Back
 OJVM  Vol.4 No.10 , October 2014
Differentiation of Avian Pathogenic Escherichia coli Strains from Broiler Chickens by Multiplex Polymerase Chain Reaction (PCR) and Random Amplified Polymorphic (RAPD) DNA
Dirgam Ahmad Roussan1*, Hana Zakaria2, Ghassan Khawaldeh1, Ibrahim Shaheen1
Show more
Abstract: We examined 50 Escherichia coli (E. coli) strains isolated from broiler chickens between January 2013 to March 2014 in order to evaluate the epidemiological prevalence of avian pathogenic E. coli (APEC) in Jordan by multiplex PCR and random amplification of polymorphic DNA (RAPD) tests. The multiplex polymerase chain reaction (PCR) which was used as tentative criteria of APEC targets 8 virulence associated genes; enteroaggregative toxin (astA), Type 1 fimbria adhesion (fimH), iron-repressible protein (irp2), P fimbriae (papC), aerobactin (iucD), temperature-sensitive hemagglutinin (tsh), vacuolating autotransporter toxin (vat), and colicin V plasmid operon (cva/cvi) genes. The number of detected genes could be used as a reliable index of their virulence. E. coli strains already typed as an APEC always harbor 5 to 8 genes, but non-APEC strains harbor less than 4 genes. Assuming the criteria of an APEC is possession of 5 or more virulence associated genes; we found that all 50 E. coli strains were classified as APEC strains. The RAPD analysis showed that the E. coli strains could be grouped into 35 of RAPD types by using these two different RAPD primer sets, RAPD analysis primer 4 5'AAGAGCCCGT5', and RAPD analysis primer 6 5'CCCGTCAGCA3'. The current study confirmed the endemic nature of APEC in broiler flocks in Jordan. It is essential that the biosecurity on poultry farms should be improved to prevent the introduction and dissemination of APEC and other agents. Furthermore, farmers need to be educated about the signs, lesions, and the importance of this agent.
Keywords: Avian Pathogenic Escherichia coli, Broiler, Multiplex PCR, Random Amplification of Polymorphic DNA
Cite this paper: Roussan, D. , Zakaria, H. , Khawaldeh, G. and Shaheen, I. (2014) Differentiation of Avian Pathogenic Escherichia coli Strains from Broiler Chickens by Multiplex Polymerase Chain Reaction (PCR) and Random Amplified Polymorphic (RAPD) DNA. Open Journal of Veterinary Medicine, 4, 211-219. doi: 10.4236/ojvm.2014.410025.
References

[1]   Hirsh, D.C. (2004) Enterobacteriaceaae: Escherichia. In: Hirsh, D.C, Maclachlan, N.J. and Walker, R.L., Eds., Veterinary Microbiology, Blackwell Publishing, London, 61-68.

[2]   Barnes, J.H., Nolan, L.K. and Vaillancourt, J.P. (2008) Colibacillosis. In: Saif, Y.M., Saif, A.M., Fadly, J.R., Glisson, L.R., McDougald, L.K., Nolan, and Swayne, D.E., Eds., Diseases of Poultry, 12th Edition, Blackwell Publishing, Ames, 691-737.

[3]   Dozois, C.M., Dho-Moulin, M., Brée, A., Fairbrother, J.M., Desautels, C. and Curtiss, R. (2000) Relationship between the tsh Autotransporter and Pathogenicity of Avian Escherichia coli and Localization and Analysis of tsh Genetic Region. Infection and Immunity, 68, 4145-4154.
http://dx.doi.org/10.1128/IAI.68.7.4145-4154.2000

[4]   Tivendale, K.A., Allen, J.L., Ginns, C.A., Crabb, B.S. and Browning, G.F. (2004) Association of iss and iucA, but Not tsh, with Plasmid-Mediated Virulence of Avian Pathogenic Escherichia coli. Infection and Immunity, 72, 6554-6560. http://dx.doi.org/10.1128/IAI.72.11.6554-6560.2004

[5]   Johnson, T.J., Siek, K.E., Johnson, S.J. and Nolan, L.K. (2006) DNA Sequence of a ColV Plasmid and Prevalence of Selected Plasmid-Encoded Virulence Genes among Avian Escherichia coli Strains. Journal of Bacteriology, 188, 745-758. http://dx.doi.org/10.1128/JB.188.2.745-758.2006

[6]   Janben, T., Schwarz, C., Preikschat, P., Voss, M., Philipp, H.C. and Wieler, L.H. (2001) Virulence-Associated Genes in Avian Pathogenic Escherichia coli (APEC) Isolated from Internal Organs of Poultry Having Died from Colibacillosis. International Journal of Medical Microbiology, 291, 371-378. http://dx.doi.org/10.1078/1438-4221-00143

[7]   Ewers, C., Janben, T., Kiebling, S., Philipp, H.C. and Wieler, L.H. (2005) Rapid Detection of Virulence-Associated Genes in Avian Pathogenic Escherichia coli by Multiplex Polymerase Chain Reaction. Avian Diseases, 49, 269-273. http://dx.doi.org/10.1637/7293-102604R

[8]   Provence, D.L. and Curtiss, R. (1994) Isolation and Characterization of a Gene Involved in Hemagglutination by an Avian Pathogenic Escherichia coli Strain. Infection and Immunity, 62, 1369-1380.

[9]   Wooley, R.E., Gibbs, P.S., Brown, T.B. and Maurer, J.J. (2000) Chicken Embryo Lethality Assay for Determining the Virulence of Avian Escherichia coli Isolates. Avian Diseases, 44, 318-324.
http://dx.doi.org/10.2307/1592546

[10]   Kwon, S.G., Cha, S.Y., Choi, E.J., Kim, B., Song, H.J. and Jang, H.K. (2008) Epidemiological Prevalence of Avian Pathogenic Escherichia coli Differentiated by Multiplex PCR from Commercial Chickens and Hatchery in Korea. Journal of Bacteriology and Virology, 38, 179-188.
http://dx.doi.org/10.4167/jbv.2008.38.4.179

[11]   van der Westhuizen, W.A. and Bragg, R.R. (2012) Multiplex Polymerase Chain Reaction for Screening Avian Pathogenic Escherichia coli for Virulence Genes. Avian Pathology, 41, 33-40.
http://dx.doi.org/10.1080/03079457.2011.631982

[12]   Chansiripornchai, N., Ramasoota, P., Sasipreeyajan, J. and Svenson, S.B. (2001) Differentiation of Avian Pathogenic Escherichia coli (APEC) Strains by Random Amplified Polymorphic DNA (RAPD) Analysis. Veterinary Microbiology, 80, 75-83. http://dx.doi.org/10.1016/S0378-1135(00)00380-1

[13]   Salehi, T.Z., Madani, S.A, Karimi, V. and Khazaeli, F.A. (2008) Molecular Genetic Differentiation of Avian Escherichia coli by RAPD-PCR. Brazilian Journal of Microbiology, 39, 494-497.
http://dx.doi.org/10.1590/S1517-83822008000300015

[14]   Nei, M. and Li, W.H. (1979) Mathematical Model for Studying Genetic Variation of Restriction Endonucleases. Proceedings of the National Academy of Science of the United States of America, 10, 5269-5273. http://dx.doi.org/10.1073/pnas.76.10.5269

[15]   Hillis, D.M. and Moritz, C. (1990) Molecular Systematics. Sinauer Associates, Sunderland.

[16]   Hunter, P.R. and Gaston, M.A. (1988) Numerical Index of the Discriminatory Ability of Typing Systems: An Application of Simpson’s Index of Diversity. Journal of Clinical Microbiology, 11, 2465-2466.

[17]   da Silveira, W.D., Ferreira, A., Brocchi, M., de Hollanda, L.M., de Castro, A.F.P., Yamada, A.T. and Lancellotti, M. (2002) Biological Characterization and Pathogenicity of Avian Escherichia coli Strains. Veterinary Microbiology, 85, 47-53. http://dx.doi.org/10.1016/S0378-1135(01)00482-5

[18]   La Ragione, R.M. and Woodward, M.J. (2002) Virulence Factors of Escherichia coli Serotypes Associated with Avian Colisepticaemia. Research in Veterinary Science, 73, 27-35.
http://dx.doi.org/10.1016/S0034-5288(02)00075-9

[19]   Delicato, E.R., De Brito, B.G., Gaziri, L.C. and Vidotto, M.C. (2003) Virulence Associated Genes in Escherichia coli Isolates from Poultry with Colibacillosis. Veterinary Microbiology, 94, 97-103. http://dx.doi.org/10.1016/S0378-1135(03)00076-2

[20]   Maurer, J.J., Lee, M.D., Lobsinger, C., Brown, T., Maier, M. and Thayer, S.G. (1998) Molecular Typing of Avian Escherichia coli Isolates by Random Amplification of Polymorphic DNA. Avian Diseases, 42, 431-451. http://dx.doi.org/10.2307/1592670

[21]   Dozois, C.M., Fairbrother, J.M., Harel, J. and Bosse, M. (1992) Pap-and Pil-Related DNA Sequences and Other Virulence Determinants Associated with Escherichia coli Isolated from Septicemic Chickens and Turkeys. Infection and Immunity, 60, 2648-2656.

[22]   Pourbakhsh, S.A., Dho-Moulin, M., Brée, A., Desautels, C., Martineau-Doize, B. and Fairbrother, J.M. (1997) Localization of the in Vivo Expression of P and F1 Fimbriae in Chickens Experimentally Inoculated with Pathogenic Escherichia coli. Microbial Pathogenesis, 22, 331-341.
http://dx.doi.org/10.1006/mpat.1996.0116

[23]   Vidotto, M.C., Navarro, H.R. and Gaziri, L.C. (1997) Adherence Pili of Pathogenic Strains of Avian Escherichia coli. Veterinary Microbiology, 59, 79-87.
http://dx.doi.org/10.1016/S0378-1135(97)00124-7

[24]   Stathopoulos, C., Provence, D.L. and Curtiss, R. (1999) Characterization of the Avian Pathogenic Escherichia coli Hemagglutinin Tsh, a Member of the Immunoglobulin A Protease-Type Family of Autotransporters. Infection and Immunity, 67, 772-781.

[25]   Savarino, S.J., MaVeigh, A., Watson, J., Gravioto, A., Molina, J., Echeverria, P., Bhan, M.K., Levine, M.M. and Fasano, A. (1996) Enteroaggregative Escherichia coli Heat-Stable Enterotoxin Is Not Restricted to Enteroaggregative E. coli. The Journal of Infectious Diseases, 73, 1019-1022.
http://dx.doi.org/10.1093/infdis/173.4.1019

[26]   Nataro, J.P. and Kaper, J.B. (1998) Diarrheagenic Escherichia coli. Clinical Microbiology Reviews, 11, 142-201.

[27]   Gilson, L., Mahanty, H.K. and Kolter, R. (1987) Four Plasmid Genes Are Required for Colicin V Synthesis, Export, and Immunity. Journal of Bacteriology, 169, 2466-2470.

[28]   Vandekerckhove D., Van De Maele, F., Adriaensen, C., Zaleska, M., Hernalsteens, J.P., De Baets, L., Butaye, P., Van Immerseel, F., Wattiau, P., Laevens, H., Mast, J., Goddeeris, B. and Pasmans, F. (2005) Virulence Associated Traits in Avian Escherichia coli: Comparison between Isolates from Colibacillosis Affected and Clinically Healthy Layer Flocks. Veterinary Microbiology, 108, 75-87. http://dx.doi.org/10.1016/j.vetmic.2005.02.009

[29]   Kapur, V., Sischo, W.M., Greer, R.S., Whittam, T.S. and Musser, J. (1995) Molecular Population Genetic Analysis of Staphylococcus aureus Recovered from Cows. Journal of Clinical Microbiology, 33, 376-380.

[30]   Peigramhari, S.M., Vaillancourt, J.P., Wilson, R.A. and Gyles, C.L. (1995) Characteristics of Escherichia coli Isolates from Avian Cellulitis. Avian Diseases, 39, 116-124. http://dx.doi.org/10.2307/1591990

[31]   Frank, S.M., B. Bosworth, B.T. and Moon, H.W. (1998) Multiplex PCR for Enterotoxigenic, Attaching and Effacing, and Shiga Toxin-Producing Escherichia coli Strains from Calves. Journal of Clinical Microbiology, 36, 1795-1797.

[32]   Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA Sequencing with Chain Terminating Inhibitors. Proceedings of the National Academy of Science of the United States of America, 74, 5463-5470. http://dx.doi.org/10.1073/pnas.74.12.5463

 
 
Top