An assessment for comparing virulent factors of enterotoxigenic E.coli k99 isolates by PCR and SDS- PAGE methods
Subject Areas : Molecular MicrobiologyYasaman Kiani 1 , Yahya Tahamtan 2 , Mohammad Hosein Hoseini 3 , Masumeh Hayati 4
1 - Department of Microbiology, Jahrom Branch, Islamic Azad University, Jahrom, Iran
2 - Department of Bacteriology, Razi Vaccine and Serum Reserch Institute, Shiraz, Iran
3 - Department of Bacteriology, Razi Vaccine and Serum Reserch Institute, Shiraz, Iran
4 - Department of Bacteriology, Razi Vaccine and Serum Reserch Institute, Shiraz, Iran
Keywords: SDS-PAGE, PCR, E. coli K99, Calf,
Abstract :
Background and Objective: Since E. coli K99 is a causative agent of diarrhea and casualties in calf, it has adverse impacts on economy. This research studied the pathogenic factors of the E. coli by Multiplex PCR, and determined protein pattern of different E. coli isolates by SDS-PAGE (sodium dodsil sulfate polyacryl amid gel electroforesis). Materials and methods: 300 rectal swabs were collected from 1-30 days-old calf during a period between 2010 to 2011. One part of the samples was studied with Multiplex PCR method for determination of the K99, F41 as well as STa genes, and another part was cultured for E. coli isolation and SDS-PAGE assay. Results: The PCR results indicated that 16 of the 200 isolated E. coli had K99, F41, and STa pathogenic factors. LT was not found in the isolates. SDS-PAGE results showed different protein pattern in the isolated E. coli strains. The results of SDS-PAGE column showed only one band for K99 antigen. Conclusion: Based on the found virulent factors in the isolated E. coli K99, it has been shown that the isolates contain at least three pathogenic genes (K99, F41, and STa). Although no significant difference has been seen between the isolates in terms of gene investigations, there was a meaningful difference in the protein patterns of the isolates. As a conclusion, different bacteria showed different protein patterns and consequently had different pathogenic ability.
1. Levine MM. Escherichia coli that cause diarrhea: enterotoxigenic, enteropathogenic, enteroinvasive, enterohemorrhagic and enteroadherant. J Infect Dis. 1987; 155(3): 377-389.
2. Schoonderwoerd M, Clarke RC, van Dreumel AA, Rawluk SA. Colitis in calves: natural and experimental infection with a verotoxin-producing strain of Escherichia coli O111:NM. Can J Vet Res. 1988; 52(4): 484-487.
3. Johnson MW, Fitzgerald GR, Welter MW, Welter CJ. The six most common pathogens responsible for diarrhea in newborn pig. Vet Med. 1992; 87(4): 382-386.
4. Hacker J. Role of fimbrial adhesions in the pathgenesis of Escherichia coli infections. Can J Microbiol. 1992; 38(7): 720-727.
5. Klemm P. Fimbrial adhesions of Escherichia coli. Rev Infect Dis. 1985; 7(3): 321-340.
6. Nagy B, Fekete PZ. Enterotoxigenic Escherichia coli in veterinary medicine. Int J Med Microbiol. 2005; 295(6-7): 443–454.
7. Paton AW, Paton JC. Detection and characterization of shiga toxigenic Esherishia Coli by using multiplex PCR assay for stx(1), stx (2), eaeA, enterohemorragic E.coli hlyA , rfbom. J Clin Microbiol. 1998; 36(2): 598- 602.
8. Elwell LP. Plasmid- mediated factors associated with virulence of bacteria to animals. Ann Rev Microbiol. 1980; 34: 465-496.
9. Shimizu M, Sakano T, Yamamoto J, Kitajima K. Incidence and some characteristics of fimbriae FY and 31A of Esherishia coli isolated from calves with diarrhea in Japan. Microbiol Immunol. 1987; 31(5): 417-426.
10. Acha SJ, Kuhn I, Jonsson P, Mbazima G, Katouli M, Mollby R. Studies on calf diarrhea in Mozambique: prevalence of bacterial pathogens. Acta Vet Scand. 2004; 45(1-2): 27-36.
11- Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev. 1998; 11(1): 142-201.
12. Akam A, Khelef D, Kaidi R, Othmani A, Lafri M, Tali-Maamar H, Rahal K, Tahrat N, Chirila F, Cozma V, Abdul Hussain MS. Frequency of Cryptosporidium parvum, Escherichia coli K99 and Salmonella spp. isolated from healthy and unhealthy calves in six breeding farms from Mitidja, Algeria (Preliminary results). Rev Sci Parasitol. 2004; 5(1-2): 13-21.
13. Li SG, Shen ZQ, Shan H. Cloning and nucleotide sequencing of fusion gene of K99 antigen and heat stable toxin I of Escherichia coli. Zhongguo Shou Yao Zazhi. 2007; 4: 11-14.
14. Sunderland SJ, Sarasola P, Rowan TG, Giles CJ, Smith DG. Efficacy of danofloxacin 18% injectable solution in the treatment of Escherichia coli diarrhoea in young calves in Europe. Res Vet Sci. 2003; 74(2): 171-178.
15. Guler L, Gunduz K, Ok U. Virulence factors and antimicrobial susceptibility of Escherihia coli isolated from calves in Turky. Zoonoses Public Health. 2008; 55(5): 249-257.
16. Hariharan H, Coles M, Poole D, Page R. Antibiotic resistance among enterotoxigenic Escherichia coli from piglets and calves from piglets and calves from piglts and calves with diarrhea. Can Vet J. 2004; 45(7): 605-606.
17. Khai LTL, Voung PQ, Kobayashi H, Phan TT, Loc CB, Yamasaki S, Taniguchi T. Isolation,identification and treatment of piglet'diarrhea caused by enterotoxigenic Escherichia coli K88,K99,987P in Cantho Province,Vietnam. Res Microbiol. 2008; 24: 210-21.
18. Guinee PAM, Jansen WH, Agterberg CM. Detection of the K99 antigen by means of agglutination and immunoelectrophoresis in Escherichia coli isolates from calves and its correlation with enterotoxigenicity. Infect Immun. 1976; 13(5): 1369-1377.
19. Guinee PAM, Veldkamp MJ, Jansen WH. Improved minca medium for the detection of K99 antigen in calf enterotoxigenic strains of Escherichia coli. Infect Immun. 1977; 15(2): 676-678.
20. Evans DG, Evans DJ, Tjoa WS, DuPont HL. Detection and characterization of colonization factor enterotoxigenic Escheriachia coli isolated from adults with diarrhea. Infect Immun. 1978; 19(2): 727-736.
21. Franck SM, Bosworth BT, Moon HW. Multiplex PCR for enterotoxigenic, attaching and effacing, and Shiga toxin-producing Escherichia coli strains from calves. J Clin Microbiol. 1998; 36(6): 1795-1797.
22. Bourgouin H. The place of cryptosporidium among the diseases of new born claves in correze. Bulletin-des- G.T.V. 1996; 2: 19-41
23. Isaacson RE. K99 surface antigen of Esherichia coli: purification and partial characterization . Infect Immun. 1977; 15(1): 272-279.
