Journal of Genetics

, 99:14 | Cite as

Single-nucleotide polymorphisms in CLEC7A, CD209 and TLR4 gene and their association with susceptibility to paratuberculosis in Indian cattle

  • B. Gopi
  • Ran Vir SinghEmail author
  • Satish Kumar
  • Sushil Kumar
  • Anuj Chauhan
  • Amit Kumar
  • Shoor Vir Singh
Research Article


The aim of this study was to identify the single-nucleotide polymorphisms (SNPs) in bovine candidate genes CLEC7A, CD209 and TLR4, and explore the association between these SNPs with the occurrence of bovine paratuberculosis (PTB) disease. For this purpose, 549 animals were screened by a panel of four diagnostic tests, namely Johnin PPD test, ELISA test, faecal microscopy and IS900 blood PCR against Mycobacterium avium ssp. paratuberculosis (MAP) to develop case–control populations. SNPs were genotyped by polymerase chain reaction-restriction fragment length polymorphism method. Genotypic–phenotypic associations were assessed by the PROC-LOGISTIC procedure of SAS 9.3. Of the seven SNPs; rs110353594 in CLEC7A gene and rs8193046 in TLR4 gene were found to be associated with PTB. For SNP rs110353594, odds of CC and CT genotypes vs TT genotype was 1.543 (0.420–5.667; 95% CI) and 0.284 (0.104–0.774; 95% CI), respectively which means that CT genotype was more resistant than TT and CC genotypes against bovine PTB. For SNP rs8193046, odds of AA and AG genotypes versus GG genotype was 0.947 (0.296–3.034; 95% CI) and 3.947 (1.555–10.022; 95% CI), respectively, i.e. probability for getting an infection in animals with AG genotype was 3.94 times more as compared to GG genotype. Hence, a selection programme favouring CT genotype for rs110353594 and against AG genotype for rs8193046 may be beneficial for conferring resistance against bovine PTB.


immune response paratuberculosis resistance single-nucleotide polymorphism TLR4 gene. 



We thank Director, ICAR-Indian Veterinary Research Institute, Izatnagar, India for providing necessary facilities and funding of this work with grant no. IXX09774.


  1. Attalla S. A., Seykora A. J., Cole J. B. and Heins B. J. 2010 Genetic parameters of milk ELISA scores for Johne’s disease. J. Dairy Sci. 93, 1729–1735.PubMedCrossRefGoogle Scholar
  2. Bastida F. and Juste R. A. 2011 Paratuberculosis control: a review with a focus on vaccination. J. Immune Based Ther. Vaccines 9, 8.PubMedPubMedCentralCrossRefGoogle Scholar
  3. Benedictus G., Dijkhuizen A. A. and Stelwagen J. 1987 Economic losses due to paratuberculosis in dairy cattle. Vet. Rec. 121, 142–146.PubMedCrossRefGoogle Scholar
  4. Bishop S. C. and MacKenzieK. M. 2003 Genetic management strategies for controlling infectious diseases in livestock populations. Genet. Sel. Evol. 35, S3.PubMedPubMedCentralCrossRefGoogle Scholar
  5. Bharati J., Dangi S. S., Mishra S. R., Chouhan V. S., Verma V., Shankar O. et al. 2017 Expression analysis of toll like receptors and interleukins in Tharparkar cattle during acclimation to heat stress exposure. J. Therm. Biol. 65, 48–56.PubMedCrossRefGoogle Scholar
  6. Brown G. D., Herre J., Williams D. L., Willment J. A., Marshall A. S. and Gordon S. 2003 Dectin-1 mediates the biological effects of β-glucans. J. Exp. Med. 197, 1119–1124.PubMedPubMedCentralCrossRefGoogle Scholar
  7. Buza J. J., Mori Y., Bari A. M., Aodon-geril H. H., Hirayama S., Shu Y. et al. 2003 Mycobacterium avium subsp. paratuberculosis infection causes suppression of RANTES, monocyte chemoattractant protein 1, and tumor necrosis factor alpha expression in peripheral blood of experimentally infected cattle. Infect. Immun. 71, 7223–7227.PubMedPubMedCentralCrossRefGoogle Scholar
  8. Chang K., Deng S., Lu W., Wang F., Jia S., Li F et al. 2012 Association between CD209-336A/G and-871A/G polymorphisms and susceptibility of tuberculosis: a meta-analysis. PLoS One 7, e41519.PubMedPubMedCentralCrossRefGoogle Scholar
  9. Chase C. C., Hurley D. J. and Reber A. J. 2008 Neonatal immune development in the calf and its impact on vaccine response. Vet. Clin. North Am. Food Anim. Pract. 24, 87–104.PubMedCrossRefGoogle Scholar
  10. Cinar M. U., Hizlisoy H., Akyüz B., Arslan K., Aksel E. G. and Gümüşsoy K. S. 2018 Polymorphisms in toll-like receptor (TLR) 1, 4, 9 and SLC11A1 genes and their association with paratuberculosis susceptibility in Holstein and indigenous crossbred cattle in Turkey. J. Genet. 97, 1147–1154.CrossRefGoogle Scholar
  11. Correa-Valencia N. M., Ramírez N. F., Olivera M. and Fernández-Silva J. A. 2016 Milk yield and lactation stage are associated with positive results to ELISA for Mycobacterium avium subsp. paratuberculosis in dairy cows from Northern Antioquia, Colombia: a preliminary study. Trop. Anim. Health Prod. 48, 1191–1200.PubMedCrossRefGoogle Scholar
  12. Coussens P. M., Verman N., Coussens M. A., Elftman M. D. and McNulty A. M. 2004 Cytokine gene expression in peripheral blood mononuclear cells and tissues of cattle infected with Mycobacterium avium subsp. paratuberculosis: evidence for an inherent proinflammatory gene expression pattern. Infect. Immun. 72, 1409–1422.PubMedPubMedCentralCrossRefGoogle Scholar
  13. Ellingson J. L., Anderson J. L., Koziczkowski J. J., Radcliff R. P., Sloan S. J., Allen S. E. et al. 2005 Detection of viable Mycobacterium avium subsp. paratuberculosis in retail pasteurized whole milk by two culture methods and PCR. J. Food Prot. 68, 966–972.PubMedCrossRefGoogle Scholar
  14. Ferwerda G., Meyer‐Wentrup F., Kullberg B. J., Netea M. G. and Adema G. J. 2008 Dectin‐1 synergizes with TLR2 and TLR4 for cytokine production in human primary monocytes and macrophages. Cell Microbiol. 10, 2058–2066.PubMedCrossRefGoogle Scholar
  15. Fridriksdottir V., Gunnarsson E., Sigurdarson S. and Gudmundsdottir K. B. 2000 Paratuberculosis in Iceland: epidemiology and control measures, past and present. Vet. Microbiol. 77, 263–267.PubMedCrossRefGoogle Scholar
  16. Gantner B. N., Simmons R. M., Canavera S. J., Akira S. and Underhill D. M. 2003 Collaborative induction of inflammatory responses by dectin-1 and Toll-like receptor 2. J. Exp Med. 197, 1107–1117.PubMedPubMedCentralCrossRefGoogle Scholar
  17. Gao Y., Jiang J., Yang S., Cao J., Han B., Wang Y. et al. 2018 Genome-wide association study of Mycobacterium avium subspecies paratuberculosis infection in Chinese Holstein. BMC genomics. 19, 972.PubMedPubMedCentralCrossRefGoogle Scholar
  18. Herre J., Gordon S. and Brown G. D. 2004. Dectin-1 and its role in the recognition of β-glucans by macrophages. Mol. Immunol. 40, 869–876.PubMedCrossRefGoogle Scholar
  19. Jensen K., Makins G. D., Kaliszewska A., Hulme M. J., Paxton E. and Glass E. J. 2009 The protozoan parasite Theileria annulata alters the differentiation state of the infected macrophage and suppresses musculoaponeurotic fibrosarcoma oncogene (MAF) transcription factors. Int. J. Parasitol. 39, 1099–1108.PubMedPubMedCentralCrossRefGoogle Scholar
  20. Juste R. A. and Perez V. 2011 Control of paratuberculosis in sheep and goats. Vet. Clin. North Am. Food Anim. Pract. 27, 127–138.PubMedCrossRefGoogle Scholar
  21. Koets A. P., Koets G., Adugna L. L., Janss H. J., Van waeering C. H., Kalis G. H. et al. 2000 Genetic variation of susceptibility to Mycobacterium avium subsp. paratuberculosis infection in dairy cattle. J. Dairy Sci. 83, 2702–2708.CrossRefGoogle Scholar
  22. Koets A., Santema W., Mertens H., Oostenrijk D., Keestra M., Overdijk M. et al. 2010 Susceptibility to paratuberculosis infection in cattle is associated with single nucleotide polymorphisms in toll-like receptor 2 which modulate immune responses against Mycobacterium avium subspecies paratuberculosis. Prev. Vet. Med. 93, 305–315.PubMedCrossRefGoogle Scholar
  23. Kumar S. 2015 Single nucleotide polymorphism in candidate genes and their association with occurrence of paratuberculosis in cattle. M.V.Sc. Thesis. IVRI (Deemed University), Bareilly, UP, India.Google Scholar
  24. Kumar N., Ganguly I., Singh R., Deb S. M., Kumar S., Sharma A. et al. 2011 DNA polymorphism in the SLC11A1 gene and its association with brucellosis resistance in Indian zebu (Bos indicus) and crossbred (Bos indicus× Bos taurus) Cattle. Asian-Australas. J. Anim. Sci. 24, 898–904.CrossRefGoogle Scholar
  25. Kumar S., Kumar S., Singh R., Chauhan A., Agrawal S., Kumar A. et al. 2017 Investigation of genetic association of single nucleotide polymorphisms in SP110 Gene with occurrence of paratuberculosis disease in Cattle. Int. J. Livest. Res. 7, 81–88.Google Scholar
  26. Kumar S., Kumar S., Singh R. V., Chauhan A., Kumar A., Bharati J. et al. 2019a Association of Bovine CLEC7A gene polymorphism with host susceptibility to paratuberculosis disease in Indian cattle. Res. Vet. Sci. 123, 216–222.PubMedCrossRefGoogle Scholar
  27. Kumar S., Kumar S., Singh, R. V., Chauhan A., Kumar A., Sulabh S. et al. 2019b. Genetic association of polymorphisms in bovine TLR2 and TLR4 genes with Mycobacterium avium subspecies paratuberculosis infection in Indian cattle population. Vet. Res. Commun. 43, 105–114.PubMedCrossRefGoogle Scholar
  28. Küpper J., Brandt H., Donat K. and Erhardt G. 2012 Heritability estimates for Mycobacterium avium subspecies paratuberculosis status of German Holstein cows tested by fecal culture. J. Dairy Sci. 95, 2734–2739.PubMedCrossRefGoogle Scholar
  29. Mucha R., Bhide M. R., Chakurkar E. B., Novak M. and Mikula Sr I. 2009 Toll-like receptors TLR1, TLR2 and TLR4 gene mutations and natural resistance to Mycobacterium avium subsp. paratuberculosis infection in cattle. Vet. Immunol. Immunopathol. 128, 381–388.PubMedCrossRefGoogle Scholar
  30. Nielsen S. S. and Toft N. 2008 Ante mortem diagnosis of paratuberculosis: a review of accuracies of ELISA, interferon-γ assay and faecal culture techniques. Vet. Microbiol. 129, 217–235.PubMedCrossRefGoogle Scholar
  31. Pant S. D., Verschoor C.P., Schenkel F. S., You Q., Kelton D. F. and Karrow N. A. 2011 Bovine PGLYRP1 polymorphisms and their association with resistance to Mycobacterium avium ssp. paratuberculosis. Anim. Genet. 42, 354–360.PubMedCrossRefGoogle Scholar
  32. Pant S.D., Verschoor C. P., Schenkel F. S., You Q., Kelton D.F. and Karrow N. A. 2014 Bovine CLEC7A genetic variants and their association with seropositivity in Johne’s disease ELISA. Gene 537, 302–307.PubMedCrossRefGoogle Scholar
  33. Pieper L., Sorge U. S., DeVries T. J., Godkin A., Lissemore K. and Kelton D. F. 2015 Evaluation of the Johne’s disease risk assessment and management plan on dairy farms in Ontario, Canada. J. Dairy Sci. 98, 6792–6800.PubMedCrossRefGoogle Scholar
  34. Pinedo P. J., Buergelt C. D., Donovan G. A., Melendez P., Morel L., Wu R. et al. 2009 Candidate gene polymorphisms (BoIFNG, TLR4, SLC11A1) as risk factors for paratuberculosis infection in cattle. Prev. Vet. Med. 91, 189–196.PubMedCrossRefGoogle Scholar
  35. Prajapati B. M., Gupta J. P., Pandey D. P., Parmar G. A. and Chaudhari J. D. 2017 Molecular markers for resistance against infectious diseases of economic importance. Vet. World. 10, 112–120.PubMedPubMedCentralCrossRefGoogle Scholar
  36. Prakash O., Kumar A., Sonwane A., Rathore R., Singh R. V., Chauhan A. et al. 2014 Polymorphism of cytokine and innate immunity genes associated with bovine brucellosis in cattle. Mol. Biol. Rep. 41, 2815–2825.PubMedCrossRefGoogle Scholar
  37. Quesniaux V., Fremond C., Jacobs M., Parida S., Nicolle D., Yeremeev V. et al. 2004 Toll-like receptor pathways in the immune responses to mycobacteria. Microbes Infect. 6, 946–959.PubMedCrossRefGoogle Scholar
  38. Ruiz‐Larrañaga O., Garrido J. M., Iriondo M., Manzano C., Molina E., Koets A. P. et al. 2010a Genetic association between bovine NOD2 polymorphisms and infection by Mycobacterium avium subsp. paratuberculosis in Holstein‐Friesian cattle. Anim. Genet. 41, 652–655.PubMedCrossRefGoogle Scholar
  39. Ruiz-Larrañaga O., Garrido J. M., Iriondo M., Manzano C., Molina E., Montes I. et al. 2010b SP110 as a novel susceptibility gene for Mycobacterium avium subspecies paratuberculosis infection in cattle. J. Dairy Sci. 93, 5950–5958.PubMedCrossRefGoogle Scholar
  40. Ruiz-Larrañaga O., Manzano C., Iriondo M., Garrido J. M., Molina E., Vazquez P. et al. 2011 Genetic variation of toll-like receptor genes and infection by Mycobacterium avium ssp. paratuberculosis in Holstein-Friesian cattle. J. Dairy Sci. 94, 3635–3641.PubMedCrossRefGoogle Scholar
  41. Ruiz-Larrañaga O., Iriondo M. and Manzano C. 2012 Single-nucleotide polymorphisms in the bovine CD209 candidate gene for susceptibility to infection by Mycobacterium avium subsp. paratuberculosis. Anim Genet. 43, 646–647.PubMedCrossRefGoogle Scholar
  42. Ruiz-Larrañaga O., Vázquez P., Iriondo M., Manzano C., Aguirre M., Garrido J. M. et al. 2017 Evidence for gene-gene epistatic interactions between susceptibility genes for Mycobacterium avium subsp. paratuberculosis infection in cattle. Livest. Sci. 195, 63–66.CrossRefGoogle Scholar
  43. Sadana T., Singh R. V., Singh S. V., Saxena V. K., Sharma D., Singh P. K. et al. 2015 Single nucleotide polymorphism of SLC11A 1, CARD15, IFNG and TLR2 genes and their association with Mycobacterium avium subspecies paratuberculosis infection in native Indian cattle population. Indian J. Biotechnol. 14, 469–475.Google Scholar
  44. Sambrook J. and Russel D. W. 2001 Molecular cloning- A laboratory manual. 3rd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.Google Scholar
  45. Sharma B. S., Abo‐Ismail M. K., Schenkel F. S., You Q., Verschoor C. P., Pant S. D. et al. 2015 Association of TLR 4 polymorphisms with Mycobacterium avium subspecies paratuberculosis infection status in Canadian Holsteins. Anim. Genet. 46, 560–565.PubMedCrossRefGoogle Scholar
  46. Shinkai H., Toki D., Okumura N., Takenouchi T., Kitani H. and Uenishi H. 2016 Polymorphisms of the immune-modulating receptor dectin-1 in pigs: their functional influence and distribution in pig populations. Immunogenetics. 68, 275–284.PubMedCrossRefGoogle Scholar
  47. Singh S. V., Singh A. V., Singh R., Sharma S., Shukla N., Misra S. et al. 2000 Sero-Prevalence of Johne’s disease in buffaloes and cattle population of North India using indigenous ELISA kit based on native Mycobacterium avium subspecies paratuberculosis ‘Bison Type’ Genotype of Goat Origin, Comp. Immunol. Microbiol. Infect. Dis. 31, 419–433.CrossRefGoogle Scholar
  48. Singh S. V., Singh P. K., Gupta S., Chaubey K. K., Singh B., Kumar A. et al. 2013a Comparison of microscopy and blood-PCR for the diagnosis of clinical Johne’s disease in domestic ruminants. Iran J. Vet. Res. 14, 345–349.Google Scholar
  49. Singh S. V. Dhama K., Chaubey K. K., Kumar N., Singh P. K., Sohal J. S. et al. 2013b Impact of host genetics on susceptibility and resistance to Mycobacterium avium subspecies paratuberculosis infection in domestic ruminants. Pak. J. Biol. Sci. 16, 251–266.PubMedCrossRefGoogle Scholar
  50. Singh S. V., Singh A. V., Singh P. K., Gupta S., Singh H., Singh B. et al. 2013c Evaluation of ‘Indigenous Vaccine’ developed using ‘Indian Bison Type’Genotype of Mycobacterium avium subspecies paratuberculosis Strain ‘S5’of goat origin in a sheep flock endemic for johne’s disease: a three years trial in India. World J. Vaccines. 3, 52–59.CrossRefGoogle Scholar
  51. Slana I., Paolicchi F., Janstova B., Navratilova P. and Pavlik I. 2008 Detection methods for Mycobacterium avium subsp paratuberculosis in milk and milk products: a review. Vet. Med. (Praha). 53, 283.Google Scholar
  52. Tiwari A., VanLeeuwen J. A., McKenna S.L., Keefe G. P. and Barkema H. W. 2006 Johne’s disease in Canada: part I: clinical symptoms, pathophysiology, diagnosis, and prevalence in dairy herds. Can. Vet. J. 47, 874–882.PubMedPubMedCentralGoogle Scholar
  53. Tiwari A., VanLeeuwen J. A., Dohoo I. R., Keefe G. P., Haddad J. P., Scott H. M. et al. 2009 Risk factors associated with Mycobacterium avium subspecies paratuberculosis seropositivity in Canadian dairy cows and herds. Prev. Vet. Med. 88, 32–41.PubMedCrossRefGoogle Scholar
  54. Vary P. H., Andersen P. R., Green E., Hermon-Taylor J. and McFadden J. J. 1990 Use of highly specific DNA probes and the polymerase chain reaction to detect Mycobacterium paratuberculosis in Johne’s disease. J. Clin. Microbiol. 28, 933–937.PubMedPubMedCentralCrossRefGoogle Scholar
  55. Vazquez P., Ruíz-Larrañaga O., Garrido J. M., Iriondo M., Manzano C., Agirre M. et al. 2014 Genetic association analysis of paratuberculosis forms in Holstein-Friesian Cattle, HindawiPublishing Corporation. Vet. Med. Int. 2014, Article ID 321327.Google Scholar
  56. Verschoor C. P., Pant S. D., You Q., Schenkel F. S., Kelton D. F. and Karrow N. A. 2010 Polymorphisms in the gene encoding bovine interleukin-10 receptor alpha are associated with Mycobacterium avium ssp. paratuberculosis infection status. BMC Genet. 11, 23.PubMedPubMedCentralCrossRefGoogle Scholar
  57. Vincze T., Posfai J. and Roberts R. J. 2003 NEBcutter: a program to cleave DNA with restriction enzymes. Nucleic Acids Res. 31, 3688–3691.PubMedPubMedCentralCrossRefGoogle Scholar
  58. Weiss D. J., Evanson O. A., Deng M. and Abrahamsen M. S. 2004 Sequential patterns of gene expression by bovine monocyte-derived macrophages associated with ingestion of mycobacterial organisms. Microb. Pathog. 37, 215–224.PubMedCrossRefPubMedCentralGoogle Scholar
  59. Willcocks S., Yamakawa Y., Stalker A., Coffey T. J., Goldammer T. and Werling D. 2006 Identification and gene expression of the bovine C-type lectin Dectin-1. Vet. Immunol. Immunopathol. 113, 234–242.PubMedCrossRefGoogle Scholar
  60. Yadav M. and Schorey J. S. 2006 The β-glucan receptor dectin-1 functions together with TLR2 to mediate macrophage activation by mycobacteria. Blood 108, 3168–3175.PubMedPubMedCentralCrossRefGoogle Scholar
  61. Yadav R., Sharma A. K., Singh R., Sonwane A., Kumar A., Chauhan A. et al. 2014 An association study of SNPs with susceptibility to bovine paratuberculosis infection in cattle. Indian J. Anim. Sci. 84, 490–493.Google Scholar
  62. Yang G., Zhou H., Hu J., Luo Y. and Hickford J. G. 2011 Variation in the yak dectin-1 gene (CLEC7A). DNA Cell Biol. 30, 1069–1071.PubMedCrossRefGoogle Scholar
  63. Zhou H., Hu J., Luo Y. and Hickford J. G. 2010 Variation in the ovine C-type lectin dectin-1 gene (CLEC7A). Dev. Comp. Immunol. 34, 246–249.PubMedCrossRefGoogle Scholar

Copyright information

© Indian Academy of Sciences 2020

Authors and Affiliations

  1. 1.Division of Animal GeneticsICAR-Indian Veterinary Research InstituteIzatnagarIndia
  2. 2.ICAR-National Research Centre on PigRani, GuwahatiIndia
  3. 3.ICAR-Central Institute for Research on GoatMakhdoomIndia

Personalised recommendations