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Research Article

volume 53 issue 5 (may 2019) : 667-671,   Doi: 10.18805/ijar.B-3568

Prevalence and molecular characterization of cryptosporidium species in buffalo calves of western Haryana

K
Krutanjali Swain
S
Sukhdeep Vohra
A
A.K. Sangwan
A
Abhilash Routray
S
Sumitra Panigrahi
S
Subha Ganguly
1Department of Veterinary Parasitology, Faculty of Veterinary Science and Animal Husbandry, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar - 125 004, Haryana, India.
Cite article:- Swain Krutanjali, Vohra Sukhdeep, Sangwan A.K., Routray Abhilash, Panigrahi Sumitra, Ganguly Subha (2018). Prevalence and molecular characterization of cryptosporidium species in buffalo calves of western Haryana. Indian Journal of Animal Research. 53(5): 667-671. doi: 10.18805/ijar.B-3568.

ABSTRACT

A sum total of 402 faecal samples from buffalo calves belonging to the age group of three months were collected randomly without any clinical symptom from various livestock farms and small holdings in four districts of Haryana. The presence or absence of Cryptosporidium spp. oocysts in faecal samples was confirmed by the standard microscopic examination of the modified Ziehl-Neelsen (MZN) stained faecal smears. The overall prevalence in buffalo calves was found to be 8.7%. The seasonal prevalence study revealed a maximum occurrence of Cryptosporidium infection during rainy followed by autumn, winters and minimum in summer season. Age related prevalence showed highest infection in calves between 16-30 days age which gradually decreased with increase in age. The DNA of representative positive faecal samples was extracted using standard phenol-chloroform-isoamylalcohol (PCI) method and then was subjected to nested PCR. All the extracted DNA were amplified for a 1325 bp primary fragment followed by a 834 bp nested fragment in a nested PCR of 18S rRNA gene, thereby confirming the presence of Cryptosporidium spp. The restricted enzyme digestion pattern (restricted fragment length polymorphism) of the 834 bp nested fragment using the restriction enzymes SspI and VspI confirmed the presence of C. parvum. Sequencing of Cryptosporidium DNA samples were carried out which also proved all samples to be of C. parvum. This is the first report of molecular identification of C. parvum in buffalo calves of Haryana and these Haryana isolates of Cryptosporidium parvum isolated from diarrhoeic buffalo calves are divergently related to rest of the part of the country isolates and indicates the zoonotic potential.

REFERENCES

  1. Abeywardena, H., Jex, A. R., Samson-Himmelstjerna, G. V., Haydon, S. R., Stevens, M. A and Gasser, R. B. (2013). First molecular characterization of Cryptosporidium and Giardia from Bubalus bubalis (water buffalo) in Victoria, Australia. Infect. Genet. Evol. 20: 96-102. 
  2. Bhat, S.A., Juyal, P.D and Singla L.D. (2013). Distribution of bovine cryptosporidiosis in India. Trends in Parasitology Research. 2(2). 5-13X.
  3. Bhat, S.A., Juyal, P.D. and Singla, L.D. (2012). Prevalence of cryptosporidiosis in neonatal buffalo calves in Ludhiana district of Punjab. India. Asian J. Anim. Vet. Adv. 7: 512-520. 
  4. Chai, J.Y., Kim, N.Y, Guk, S.M., Park, Y.K., Seo, M., Han, E.T and Lee, S.H. (2001). High prevalence and seasonality of cryptosporidiosis in a small rural village occupied predominantly by aged people in the Republic of Korea. Am J. Trop. Med. Hyg. 65:518–522.
  5. Dubey, J.P., Fayer, R. and Rao, J.R. (1992). Cryptosporidial oocysts in faeces of water buffalo and zebu calves in India. J. Vet. Parasitol. 6: 55-56. 
  6. Fayer, R., Santin, M, and Trout, J.M. (2007). Prevalence of Cryptosporidium species and genotypes in mature dairy cattle on farms in eastern United States compared with younger cattle from the same locations. Vet Parasitol.145:260–266. 
  7. Feng, Y., Ortega, Y., He, G., Das, P., Xu, M., Zhang, X., Fayer, R., Gatei, W., Cama, V. and Xiao, L. (2007). Wide geographic distribution of Cryptosporidium bovis and the deer-like genotype in bovines. Vet. Parasitol.144: 1–9.
  8. Hingole, A.C, Gudewar, J.G., Pednekar, R.P and Gatne, M.L. (2016). Prevalence and molecular characterization of Cryptosporidium species in cattle and buffalo calves in Mumbai region of India. J Parasit Dis. 41: 1-6.
  9. Karanis, P., Eiji, T., Palomino, L., Boonrod, K., Plutzer, J., Ongerth J. and Igarashi, I. (2010). First description of Cryptosporidium bovis in Japan and diagnosis and genotyping of Cryptosporidium spp. in diarrhoeic pre-weaned calves in Hokkaido. Vet Parasitol. 169: 387–390.
  10. Khan, S.M., Debnath, C., Pramanik, A.K., Xiao, L., Nozaki, T. and Ganguly, S. (2010). Molecular characterization and assessment of zoonotic transmission of Cryptosporidium from dairy cattle in West Bengal, India. Vet. Parasitol. 171: 41–47.
  11. Kumar, D., Sreekrishnan, R. and Das, S.S. (2004). Cryptosporidiosis in man and animals in Pondicherry. Indian. J. Ani. Sci. 74: 261-263. 
  12. Mallinath, R.H.K., Chikkachowdappa, P.G., Gowda, A.K.J. and D’Souza, P.E. (2009). Studies on the prevalence of cryptosporidiosis in bovines in organized dairy farms in and around Bangalore, South India.Veterinarski. Arhiv.79: 461-470. 
  13. Maurya, P.S., Rakesh, R.L., Pradeep, B., Kumar, S., Kundu, K., Garg, R., Ram, H., Kumar, A. and Banerjee P.S. (2013). Prevalence and risk factors associated with Cryptosporidium spp. infection in young domestic livestock in India. Trop. Anim. Health Pro. 45: 941-946. 
  14. Mohanty, B.N. and Panda, M.R. (2012). Prevalence of cryptosporidiosis in buffaloes in and around Bhubaneswar, Odisha. Indian J. Field Veterinarian. 8: 55-58. 
  15. Nasir, A., Avais, M., Khan, M.S. and Ahmad, N. (2009). Prevalence of Cryptosporidium parvum infection in Lahore (Pakistan) and its association with diarrhoea in dairy calves. Inter. J. Agri. Biology. 11: 221–224. 
  16. Navin, T.R. and Juranek, D.D. (1984). Cryptosporidiosis: Clinical, epidemiologic and parasitologic review. Rev. Infec. Dis. 6: 313.
  17. Nooruddin M. and Sarma, D.K. (1987). Role of Cryptosporidium in calf diarrhoea. Livestock Advisor: 12: 49–51. 
  18. OIE Terrestrial Manual .(2008). Cryptosporidiosis, Chapter 2.9.4, PP 1201
  19. Olson, M.E., Ralston, B.J., O’Handley, R., Guselle N. J. and Appelbee, A.J. (2003). What is the clinical and zoonotic significance of cryptosporidiosis in domestic animals and wildlife. In: Cryptosporidium: From Molecules to Disease.( eds R. C. A. Thompson A, Armson and Ryan U M ). Elsevier. Pp 51-68.
  20. Paul, S., Chandra, D., Tewari, A.K., Banerjee, P.S., Ray, D.D., Raina, O.K. and Rao, J.R. (2009). Prevalence of Cryptosporidium andersoni: a molecular epidemiological survey among cattle in India. Vet. Parasitol. 161: 31–35.
  21. Paul, S., Chandra, D., Ray, D.D., Tewari, A.K., Rao, J.R., Banerjee, P.S., Baidya, S. and Raina, O.K. 2008. Prevalence and molecular characterization of bovine Cryptosporidium isolates in India. Vet. Parasitol. 153: 143– 146. 
  22. Roy, S.S., Sarkar, S., Batabyal, S., Pramanik, A.K. and Das, P. (2006). Observation on the epidemiology of bovine Cryptosporidiosis in India. Vet. Parasitol.141: 330–333. 
  23. Sabry, A., Khodery, E. and Osman, S.A. (2008). Cryptosporidiosis in buffalo calves (Bubalus bubalis): Prevalence and potential risk factors. Trop. Anim. Health Prod.40: 419–426. 
  24. Sambrook, J. and Russell, D.W. (2001). Molecular cloning – a laboratory manual. Third Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, Newyork
  25. Shobhamani, B., Singari, N.A. and Syamasundar, N. (2006). A study on clinical manifestations of cryptosporidiosis in calves. Indian. Vet. J. 83: 677-678. 
  26. Snedecor, G.W. and Cochran, W.G. (1994). Statistical Methods, 8th edn. Iowa State University Press, Ames 505. 
  27. Swain, K., Routray, A., Sahoo, S., Ganguly, S. (2018). Cryptosporidium oocyst shedding in buffalo calves in Haryana: a case study. I    ndian J. Anim. Res. DOI: 10.18805/ijar.B-3487. 
  28. Venu. R., Latha B. R., Basith S. A., Sreekumar, C., Raj, G.D. and Raman, M. (2012). Factors influencing on prevalence of Cryptosporidium infection in south Indian dairy calves. J. Parasit. Dis. 37: 168-172.
  29. Xiao, L., Escalante, L., Yang, C., Sulaiman, I., Escalante, A. A., Monsali, R.J., Fayer, R. and Lal, A. A. (1999). Phylogenetic analysis of Cryptosporidium parasites based on the SSU rRNA gene locus. Appl. Environ. Microbiol. 65: 1578-1583. 
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Indian Journal of Animal Research
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    Research Article

    volume 53 issue 5 (may 2019) : 667-671,   Doi: 10.18805/ijar.B-3568

    Prevalence and molecular characterization of cryptosporidium species in buffalo calves of western Haryana

    K
    Krutanjali Swain
    S
    Sukhdeep Vohra
    A
    A.K. Sangwan
    A
    Abhilash Routray
    S
    Sumitra Panigrahi
    S
    Subha Ganguly
    1Department of Veterinary Parasitology, Faculty of Veterinary Science and Animal Husbandry, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar - 125 004, Haryana, India.
    Cite article:- Swain Krutanjali, Vohra Sukhdeep, Sangwan A.K., Routray Abhilash, Panigrahi Sumitra, Ganguly Subha (2018). Prevalence and molecular characterization of cryptosporidium species in buffalo calves of western Haryana. Indian Journal of Animal Research. 53(5): 667-671. doi: 10.18805/ijar.B-3568.

    ABSTRACT

    A sum total of 402 faecal samples from buffalo calves belonging to the age group of three months were collected randomly without any clinical symptom from various livestock farms and small holdings in four districts of Haryana. The presence or absence of Cryptosporidium spp. oocysts in faecal samples was confirmed by the standard microscopic examination of the modified Ziehl-Neelsen (MZN) stained faecal smears. The overall prevalence in buffalo calves was found to be 8.7%. The seasonal prevalence study revealed a maximum occurrence of Cryptosporidium infection during rainy followed by autumn, winters and minimum in summer season. Age related prevalence showed highest infection in calves between 16-30 days age which gradually decreased with increase in age. The DNA of representative positive faecal samples was extracted using standard phenol-chloroform-isoamylalcohol (PCI) method and then was subjected to nested PCR. All the extracted DNA were amplified for a 1325 bp primary fragment followed by a 834 bp nested fragment in a nested PCR of 18S rRNA gene, thereby confirming the presence of Cryptosporidium spp. The restricted enzyme digestion pattern (restricted fragment length polymorphism) of the 834 bp nested fragment using the restriction enzymes SspI and VspI confirmed the presence of C. parvum. Sequencing of Cryptosporidium DNA samples were carried out which also proved all samples to be of C. parvum. This is the first report of molecular identification of C. parvum in buffalo calves of Haryana and these Haryana isolates of Cryptosporidium parvum isolated from diarrhoeic buffalo calves are divergently related to rest of the part of the country isolates and indicates the zoonotic potential.

    REFERENCES

    1. Abeywardena, H., Jex, A. R., Samson-Himmelstjerna, G. V., Haydon, S. R., Stevens, M. A and Gasser, R. B. (2013). First molecular characterization of Cryptosporidium and Giardia from Bubalus bubalis (water buffalo) in Victoria, Australia. Infect. Genet. Evol. 20: 96-102. 
    2. Bhat, S.A., Juyal, P.D and Singla L.D. (2013). Distribution of bovine cryptosporidiosis in India. Trends in Parasitology Research. 2(2). 5-13X.
    3. Bhat, S.A., Juyal, P.D. and Singla, L.D. (2012). Prevalence of cryptosporidiosis in neonatal buffalo calves in Ludhiana district of Punjab. India. Asian J. Anim. Vet. Adv. 7: 512-520. 
    4. Chai, J.Y., Kim, N.Y, Guk, S.M., Park, Y.K., Seo, M., Han, E.T and Lee, S.H. (2001). High prevalence and seasonality of cryptosporidiosis in a small rural village occupied predominantly by aged people in the Republic of Korea. Am J. Trop. Med. Hyg. 65:518–522.
    5. Dubey, J.P., Fayer, R. and Rao, J.R. (1992). Cryptosporidial oocysts in faeces of water buffalo and zebu calves in India. J. Vet. Parasitol. 6: 55-56. 
    6. Fayer, R., Santin, M, and Trout, J.M. (2007). Prevalence of Cryptosporidium species and genotypes in mature dairy cattle on farms in eastern United States compared with younger cattle from the same locations. Vet Parasitol.145:260–266. 
    7. Feng, Y., Ortega, Y., He, G., Das, P., Xu, M., Zhang, X., Fayer, R., Gatei, W., Cama, V. and Xiao, L. (2007). Wide geographic distribution of Cryptosporidium bovis and the deer-like genotype in bovines. Vet. Parasitol.144: 1–9.
    8. Hingole, A.C, Gudewar, J.G., Pednekar, R.P and Gatne, M.L. (2016). Prevalence and molecular characterization of Cryptosporidium species in cattle and buffalo calves in Mumbai region of India. J Parasit Dis. 41: 1-6.
    9. Karanis, P., Eiji, T., Palomino, L., Boonrod, K., Plutzer, J., Ongerth J. and Igarashi, I. (2010). First description of Cryptosporidium bovis in Japan and diagnosis and genotyping of Cryptosporidium spp. in diarrhoeic pre-weaned calves in Hokkaido. Vet Parasitol. 169: 387–390.
    10. Khan, S.M., Debnath, C., Pramanik, A.K., Xiao, L., Nozaki, T. and Ganguly, S. (2010). Molecular characterization and assessment of zoonotic transmission of Cryptosporidium from dairy cattle in West Bengal, India. Vet. Parasitol. 171: 41–47.
    11. Kumar, D., Sreekrishnan, R. and Das, S.S. (2004). Cryptosporidiosis in man and animals in Pondicherry. Indian. J. Ani. Sci. 74: 261-263. 
    12. Mallinath, R.H.K., Chikkachowdappa, P.G., Gowda, A.K.J. and D’Souza, P.E. (2009). Studies on the prevalence of cryptosporidiosis in bovines in organized dairy farms in and around Bangalore, South India.Veterinarski. Arhiv.79: 461-470. 
    13. Maurya, P.S., Rakesh, R.L., Pradeep, B., Kumar, S., Kundu, K., Garg, R., Ram, H., Kumar, A. and Banerjee P.S. (2013). Prevalence and risk factors associated with Cryptosporidium spp. infection in young domestic livestock in India. Trop. Anim. Health Pro. 45: 941-946. 
    14. Mohanty, B.N. and Panda, M.R. (2012). Prevalence of cryptosporidiosis in buffaloes in and around Bhubaneswar, Odisha. Indian J. Field Veterinarian. 8: 55-58. 
    15. Nasir, A., Avais, M., Khan, M.S. and Ahmad, N. (2009). Prevalence of Cryptosporidium parvum infection in Lahore (Pakistan) and its association with diarrhoea in dairy calves. Inter. J. Agri. Biology. 11: 221–224. 
    16. Navin, T.R. and Juranek, D.D. (1984). Cryptosporidiosis: Clinical, epidemiologic and parasitologic review. Rev. Infec. Dis. 6: 313.
    17. Nooruddin M. and Sarma, D.K. (1987). Role of Cryptosporidium in calf diarrhoea. Livestock Advisor: 12: 49–51. 
    18. OIE Terrestrial Manual .(2008). Cryptosporidiosis, Chapter 2.9.4, PP 1201
    19. Olson, M.E., Ralston, B.J., O’Handley, R., Guselle N. J. and Appelbee, A.J. (2003). What is the clinical and zoonotic significance of cryptosporidiosis in domestic animals and wildlife. In: Cryptosporidium: From Molecules to Disease.( eds R. C. A. Thompson A, Armson and Ryan U M ). Elsevier. Pp 51-68.
    20. Paul, S., Chandra, D., Tewari, A.K., Banerjee, P.S., Ray, D.D., Raina, O.K. and Rao, J.R. (2009). Prevalence of Cryptosporidium andersoni: a molecular epidemiological survey among cattle in India. Vet. Parasitol. 161: 31–35.
    21. Paul, S., Chandra, D., Ray, D.D., Tewari, A.K., Rao, J.R., Banerjee, P.S., Baidya, S. and Raina, O.K. 2008. Prevalence and molecular characterization of bovine Cryptosporidium isolates in India. Vet. Parasitol. 153: 143– 146. 
    22. Roy, S.S., Sarkar, S., Batabyal, S., Pramanik, A.K. and Das, P. (2006). Observation on the epidemiology of bovine Cryptosporidiosis in India. Vet. Parasitol.141: 330–333. 
    23. Sabry, A., Khodery, E. and Osman, S.A. (2008). Cryptosporidiosis in buffalo calves (Bubalus bubalis): Prevalence and potential risk factors. Trop. Anim. Health Prod.40: 419–426. 
    24. Sambrook, J. and Russell, D.W. (2001). Molecular cloning – a laboratory manual. Third Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, Newyork
    25. Shobhamani, B., Singari, N.A. and Syamasundar, N. (2006). A study on clinical manifestations of cryptosporidiosis in calves. Indian. Vet. J. 83: 677-678. 
    26. Snedecor, G.W. and Cochran, W.G. (1994). Statistical Methods, 8th edn. Iowa State University Press, Ames 505. 
    27. Swain, K., Routray, A., Sahoo, S., Ganguly, S. (2018). Cryptosporidium oocyst shedding in buffalo calves in Haryana: a case study. I    ndian J. Anim. Res. DOI: 10.18805/ijar.B-3487. 
    28. Venu. R., Latha B. R., Basith S. A., Sreekumar, C., Raj, G.D. and Raman, M. (2012). Factors influencing on prevalence of Cryptosporidium infection in south Indian dairy calves. J. Parasit. Dis. 37: 168-172.
    29. Xiao, L., Escalante, L., Yang, C., Sulaiman, I., Escalante, A. A., Monsali, R.J., Fayer, R. and Lal, A. A. (1999). Phylogenetic analysis of Cryptosporidium parasites based on the SSU rRNA gene locus. Appl. Environ. Microbiol. 65: 1578-1583. 
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