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.

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 verotoxin-

producing Escherichia coli O157:H7.1987. Lancet, 8524:98.

Bean, NH, Griffin DE. 1990 Food-borne disease outbreaks in the United States, 1973–1987:

pathogens, vehicles and trends. Journal of Food Protection, 53:804–817.

Beutin,L. Geier D, Steinrück 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 Clinical Microbiology,

31:2483–2488.

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. Gonza´lez, A. Mora, C. Prado, L. Ferna´ndez, 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, 117:251–257.

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 coli O157:H7 infection Associated with the

consumption of unpasteurized goat’s milk. Epidemiological

Infection,119:299–305.

Blanco, M., J. E. Blanco, E. A. Gonza´lez, 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 and verotoxins in porcine Escherichia coli strains

belonging to different O:K:H serotypes: relationship with toxic

phenotypes. Journal of Clinical Microbiology, 35:2958–2963.

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 toxin-

producing) Escherichia coli isolated from healthy sheep. Comprehensive

Immunological Microbiological Infectionious Diseases, 23:1–7.

Blanco, J., M. Blanco, J. E. Blanco, A. Mora, M. P. Alonso, E. A. Gonza´lez, and

M. I. Berna´rdez. 2001. Epidemiology of verocytotoxigenic Escherichia coli

(VTEC) in ruminants, Journal of Veterinary Research,54: 113–148.

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, 1992–1993. Morbid

Mortal Wkly Rep 42:258–263.

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, 111:439–447.

Chapman, P. A., C. A. Siddons, A. T. Cerdan Malo, and M. A. Harkin. 1997. A 1-

year study of Escherichia coli O157 in cattle, sheep, pigs and poultry. Epidemiological

Infection, 119:245–250.

Chapman, P. A., A. T. Cerda´n 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, 64:139–150.

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

and characterization of verocytotoxin-producing Escherichia coli O157

strains from Dutch cattle and sheep. Journal of Clinical Microbiology,

36:878–882.

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,

16:94–101.

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, 2:5–38

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, 35:892–899.

March SB, Ratnam S. 1986. Sorbitol-MacConkey medium for detection of Escherichia coli

O157:H7 associated with hemorrhagic colitis. Journal of Clinical Microbiology, 23:869-

872.

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, 11:450–479.

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, 308:681–685, 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, 348:831–832.

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, 31:203–208.

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.