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Current IssueEditorial BoardOnline First ArticlesArchive

Research Article

volume 54 issue 8 (august 2020) : 1024-1028,   Doi: 10.18805/ijar.B-3860

Study of Leptospira Infection in Buffaloes through Molecular and Bacteriological Techniques

P
Pande Dushyant
K
Khan Wiqar
C
Chaudhari Sandeep
S
Shinde Shilpshri
P
Patil Archana
L
Likhite Amrut
A
Allai Rakesh
1Department of Veterinary Public Health and Epidemiology Nagpur Veterinary College, Nagpur-440 006, Maharashtra, India.
Cite article:- Dushyant Pande, Wiqar Khan, Sandeep Chaudhari, Shilpshri Shinde, Archana Patil, Amrut Likhite, Rakesh Allai (2020). Study of Leptospira Infection in Buffaloes through Molecular and Bacteriological Techniques. Indian Journal of Animal Research. 54(8): 1024-1028. doi: 10.18805/ijar.B-3860.

ABSTRACT

In India buffaloes are reared for milk, meat and draft purposes. The economic importance of buffalo rearing in Nagpur is more than cow and contribute major share to the milk production of the district. Apart from its zoonotic implications; Leptospirosis is one of the major causes of abortion, still births, infertility, repeat breeding, decreased milk production, mastitis in ruminants. Considering its endemic nature in India, the present study is aimed at estimating the prevalence of leptospirosis in buffaloes and to establish the possible route of transmission through related canine, rodent and environment. A total of 621(299 each blood & sera and 23 urine) samples collected from 196 apparently healthy and 103 clinically suspected buffaloes and blood samples of 21 stray dogs along with 27 environmental samples were subjected to PCR targeting sec Y gene (202bp) and 16SrRNA gene (331bp).A total of 92 samples including 53 blood samples (buffalo= 45, rodent= 5, canine= 3), 30 buffalo sera samples, four urine samples (buffalo=2 and canine=2) and five environmental samples comprising of 3 water and 2 feed,  were processed for isolation of Leptospira in the EMJH medium. Out of 196 blood samples of apparently healthy buffaloes a total of 27 (13.77%) animals were found positive for 16S rRNA gene and 3(1.53%) for sec Y gene. Similarly, 17.47% (18/103) animals were positive for 16S rRNA gene while 7(6.79%) for sec Y gene. A total of 30 (15.30%) apparently healthy animals were positive for either 16SrRNA gene or secY gene. Whereas 25(24.27%) samples from suspected animals were positive for either of the gene. All the urine samples from buffaloes, dogs and environmental samples were negative for presence of both the genes. All the samples processed for isolation of organism turned negative. The accurate and rapid diagnosis of leptospirosis is important and beneficial for surveillance and investigation of transmission dynamics of the disease. Even though the active infection can be readily diagnosed through blood samples it is essential to screen each sample by various tests for proper understandings about the disease distribution.

REFERENCES

  1. Ayral F.C., Bicout D.J., Pereira H., Artois M. and Kodjo A. (2014). Short Report: Distribution of leptospira serogroups in cattle herds and dogs in France. Am. J. Trop. Med. Hyg. 91(4): 756–759.
  2. Balakrishnan G., Roy P. and Chandran D. J. (2015). Prevalence of leptospirosis among buffaloes. Buffalo bulletein. 14 (3): 313-317.
  3. Balakrishnan, G., Roy, G.P., Govindarajan, R., Ramaswamy, V. and Murali Manohar, B. (2011). Seroepidemiological studies on leptospirosis among bovines in organized farm. Int. J. Agro Vet. Med. Sci. 5(6): 511-519.
  4. Balamurugan V, Gangadhar N. L., Mohandoss N., S. R. A. Thirumalesh, M. Dhar, R. et al (2013). Characterization of Leptospira isolates from animals and humans: phylogenetic analysis identifies the prevalence of intermediate species in India, Springer Plus. 2:362.
  5. Balamurugan V., S. R. A. Thirumalesh, R. Sridevi, G. Govindaraj, M. Nagalingam, D. et al (2016 a). Microscopic Agglutination Test Analysis Identifies Prevalence of Intermediate Species Serovars in Ruminants in Endemic States of India. Proc. Natl. Acad. Sci. India, Sect. B Biol. Sci. 86 (2): 469–475.
  6. Balamurugan V., S. Veera., S.R.A. Thirumalesh., A. Alamuri., R. Sridevi., P.P. et al (2017). Distribution of serogroup specific antibodies against Leptospirosis in Livestock in Odisha. Indian Journal of Animal Sciences. 87 (5): 546–551.
  7. Benacer. D., Zain S. N.M., Amran F., Galloway R. L. and Thong K. L. (2013). Isolation and Molecular Characterization of Leptospira interrogans and Leptospir aborgpetersenii Isolates from the Urban Rat Populations of Kuala Lumpur, Malaysia. Am. J. Trop. Med. Hyg. 88(4): 704–709.
  8. Boonslip S., J. Thaipadungpanit., P. Amorchai., V. Wutheikanun., W. Cheirakul., D. et al (2011). Molecular detection and speciation of pathogenic Leptospira spp.in blood from patients with culture- negative leptospirosis, BMC Infectious Diseases. 11: 338.
  9. Bourhy P., S. Bremont, F. Zinini, C. Giry and M. Picardeau (2011). Comparison of Real-Time PCR Assays for Detection of Pathogenic Leptospira spp. in Blood and Identification of Variations in Target Sequences. Journal of Clinical Microbiology.49 (6): 2154–2160.
  10. Burhan C. H., E. Basri, O. Hasan, M Adile (2000). Detection of leptospira species by polymerase chain reaction (PCR) in urine of cattle. Turk. J. Vet. Anim. Sci. 24: 123–130.
  11. Chowdry A.K. (1903) Jaundice at Port Blair, Andaman Islands; India Med. Gaz. 38 pp 409-412.
  12. Costa, J., E. Hagan, J.C.,Michael., K.Paul., T.,Martha S. M.-S. Claudia., S. Bernadette A-R. Albert I. Ko (2015). Global morbidity and mortality of leptospirosis: a systematic Review. PLoS Negl Trop Dis. 9(6):e0003819.
  13. Director A., B. Penna., C. Hamond., A. P. Louriero., G. Martins., M.A. Medeiros and W. Lilenbaum (2014) Isolation of Leptospira interrogans Hardjoprajitno from vaginal fluid of clinically healthy ewe suggests potential for Venereal transmission. Journal of Medical Microbiology. 63: 1234 to 1236.
  14. Fearnley C., P.R. Wakeley, J. Gallego-Beltran, C. Dalley, S. Williamson, C. Gaudie, M.J. Woodward. (2008). The development of a real-time PCR to detect pathogenic Leptospira species in kidney tissue. ResVet Sci. 85(1):8–16.
  15. Fornazari F., Rodrigo Costa da Silva, Virginia Bodelão Richini-    Pereira, Hugo Enrique et al (2012). Comparison of conventional PCR, quantitative PCR, bacteriological culture and the Warthin Starry technique to detect Leptospira spp. in kidney and liver samples from naturally infected sheep from Brazil. Journal of Microbiological Methods. 90: 321–326.
  16. Ganoza C.A., Matthias M. A., Richards D.C., K.C. Brouwer., C.B. Cunningham.,E.R. Segura., R.H.Gilman., E.Gotuzzo., J.M.Vinetz (2006). Determining risk for severe leptospirosis by molecular analysis of environmental surface waters for pathogenic leptospira. PLOS Med. 3(8):1329- 1340.
  17. Ghatak,S., R. B. Muthukumaran and S. K.Nachimuthu (2013). A Simple Method of Genomic DNA Extraction from Human Samples for PCR-RFLP Analysis. Journal of Biomolecular Techniques. 24:224–231.
  18. Hamond C., G. Martins., W. Lilenbaum., M. Pinna and M.A. Medeiros (2015). Infection by Leptospira spp. In Cattle in Tropical Region, Rio de Janerio, Brazil. Am. J. Trop. Med. Hyg., 92(1): 210.
  19. Harkin K.R., Roshto Y.M. and Sullivan J.T. (2003). Clinical application of polymerase chain reaction assay for diagnosis of leptospirosis in dogs. JAVMA. 222: No.(9).
  20. Hazikolaei M. R. H., M. Ghorbanpour and G. Abdollapour (2006). Seroprevalence of Leptospiral infection in Buffalo (Bubalusbubalis). Bull Vet InstPulawy. 50: 341-344.
  21. Higgins, R.J., J.F Harbourne, T.W.A Little and A.E Stevens, (1980). Mastitis and abortus in dairy cattle associated with Leptospira of the serotype hardjo. Vet. Rec., 107:307 310.
  22. Hoyos C.E., V.R. Rodriguez., A.P. Doria., O. Vegara and A.C. Rangel (2017). Epidemiology behavior of leptospirosis in Cienaga de Oro, Cordoba (Colombia). Trop Anim Health Prod. 49:1345–1351.
  23. Koizumi N., Muto M., Tanikawa T., Mizutani H., Sohmura Y., Hayashi E., Akao N., Hoshino M., Kawabata H. and Watanabe H. (2009). Human leptospirosis cases and the Journal of Medical Microbiology. 58: 1227–1230.
  24. Kurilung. A., Chanchaithong P., Lugsomya K., Niyomthama W., Wuthiekanun V., Prapasarakul N. (2017). Molecular detection and isolation of pathogenic Leptospira from asymptomatic humans, domestic animals and water sources in Nan province, a rural area of Thailand. Research in Veterinary Science. 115: 146–154.
  25. Marianelli. C., M. Tarantino, S. Astarita, A. Martucciello, F. Capuanoand G. Galiero (2007). Molecular detection of Leptospiraspecies in aborted fetuses of water buffalo. Veterinary Record. 161: 310-311.
  26. Martin L.F., L. Philippot, S. Hallet, R. Chaussod, J.C. German, G. Soulas and G. Cautroux (2001). DNA extraction from soils: old bias for new microbial diversity analysis methods. Appl Environ Microbiol. 67: 2354-2359.
  27. Martins G, Loureiro AP, Hamond C, Pinna MH, Bremont S, Bourhy P, (2015). First isolation of Leptospira noguchii serogroups Panama and Autumnalis from cattle. Epidemiol Infect. 143(7):1538-41.
  28. Merien F., P. Amouriaux, P. Perolat, G. Barantan and I.S. Girons (1992). Polymearse Chain Reaction for Detection of Leptospira Spp.in Clinical samples. Journal of clinical microbiology. (30) 9: 2219-2224.
  29. Pandian S. J., Ray P. K., Chandran P.C. and Kumar M. (2015). Seroprevalence of Brucellaabortus and Leptospirahardjo in cattle. Veterinary World, EISSN: 2231-0916.
  30. Patel J. M., Vihol P.D., Raval J.K., Patel K.M., Chaudhari N.F., Rathodand P.H., Patel J. H. (2014). Seroprevalence of Leptospirosis in clinically ailing bovine. Journal of Animal Research. 5: 31-35.
  31. Patel J.M., Vihol P.D., Prasad M.C., Patel J.H., Raval J.K. and Kalyani I.H. (2017). Polymerase Chain Reaction in the Diagnosis of Spontaneous Leptospirosis in Bovines. International Journal of Current Microbiology and Applied Sciences. 6(12): 1723-1728.
  32. Rawlins J., Portanova. A., Zuckerman I., Loftis A., Ceccato P., Willingham A.L. and Verma A. (2014). Molecular detection of Leptospiral DNA in environmental water on St. Kitts. Int. J. Environ. Res. Public Health.11: 7953-7960.
  33. Ridzlan, F.R., Bahaman A.R., Bejo S. K., andMutalib A.R. (2010). Detection of pathogenic Leptospira from selected environment in Kelantan and Terengganu, Malaysia. Tropical Biomedicine. 27(3): 632–638.
  34. Saito M.,S. Miyahara., S.Y. A. M. Villanueva., N. Aramaki., M Ikejiri., Y. Kobayashi., J.P. et al (2014). PCR and culture identification of pathogenic Leptospira spp. from coastal soil in Leyte, Philippines, after a storm surge during super typhoon Haiyan (Yolanda). Applied and Environmental Microbiology. (80): 6926–6932.
  35. Salgado. M., B. Otto., M. Moroni., E. Sandoval., G. Reinhardt., S. Boqvist., C. Encina. and C. Muñoz-Zanzi (2015). isolation of Leptospira interrogans serovar Hardjoprajitno from a calf with clinical leptospirosis in Chile. BMC Veterinary Research. 11:66.
  36. SelvarajJ., B. MuraliManohar, R. Govindarajan, Vajiravelu Jayakumar, T.V. Meenambigai and Balachandran C.(2010). Seroprevalence of leptospirosis in she-buffaloes (Bosbubalis) at slaughter in Chennai, India. Buffalo BulletinVol. 29: 2.
  37. Shafighi, T., Zahraei T. S., Abdollahpour G., Asadpour L., Akbarein H. and, Salehzadeh A. (2014). Molecular detection of Leptospira spp. in the urine of cattle in northern Iran. Iranian Journal of Veterinary Research.15 (4): 402-405.
  38. Sharma, S., P. Vijayachari, A.P. Sugunan and S.C. Sehgal (2003). Leptospiral carrier state and seroprevalence among animal population – a cross-sectional sample survey in Andaman and Nicobar Islands. Epidemiol. Infect. 131: 985–989.
  39. Sivaseelan, S., Rani U., R. and Kathiresan D., (2003). Sero-prevalence of Leptospirosis in sheep and goats in Madurai District-    Tamil Nadu. Indian Veterinary Journal. 80:375- 376.
  40. Suepaul S.M., Carrington C.V.F., Campbell M., Borde G. and Adesiyun A. A. (2010). Serovars of Leptospira isolated from dogs and rodents. Epidemiol. Infect. 138: 1059–1070.
  41. Thaipadungpanit J., Cheirakul W., Wutheikanun V., Limmathurotsakul D., Amorchai P., et al (2011) Diagnostic accuracy of real time PCR assays targeting 16S rRNA and lipl32 genes for Human Leptospirosis in Thailand: A case- control study. PLOS ONE. 6 (1): 1-6.
  42. Varma A., Rai R.B., Balakrishnan P., Gupta A. and Naveen K. A. (2001). Seroprevalence of leptospirosis in animals of Andaman and Nicobar Islands. Indian Veterinary Journal. 78: 936.
  43. Vasconcellos S. A., João C.F. Oliveira, Zenáide M. Morais, Pietro S. Baruselli, Renato et al (2001). Isolation of Leptospira Santarosai, Serovar Guaricura from buffaloes (Bubalus Bubalis) In Vale Do Ribeira, São Paulo, Brazil. Brazilian Journal of Microbiology. 32:298-300.
  44. Wilson K (1997). Preparation of genomic DNA from Bacteria. Current Protocols In Molecular Biology. 2:4.1- 2.4.5.
  45. Woolley J. M. (1911). Malaria in Andamans; Indian Med. Gaz. 46: 409-411.
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    Research Article

    volume 54 issue 8 (august 2020) : 1024-1028,   Doi: 10.18805/ijar.B-3860

    Study of Leptospira Infection in Buffaloes through Molecular and Bacteriological Techniques

    P
    Pande Dushyant
    K
    Khan Wiqar
    C
    Chaudhari Sandeep
    S
    Shinde Shilpshri
    P
    Patil Archana
    L
    Likhite Amrut
    A
    Allai Rakesh
    1Department of Veterinary Public Health and Epidemiology Nagpur Veterinary College, Nagpur-440 006, Maharashtra, India.
    Cite article:- Dushyant Pande, Wiqar Khan, Sandeep Chaudhari, Shilpshri Shinde, Archana Patil, Amrut Likhite, Rakesh Allai (2020). Study of Leptospira Infection in Buffaloes through Molecular and Bacteriological Techniques. Indian Journal of Animal Research. 54(8): 1024-1028. doi: 10.18805/ijar.B-3860.

    ABSTRACT

    In India buffaloes are reared for milk, meat and draft purposes. The economic importance of buffalo rearing in Nagpur is more than cow and contribute major share to the milk production of the district. Apart from its zoonotic implications; Leptospirosis is one of the major causes of abortion, still births, infertility, repeat breeding, decreased milk production, mastitis in ruminants. Considering its endemic nature in India, the present study is aimed at estimating the prevalence of leptospirosis in buffaloes and to establish the possible route of transmission through related canine, rodent and environment. A total of 621(299 each blood & sera and 23 urine) samples collected from 196 apparently healthy and 103 clinically suspected buffaloes and blood samples of 21 stray dogs along with 27 environmental samples were subjected to PCR targeting sec Y gene (202bp) and 16SrRNA gene (331bp).A total of 92 samples including 53 blood samples (buffalo= 45, rodent= 5, canine= 3), 30 buffalo sera samples, four urine samples (buffalo=2 and canine=2) and five environmental samples comprising of 3 water and 2 feed,  were processed for isolation of Leptospira in the EMJH medium. Out of 196 blood samples of apparently healthy buffaloes a total of 27 (13.77%) animals were found positive for 16S rRNA gene and 3(1.53%) for sec Y gene. Similarly, 17.47% (18/103) animals were positive for 16S rRNA gene while 7(6.79%) for sec Y gene. A total of 30 (15.30%) apparently healthy animals were positive for either 16SrRNA gene or secY gene. Whereas 25(24.27%) samples from suspected animals were positive for either of the gene. All the urine samples from buffaloes, dogs and environmental samples were negative for presence of both the genes. All the samples processed for isolation of organism turned negative. The accurate and rapid diagnosis of leptospirosis is important and beneficial for surveillance and investigation of transmission dynamics of the disease. Even though the active infection can be readily diagnosed through blood samples it is essential to screen each sample by various tests for proper understandings about the disease distribution.

    REFERENCES

    1. Ayral F.C., Bicout D.J., Pereira H., Artois M. and Kodjo A. (2014). Short Report: Distribution of leptospira serogroups in cattle herds and dogs in France. Am. J. Trop. Med. Hyg. 91(4): 756–759.
    2. Balakrishnan G., Roy P. and Chandran D. J. (2015). Prevalence of leptospirosis among buffaloes. Buffalo bulletein. 14 (3): 313-317.
    3. Balakrishnan, G., Roy, G.P., Govindarajan, R., Ramaswamy, V. and Murali Manohar, B. (2011). Seroepidemiological studies on leptospirosis among bovines in organized farm. Int. J. Agro Vet. Med. Sci. 5(6): 511-519.
    4. Balamurugan V, Gangadhar N. L., Mohandoss N., S. R. A. Thirumalesh, M. Dhar, R. et al (2013). Characterization of Leptospira isolates from animals and humans: phylogenetic analysis identifies the prevalence of intermediate species in India, Springer Plus. 2:362.
    5. Balamurugan V., S. R. A. Thirumalesh, R. Sridevi, G. Govindaraj, M. Nagalingam, D. et al (2016 a). Microscopic Agglutination Test Analysis Identifies Prevalence of Intermediate Species Serovars in Ruminants in Endemic States of India. Proc. Natl. Acad. Sci. India, Sect. B Biol. Sci. 86 (2): 469–475.
    6. Balamurugan V., S. Veera., S.R.A. Thirumalesh., A. Alamuri., R. Sridevi., P.P. et al (2017). Distribution of serogroup specific antibodies against Leptospirosis in Livestock in Odisha. Indian Journal of Animal Sciences. 87 (5): 546–551.
    7. Benacer. D., Zain S. N.M., Amran F., Galloway R. L. and Thong K. L. (2013). Isolation and Molecular Characterization of Leptospira interrogans and Leptospir aborgpetersenii Isolates from the Urban Rat Populations of Kuala Lumpur, Malaysia. Am. J. Trop. Med. Hyg. 88(4): 704–709.
    8. Boonslip S., J. Thaipadungpanit., P. Amorchai., V. Wutheikanun., W. Cheirakul., D. et al (2011). Molecular detection and speciation of pathogenic Leptospira spp.in blood from patients with culture- negative leptospirosis, BMC Infectious Diseases. 11: 338.
    9. Bourhy P., S. Bremont, F. Zinini, C. Giry and M. Picardeau (2011). Comparison of Real-Time PCR Assays for Detection of Pathogenic Leptospira spp. in Blood and Identification of Variations in Target Sequences. Journal of Clinical Microbiology.49 (6): 2154–2160.
    10. Burhan C. H., E. Basri, O. Hasan, M Adile (2000). Detection of leptospira species by polymerase chain reaction (PCR) in urine of cattle. Turk. J. Vet. Anim. Sci. 24: 123–130.
    11. Chowdry A.K. (1903) Jaundice at Port Blair, Andaman Islands; India Med. Gaz. 38 pp 409-412.
    12. Costa, J., E. Hagan, J.C.,Michael., K.Paul., T.,Martha S. M.-S. Claudia., S. Bernadette A-R. Albert I. Ko (2015). Global morbidity and mortality of leptospirosis: a systematic Review. PLoS Negl Trop Dis. 9(6):e0003819.
    13. Director A., B. Penna., C. Hamond., A. P. Louriero., G. Martins., M.A. Medeiros and W. Lilenbaum (2014) Isolation of Leptospira interrogans Hardjoprajitno from vaginal fluid of clinically healthy ewe suggests potential for Venereal transmission. Journal of Medical Microbiology. 63: 1234 to 1236.
    14. Fearnley C., P.R. Wakeley, J. Gallego-Beltran, C. Dalley, S. Williamson, C. Gaudie, M.J. Woodward. (2008). The development of a real-time PCR to detect pathogenic Leptospira species in kidney tissue. ResVet Sci. 85(1):8–16.
    15. Fornazari F., Rodrigo Costa da Silva, Virginia Bodelão Richini-    Pereira, Hugo Enrique et al (2012). Comparison of conventional PCR, quantitative PCR, bacteriological culture and the Warthin Starry technique to detect Leptospira spp. in kidney and liver samples from naturally infected sheep from Brazil. Journal of Microbiological Methods. 90: 321–326.
    16. Ganoza C.A., Matthias M. A., Richards D.C., K.C. Brouwer., C.B. Cunningham.,E.R. Segura., R.H.Gilman., E.Gotuzzo., J.M.Vinetz (2006). Determining risk for severe leptospirosis by molecular analysis of environmental surface waters for pathogenic leptospira. PLOS Med. 3(8):1329- 1340.
    17. Ghatak,S., R. B. Muthukumaran and S. K.Nachimuthu (2013). A Simple Method of Genomic DNA Extraction from Human Samples for PCR-RFLP Analysis. Journal of Biomolecular Techniques. 24:224–231.
    18. Hamond C., G. Martins., W. Lilenbaum., M. Pinna and M.A. Medeiros (2015). Infection by Leptospira spp. In Cattle in Tropical Region, Rio de Janerio, Brazil. Am. J. Trop. Med. Hyg., 92(1): 210.
    19. Harkin K.R., Roshto Y.M. and Sullivan J.T. (2003). Clinical application of polymerase chain reaction assay for diagnosis of leptospirosis in dogs. JAVMA. 222: No.(9).
    20. Hazikolaei M. R. H., M. Ghorbanpour and G. Abdollapour (2006). Seroprevalence of Leptospiral infection in Buffalo (Bubalusbubalis). Bull Vet InstPulawy. 50: 341-344.
    21. Higgins, R.J., J.F Harbourne, T.W.A Little and A.E Stevens, (1980). Mastitis and abortus in dairy cattle associated with Leptospira of the serotype hardjo. Vet. Rec., 107:307 310.
    22. Hoyos C.E., V.R. Rodriguez., A.P. Doria., O. Vegara and A.C. Rangel (2017). Epidemiology behavior of leptospirosis in Cienaga de Oro, Cordoba (Colombia). Trop Anim Health Prod. 49:1345–1351.
    23. Koizumi N., Muto M., Tanikawa T., Mizutani H., Sohmura Y., Hayashi E., Akao N., Hoshino M., Kawabata H. and Watanabe H. (2009). Human leptospirosis cases and the Journal of Medical Microbiology. 58: 1227–1230.
    24. Kurilung. A., Chanchaithong P., Lugsomya K., Niyomthama W., Wuthiekanun V., Prapasarakul N. (2017). Molecular detection and isolation of pathogenic Leptospira from asymptomatic humans, domestic animals and water sources in Nan province, a rural area of Thailand. Research in Veterinary Science. 115: 146–154.
    25. Marianelli. C., M. Tarantino, S. Astarita, A. Martucciello, F. Capuanoand G. Galiero (2007). Molecular detection of Leptospiraspecies in aborted fetuses of water buffalo. Veterinary Record. 161: 310-311.
    26. Martin L.F., L. Philippot, S. Hallet, R. Chaussod, J.C. German, G. Soulas and G. Cautroux (2001). DNA extraction from soils: old bias for new microbial diversity analysis methods. Appl Environ Microbiol. 67: 2354-2359.
    27. Martins G, Loureiro AP, Hamond C, Pinna MH, Bremont S, Bourhy P, (2015). First isolation of Leptospira noguchii serogroups Panama and Autumnalis from cattle. Epidemiol Infect. 143(7):1538-41.
    28. Merien F., P. Amouriaux, P. Perolat, G. Barantan and I.S. Girons (1992). Polymearse Chain Reaction for Detection of Leptospira Spp.in Clinical samples. Journal of clinical microbiology. (30) 9: 2219-2224.
    29. Pandian S. J., Ray P. K., Chandran P.C. and Kumar M. (2015). Seroprevalence of Brucellaabortus and Leptospirahardjo in cattle. Veterinary World, EISSN: 2231-0916.
    30. Patel J. M., Vihol P.D., Raval J.K., Patel K.M., Chaudhari N.F., Rathodand P.H., Patel J. H. (2014). Seroprevalence of Leptospirosis in clinically ailing bovine. Journal of Animal Research. 5: 31-35.
    31. Patel J.M., Vihol P.D., Prasad M.C., Patel J.H., Raval J.K. and Kalyani I.H. (2017). Polymerase Chain Reaction in the Diagnosis of Spontaneous Leptospirosis in Bovines. International Journal of Current Microbiology and Applied Sciences. 6(12): 1723-1728.
    32. Rawlins J., Portanova. A., Zuckerman I., Loftis A., Ceccato P., Willingham A.L. and Verma A. (2014). Molecular detection of Leptospiral DNA in environmental water on St. Kitts. Int. J. Environ. Res. Public Health.11: 7953-7960.
    33. Ridzlan, F.R., Bahaman A.R., Bejo S. K., andMutalib A.R. (2010). Detection of pathogenic Leptospira from selected environment in Kelantan and Terengganu, Malaysia. Tropical Biomedicine. 27(3): 632–638.
    34. Saito M.,S. Miyahara., S.Y. A. M. Villanueva., N. Aramaki., M Ikejiri., Y. Kobayashi., J.P. et al (2014). PCR and culture identification of pathogenic Leptospira spp. from coastal soil in Leyte, Philippines, after a storm surge during super typhoon Haiyan (Yolanda). Applied and Environmental Microbiology. (80): 6926–6932.
    35. Salgado. M., B. Otto., M. Moroni., E. Sandoval., G. Reinhardt., S. Boqvist., C. Encina. and C. Muñoz-Zanzi (2015). isolation of Leptospira interrogans serovar Hardjoprajitno from a calf with clinical leptospirosis in Chile. BMC Veterinary Research. 11:66.
    36. SelvarajJ., B. MuraliManohar, R. Govindarajan, Vajiravelu Jayakumar, T.V. Meenambigai and Balachandran C.(2010). Seroprevalence of leptospirosis in she-buffaloes (Bosbubalis) at slaughter in Chennai, India. Buffalo BulletinVol. 29: 2.
    37. Shafighi, T., Zahraei T. S., Abdollahpour G., Asadpour L., Akbarein H. and, Salehzadeh A. (2014). Molecular detection of Leptospira spp. in the urine of cattle in northern Iran. Iranian Journal of Veterinary Research.15 (4): 402-405.
    38. Sharma, S., P. Vijayachari, A.P. Sugunan and S.C. Sehgal (2003). Leptospiral carrier state and seroprevalence among animal population – a cross-sectional sample survey in Andaman and Nicobar Islands. Epidemiol. Infect. 131: 985–989.
    39. Sivaseelan, S., Rani U., R. and Kathiresan D., (2003). Sero-prevalence of Leptospirosis in sheep and goats in Madurai District-    Tamil Nadu. Indian Veterinary Journal. 80:375- 376.
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