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Molecular characterization of Streptococcus agalactiae and Streptococcus uberis isolates from bovine milk

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Abstract

Streptococci are one among the major mastitis pathogens which have a considerable impact on cow health, milk quality, and productivity. The aim of the present study was to investigate the occurrence and virulence characteristics of streptococci from bovine milk and to assess the molecular epidemiology and population structure of the Indian isolates using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Out of a total of 209 bovine composite milk samples screened from four herds (A–D), 30 Streptococcus spp. were isolated from 29 milk samples. Among the 30 isolates, species-specific PCR and partial 16S rRNA gene sequence analysis identified 17 Streptococcus agalactiae arising from herd A and 13 Streptococcus uberis comprising of 5, 7, and 1 isolates from herds B, C, and D respectively. PCR based screening for virulence genes revealed the presence of the cfb and the pavA genes in 17 and 1 S. agalactiae isolates, respectively. Similarly, in S. uberis isolates, cfu gene was present in six isolates from herd C, the pau A/skc gene in all the isolates from herds B, C, and D, whereas the sua gene was present in four isolates from herd B and the only isolate from herd D. On MLST analysis, all the S. agalactiae isolates were found to be of a novel sequence type (ST), ST-483, reported for the first time and is a single locus variant of the predicted subgroup founder ST-310, while the S. uberis isolates were found to be of three novel sequence types, namely ST-439, ST-474, and ST-475, all reported for the first time. ST-474 was a double locus variant of three different STs of global clonal complex ST-143 considered to be associated with clinical and subclinical mastitis, but ST-439 and ST-475 were singletons. Unique sequence types identified for both S. agalactiae and S. uberis were found to be herd specific. On PFGE analysis, identical or closely related restriction patterns for S. agalactiae ST-483 and S. uberis ST-439 in herds A and B respectively, but an unrelated restriction pattern for S. uberis ST-474 and ST-475 isolates from herds D and C respectively, were obtained. This signifies that the isolates of particular ST may exhibit related PFGE patterns suggesting detection of a faster molecular clock by PFGE than MLST. Since all the isolates of both the species belonged to novel sequence types, their epidemiological significance in global context could not be ascertained, however, evidence suggests that they have uniquely evolved in Indian conditions. Further research would be useful for understanding the role of these pathogens in bovine sub-clinical mastitis and implementing effective control strategies in India.

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References

  • Almeida, R.A., Luther, D.A., Park, H.M., and Oliver, S.P., 2006. Identification, isolation, and partial characterization of a novel Streptococcus uberis adhesion molecule (SUAM). Veterinary Microbiology, 115, 183–191

    Article  PubMed  CAS  Google Scholar 

  • Almeida, R.A., Luther, D.A., Patel, D., and Oliver, S.P., 2011. Predicted antigenic regions of Streptococcus uberis adhesion molecule (SUAM) are involved in adherence to and internalization into mammary epithelial cells. Veterinary Microbiology, 148, 323–328

    Article  PubMed  CAS  Google Scholar 

  • Bansal, B.K., and Gupta, D.K., 2009. Economic analysis of bovine mastitis in India and Punjab–A Review. Indian Journal of Dairy Science, 62, 337–344

    Google Scholar 

  • Chen, L., Yang, J., Yu, J., Yao, Z., Sun, L., Shen, Y., and Jin, Q., 2005. VFDB: A reference database for bacterial virulence factors. Nucleic Acid Research, 33, D325-D328

    Article  CAS  Google Scholar 

  • Chen, X., Dego, O.K., Almeida, R.A., Fuller, T.E., Luther, D.A., and Oliver, S.P., 2010. Deletion of sua gene reduces the ability of Streptococcus uberis to adhere to and internalize into bovine mammary epithelial cells. Veterinary Microbiology, 147, 426–434

    Article  PubMed  Google Scholar 

  • Coffey, T.J., Pullinger, G.D., Urwin, R., Jolley, K.A., Wilson, S.M., Maiden, M.C., and Leigh, J.A., 2006. First insights into the Evolution of Streptococcus uberis: a multilocus sequence typing scheme that enables investigation of its population biology. Applied and Environmental Microbiology, 72, 1420–1428

    Article  PubMed  CAS  Google Scholar 

  • Dmitriev, A., Hu, Y.Y., Shen, A.D., Suvorov, A., Katokova, M., Mikula, I., and Totolyan, A., 1999. Comparative genetic study of group B Streptococcus strains if human and bovine origin. Folia Microbiologica (Praha), 44, 449–453

    Article  CAS  Google Scholar 

  • Dmitriev, A., Bhide, M., and Mikula, I., 2006. cpn60 gene-based multiplex-PCR assay for simultaneous identification of Streptococcal species. Acta Veterinaria Brunensis, 75, 235–240

    Article  CAS  Google Scholar 

  • Douglas, V.L., Fenwick, S.G., Pfeiffer, D.U., Williamson, N.B., and Holmes, C.W., 2000. Genomic typing of Streptococcus uberis isolates from cases of mastitis, in New Zealand dairy cows, using pulsed-field gel electrophoresis. Veterinary Microbiology, 75, 27–41

    Article  PubMed  CAS  Google Scholar 

  • Duarte, R.S., Miranda, O.P., Bellei, B.C., Brito, M.A., and Teixeira, L.M., 2004. Phenotypic and molecular characteristics of Streptococcus agalactiae isolates recovered from milk of dairy cows in Brazil. Journal of Clinical Microbiology, 42, 4214–4222

    Article  PubMed  CAS  Google Scholar 

  • Enright, M. C., and Spratt, B.G., 1999. Multilocus sequence typing. Trends in Microbiology, 7, 482–487

    Article  PubMed  CAS  Google Scholar 

  • Fang, W., Almeida, R.A., and Oliver, S.P., 2000. Effects of lactoferrin and milk on adherence of Streptococcus uberis to bovine mammary epithelial cells. American Journal of Veterinary Research, 61, 275–279

    Article  PubMed  CAS  Google Scholar 

  • Franken, C., Haase, G., Brandt, C., Weber-Heynemann, J., Mastin, S., Lammler, C., Podbielski, Lutticken, A. R., and Spellerberg, B., 2001 Horizontal gene transfer and host specificity of beta-haemolytic streptococci: the role of a putative composite transposon containing scpB and lmb. Molecular Microbiology, 41, 925–935

    Article  PubMed  CAS  Google Scholar 

  • Gase, K., Ferretti, J.J., Primeaux, C., and McShan, W.M., 1999. Identification, cloning, and expression of the CAMP factor gene (cfa) of Group A Streptococci. Infection and Immunity, 67, 4725–4731

    PubMed  CAS  Google Scholar 

  • Gianneechini, R., Concha, C., Rivero, R., Delucci, I., and Moreno López, J., 2002. Occurrence of clinical and sub-clinical mastitis in dairy herds in the West Littoral Region in Uruguay. Acta Veterinaria Scandinavica, 43, 221–230

    Article  PubMed  CAS  Google Scholar 

  • Jiang, M., Babiuk, L.A., and Potter, A.A., 1996. Cloning, sequencing and expression of the CAMP factor gene of Streptococcus uberis. Microbial Pathogenesis, 20, 297–307

    Article  PubMed  CAS  Google Scholar 

  • Jolley, K.A., Chan, M., and Maiden, M.C.J., 2004. mlstdbNet—distributed multi-locus sequence typing (MLST) databases. BMC Bioinformatics, 5, 86

    Article  PubMed  Google Scholar 

  • Keefe, G.P., 1997. Streptococcus agalactiae mastitis: A review. Canadian Veterinary Journal, 38, 429–437

    CAS  Google Scholar 

  • Khan, I.U., Hassan, A.A., Abdulmawjood, A., Lämmler, C., Wolter, W., and Zschöck M., 2003. Identification and epidemiological characterization of Streptococcus uberis isolated from bovine mastitis using conventional and molecular methods. Journal of Veterinary Science, 4, 213–224

    PubMed  CAS  Google Scholar 

  • Lasagno, M.C., Reinoso, E.B., Dieser, S.A., Calvinho, L.F., Buzzola, F., Vissio, C., Bogni, C.I., and Odierno, L.M., 2011. Phenotypic and genotypic characterization of Streptococcus uberis isolated from bovine sub-clinical mastitis in Argentinean dairy farms. Revista Argentina de Microbiologia, 43, 212–217

    PubMed  Google Scholar 

  • Lindahl, G., Stalhammar-Carlemalm, M., and Areschoug, T., 2005. Surface proteins of Streptococcus agalactiae and related proteins in other bacterial pathogens. Clinical Microbiology Review, 18, 102–127

    Article  CAS  Google Scholar 

  • Maiden, M.C.J., Bygraves, J.A., Feil, E., Morelli, G., Russell, J.E., Urwin, R., Zhang,Q., Zhou,J., Zurth, K., Caugant, D.A., Feavers, I.M., Achtman, M., and Spratt, B.G., 1998. Multilocus sequence typing: A portable approach to the identification of clones within populations of pathogenic microorganisms. Proceedings of National Academy of Sciences, USA, 95, 3140–3145

    Article  CAS  Google Scholar 

  • Manning, S.D., Springman, A.C., Million, A.D., Milton, N.R., McNamara, S.E., Somse, P.A., Bartlett, P., and Davies, H.D., 2010. Association of Group B Streptococcus Colonization and Bovine Exposure: A Prospective Cross-Sectional Cohort Study. Plos One, 5, 1–6

    Google Scholar 

  • McDougall, S., Parkinson, T., Leyland, M., Anniss, F., and Fenwick, S., 2004. Duration of infection and strain variation in Streptococcus uberis isolated from cow's milk. Journal of Dairy Science, 87, 2062–72

    Article  PubMed  CAS  Google Scholar 

  • Merl, K., Abdulmawjood, A., Lammler, C., and Zschock, M., 2003. Determination of epidemiological relationships of Streptococcus agalactiae isolated from bovine mastitis. FEMS Microbiology Letters, 229, 87–92

    Article  Google Scholar 

  • Oliver, S.P., Almeida, R.A., and Calvinho, L.F., 1998. Virulence Factors of Streptococcus uberis isolated from cows with mastitis. Journal of Veterinary Medicine, B, 45, 461–471

    Article  CAS  Google Scholar 

  • Patel, D., Almeida, R.A., Dunlap, J.R., Oliver, S.P., Petti, C.A., Polage, C.R., and Schreckenberger, P., 2009. Bovine lactoferrin serves as a molecular bridge for internalization of Streptococcus uberis into bovine mammary epithelial cells. Veterinary Microbiology, 137, 297–301

    Article  PubMed  CAS  Google Scholar 

  • Phuektes, P., Mansell, P.D., Dyson, R.S., Hooper, N.D., Dick, J.S., and Browning, G.F., 2001. Molecular epidemiology of Streptococcus uberis isolates from dairy cows with mastitis. Journal of Clinical Microbiology, 39, 1460–1466

    Article  PubMed  CAS  Google Scholar 

  • Pullinger, G.D., López-Benavides, M., Coffey, T.J., Williamson, J.H., Cursons, R.T., Summers, E., Lacy-Hulbert, J., Maiden, M. C., and Leigh, J. A., 2006. Application of Streptococcus uberis multilocus sequence typing: Analysis of the population structure detected among environmental and bovine isolates from New Zealand and the United Kingdom. Applied and Environmental Microbiology, 72, 1429–1436

    Article  PubMed  CAS  Google Scholar 

  • Pullinger, G. D., Coffey, T. J., Maiden, M. C., and Leigh, J. A., 2007. Multilocus-sequence typing analysis reveals similar populations of Streptococcus uberis are responsible for bovine intramammary infections of short and long duration. Veterinary Microbiology, 119, 194–204

    Article  PubMed  CAS  Google Scholar 

  • Rao, P., Birthal, P.S., Kar, D., Wickramaratne., and Shreshta, H.R., 2004. Increasing livestock productivity in mixed crop livestock systems in south Asia, ICRISAT, Hyderabad, India

    Google Scholar 

  • Rato, M.G., Bexiga, R., Nunes, S.F., Cavaco, L.M., Vilela, C.L., and Sanches, S., 2008. Molecular epidemiology and population structure of bovine Streptococcus uberis. Journal of Dairy Science, 91, 4542–4551

    Article  PubMed  CAS  Google Scholar 

  • Sharif, A., Umer, M., and Muhammad, G., 2009. Mastitis control in dairy production. Journal of Agriculture and Social Science, 5, 102–105

    Google Scholar 

  • Sharma, A., Dhingra, P., Pander, B.L., and Kumar, R., 2006. Bovine subclinical mastitis: prevalence and treatment with homeopathic medicine. International Journal of Cow Science, 2, 40–44

    Google Scholar 

  • Shome, B. R., Mitra, S. D., Bhuvana, M., Krithiga, N., Velu, D., Shome, R., Isloor, S., Barbuddhe, S. B., and Rahman, H., (2011). Multiplex PCR assay for species identification of bovine mastitis pathogens. Journal of Applied Microbiology 111, 1349–56

    Article  PubMed  CAS  Google Scholar 

  • Shum, L.W.C., McConnel, C.S., Gunna, A.A., and Housea, J.K., 2009. Environmental mastitis in intensive high-producing dairy herds in New South Wales. Australian Veterinary Journal, 87, 469–475

    Article  PubMed  CAS  Google Scholar 

  • Smith, T.C., Roehl, S.A., Pillai, P., Li, S., Marrs, C.F., and Foxman, B., 2007. Distribution of novel and previously investigated virulence genes in colonizing and invasive isolates of Streptococcus agalactiae. Epidemiology and Infection, 135, 1046–1054

    Article  PubMed  CAS  Google Scholar 

  • Sullivan, C. B., Diggle, M. A., and Clarke, S.C., 2005. Review—Multilocus sequence typing—Data analysis in clinical microbiology and public health. Molecular Biotechnology, 29, 245–254

    Article  PubMed  CAS  Google Scholar 

  • Sumathi, B.R., Veeregowda, B.M., and Gomes, A. R., 2008. Prevalence and antibiogram profile of bacterial Isolates from clinical bovine mastitis. Veterinary World, 1, 237–238

    Google Scholar 

  • Tamilselvam, B., Almeida, R.A., Dunlap, J.R., and Oliver, S.P., 2006. Streptococcus uberis internalizes and persists in bovine mammary epithelial cells. Microbial Pathogenesis, 40, 279–285

    Article  PubMed  Google Scholar 

  • Tenhagen, B.A., Koster, G., Wallmann, J., and Heuwieser, W., 2006. Prevalence of mastitis pathogens and their resistance against antimicrobial agents in dairy cows in Brandenburg, Germany. Journal of Dairy Science, 89, 2542–2551

    Article  PubMed  CAS  Google Scholar 

  • Tenover, F.C., Arbeit, R.D., Goering, R.V., Mickelsen, P.A., Murray, B. E., Persing, D. H., and Swaminathan, B., 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. Journal of Clinical Microbiology, 33, 2233–2239

    PubMed  CAS  Google Scholar 

  • Tomita, T., Meehan, B., Wongkattiya, N., Malmo, J., Pullinger, G., Leigh, J., and Deighton, M., 2008. Identification of Streptococcus uberis multilocus sequence types highly associated with mastitis. Applied and Environmental Microbiology, 74, 114–124

    Article  PubMed  CAS  Google Scholar 

  • Ward, P.N., and Leigh, J.A., 2004. Genetic analysis of Streptococcus uberis plasminogen activators. Indian Journal of Medical Research, 119, 136–140

    PubMed  CAS  Google Scholar 

  • Zadoks, R.N., and Fitzpatrick, J.L., 2009. Changing trends in mastitis. Irish Veterinary Journal, 62, 59–70

    Article  Google Scholar 

  • Zadoks, R.N., Gillespie, B.E., Barkema, H.W., Sampimon, O.C., Oliver, S.P., and Schukken, Y.H., 2003. Clinical, epidemiological and molecular characteristics of Streptococcus uberis infections in dairy herds. Epidemiology and Infection, 130, 335–349

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

The research was supported by grants from the National Agricultural Innovation Project (NAIP), Indian Council of Agricultural Research, Govt. of India, New Delhi, under component four: Basic and Strategic Research through NAIP to BRS (NAIP/Comp-4/C-30017/2008-09).

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Authors and Affiliations

  1. Project Directorate on Animal Disease Monitoring and Surveillance, Hebbal, Bangalore, 560 024, India

    Bibek Ranjan Shome, Mani Bhuvana, Susweta Das Mitra, Natesan Krithiga, Rajeswari Shome, Dhanikachalam Velu, Apala Banerjee, Krishnamshetty Prabhudas & Habibar Rahman

  2. ICAR Research Complex for Goa, Old Goa, 403 402, India

    Sukhadeo B. Barbuddhe

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  1. Bibek Ranjan Shome
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  2. Mani Bhuvana
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Correspondence to Sukhadeo B. Barbuddhe.

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Shome, B.R., Bhuvana, M., Mitra, S.D. et al. Molecular characterization of Streptococcus agalactiae and Streptococcus uberis isolates from bovine milk. Trop Anim Health Prod 44, 1981–1992 (2012). https://doi.org/10.1007/s11250-012-0167-4

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