Bacteriology Rev. Medicine Veterinary
Rays trop. 45: 137-138.
Mandel, G. L.; Hook, E. W. and Douglas, R. G. (1985). Evaluation of
fever in returned travelers. J. Emerging Infec. Dis. 1:
54-57.
Marx, M.B. (1969). Two surveys of Salmonella infection among
certain species of wildlife in northern Virginia (1963-
1966). Ameri. J. Vet. Res. 30: 2003-2006.
Mastroeni, P., J. A. Chabalgoity, S. J. Dunstan, D. J. Maskell, and
G.Dougan. (2001). Salmonella: immune responses and
vaccines. Vet. J. 161:132–164.
Mead, P.S.; Sluisker, M.; Dietz, V.; Mccaig, L.F.; Bresee, J.S. and
Tauxe, R.V. (1999). Food related illness and death in the
United States. Emer. Infec. Dis. 134: 607-623.
Mellory, S.G.; Mccracken, S.D.; Neill and Brien, J.J. (1989). Control,
prevention and eradication of Salmonella enteritidis
77
infection in broiler and broiler breeder flocks. J. Vet.
Res. 125:548.
Menzies, F. D.; Neill, S. D.; Goodall, E. A. and Mcllory, S. G. (1994).
Avian Salmonella infection in Northern Ireland, 1979-
1991. Prev. Vet. Med. 19(2) 119-128.
Minor, L.L.E. (1984). Genus Ш Salmonella lignieres in Kreig and
Holt (editors), Bergery's Manual of Systematic
Bacteriology, vol. 1 (1984). Williams and Wilkins,
Baltimor, London, 20: 427-458.
Miragou, V., P. T. Tassios, N. J. Legalis, and L. S. Tzouvelekis.
(2004). Expanded-spectrum cephalosporin resistance in
non-typhoid Salmonella. Int. J. Antimicrob Agents
23:547-555.
Mizumoto, N., Y. Toyota-Hanatani, K. Sasai, H. Tani, T. Ekawa, H.
Ohta, and E. Baba. (2004). Detection of specific
antibodies against deflagellated Salmonella enteritidis
and S. enteritidis FliC-specific 9kDa polypeptide. Vet.
Microbiol. 99:113–120.
Morales, R.A. and McDowell, R.M. (1999). Economic consequence
of Salmonella enterica serovar enteritidis in humans and
animals. Iowa State University, 7: 271-299.
Morgan E, Campbell JD, Rowe SC, Bispham J, Stevens MP, Bowen
AJ, Barrow PA, Maskell DJ, Wallis TS. (2004).
Identification of host-specific colonization factors of
78
Salmonella enterica serovar Typhimurium. Mol
Microbiol. 54: 994–1010
Mrdjen, M.;Kovincic, I.; Gagic, N.; Babic,M.; Glavicic, M. and
Velhner, M. (1987). Salmonella serotypes in chicks in the
first days of life. J. Vet. Res. 22: 247-249.
Murray, C.J. (2000). Salmonella in the environment. Review Science
and Technical Office of International Epizootics, 22:
247-249.
Murray, P. R; Baron, E. J; Jorgensen, J. H; Pfaller, M. A and Yolken,
R. H. (2005). Manual of Clinical Microbiology 8th ed.
ASM press, Washington. Vol. 1.
Nicklas, W. (1987). Introduction of Salmonella into a centralized
laboratory animal facility by infected day-old chicks.
Lab. Anim. 21(2) 161-163.
Nielsen, B.B. and Clausen, B. (1975). The incidence of Salmonella
bacteria in Danish Wildlife and in imported animals.
Nordisk Veterinary Medicine, 27: 633-640.
Olesiuk, O. M.; Carlson, V. L.; Snoeyenbos, G. H. and Smyser, C. F.
(1969). Experimental Salmonella typhimurium infection
in two chicken flocks. Avian Dis. 13: 500-508.
Orhan, G. and Guler, L. (1993). Bacteriological and serological
identification of Salmonella species in the organs and
faces of chickens eggs and feed. Poult. Sci. 4: 15-20.
79
Pacini, R.; Panizzi, L.; Quagli, E.; Galassi, R.; Marinari, M. G. and
Lenco M. E. (1993). Epidemiology of Salmonella
enteritidis in the Livorono area during 1992. Industrie-
Alimentari, 32(318) 836-841.
Pan, G.Y.; Liu, L.; Chen, J.D. and Luo, D.Y. (1993). Isolation and
identification of enterobacterial strains from chicks.
Avian Dis. 28: 272-275.
Paniker, C. K. J. and Vilma, K. N. (1997). Transferable
Chloramphenicol resistance in Salmonella Typhi. Nature.
239: 109-110.
Phillips, R.A. and Optiz, H.M. (1995). Pathogenicity and persistence
of Salmonella enteritidis and egg contamination in
normal and infectious bursal disease virus infected
leghom chicks. Avian Dis. 39: 778-787.
Pomeroy, B. S. and Fenstermacher, R. 1941. Paratyphoid infections of
turkeys. Am. J. Vet. Res. 2: 285.
Poppe, C. (1994). Salmonella enteritidis in Canada. Intern. J. Food
Microbiol. 22: 1-5.
Porter SB, Curtiss R. (1997). III Effect of inv mutations on Salmonella
virulence and colonization in 1-day-old White Leghorn
chicks. Avian Dis. 41:45–57.
Rabsch, W.; Rabsh, B.M.; Hargis, R.M.; Tsolis, R.A. Kingsley, K.H.;
Hinz, H.: Tschape, and Baumler, (2000). Competive
80
exclution of Salmonella enteritidis by Salmonella
gallinarum in poultry. Emerging Infec. Dis. 7: 443-448.
Reddy, K. S.; Madokhot, U. V. and Uppal, R. P. (1987). Efficacy of
sulphadiazine- trimethoprim combination against
Salmonella gallinarum. Ind. Vet J. 64(3) 318-322.
Reynolds, D.J.; Davies, M. and Wray, C. (1997). Evaluation of
combined antibiotics and competitive exclusion treatment
in broiler breeder flocks infected with Salmonella
enterica serovar enteritidis. Avian Pathol. 26: 83-93.
Roliniski, Z.; Wernicka-Furmaga, R. and Kowalski, C. (1994).
Activity of flumaquine Salmonella species in Vitro and
the treatment of experimental infection in mice.
Medycyna-Weterynaryjna, 44: 416-465.
Ryan, K.J. and Ray, C.G. (2004). Antimicrobial agents. Medical
Microbiology, 4th ed. McGraw Hill.
Santos RL, Zhang S, Tsolis RM, Kingsley RA, Adams LG, Baumler
AJ. (2001). Animal models of Salmonella infections:
enteritis versus typhoid fever. Microbes Infect. 3:1335–
1344.
Sbrogio-Almeida, M. E., and L. C. Ferreira. (2001). Flagellin
expressed by live Salmonella vaccine strains induces
distinct antibody responses following delivery via
systemic or mucosal immunization routes. FEMS
Immunol. Med.
81
Scherer, C.A. and Miller, S.I. Molecular pathogenesis of Salmonellae
(2001). In: principles of bacteria pathogenesis. Grosimar,
S.A. (editor) pp: 265-333. Academic press USA.
Schnurrenberger, P.R.; Held, L.J.; Quist, K.D. and Galton, M.M.
(1968). Prevalence of Salmonella spp. In Domestic
animals and wildlife on selected Hinois farms. J. Appl.
Med. Assoc. 5: 442-445.
Shivaprasad, H.L.; Nagarja, B.S. and Williams, J.E. (1998).
Pathogenesis of Salmonella eneritidis infection in laying
Chickens. Avian Dis. 34: 548-557.
Singer, J.T.; Opitz, H.M.; Gershman, M.; Hali, M.M. and Raw, S.V.
(1992). Molecular characterization of Salmonella
enteritidis isolates from Maine poultry and poultry farm
environments. Avian Dis. 36: 324-333.
Siqueira, R.S.; Siqueira, C.E.R.; Dodd and Rees, C.E. (2003). Phage
amplification assay as rapid method for Salmonella
detection. J. Microbiol. 34: 118-120.
Soumet, G.; Ermel, N.;Rose, P.; Drouin. G.; Salvat, and Colin, P.
(1999). Evaluation of multiplex assay for simultaneous
identification of Salmonella spp., Salmonella enteritidis
and Salmonella typhimurium from environmental swabs
of poultry houses. Appl. Microbiol. 28: 113-117.
Steele-Mortimer O, Brumell JH, Knodler LA, Meresse S, Lopez A,
Finlay BB. (2002). The invasion-associated type III
secretion system of Salmonella enterica serovar
82
Typhimurium is necessary for intracellular proliferation
and vacuole biogenesis in epithelial cells. Cell.
Microbiol. 4:43–54
Strindelius, L., M. Filler, and I. Sjoholm. (2004). Mucosal
immunization with purified flagellin from Salmonella
induces systemic and mucosal immune responses in
C3H/HeJ mice. Vaccine 22:3797–3808.
Su, L. H.; Chiu, C.H; Chu, C.; Wang, M. H.; Chia, J. H. and Wu, T. L.
(2003). In vivo acquisition cefriaxone resistance in S.
enterica serotype Anatum. Antimicrib. Agents
Chemother. 47: 563-567.
Thijs IM, De Keersmaecker SC, Fadda A, Engelen K, Zhao H,
McClelland M, Marchal K, Vanderleyden J. (2007).
Delineation of the Salmonella enterica serovar
Typhimurium HilA regulon through genome-wide
location and transcript analysis. J. Bacteriol. 189:4587–
4596.
Threlfall, E. J.; Frost, J. A.; Ward, L. R. and Rowe, B. (1996).
Increasing spectrum of resistance in multiresistant
Salmonella typhimurium [Letter]. Lancet. 347: 1053-
1045.
Turner AK, Lovell MA, Hulme SD, Zhang-Barber L, Barrow PA.
(1998). Identification of Salmonella typhimurium genes
required for colonization of the chicken alimentary tract
83
and for virulence in newly hatched chicks. Infect Immun.
66: 2099–2106.
Van Asten, A. J., K. A. Zwaagstra, M. F. Baay, J. G. Kusters, J. H.
Huis in’t Veld, and B. A. van der Zeijst. (1995).
Identification of the domain which determines the g,m
serotype of the flagellin of Salmonella enteritidis. J.
Bacteriol. 177: 1610–1613.
Van Zijderveld, F. G., A. M. Zijderveld-van Bemmel, and J. Anakotta.
(1992). Comparison of four different enzyme-linked
immunosorbent assays for serological diagnosis of
Salmonella enteritidis infections in experimentally
infected chickens. J. Clin. Microbiol. 30: 2560–2566.
Waltman, W.D.; Horne, A.M. and Pirkle, C. (1993). Influence of
enrichment incubation time in the isolation of
Salmonella. Avian Dis. 37: 884- 887.
Waterman SR, Holden DW. (2003). Functions and effectors of the
Salmonella pathogenicity island (2) type III secretion
system. Cell. Microbiol. 5: 501–511.
Wieliczko, A. (1994). Occurrence of Campylobacter spp. and
Salmonella in slaughter poultry-correlation to
pathological changes in liver. Berliner und Munchener
Tierartliche Wochenschrift, 107(4) 115-121.
Williams, J.F. and Benson, S.T. (1978). Survival of Salmonella
typhimurium in poultry feed and litter at three
temperatures. Avian Dis. 22: 742-747.
84
Wray, C. and Wray, A. (eds) (2000). Salmonella in domestic animals.
CAB international, Wallingford, Oxon, UK, 32: 407-427.
Yagoub, I.A. and Mohamed, T.E. (1987). Isolation and identification
of Salmonella from chickens in Khartoum province of the
Sudan. Brit. Vet. J. 143: 537-540.
Zhang-Barber, A.K. and Turner, P.A. (1999). Vaccination for control
of Salmonella in poultry. Poult. Sci. 17: 2538-2545.
Zhao, B.; Villena, R.; Sudler, E.; Yeh, S.; Zhao, D.G.; White, D.;
Wagner and Meng, J. (2001). Prevalence of
Camplobacter spp., Escherichia coli, and Salmonella
serovars in retail chicken, turkey, pork, and beef from the
greater Washington. Appl. Envi. Microbiol. 67: 5431-
5436.