Gene expression study of pathogenic hemolysin producing E. coli isolated from cattle by using reverse transcription real-time PCR

  • Hassan Hachim Nasser College of Veterinary Medicine, University of Al-Qadisiyah, Iraq.
  • Ghassan Khudhair Ismaeel Department of Internal Veterinary Medicine, College of Veterinary Medicine, University of Al-Qadisiyah, Iraq.
  • Noor Mahmood Majeed College of Dentistry, University of Al-Qadisiyah, Iraq


The main goal of current study is an investigation of detection and expression of hemolysin toxin in E.coli isolates in clinical veterinary infection cases included Mastitis milk, Abortion samples and diarrhea samples from cattle in Al-Diwaniyah city by using real time-polymerase chain reaction technique. Total samples that used in this study are (24) isolates, included (9) isolates from mastitis cases, (5) isolates from abortion cases, and (10) isolates have taken from Diarrhea cases in cattle. The total prevalence of hemolysin in E.coli isolates is 18/24 (75%), where the percentage of occurrence of hemolysin production E.coli isolates by using real-time PCR was 7/9 (77%), 5/5 (100%), and 6/10 (60%) in Mastitis milk, Abortion and Diarrhea cases in cattle respectively. In concluding, production of hemolysin toxin by E.coli isolates have contrast activity depend on the type of sample; the study found the abortion isolates have high gene expression followed by diarrhea isolates, then milk isolates.


1-Ernest Hovingh. Extension Veterinarian, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech. Abortions in Dairy Cattle-I Common Causes of Abortions publication.(2009);404-288.
2-EJ Bicknell, C Reggiardo, TH Noon, GA Bradley, F Lozano-Alarcon. abortion diseases of range cattle. Animal Care and Health Maintenance (2005).
3-Cristina Bogni, Liliana Odierno, Claudia Raspanti, José Giraudo, Alejandro Larriestra, Elina Reinoso, Mirta Lasagno, Mirian Ferrari, Edith Ducrós, Cecilia Frigerio, Susana Bettera, Matías Pellegrino, Ignacio Frola, Silvana Dieser and Claudina Vissio.(2011).War against mastitis: Current concepts on controlling bovine mastitis pathogens. Science against microbial pathogens: communicating current research and technological advances.
4-A Villarroel. Scours in Beef Calves Causes and Treatments. EM 8977-E Aurora Villarroel, Extension veterinarian, Oregon State University. (2009).
5-Wang MC, Tseng CC, WU AB, Huang JJ, Shew BS, WU JJ. Different roles of host and bacterial factors in Escherichia coli extra-intestinal infections. Clin Microbiol Infec. 15: (2009); 372-379.
6-Bentancor A, Rumi MV, Carbonari C, Gerhardt E, Larza M, Vilte DA, Creydt VP, Chinen I. Profile of Shiga toxin-producing Escherichia coli strains isolated from dogs, cats, and genetic relationships with isolates from cattle, meat and humans. Vet Microbiol. (2012); 156, 336–342.
7-Rey J, Sa′nchez S, Blanco JE, Hermoso de Mendoza J, Hermoso de Mendoza M, Garcı′a A, Gil C, Tejero Prevalence, serotypes and virulence genes of Shiga toxin-producing Escherichia coli isolated from ovine and caprine milk and other dairy products in Spain. Int J Food Microbiol (2006); 107, 212-217.
8-Felmlee TS, Pellett RA Welch. The nucleotide sequence of an E. coli chromosomal hemolysin. J. Bacteriol. 163: (1985); 94-105.
9-Hess J, W Wels, M Vogel, W Goebel. Nucleotide sequence of a plasmid encoded hemolysin determinant and its comparison with a corresponding chromosomal hemolysin sequence. FEMS Microbiol. Lett. 34: (1986); 1-11.
10-Bhakdi S, S Greulich, M Muhly, B Eberspacher, H Becker, A Thiele, F Hugo. The potent leukocidal action of Escherichia coli hemolysin mediated by permeabilization of target cell membranes. J. Exp. Med. 169: (1989); 737–754.
11-Gadeberg OV, I Orskov. In vitro cytotoxic effect of a-hemolytic Escherichia coli on human blood granulocytes. Infect. Immun. 45: (1984); 255-260.
12-Bhakdi S, M Muhly, S Korom, G Schmidt. Effects of Escherichia coli hemolysin on human monocytes: cytocidal action and stimulation of interleukin 1 release. J. Clin. Invest. 85: (1990); 1746–1753.
13-Suttorp N, B Floer, H Schnittler, W Seeger, S Bhakdi. Effects of Escherichia coli hemolysin on endothelial cell function. Infect. Immun. 58: (1990); 3796–3801.
14-Keane WF, R Welch, G Gekker, PK Peterson. Mechanism of Escherichia coli a-hemolysin induced injury to isolated renal tubular cells. Am. J. Pathol. 126: (1987); 350-357.
15-Mobley HLT, DM Green, AL Trifillis, DE Johnson, GR Chippendale, CV Lockatell, BD Jones, JW Warren. Pyelonephritogenic Escherichia coli and killing of cultured human renal proximal tubular epithelial cells: role of hemolysin in some strains. Infect. Immun. 58: (1990); 1281-1289.
16-Rey J, Sa′nchez S, Blanco JE, Hermoso de Mendoza J, Hermoso de Mendoza M, Garcı′a A, Gil C, Tejero N. Prevalence, serotypes and virulence genes of Shiga toxin-producing Escherichia coli isolated from ovine and caprine milk and other dairy products in Spain. Int J Food Microbiol 107, (2006); 212-217.
17-Müller D, Hughes C, Goebel W.J Baeteriol (1983); 153:846.
18-Beutin L, Alexksic S, Zimmerman 5, Gleier K. Virulence factors and phenotypic traits of Vero toxigenic strains of Escherichia coli isolated from human patients in Germany. Med Microbiol lmmunol 183, (1994); 13-21.
19-Emody L, Kerenyi M, Nagy G. Virulence factors of uropathogenic Escherichia coli. Int. J. Antimicrob. Agents. 22: (2003);29-33.
20-Raksha R, Srinivasa H, Macaden RS. Occurrence and characterization of uropathogenic E. coli in urinary tract infection. Indian Med. Microbiol. 21: (2003); 102-107.
21-Siegfried L, Kmetova M, puzova H, Molokacova M, Filka J. Virulence associated factors in E. coli strains isolated from children with Urinary tract infections. J. Med Microbiol . (1994); 41: 127-32.
22-Anwar Kadhim AL–Saffar, Wejdan R Taj Al-daan, Shahad Abed Al Redha Ali. Effect of Piper cubeba fruits extract on Hemolysin production of E. coli isolated from urinary tract infection. International Journal of ChemTech Research CODEN (USA), (2016); PP: 2455-9555Vol.9, No.12, pp 476-482,
23- Vaish, R, Padeep MMS, Setty CR, Kandi V. Evolution of virulence factors and antibiotic sensitivity pattern of E .coli isolated from extra intestinal infection.J. CureusV.(2016);8(5) e604.
24-Kausar Y, Chunchanur SK, Nadagir SD, Halesh LH, Chandrasekhar MR.Virulence factors, Serotypes and Antimicrobial susceptibility pattern of Escherichia coli in urinary tract infections. Al amen.J .Med Sciences. (2009); 02:4751.
25-Ebrahim Rahimi, Faham KhamesipouR, Farzad Yazdi, Hassan Momtaz. Isolation and Characterization of Enterohaemorragic Escherichia coli O157:H7 and EHEC O157:NM from Raw Bovine, Camel, Water Buffalo, Caprine and Ovine Milk in Iran . Kafkas Univ Vet Fak Derg ,(2012);18 (4): 559-564.
26-Jwan Ahmed Ali. (2012). Hemolysin and Bacteriocin Production of E. coli Isolated from Urinary Tract Infection. Journal of Babylon University/Pure and Applied Sciences/ No.(5)/ Vol.(20).
27-Sharma S, Bhat GK, Shenoy S. Virulence factors and drug resistance in Escherichia coli isolated from extra intestinal infections. Indian Journal of Medical Microbiology. (2007);25 (4): 369-373.
28-Kazunori Murase, Tadasuke Ooka, Atsushi Iguchi, Yoshitoshi Ogura, Keisuke Nakayama, Md Asadulghani, Md Rakibul Islam, Hirotaka Hiyoshi, Toshio Kodama, Lothar Beutin and Tetsuya Hayashi.(2012). Haemolysin E- and enterohaemolysin-derived haemolytic activity of O55/O157 strains and other Escherichia coli lineages. Microbiology, 158, 746–758 DOI 10.1099/mic.0.054775-0.
29-Yasmeen, K., Sneha, K.C., Shobha, D.N., Halesh, L.H. and Chnder, M.R. (2009). Virulence factors serotypes and antimicrobial susceptibility pattern of E. coli in urinary tract infections. Alamen J. Med. Sci. 2 (1): 47-51.
30-Marques LR1, Abe CM, Griffin PM, Gomes TA.(1995) . Association between alpha-hemolysin production and HeLa cell-detaching activity in fecal isolates of Escherichia coli. J Clin Microbiol. São Paulo, Brazil.;33(10):2707-9.
31- Karns J. S., J. S. Van Kessel,B. J. McClusky,and M. L. Perdue. (2007). The incidence of Escherichia coli O157:H7 and E. coli Virulence Factors in US Bulk Tank Milk as Determined by Polymerase Chain Reaction.American Dairy Science Association. J. Dairy Sci. 90:3212–3219.
32-Jorge blanco,miguel blanco, jesus e. blanco, azucena mora,nrique a. gonzalez, maria i. bernardez, maria p. alonso, amparo coira, asuncion rodriguez, joaquin rey,:j: juan m. alonso,:j: and miguel a. usera.(2003).Verotoxin-Producing Escherichia coli in Spain: Prevalence, Serotypes, and Virulence Genes of 0157 H7 and Non-0157 VTEC in Ruminants, Raw Beef Products, and Humans. The Society for Experimental Biology and Medicine.
33-Yaghobzadeh N, Ownagh A, Mardani K, Khalili M. Prevalence, molecular characterization and serology of Shiga toxin producing Escherichia coli isolated from buffaloes in West Azerbaijan, Iran. (2011).
34-Blanco J, Blanco M. Blanco JE. Mora A. Alonso MP. Gonzalez EA. Bernardez MI. Epidemiology of verocytotoxigenic Escherichia coli (VTEC) in ruminants. In: Duffy G. Garvey P. McDowell DA. Eds. Verocytotoxigenic E. coli. Trumbull. CT: Food and Nutrition Press. Pp: ll3-148.2001.
35-El-Jakee -JK, RM Mahmoud, AA Samy, Mona A El-Shabrawy, MM. Effect and W.A. Gad El-Said.Molecular Characterization of E. coli Isolated from Chicken, Cattle and Buffaloes. International Journal of Microbiological Research. (2012); 3 (1): 64-74.
36-Gehua Wang, Clifford G Clark, Frank G Rodgers. Detection in Escherichia coli of the Genes Encoding the Major Virulence Factors, the Genes Defining the O157:H7 Serotype, and Components of the Type 2 Shiga Toxin Family by Multiplex PCR. Journal of clinical microbiology, (2002); P. 3613–3619.
37-Griffiths BB, McClain O. "The role of iron in the growth and hemolysin (Streptolysin S) production in Streptococcus pyogenes". J. Basic Microbiol. (1988); 28 (7): 427–36. PMID 3065477.
38-Goñi FM1, Ostolaza H. E. coli alpha-hemolysin: a membrane-active protein toxin. Braz J Med Biol Res. ; (1998); 31(8):1019-34.
39-Burnside K, Lembo A, de Los Reyes M, Iliuk A, Binhtran NT, Connelly JE, Lin WJ, Schmidt BZ, Richardson AR, Fang FC, Tao WA, Rajagopal L (2010). "Regulation of hemolysin expression and virulence of Staphylococcus aureus by a serine/threonine kinase and phosphatase". PLoS ONE. 5 (6): e11071.
40-everett c. short, jr., and harold j. kurtz.(1971). Properties of the Hemolytic Activities of Escherichia coli. Infection and Immunity, p. 678-687 Vol. 3, No. 5
41- Snyder, I. S., and P. Zwadyk. (1969). Some factors affecting the production and assay of Escherichia coli haemolysins. J. Gen. Microbiol. 55:139-143.
42-Henrik Chart, Claire Jenkins, Henry R. Smith, Dawn Hedges and Bernard Rowe. (1998). Haemolysin production by strains of Verocytotoxin-producing Escherichia coli . MivObiolOgy , 144,103-1 07 Printed in Great Britain.
43-Beutin, L., Geier, D., Zimmerman, 5. & Karch, H. (1995). Virulence markers of Shiga-like toxin-producing Escherichia coli strains originating from healthy domestic animals of different species. J Clin Microbiol33, 631-635.
44-Lebek G, Gruening HM. The relationship between the hemolytic property and iron metabolism in Escherichia coli. lnfect lmmun (1985); 50, 682-686.
45-Neil R, Wyborn Melanie R, Stapleton Valia A Norte, Ruth E. Roberts, Jamie Grafton, and Jeffrey Green. Regulation of Escherichia coli Hemolysin E Expression by H-NS and Salmonella SlyA. Journal of Bacteriology, Mar.(2004); P:1620–1628 Vol. 186, No. 6
46-W. Springer AND W. Goebelt. (1980).Synthesis and Secretion of Hemolysin by Escherichia coli. Journal of Bacteriology, Oct. P. 53-59 Vol. 144, No. 1.