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UNIVERSITY OF AGRICULTURE, FAISALABAD
Department of Microbiology
(Synopsis for M. Phil Degree in Microbiology)
TITLE: Prevalence of shiga toxin producing Escherichia coli in sheep and
goats in Faisalabad
Name: Hira Hameed
Regd. No. 2003-ag-2044
Supervisor Prof. Dr. Iftikhar Hussain
ABSTRACT
Shiga toxin producing Escherichia-coli (STEC) is an important cause of human diseases
including diarrhea, hemorrhagic colitis (HC), the hemolytic uremic syndrome (HUS), and
thrombotic thrombocytopenia purpura (TTP). The natural reservoirs of this pathogen are sheep,
goat, cattle, buffalo and wild animals. Consumption of contaminated and improperly cooked
meat is the cause of food borne outbreaks of E. coli O157:H7. The aim of present study is to see
the prevalence of Shiga toxin producing E. coli 0157:H7 in sheep and goats in Faisalabad. For
this purpose, 200 meat and fecal samples of sheep and goat will be obtained from slaughter
house and meat market of District Faisalabad. The isolated E. coli positive samples if any will be
identified through conventional methods. Specific culture media and latex agglutination test will
be used to further identify STEC isolates.
UNIVERSITY OF AGRICULTURE, FAISALABAD
Department of Microbiology
(Synopsis for M. Phil Degree in Microbiology)
TITLE: Prevalence of Shiga toxin producing Escherichia coli in sheep and
goats in Faisalabad
a) Date of Admission 18-09-2008
b) Date of Initiation October, 2009
c) Probable Duration 4 Months
Personnel:
Name Hira Hameed
Regd. No. 2003-ag-2044
Supervisor Prof. Dr. Iftikhar Hussain
Supervisory Committee:
1) Prof. Dr. Iftikhar Hussain (Chairman)
2) Dr. Muhammad Arshad (Member)
3) Dr. Irfan Yousaf (Member)
NEED FOR THE PROJECT
Shiga toxin producing Escherichia coli (STEC) is among pathogenic strains of E. coli.
(Kudva et al., 1997). Domestic ruminants especially cattle, sheep and goat have been implicated
as principle reservoir of STEC strains that cause human infections, although other domestic non-
ruminants animals including pigs, poultry, cat and dogs can also harbor these bacteria (Blanco et
al., 2003). E. coli O157:H7 was first recognized as pathogen in 1982 after two human illness
outbreaks in Oregon and Michigan (Riley et al., 1983). STEC O157:H7 has been the cause of
series of outbreaks, especially in Canada, Japan (Watanabe et al., 1996) and the United States
(CDC, 1993; Bean and Griffin 1990) and the United Kingdom (Karmali, 1989; Paton, and Paton
1998).
STEC is a major cause of gastroenteritis that may be complicated by hemorrhagic colitis,
thrombocytopenia and the hemolytic uremic syndrome, which is the main cause of acute renal
failure in children (Blanco et al., 2003). Most outbreaks and sporadic cases of HC and HUS have
been attributed to strains of entrohemorrhagic serotype O157:H7 (Boerlin et al., 1999; Karmali,
1989). Transmission of STEC occurs through consumption of undercooked meat (Chapman et
al., 2001; Chapman et al., 1993), unpasteurized dairy products (Zschock et al., 2000) and
vegetables or water contamination by the feces of carrier animals (Dev et al., 1991). Person to
person transmission has been documented (Blanco et al., 1997). Ruminant animals, especially
cattle, are thought to be the most important reservoir of STEC (Borczk et al., 1987) which are
carried in the gastrointestinal tract and shed in the feces but recently STEC strains have been
detected in sheep and goat feces or at slaughter, showing that small ruminants may also represent
a source of contamination for humans (Blanco et al., 2003). Transmission of STEC O157:H7 and
other STEC serotypes to humans by raw goat milk or homemade cheese made from raw milk of
goat has been demonstrated (Bielaszewska et al., 1997; Blanco et al., 2001). Recent evidence
indicated that sheep harbor O157:H7 (Chapman et al., 1997; Kudva et al., 1997) at rate similar to
or higher than those reported for cattle and suggested their potential role as a food safety risk
factor (Hussein et al., 2000). A prevalence rate of 67% was reported for sheep in Germany
(Beutin et al., 1993) and Australia (Bettethein et al., 2000). Therefore, lamb, mutton and their
product share a food safety risk similar to that of beef. The importance of food safety has
increased dramatically in recent years. It is important to understand the types and trends of STEC
shedding by meat animals, including sheep. Small ruminants have been subjected to fewer
survey compared with the number of surveys done with cattle.
OBJECTIVE OF PRESENT STUDY :
To isolate E. coli from meat and feces of sheep and goats in Faisalabad.
To measure the prevalence of STEC in meat and feces of sheep and goat in Faisalabad
To confirm the STEC isolates through selective culturing and latex agglutination test.
REVIEW OF LITERATURE
Kudva et al. (1995) found naturally occurring, potentially virulent E.
coli O157:H7 strains in sheep. The incidence of E. coli O157:H7 was transient
and ranged from 31% of sheep in June to none in November. The use of
sensitive culture technique and the choice of the proper sampling season were both essential for
detecting this bacterium in sheep.
Heuvelink et al. (1997) collected fecal samples from Dutch cattle and sheep examined the
presence of verocytotoxin producing E. coli of serogroup O157 during the period from July to
November 1995 and 1996 at the main slaughterhouse of the Netherlands. E. coli O157 strains
could be isolated from 57 (10.6%) of 540 adult cattle, 2 (0.5%) of 397 veal calves, 2 (3.8%) of
52 ewes, and 2 (4.1%) of 49 lambs. All animal isolates were positive for both VT (VT1 and/or
VT2) and E. coli attaching-and-effacing gene sequences, and therefore, they were regarded as
potential human pathogens. He compared 63 animal isolates with 33 fecal O157 VTEC strains
previously isolate from humans with the diarrhea associated form of the hemolytic- uremic
syndrome. He noted that they have a marked similarity between animal and human isolates,
30(90.9%) of the 33 human isolates appeared to be of E. coli O157 strain types also isolated
from cattle and sheep. It was concluded that Dutch cattle and sheep are an important reservoir of
E. coli O157 strains that are potentially pathogenic for humans.
Chapman et al. (2001) conducted a one-year study of E. coli O157 in cattle and sheep at
slaughter, on beef and lamb carcasses and in raw beef and lamb product from retail butcher’s
shop in the Sheffield area. Each month, fecal samples were collected immediately after slaughter
from 400 cattle and 600 sheep and 400-430 samples of raw meat products from butcher’s shop.
Meat samples were also obtained from 1500 beef and 1500 lamb carcasses. All samples were
analyzed for E. coli O157 through conventional method and raw meat products were also
examined for number of generic E. coli by a standard membrane culture method. E. coli O157
was isolated from 620(12.9%) of 4800 cattle, 100(7.4%) of 600 sheep, 21(1.4%) of 1500 beef
carcasses, 10(0.7%) of 1500 lamb carcasses and from 22(0.44%) of 4983 raw meat products. E.
coli O157 was isolated more frequently from lamb products (0.4%). Numbers of generic E. coli
in meat products reached seasonal peaks in July and August and more frequently in lamb
products (50.8 and 42.4% respectively) than in beef products (19.3 and 23.8% respectively). The
majority of E. coli O157 strains from animal carcasses and meat samples were isolated during
summer. Most were verotoxigenic as determined by Vero cell assay and DNA hybridization,
eaeA gene positive and contained a 92 kb plasmid. The isolates were compared with 66 isolates
from human cases over the same period. A combination of phage type, toxin genotype and
plasmid analysis allowed subdivision of all the E. coli O157 isolates into 96 subtypes. Of these
subtypes, 53(55%) were isolated only from bovine fecal samples. However, 61 (92%) of the 66
isolates from human belonged to 13 subtypes, which were also found in the animal population.
Blanco et al. (2003) examined fecal swab from 1,300 healthy lambs in 93 flocks in Spain
in 1997. STEC O157:H7 strains were isolated from 5 (0.4%) animals in 4 flocks, and non-O157
STEC strains were isolated from 462 (36%) lambs in 63 flocks. A total of 384 ovine STEC
strains through PCR showed that 213 (55%) strains carried the stx1 gene, 10 (3%) possessed the
stx2 gene, and 161 (42%) carried both the stx1 and the stx2 genes. Enterohemolysin (ehxA)
and intimin (eae) virulence genes were detected in 106 (28%) and 23 (6%) of the STEC strains,
respectively. The STEC strains belonged to 35 O serogroups and 64 O: H serotypes. The
majority (82%) of ovine STEC strains belonged to serotypes previously found
to be associated with human STEC strains, and 51% belonged to serotypes
associated with STEC strains isolated from patients with hemolytic-uremic
syndrome. Thus, this study confirms that healthy sheep are a major reservoir
of STEC strains pathogenic for humans.
Bopp et al. (2003) reported largest outbreaks of waterborne
Escherichia coli O157:H7 in the United States during August 1999. Culture
methods were used to isolate E. coli O157:H7 from specimen 128 of 775
patients with suspected infections. The Plus field gel electrophoresis results
implicated the water distribution system as the main source of the E. coli
O157:H7 outbreak.
Novtan et al. (2005) examined ninety-three rectal swabs of lambs and
young goats from two extensively and two intensively managed herds in
Jordan for STEC. The STEC O157:H7 strains were demonstrated in 8 of 32
diarrheic lambs 1-3 weeks old in one sheep herd with intensive milk
production. In the remaining three herds, serogroups O128, O78, O15 and
serotypes O128:K85 of STEC strains were the most frequent findings. The
presence of stx2, ehlyA and eaeA genes in all STEC O157:H7 isolates was
confirmed by PCR. In untypable STEC isolates, stx2 and ehlyA genes were
detected. In other STEC non-O157 isolates, only stx1 an ehlyA genes were
found. All STEC O157:H7 isolates were resistant against sulphonamides and
chloramphenicol; five were also resistant against ampicillin and
streptomycin, one against co-trimoxazole. STEC isolates resistant against
antimicrobial agents were demonstrated only in herds with intensive
management.
Mohsin et al. (2007) investigated the prevalence of STEC in stool samples of 200
children during the summer season of 2005, in Faisalabad, Pakistan. Multiplex PCR results
showed that 22 (11%) of the patient were positive for stx genes. Among other virulence factors,
plasmid borne hly and eae genes were detected in 13 (6.5%) and 16 (0.8%) patients respectively.
However, only 14 STEC isolates were obtained. Among these 11 (78.5%), 11 (78.5%), 3
(21.4%) and 6 (42.8%) were positive for stx1, stx2, hly and eae genes respectively. Only 3 of
these isolates belonged to serotype O157. It was concluded that serotype O157 was not the major
pathogen.
Hassanain (2008) examined 185-meat samples collected from 13 restaurants revealed that
16.75% were infected. The commonest bacterial isolates were non-tyhoidal Salmonella (41.93%)
and E. coli (32.25%) and 60% of the E. coli meat isolates were hemolytic. Blood samples of
symptomatic 63 food handlers were collected with the aim of detecting antibodies in their sera
against the prepared whole cell protein antigen of the commonest zoonotic bacterial isolates.
ELISA results showed that 17.46 and 22.22% of the examined 63 food handler’s sera were
positive for the hemolytic E. coli and non- typhoidal Salmonella whole cell protein antigens,
respectively.
Hiko et al. (2008) conducted a study over a period of 7 months (October 2006 to April
2007), during which a total of 738 raw meat samples were collected from bovines (n= 250),
sheep (n=243) and goat (n=245) and for the presence of E. coli O157: H7 was investigated.
Antimicrobial susceptibility testing was performed using the disk diffusion method. E. coli
O157:H7 were isolated from 31 (4.2%) out of 738 meat samples examined. Among meat
samples examined, the highest prevalence (8%) was recorded in beef, followed by lamb mutton
(2.5%) and goat meat (2%). The isolated strains were found to be susceptible (100%) to
amikacin, chloramphenicol, gentamicin, kanamycin, nalidixic acid, norfloxacin, polymyxin B
and trimethoprim-sulfamethoxazole. Multidrug resistance to three or more drugs was detected in
7/31 (22.6%) strains. The results of this study revealed the presence of E. coli O157:H7 in retail
raw meats reaching consumers, indicating possible risks of infection to people through the
consumption of raw/under-cooked meat or cross-contamination of other food products. Multiple
drug resistant isolates detected in the present study may pose a threat to humans and further limit
therapeutic options.
Abongo and Maggy. (2008) investigated the prevalence of E. coli O157: H7 isolates from
meat and meat products (45 samples each of bilong, cold meat, mince meat and polony) sold in
the Amathole district Municipality of the Eastern Cape Province of South Africa. Five (2.8%)
out of 180 meat and meat products examined were positive for E. coli O157:H7 that carried the
flicH7, rfbEO157 and eaeA genes. Two of the E. coli O157:H7 isolates were resistant against all
the 8 antibiotics tested.
MATERIAL AND METHODS
SAMPLE COLLECTION
Total 200 meat and fecal samples of sheep and goats from Faisalabad will be randomly
collected as mentioned below in Table 1. Sex and age of the animal will be recorded.
Table 1: Protocol of meat and fecal samples collection
Species Type of sample Source of samples No. of samples
Sheep Feces Slaughter house in
Faisalabad
50
Meat Meat shops in
Faisalabad
50
Goat Feces Slaughter house in
Faisalabad
50
Meat Meat shops in
Faisalabad
50
TOTAL 200
PROCEDURE
A. Isolation and identification of E. coli through conventional methods.
1. Pre-enrichment of the samples in selective broth
(Islam et al., 2006)
Samples will be pre-enriched in STEC modified tryptone soy broth (oxoid).
2. STEC isolation through selective medium
(Heuvelink et al., 1998)
Sorbitol MacConkey agar (Oxoid) supplemented with cefixime (0.05mg/litter) and potassium
tellurite (2.5mg/litter) (CT-SMAC) will be used for selective isolation of E. coli O157:H7.
B. Biochemical identification of E. coli
(Elmali et al., 2005)
The isolates will be biochemically characterized by sugar fermentation and IMViC
(Indole, Methyl red, Voges Proskauser and citrate
C. Serological identification of E. coli O157:H7.
(Islam et al., 2006)
E. coli O157 latex test kit (Oxoid) will be used for identification and confirmation of
E. coli O157 through latex agglutination test.
D. Statistical Analysis
(Thrusfield, 2007)
Prevalence of STEC in sheep and goat will be measured and prevalence data will be
analyzed through Chi- square test.
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SIGNATURES
Student’s Name: Hira Hameed _________________
Supervisor: Prof. Dr. Iftikhar Hussain _________________
SUPERVISORY COMMITTEE:
i) Prof. Dr. Iftikhar Hussain (Chairman) __________________
ii) Dr. Muhammed Arshad (Member) __________________
iii) Dr. Irfan Yousaf (Member) __________________
FORWARDED:
Chairman, Department of Microbiology,University of Agriculture, Faisalabad.
FACULTY SCRUTINY COMMITTEE
Prof. Dr. M. Tariq Javed (Convener) ______________________
Dr. Muhammad Arshad (Member) ______________________
Dr. Ijaz Ahmad (Member) ______________________
Dr. Fqir Muhammad (Member) ______________________
REVIEWED AND WITNESSED:
Dean, Director,
Faculty of Veterinary Sciences, Advanced Studies,
University of Agriculture, University of Agriculture,
Faisalabad. Faisalabad.