Molecular detection of inv A and spv virulence genes in Salmonella typhimurium isolated from human and animals in Iran
Keywords:
Salmonella typhimurium, Multiplex PCR, Genes, spvA, spvB, spvCAbstract
In the present study, multiplex Polymerase Chain Reaction (m-PCR), simple PCR assay was used to confirm Salmonella typhimurium and detect Salmonella plasmid virulence (spvA, spvB, spvC) genes in human, bovine and poultry isolates. Fifty-five S. typhimurium isolates from bovine (n=15), poultry (n=20) and human (n=20) sources were isolated and analyzed with biochemical and serological testes. Firstly, (M-PCR) assay with four sets of primers was selected: ST139 - ST141 (284bp), specific for invA gene of Salmonella spp. the RFbj (663bp), Flic(183bp), Fljb(526 bp) specific for the rfbj, fliC, and fljB genes of S. typhimurium. In the second stage, simple PCR method with one set primer for SpvA (604bp) and another set primer for SpvB (1063bp) were applied to detect spvA and spvB genes. Also an M-PCR assay with two set primers InvA (244bp) and SpvC (571bp) was carried out to simultaneously detect and identify invA and spvC genes in S. typhimurium. Analysis of the samples shows that while the presence of spvA, spvB, and spvC genes in S. typhimurium from bovine source were 100% (15/15), these same genes were present in 65% (13/20), 100% (20/20) and 65%(13/20) of the poultry sources respectively.In addition, twenty (n=20) human isolates of S. typhimurium were obtained from Baghuyatallah University of Medical Science, Tehran, Iran. The study also shows that spvA, spvB and spvC genes were present in 85%, 100% and 85% of human source respectively. The study represents the first report in Iran about the genotypic diversity of spvA, spvB and spvC genes of S.typhimurium.
References
Aabo, S., Rasmussen, F., Rossen, Sorensen, L.P.D., Olsen, J.E., 1993. Salmonella identification by the polymerase chain reaction. Mol. Cell. prob. 7, 171-178.
Asten, A.J., Dijk, J.E., 2005. Distribution of “classic” virulence factors among Salmonella Serovars Fems immunology and medical microbiology. 44, 251-259.
Ahmer. M.M.B., Tran. M., Heffron, f., 1998. The virulence plasmid of Salmonella typhimurium is self-transmissible. J. Bacteriol. 181(4), 1364-1368.
Boyed, E.F., Hartl, D.L.,. 1998. Salmonella virulence plasmid: modular acquisition of the spv virulence region by an F-Plasmid in Salmonella entrica subspecies I and Insertion into the chromosome of subspecies II, IIIa, IV and VII Isolate. Genet. Soci. Am. 149, 1183-1190.
Brian, M., Ahmer, M., Tran, M., Hefron, F., 1998. The virulence plasmid of Salmonella typhimurium is self transmissible. J. Bacteriol. 181(4), 1364-1368.
Cortez, A.L., Carvalho, A.C., Ikuno, A.A., Burger, K.P., idal-Marthins, A.M., 2006. Identification of Salmonella serovars isolate from chicken abattoirs by multiplex PCR. Res. Vet. sci. 81, 340-344.
Chiu, C.H., Ou, J.T., 1996. Rapid identification of Salmonella serovars in feces by specific detection of virulence genes invA, and spvC, by an enrichment broth culture-multiplex PCR combination assay. J. Clin.Microbiol. 96, 2619-2622.
Chiu, C.H., Su, L.H., Chu, CH., Wang, M.H., Yeh, C.M., Weill, F.X., Chu, C., 2006. Detection of multidrug- resistant Salmonella entrica serovar Typhimurium phage types DT102, DT 104 and U302 by Multiplex PCR. J. Clin. microbiol. 10, 2354-235.
Chu, C., Hong, S.F., Tsai, C., Lin, W.S., Liu, T.P., Ou, J., 1999. Comparative physical and genetic maps of the virulence plasmids of Salmonella entrica serovar Typhimurium, Enteritidis, Choleraesuis and Dublin. Infec. Immun. 99, 2611.
Chu, C., Chiu, C.H., 2006. Evolutions of the virulence plasmid of non-typhoid Salmonella and its association with antimicrobial resistance. Microb. infect. 8, 1931- 1936.
Del cerro, A., Soto, S.M., Mendoza, M.C., 2002. Virulence and antimicrobial-resistance gene profiles determined by PCR-based procedures for Salmonella isolated from samples of animal origin. Food. microbiol. 20, 431-436.
Gay, J., 1995. Bovine herd Salmonellosis. Am. J. Infect. Control. 23, 56-62.
Gebreyes, W.A., Thakur, S., Dorr, P., Tadesse, D.A., Post, K., Wolf, L., 2008. Occurrence of spvA virulence Gene and clinical significance for multidrug- resistant Salmonella strains. J. clin. Microbiol. 10, 777-780.
Gothoh, H., okada, N., kim, Y.G., Shiraishi, K., Hirami, N., Haneda, T., kurita, A., kikuchi, Y., Danbara, H., 2003. Extracellular secretion of the virulence plasmid- encoded ADP-ribosyl transferase SPVB in salmonella. Microb. Pathogen. 34, 227-238.
Heithoff, D.M., Shimp, W.R., Iau, P.W., Badie, G., Enioutina, E.Y., Daynes, Barbara R.A., Byrne, A., House, J., Mahan, M.J., 2008. Human Salmonella clinical isolates distinct from those of animal origin. Appl. Environ. Microbiol. 10, 1757-1766.
Halawani, E., Shohayeb, 2008. Molecular characterization of multiple antibiotic resistance in Salmonella enteric serovar Typhimurium and Enteritidis isolated in saudi Arabia. Word. J. Med. Sci. 3(1), 43-49.
Heithoff, D.M., Shimp, W.R., lau, P.W., Badie, G., Enioutina, E.Y., Daynes, Barbara, R.A., Byrne, A., House, J., Mahan, M.J., 2008. Human Salmonella clinical isolates distinct from those of animal origin. App. Environ. Microbiol. 10, 1757-1766.
Holger, C.S., Thorsten, A., Heike, M., Uwe, R., Anderson, H., 2001. Improvement of an invA- based PCR for the specific detection of Salmonella typhimurium in organs of pigs. Institute of Animal Hygiene and Public Veterinary Health, Veterinary Faculty, Germany. 585-589, 2001.
Hong, S.f., chiu, C.H., Chu, C., Feng, C., Ou, T.J., 2008. Complete nucleotide sequence of a virulence of a virulence plasmid of Salmonella entrica serovar Dublin and its phylogenetic relationship to the virulence plasmids of serovars Choleraesuis, Enteritidis and Typhmurium. FEMS microb. 282, 39-43.
Hughes, A.L., Shopland, S., Wigley, paul, Bardon, H., Leatherbarrow, L.A., William, N., Bennett, M., Pinna, E., Lawson, B., 2008. Characterisation of Salmonella enteric serovar Typhimurium isolates from wild bird in northern Engeland from 2005-2006. BMC. Vet. Res. 2008, 4, 4.
Jamshidi, A., Bassami, M.R., Afshari-Nic, S., 2008. Identification of Salmonella serovars And Rrium by a multiplex PCR-based assay from poultry carcasses in Mashhad-Iran. Int. J. vet. Res. 3 (1), 43-48.
Jenikova, G., Pazlarova, J., Demnerova, K., 2000. Detection of Salmonella in food sample by the combination of immunomagnetic separation and PCR assay. Int. Microbiol. 3, 225-229.
Khan, A.A., Nawaz, M.S. Khan, S., Sernigelia, C.E., 1999. Detection of multidrug resistant Salmonella typhimurium DT104 by multiplex polymerase chain reaction. FEEMS. Microliol. Lett. 182, 355-360.
Kurita, A.I., Gotoh, H., Eguchi, M., Okada, N., Matsuura, S., Matsui, H., Danbara, H., Kikuchi, Y., 2003. Intracellular expression of the Salmonella plasmid virulence protein, SPVB, causes apoptotic cell death in eukaryotic Cells. Microb. Pathogen. 35, 43-48.
Lim, Y.H., Hirose, K., Izumiya, H., Arakawa, Shi, H.T., Terajima, J., Itoh, K., Tamura, K., Kim, S., watanabe, H., 2003. Multiplex polymerase chain reaction assay for selective detection of Salmonella entrica serovar Typhimurium. Jpn. J. Infect. Dis. 56, 151-155.
Ling, J.M.L., 2009. Rapid detection of food-borne pathogens in clinical specimens, food and environmental samples. Hong Kong. Med. J. 15(1), 26-29.
Malkawi, H.I., Gharaibeh, R., 2004. Rapid and simultaneous Identification of two Salmonella entrica serotypes Enteritidis and Typhimurium from chicken and meat product by multiplex PCR. Biotechnol. 3(1), 44-48.
Malorny, B., Hoofer, j., Bunge, C., Helmuth, R., 2003. Multicenter validation of the analytical accuracy of Salmonella PCR: Towards an international standard. Appl. Environ. Microbiol. 6, 290-296.
Malorny, B., Hoofe,r J., Bunge, C., Helmuth, R., 2001. Evaluation of Salmonella spp. Specific primer-sets for the validation within the food PCR project. Federal Institute Health protection of consurners and veterinary medicine, National Reference Laboratory for Salmonella, Diedersdorfer Weg 1, 12277 Berlin, Germany, 2001.
Majtan, V., Szaboova, M., 2005. The Virulence Markers Of Salmonella Serovar Typhimurium different phage type strains isolated in Slovakia. Folia. microbiol. 50(6), 516-523.
Matsui, H., Bacot, M.C., Garlington, A.W., Doyle, J.T., Roberts, S., Guling, A., Paul, 2001. Virulence plasmid- borne SPVB and SPVC genes can replace the 90-kilobase plasmid in conferring virulence to Salmonella entrica serovar Typhimurium in subcutaneously inculcated mice. J. Bacteriol. 183(15), 2652- 2658.
Namimatsu, T., Asai, T., Osumi, T., Imai, Y., Soto, S., 2006. Prevalence of the virulence plasmid in Salmonella typhimurium isolates from pigs. J. Vet. Med. Sci. 68(2), 187-188.
Nikbakht, G.h., Tadjbakhsh, H., 2004. Study of plasmid virulence genes (spv) in Salmonella enterica serovars isolated in Iran. J. Fac. Vet. Med. Univ. Tehran. 59(2), 137-140.
Oliviera, S.D., Santos, L.R.D., Schuch, M.T., Silva, A.B.C., Salle, T.P., Canal, C.W., 2002. Detection and identification of Salmonellas from poultry-related samples by PCR. Vet. Microbiol. 87, 25-35.
Pan, T.M., liu, Y.J., 2002. Identification of Salmonella Enteritidis isolate by polymerase chain reaction and multiplex polymerase chain reaction. J. Microbiol. Immuno. Infect. 35, 147-151.
Rahn, K., Grandees, S.A., De, Clarke, R.C., Galen, J.E., Pinocchio, C., Curtis, R., Giles, C.L., 1992. Amplification of an inv-A sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella. Mol. Cell. probes. 6, 271-279.
Rotger, R., casadesus, J., 1999. The virulence plasmids of Salmonella. Int. Microbiol. 2, 177-184.
Salehi, Z.T., Tadjbakhsh, H., Atashparvar, N., Nadalian, M.G., Mahzounieh, M.R., 2007. Detection and identification of Salmonella typhimurium in bovine diarrheic sample by immunomagnetic separation and Multiplex PCR assay. Zoon. Public. Health. 54, 231-236.
Salehi, Z.T., Mahzounieh, M.R., Vatankhah, J., 2006. Identification and diagnosis of Salmonella serotypes in cow feacal samples by polymerase reaction (PCR). J. Fac. Vet. Med. Tehran. 61,3; 245-249.
Salehi, Z.T., 1999. Salmonella. Tehran University press. PP: 146-150. Saroj, S.D., Shashidhar, R., Bandekar, J.R., 2007. Note Genetic Diversity of food isolates of Salmonella entrica serovar Typhimurium in India. Foo. Sci. Tec. Int. 14(2), 151-156.
Scuderi, G., Fantasia, M., Filetici, E., Anastasio, M.P., 1996. Food-borne outbreaks causes by Salmonella in Italy, 1991-1996. Epidemiol. Infectol. 116, 257-265.
Soumet, C., Ermel, G., Rose, N., Rose, V., Drouin, P., Salvat, G., Colin, P., 1999. Evaluation of a multiplex PCR assay for simultaneous identification of Salmonella serovars, Salmonella enteritidis and Salmonella typhimurium from environmental swabs of poultry houses. Lett. Appl. Microbiol. 25, 113-117.
Yu, H., Wang, J., Ye, J., Tang, P., Chu, C., Hu, S., Chiu, C.H., 2005. Complete nuleotide sequence of PSCV50, the virulence plasmid of Salmonella entrica server Choleraesuis SC-B67. Plasmid 55, 145-151.
Ziemer, C.J., Steadham, S.R., 2003. Evaluation of the specifity of Salmonella PCR Primers using various intestinal bacterial species. Litt. Appl. Microbiol. 37, 463-469.
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