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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: Regd. No. Supervisor

Hira Hameed 2003-ag-2044 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 b) Date of Initiation c) Probable Duration Personnel: Name Regd. No. Supervisor Supervisory Committee: 1) 2) 3) Prof. Dr. Iftikhar Hussain Dr. Muhammad Arshad Dr. Irfan Yousaf (Chairman) (Member) (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 nonruminants 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). Hira Hameed 2003-ag-2044 Prof. Dr. Iftikhar Hussain 18-09-2008 October, 2009 4 Months

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 butchers 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 butchers 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 handlers 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 Sheep Type of sample Feces Source of samples Slaughter Faisalabad Meat Meat shops in 50 house in No. of samples 50

Faisalabad Goat Feces Slaughter Faisalabad Meat Meat shops in 50 house in 50

Faisalabad 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.

LITERATURE CITED

Abongo, B.O., and M.N.B.Momba.2008.Prevalence and Characterization of Escherichia coli O157:H7 Isolates from Meat and Meat Products Sold in Amathole District, Eastern Cape Province of South Africa. Food Microbiology, 26:173-176 Borczyk AA, Karmali MA, Lior H, Duncan LMC. Bovine reservoir of verotoxinproducing Escherichia coli O157:H7.1987. Lancet, 8524:98. Bean, NH, Griffin DE. 1990 Food-borne disease outbreaks in the United States, 19731987: pathogens, vehicles and trends. Journal of Food Protection, 53:804817. Beutin,L. Geier D, Steinrck H, Zimmermann S, Scheutz F. 1993 Prevalence and some properties of verotoxin (Shiga-like toxin) producing Escherichia coli in seven different species of healthy domestic animals. Journal of 31:24832488. Blanco, M., J. Blanco, J. E. Blanco, and J. Ramos. 1993. Enterotoxigenic, verotoxigenic and necrotoxigenic Escherichia coli isolated from cattle in Spain. American Journal of Veterinary Research, 54:1446 1451. Blanco, M., J. E. Blanco, J. Blanco, E. A. Gonzalez, A. Mora, C. Prado, L. Fernandez, M. Rio, Ramos, and M. P. Alonso. 1996. Prevalence and characteristics of Escherichia coli serotype O157:H7 and other verotoxinproducing E. coli in healthy cattle. Epidemiological Infections, 117:251257. Bielaszewska, M., J. Panda, K. Blahova, H. Minakova, E. Jkova, M. A. Karmali, J. Laubova, J. Sikulova, M. A. Preston, R. Khakhria, H. Karch, H. Klazarova, and O. Nyc. 1997. Human Escherichia of coli O157:H7 infection goats Associated with milk. the consumption unpasteurized Epidemiological Clinical Microbiology,

Infection,119:299305. Blanco, M., J. E. Blanco, E. A. Gonzalez, A. Mora, W. Jansen, T. A. T. Gomes, F. Zerbini, T. Yano, A. F. Pestana de Castro, and J. Blanco. 1997. Genes coding for enterotoxins belonging to and verotoxins O:K:H in porcine Escherichia relationship coli with strains toxic different serotypes:

phenotypes. Journal of Clinical Microbiology, 35:29582963.

Boerlin, P., S. A. McEwen, F. Boerlin-Petzold, J. B. Wilson, R. P. Johnson, and C. L. Gyles. 1999. Associations between virulence factors of Shiga toxin-producing Escherichia coli and disease in humans. Journal of Clinical Microbiology,37:497 503. Bettelheim KA, Bensink JC, Tambunan SH.2000. Serotypes of verotoxinproducing (Shiga toxinproducing) Escherichia coli isolated from healthy sheep. Comprehensive Immunological Microbiological Infectionious Diseases, 23:17. Blanco, J., M. Blanco, J. E. Blanco, A. Mora, M. P. Alonso, E. A. Gonzalez, and M. I. Bernardez. 2001. Epidemiology of verocytotoxigenic Escherichia coli (VTEC) in ruminants, Journal of Veterinary Research,54: 113148. Blanco. M., J. E. Blanco, A. Mora, J. Rey, J.M.Alonso, M. Hermoso, J. Hermoso, M.P.Alonso, G. Dahbi, E. A. Gonzalez, M.I.Bernardez and J. Blanco.2003. Serotype, Virulence Genes and Intimin Type of Shiga Toxin (verotoxin)-Producing Escherichia coli isolate from Healthy Sheep in Spain. Journal of Clinical Microbiology.p.1351-1356. Bopp, D.J. B.D. Sauders, A. L. Waring, J. Ackelsberg, N. Dumas, E. Braun-Howland, D. Dziewulski, B.J.Wallace, M. Kelly, T.Halse, K.A.Musser, P.F.Smith, D.L.Morse, and R.J.Limberger. 2003.Detection, isolation, and molecular subtyping of Escherichia coli 0157:H7 and campylobacter jejuni associated with a large waterborne outbreak. Journal of Clinical Microbiology, 1:174-80 Centers for Disease Control and Prevention. 1993 Update: multistate outbreak of Escherichia coli O157:H7 infections from hamburgers: Western United States, 19921993. Morbid Mortal Wkly Rep 42:258263. Chapman, P. A., C. A. Siddons, D. J. Wright, P. Norman, J. Fox, and E.Crick. 1993. Cattle as a possible source of verocytotoxin-producing Escherichia coli O157 infections in man. Epidemiological infection, 111:439447. Chapman, P. A., C. A. Siddons, A. T. Cerdan Malo, and M. A. Harkin. 1997. A 1year study of Escherichia coli O157 in cattle, sheep, pigs and poultry. Epidemiological Infection, 119:245250. Chapman, P. A., A. T. Cerdan Malo, M. Ellin, R. Ashton, and M. A. Harkin. 2001.Escherichia coli O157 in cattle and sheep at slaughter, on beef and lamb

carcasses and in raw beef and lamb products in South Yorkshire, UK. International Journal of Food Microbiology, 64:139150.

Dev, V. J., Main M., Gould I.1991.Waterborne outbreak of Escherichia coli O157:H7. Lancet, 337:1412 Elmali. M., Ulukanli, H. Yaman, M. Tuzcu, K. Genctav and P. Cavli. 2005. A seven Month Survey for the Detection of E. coli O157:H7 from Ground Beef Sample in the Markets of Turkey. Pakistan Journel of Nutrition, 4:158-161. Heuvelink, A.E., F.L.A.M. van den Biggelaar, E. de. Boer, R. G. Herbes, W. J. G.Mwlchers, J . H. J. Huis In`t Veld, and L.A.H.Monnens.1997.Isolation and Characterization of Verocytotoxin-Producting Escherichia coli O157 Strains from Dutch Cattle and Sheep. Journal of Clinical Microbiology, 36:878-882. Heuvelink, A. E., F. L. A. M. Van Den Biggelaar, E. De Boer, R. G. Herbes, W. J. G. Melchers, J. H. J. Huis In_T Veld, and L. A. H. Monnens. 1998.Isolation Journal of Clinical Microbiology, and characterization of verocytotoxin-producing Escherichia coli O157 strains from Dutch cattle and sheep. 36:878882. Hussein HS, Lake SL, Glimp H. A. 2000. Sheep as a reservoir for Shiga-like toxin producing Escherichia coli including O157:H7 (a review).journal of Sheep Goats Research, 16:94101. Hiko,A., D.Asrat and G.Zewda. 2008.Occurance of Escherichia O157:H7 in retail raw meat products in Ethiopia. Journel of Infection of Developing Countries, 2: 389-393. Hassanain, N. A. 2008. Detection of Antibodies against Zoonotic Food Borne Pathogens in Sera of Food Handlers. Journel of Global Veterinarians, 2:285-289 Islam, M. A., A. E. Heuvelink, E. de Boer, P. D. Sturm, R. R. Beumer, M. H. Zwietering, A. S. G. Faruque, R. Haque, D. A. Sack and K. A. Taulkder.2006. Shiga toxin-producing Escherichia coli isolated from patients with diarrhea in Bangladesh. Journal of Medical Microbiology,56:380-385. Karmali, M. A. 1989. Infection by verocytotoxin-producing Escherichia coli. Clinical Microbiology Review, 2:538

Kudva IT, Hatfield PG, Hovde CJ.1997 Characterization of Escherichia coli O157:H7 and other Shiga toxin-producing E. coli serotypes isolated from sheep. Journal of Clinical Microbiology, 35:892899. March SB, Ratnam S. 1986. Sorbitol-MacConkey medium for detection of Escherichia coli O157:H7 associated with hemorrhagic colitis. Journal of Clinical Microbiology, 23:869872. Mohsin. M., A. Hussain, M. Asghar Butt, S. Bashir, A. Tariq, S. Babar, A. Ali, A. Haque, Y. Sarwar and A. Haque.2007. Prevalence of Shiga Toxin-Producing Escherichia coli in diarrheal patients in Faisalabad region of Pakistan as determined by multiplex PCR. Journal of infections of developing Countries, 1:164-169 Thrusfield, M., 2007. Veterinary epidemiology, Third edition, Blackwell publisher. Novota. R., P. Alexa, J. Hamrik, A. Madanat, J. Smola and A. Cizek. 2005. Isolation and characterization shiga toxin producing Escherichia coli from sheep and goat in Jordan with evidence of multiresistnt serotype O157:H7 . Journal of veterinary Medicine, 50:111-118. Paton, J. C., and A. W. Paton. 1998. Pathogenesis and diagnosis of Shiga toxin-producing Escherichia coli infections. Clinical Microbiology Review, 11:450479. Riley LW, Remis RS, Helgerson SD, McGee HB, Well JG, Davis BR, Hebert RJ, Olcott ES, Johnson LM, Hargrett NT, Blake PA, Cohen ML. Hemorrhagic colitis associated with a rare Escherichia coli serotype. Journal of Medicine, 308:681685, 1983. Watanabe H, Wada A, Inagaki Y, Itoh K, Tamara K.1996. Outbreaks of enterohaemorrhagic Escherichia coli O157:H7 infection by two different genotype strains in Japan. Lancet, 348:831832. Thrusfield, M., 2007. Veterinary epidemiology, Third edition, Blackwell publisher. Zschock, M., H. P. Hamann, B. Kloppert, and W. Wolter. 2000. Shiga-toxin producing Escherichia coli in faeces of healthy dairy cows, sheep and goats: prevalence and virulence properties. Applied Microbiology, 31:203208.

SIGNATURES
Students Name: Hira Hameed
Supervisor: i) ii) iii) Prof. Dr. Iftikhar Hussain (Chairman) (Member) (Member)

_________________
_________________ __________________ __________________ __________________

SUPERVISORY COMMITTEE:
Prof. Dr. Iftikhar Hussain Dr. Muhammed Arshad Dr. Irfan Yousaf

FORWARDED: Chairman, Department of Microbiology, University of Agriculture, Faisalabad. FACULTY SCRUTINY COMMITTEE
Prof. Dr. M. Tariq Javed Dr. Muhammad Arshad Dr. Ijaz Ahmad Dr. Fqir Muhammad REVIEWED AND WITNESSED:
Dean, Faculty of Veterinary Sciences, University of Agriculture, Faisalabad. Director, Advanced Studies, University of Agriculture, Faisalabad.

(Convener) (Member) (Member) (Member)

______________________ ______________________ ______________________ ______________________

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