Open Access
Research (Published online: 25-08-2018)
22. An epidemiological investigation on occurrence of enterohemorrhagic Escherichia coli in raw milk
H. D. Vanitha, C. Sethulekshmi and C. Latha
Veterinary World, 11(8): 1164-1170

H. D. Vanitha: Department of Veterinary Public Health, College of Veterinary and Animal Sciences, Mannuthy, Thrissur - 680 651, Kerala, India.
C. Sethulekshmi: Department of Veterinary Public Health, College of Veterinary and Animal Sciences, Mannuthy, Thrissur - 680 651, Kerala, India.
C. Latha: Department of Veterinary Public Health, College of Veterinary and Animal Sciences, Mannuthy, Thrissur - 680 651, Kerala, India.

doi: 10.14202/vetworld.2018.1164-1170

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Article history: Received: 21-02-2018, Accepted: 12-07-2018, Published online: 25-08-2018

Corresponding author: H. D. Vanitha


Citation: Vanitha HD, Sethulekshmi C, Latha C (2018) An epidemiological investigation on occurrence of enterohemorrhagic Escherichia coli in raw milk, Veterinary World, 11(8): 1164-1170.

Aim: The aim of the present investigation was to study the epidemiology of enterohemorrhagic Escherichia coli (EHEC) in raw milk and molecular characterization of isolates using multiplex polymerase chain reaction (PCR).

Materials and Methods: A total of 125 raw milk samples were subjected to isolation, identification, and confirmation of virulence-associated genes by multiplex PCR (mPCR). The samples were collected from a milk cooperative society of Thrissur district, Kerala. For further epidemiological investigation, samples such as dung (126), hair coat of cow (60), udder swab (60), udder wash (60), milking utensil wash (36), Milker's hand wash (36), water (36), soil (36), and feed (36) were collected from the households from which the raw milk tested positive for EHEC.

Results: The occurrence of EHEC in individual raw milk samples was found to be 8.8%. The major source of contamination to raw milk was found to be dung (19.84%) followed by udder swab (16.67%), hair coat of cow (15%), Milker's hand and milking utensils and water (11.11% each), and udder wash and soil (8.33% each). For identification of virulence genes, all the isolates were subjected to mPCR, of 75 isolates 73.33% of isolates harbored stx 2 gene while 53.33, 36, and 36% of isolates were encoded by stx 1, eae A, and hly A genes, respectively. On epidemiological survey, the multiple risk factors accountable for occurrence of EHEC in raw milk were found to be the quality of water used, improper and inadequate udder preparation, unhygienic hands of Milker's, use of insufficiently cleaned milking utensils, and using common utensil for washings of udder and milking purposes.

Conclusion: The result of the present study signifies that raw milk was contaminated with EHEC and possesses a high public health threat. As dairy cattle and its environment serve as a potential niche for EHEC, hygienic milking practices should be adopted to curb the occurrence of EHEC in raw milk.

Keywords: Enterohemorrhagic Escherichia coli, epidemiological investigation, epidemiological survey, multiplex polymerase chain reaction, raw milk.


1. Kirk, M.D., Angulo, F.J., Havelaar, A.H. and Black, R.E. (2017) Diarrhoeal disease in children due to contaminated food. Bull. World Health Organ., 95(3): 233. [Crossref]

2. Hiraka, T., Kanoto, M., Sugai, Y., Honma, T., Makino, N., Ueno, Y. and Hosoya, T. (2015) Computed tomographic findings of enterohemorrhagic Escherichia coli O157 infection: An analysis of a 7-case regional outbreak. J. Comput. Assist. Tomo., 39(3): 406-408.

3. Garbaj, A.M., Awad, E.M., Azwai, S.M., Abolghait, S.K., Naas, H.T., Moawad, A.A. and Eldaghayes, I.M. (2016) Enterohemorrhagic Escherichia coli O157 in milk and dairy products from Libya: Isolation and molecular identification by partial sequencing of 16S rDNA. Vet. World, 9(11): 1184. [Crossref]

4. Msolo, L., Igbinosa, E.O. and Okoh, A.I. (2016) Prevalence and antibiogram profiles of Escherichia coli O157: H7 isolates recovered from three selected dairy farms in the Eastern Cape Province, South Africa. Asian Pac. J. Trop. Dis., 6(12): 990-995. [Crossref]

5. Polifroni, R., Etcheverria, A.I., Sanz, M.E., Cepeda, R.E., Kruger, A., Lucchesi, P.M. and Padola, N.L. (2012) Molecular characterization of Shiga toxin-producing Escherichia coli isolated from the environment of a dairy farm. Curr. Microbiol., 65(3): 337-343. [Crossref] [PubMed]

6. Kumar, A., Grover, S. and Batish, V.K. (2013). Application of multiplex PCR assay based on uidR and fliCH7 genes for detection of Escherichia coli O157: H7 in milk. J. Gen. Appl. Microbiol., 59(1): 11-19. [Crossref] [PubMed]

7. Hessain, A.M., Al-Arfaj, A.A., Zakri, A.M., El-Jakee, J.K., Al-Zogibi, O.G., Hemeg, H.A. and Ibrahim, I.M. (2015) Molecular characterization of Escherichia coli O157: H7 recovered from meat and meat products relevant to human health in Riyadh, Saudi Arabia. Saudi J. Biol. Sci., 22(6): 725-729. [Crossref] [PubMed] [PMC]

8. Davis, T.K., McKee, R., Schnadower, D. and Tarr, P.I. (2013) Treatment of Shiga toxin-producing Escherichia coli infections. Infect. Dis. Clin., 27(3): 577-597. [Crossref] [PubMed]

9. Meng, J., Feng, P. and Doyle, P. (2001) Pathogenic Escherichia coli. In: Doyle, I.P. and Ito, K., editors. Compendium of Methods for the Microbiological Examination of Foods. American Public Health Association, Washington. p331-341.

10. Fujisawa, T., Sata, S., Aikawa, K., Takahashi, T., Yamai, S. and Shimada, T. (2000) Modification of sorbitol MacConkey medium containing cefixime and tellurite for isolation of Escherichia coli O157: H7 from radish sprouts. Appl. Environ. Microbiol., 66(7): 3117-3118. [Crossref]

11. Meng, J., Zhao, S. and Doyle, M.P. (1998) Virulence genes of Shiga toxin producing Escherichia coli isolated from food, animals and humans. Int. J. Food Microbiol., 45: 229-235. [Crossref]

12. Paton, A.W. and Paton, J.C. (1998) Detection and characterization of Shiga toxigenic Escherichia coli by using multiplex PCR assays for stx 1, stx 2, eaeA, enterohemorrhagic E. coli hlyA, rfb O111, and rfb O157. J. Clin. Microbiol., 36(2): 598-602. [PubMed] [PMC]

13. Neher, S., Hazarika, A.K., Sharma, R.K., Barkalita, L.M., Bora, M. and Deka, P. (2015) Detection of Shiga-toxigenic Escherichia coli in milk samples of cattle by PCR. JAVS, 8: 75-78.

14. Sethulekshmi, C. and Latha, C. (2016) Occurrence and molecular characterisation of Enterohaemorrhagic E. coli in raw milk. J. Indian Vet. Assoc., 14: 30-34.

15. Solomakos, N., Govaris, A., Angelidis, A.S., Pournaras, S., Burriel, A.R., Kritas, S.K. and Papageorgiou, D.K. (2009) Occurrence, virulence genes and antibiotic resistance of Escherichia coli O157 isolated from raw bovine, caprine and ovine milk in Greece. Food Microbiol., 26(8): 865-871. [Crossref] [PubMed]

16. Ntuli, V., Njage, P.M.K. and Buys, E.M. (2016) Characterization of Escherichia coli and other Enterobacteriaceae in producer-distributor bulk milk. J. Dairy Sci., 99(12): 9534-9549. [Crossref] [PubMed]

17. Cobbold, R.N., Rice, D.H., Szymanski, M., Call, D.R. and Hancock, D.D. (2004) Comparison of Shiga-toxigenic Escherichia coli prevalences among dairy, feedlot, and cow-calf herds in Washington State. Appl. Environ. Microbiol., 70(7): 4375-4378. [Crossref] [PubMed] [PMC]

18. Wang, G., Zhao, T. and Doyle, M.P. (1996) Fate of enterohemorrhagic Escherichia coli O157: H7 in bovine feces. Appl. Environ. Microbiol., 62(7): 2567-2570. [PubMed] [PMC]

19. Abdissa, R., Haile, W., Fite, A.T., Beyi, A.F., Agga, G.E., Edao, B.M. and De Zutter, L. (2017) Prevalence of Escherichia coli O157: H7 in beef cattle at slaughter and beef carcasses at retail shops in Ethiopia. BMC Infect. Dis., 17(1): 277. [Crossref]

20. Anu, P.J. (2015) Identification of Critical Control Points in Beef Processing Line with Special Reference to Enterohaemorrhagic Escherichia coli. M.V.Sc Thesis, Kerala Veterinary and Animal Sciences University, Pookode. p86.

21. Fremaux, B., Raynaud, S., Beutin, L. and Rozand, C.V. (2006) Dissemination and persistence of Shiga toxin-producing Escherichia coli (STEC) strains on French dairy farms. Vet. Microbiol., 117(2-4): 180-191. [Crossref] [PubMed]

22. Nanu, E., Latha, C., Sunil, B., Prejit, M.T. and Menon, K.V. (2007) Quality assurance and public health safety of raw milk at the production point. Am. J. Food Technol., 2: 145-152. [Crossref]

23. Msolo, L. (2016) Studies on the Prevalence of Escherichia coli 0157: H7 in Raw Milk, Milking Machines, Cattle Udder and Hand Swabs Collected from Selected Dairy Farms in the Eastern Cape Province, South Africa (Doctoral Dissertation, University of Fort Hare).

24. Wetzel, A.N. and LeJeune, J.T. (2007) Isolation of Escherichia coli O157: H7 strains that do not produce Shiga toxin from bovine, avian and environmental sources. Lett. Appl. Microbiol., 45(5): 504-507. [Crossref] [PubMed]

25. Ateba, C.N. and Mbewe, M. (2011) Detection of Escherichia coli O157: H7 virulence genes in isolates from beef, pork, water, human and animal species in the northwest province, South Africa: Public health implications. Res. Microbiol., 162(3): 240-248. [Crossref] [PubMed]

26. Halabi, M., Orth, D., Grif, K., Wiesholzer-Pittl, M., Kainz, M., Schoberl, J. and Wurzner, R. (2008) Prevalence of Shiga toxin-, intimin-and haemolysin genes in Escherichia coli isolates from drinking water supplies in a rural area of Austria. Int. J. Hygn. Environ. Hlth., 211(3-4): 454-457. [Crossref]

27. Parul, S., Bist, B., Sharma, B. and Jain, U. (2014) Virulence-associated factors and antibiotic sensitivity pattern of Escherichia coli isolated from cattle and soil. Vet. World, 7(5): 369-372. [Crossref]

28. Das, S.C., Khan, A., Panja, P., Datta, S., Sikdar, A., Yamasaki, S. and Nair, G.B. (2005) Dairy farm investigation on Shiga toxin-producing Escherichia coli (STEC) in Kolkata, India with emphasis on molecular characterization. Epidemiol. Infect., 133(4): 617-626. [Crossref]

29. Hancock, D.D., Rice, D.H., Herriott, D.E., Besser, T.E., Ebel, E.D. and Carpenter, L.V. (1997) Effects of farm manure-handling practices on Escherichia coli O157 prevalence in cattle. J. Food Prot., 60(4): 363-366. [Crossref]

30. Neeta, P.N., Prashanth, N., Shivaswamy, M.S. and Mallapur, M.D. (2014) A study on awareness regarding milk-borne diseases in an urban community of Karnataka. Int. J. Med. Sci. Public Hlth., 3(9): 1093-1100. [Crossref]

31. Tegegne, B. and Tesfaye, S. (2017) Bacteriological milk quality: Possible hygienic factors and the role of Staphylococcus aureus in raw bovine milk in and around Gondar, Ethiopia. Int. J. Food Contam., 4(1): 1. [Crossref]