Veterinary Research Communications

, Volume 43, Issue 2, pp 105–114 | Cite as

Genetic association of polymorphisms in bovine TLR2 and TLR4 genes with Mycobacterium avium subspecies paratuberculosis infection in Indian cattle population

  • Satish Kumar
  • Subodh Kumar
  • Ran Vir SinghEmail author
  • Anuj Chauhan
  • Amit Kumar
  • Sourabh Sulabh
  • Jaya Bharati
  • Shoor Vir Singh
Original Article


Toll like receptors (TLRs) are pattern recognition molecules involved in cellular recognition of Mycobacterium avium subspecies paratuberculosis (MAP), the infectious agent causing Paratuberculosis (PTB), a notified disease of domestic and wild ruminants. The present study was undertaken to investigate the presence of single nucleotide polymorphisms (SNPs) in TLR2 and TLR4 gene and to evaluate association of these SNPs with occurrence of PTB in Indian cattle. A total of 213 cattle, were subjected to multiple diagnostic tests viz. Johnin PPD, ELISA test (Indigenous and Parachek kit method), fecal microscopy and fecal culture for detection of MAP infection. Based on screening results 51 animals each were assigned to case and control population. Two SNPs viz. rs55617172, rs41830058 in TLR2 gene and two SNPs viz. rs8193046, rs8193060 in TLR4 gene and were genotyped by PCR-RFLP method. All SNPs were found to be polymorphic except rs41830058 in the case-control population. Both SNPs in TLR4 gene but none in TLR2 genes were significantly associated with the occurrence of PTB in our population. The genotypes in SNP rs8193046 and SNP rs8193060 were significantly (P < 0.01) different in case-control population. These findings suggest that SNPs rs8193046 and rs8193060 are likely a potential marker against MAP infection and a selection programme eliminating AG genotype for rs8193046 and CT genotype for rs8193060 might be beneficial in conferring resistance to MAP infection in Indian cattle population.


Paratuberculosis SNPs TLR2 TLR4 Immune response Resistance 



We thank Director, Indian Veterinary Research Institute, Izatnagar, India for funding this work.

Compliance with ethical standards

Conflict of interest

The authors do not have any conflict of interest.


  1. Agrawal G, Borody TJ, Chamberlin W (2014) Global warming’to Mycobacterium avium subspecies paratuberculosis. Future Microbiol 9(7):829–832Google Scholar
  2. Bastida F, Juste RA (2011) Paratuberculosis control: a review with a focus on vaccination. J Immune Based Ther Vaccines 9(1):8Google Scholar
  3. Bharati J, Dangi SS, Mishra SR, Chouhan VS, Verma V, Shankar O, Bharti MK, Paul A, Mahato DK, Rajesh G, Singh G (2017) Expression analysis of toll like receptors and interleukins in Tharparkar cattle during acclimation to heat stress exposure. J Therm Biol 65:48–56Google Scholar
  4. Bishop SC, Woolliams JA (2014) Genomics and disease resistance studies in livestock. Livest Sci 166:190–198Google Scholar
  5. Bulut Y, Michelsen KS, Hayrapetian L, Naiki Y, Spallek R, Singh M, Arditi M (2005) Mycobacterium tuberculosis heat shock proteins use diverse toll-like receptor pathways to activate pro-inflammatory signals. J Biol Chem 280(22):20961–20967Google Scholar
  6. Buwitt-Beckmann U, Heine H, Wiesmuller KH, Jung G, Brock R, Akira S, Ulmer AJ (2006) TLR1- and TLR6-independent recognition of bacterial lipopeptides. J Biol Chem 281:9049–9057Google Scholar
  7. Casanova JL, Abel L (2002) Genetic dissection of immunity to mycobacteria: the human model. Annu Rev Immunol 20(1):581–620Google Scholar
  8. Chang JS, Huggett JF, Dheda K, Kim LU, Zumla A, Rook GA (2006) Myobacterium tuberculosis induces selective up-regulation of TLRs in the mononuclear leukocytes of patients with active pulmonary tuberculosis. J Immunol 176:3010–3018Google Scholar
  9. Chaubey KK, Gupta RD, Gupta S, Singh SV, Bhatia AK, Jayaraman S, Kumar N, Goel A, Rathore AS, Sahzad, Sohal JS (2016) Trends and advances in the diagnosis and control of paratuberculosis in domestic livestock. Vet Q 36(4):203–227Google Scholar
  10. Collins MT (2002) Interpretation of a commercial bovine paratuberculosis enzyme-linked immunosorbent assay by using likelihood ratios. Clin Diagn Lab Immunol 9(6):1367–1371Google Scholar
  11. Cossu A, Rosu V, Paccagnini D, Cossu D, Pacifico A, Sechi LA (2011a) MAP3738c and MptD are specific tags of Mycobacterium avium subsp. paratuberculosis infection in type I diabetes mellitus. Clin Immunol 141(1):49–57Google Scholar
  12. Cossu D, Cocco E, Paccagnini D, Masala S, Ahmed N, Frau J, Marrosu MG, Sechi LA (2011b) Association of Mycobacterium avium subsp. paratuberculosis with multiple sclerosis in Sardinian patients. PLoS One 6(4):e18482Google Scholar
  13. Cristofaro P, Opal SM (2006) Role of toll-like receptors in infection and immunity. Drugs 66(1):15–29Google Scholar
  14. Dargatz DA, Byrum BA, Barber LK, Sweeney RW, Whitlock RH, Shulaw WP, Jacobson RH, Stabel JR (2001) Evaluation of a commercial ELISA for diagnosis of paratuberculosis in cattle. J Am Vet Med Assoc 218(7):1163–1166Google Scholar
  15. Dow CT, Ellingson JL (2010) Detection of Mycobacterium avium ss. Paratuberculosis in Blau syndrome tissues. Autoimmune Dis 2010:127692Google Scholar
  16. Edmonson AJ, Lean IJ, Weaver LD, Farver T, Webster G (1989) A body condition scoring chart for Holstein dairy cows. J Dairy Sci 72:68–78Google Scholar
  17. Fecteau ME (2018) Paratuberculosis in cattle. Vet Clin North Am Food Anim Pract 34:209–222Google Scholar
  18. Gonda MG, Chang YM, Shook GE, Collins MT, Kirkpatrick BW (2006) Genetic variation of Mycobacterium avium ssp. paratuberculosis infection in US Holsteins. J Dairy Sci 89:1804–1812Google Scholar
  19. Henckaerts L, Pierik M, Joossens M, Ferrante M, Rutgeerts P, Vermeire S (2007) Mutations in pattern recognition receptor genes modulate seroreactivity to microbial antigens in patients with inflammatory bowel disease. Gut 56:1536–1542Google Scholar
  20. Hong J, Leung E, Fraser AG, Merriman TR, Vishnu P, Krissansen GW (2007) TLR2, TLR4 and TLR9 polymorphisms and Crohn’s disease in a New Zealand Caucasian cohort. J Gastroenterol Hepatol 22:1760–1766Google Scholar
  21. Kalis CHJ, Barkema HW, Hesselink JW, Van Maanen C, Collins MT (2002) Evaluation of two absorbed ELISAs and a complement fixation test as replacements for fecal culture in the detection of cows shedding Mycobacterium avium subspecies paratuberculosis. Prev Vet Med 14:219–224Google Scholar
  22. Koets A, Santema W, Mertens H, Oostenrijk D, Keestra M, Overdijk M, Labouriau R, Franken P, Frijters A, Nielen M, Rutten VPMG (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(4):305–315Google Scholar
  23. Kralik P, Pribylova-Dziedzinska R, Kralova A, Kovarcik K, Slana I (2014) Evidence of passive faecal shedding of Mycobacterium avium subsp. paratuberculosis in a Limousin cattle herd. Vet J 201:91–94Google Scholar
  24. Kumar S, Kumar S, Singh R, Chauhan A, Agrawal S, Kumar A, Singh S (2017) Investigation of genetic Association of Single Nucleotide Polymorphisms in SP110 gene with occurrence of Paratuberculosis disease in cattle. Int J Livest Res 7(3):81–88Google Scholar
  25. Kumar S, Singh RV, Chauhan A, Kumar A, Bharati J, Singh SV (2019) Association of Bovine CLEC7A gene polymorphism with host susceptibility to paratuberculosis disease in Indian cattle. Res Vet Sci 123:216–222Google Scholar
  26. Lakatos PL, Lakatos L, Szalay F, Willheim-Polli C, Österreicher C, Tulassay Z, Molnar T, Reinisch W, Papp J, Mozsik G, Ferenci P (2005) Toll-like receptor 4 and NOD2/CARD15 mutations in Hungarian patients with Crohn’s disease: phenotype-genotype correlations. World J Gastroenterol 11(10):1489–1495Google Scholar
  27. Lavers CJ, Barkema HW, Dohoo IR, McKenna SLB, Keefe GP (2014) Evaluation of milk ELISA for detection of Mycobacterium avium subspecies paratuberculosis in dairy herds and association with within-herd prevalence. J Dairy Sci 97(1):299–309Google Scholar
  28. Magombedze G, Ngonghala CN, Lanzas C (2013) Evalution of the “iceberg phenomenon” in Johne’s disease through mathematical modelling. PLoS One 8:e76636NGoogle Scholar
  29. Masala S, Cossu D, Palermo M, Sechi LA (2014) Recognition of zinc transporter 8 and MAP3865c homologous epitopes by Hashimoto's thyroiditis subjects from Sardinia: a common target with type 1 diabetes? PLoS One 9(5):e97621Google Scholar
  30. McGuire K, Jones M, Werling D, Williams JL, Glass EJ, Jann O (2006) Radiation hybrid mapping of all 10 characterized bovine toll-like receptors. Anim Genet 37(1):47–50Google Scholar
  31. McKenna SLB, Keefe GP, Tiwari A, VanLeeuwen J, Barkema HW (2006) Johne’s disease in Canada part II: disease impacts, risk factors, and control programs for dairy producers. Can Vet J 47:1089–1099Google Scholar
  32. Menzies M, Ingham A (2006) Identification and expression of toll-like receptors 1–10 in selected bovine and ovine tissues. Vet Immunol Immunopathol 109(1–2):23–30Google Scholar
  33. Merkal RS, Miller JM, Hintz AM, Bryner JH (1982) Intrauterine inoculation of Mycobacterium paratuberculosis into Guinea pigs and cattle. Am J Vet Res 43:676–678Google Scholar
  34. Mucha R, Bhide MR, Chakurkar EB, Novak M, Mikula 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(4):381–388Google Scholar
  35. Nielsen SS, Toft N (2008) Ante mortem diagnosis of paratuberculosis: a review of accuracies of ELISA, interferon-g assay and faecal culture techniques. Vet Microbiol 129:217–235Google Scholar
  36. Oshiumi H, Matsumoto M, Funami K, Akazawa T, Seya T (2003) TICAM-1, an adaptor molecule that participates in toll-like receptor 3-mediated interferon-beta induction. Nat Immunol 4:161–167Google Scholar
  37. Pant SD, Verschoor CP, Schenkel FS, You Q, Kelton DF, Karrow NA (2014) Bovine CLEC7A genetic variants and their association with seropositivity in Johne's disease ELISA. Gene 537(2):302–307Google Scholar
  38. Pieper L, Sorge US, DeVries TJ, Godkin A, Lissemore K, Kelton DF (2015) Evaluation of the Johne’s disease risk assessment and management plan on dairy farms in Ontario, Canada. J Dairy Sci 98(10):6792–6800Google Scholar
  39. Pillars RB, Grooms DL, Kaneene JB (2009) Longitudinal study of the distribution of Mycobacterium avium subsp. paratuberculosis in the environment of dairy herds in the Michigan Johne’s disease control demonstration herd project. Can Vet J 50:1039–1046Google Scholar
  40. Pinedo PJ, Buergelt CD, Donovan GA, Melendez P, Morel L, Wu R, Langaee TY, Rae DO (2009) Candidate gene polymorphhisms (BoIFNG, TLR4, SLC11A1) as risk factors for paratuberculosis infection in cattle. Prev Vet Med 91(2):189–196Google Scholar
  41. Pinedo PJ, Galvão KN, Seabury CM (2013) Innate immune gene variation and differential susceptibility to uterine diseases in Holstein cows. Theriogenology 80:384–390Google Scholar
  42. Poltorak A, He X, Smirnova I, Liu MY, Van Huffel C, Du X, Birdwell D, Alejos E, Silva M, Galanos C, Freudenberg M, Ricciardi-Castagnoli P, Layton B, Beutler B (1998) Defective LPS signaling in C3H/ HeJ and C57BL/10ScCr mice: mutations in TLR4 gene. Science 282:2085–2088Google Scholar
  43. Prakash O, Kumar A, Sonwane A, Rathore R, Singh RV, Chauhan A, Kumar P, Renjith R, Yadav R, Bhaladhare A, Baqir M (2014) Polymorphism of cytokine and innate immunity genes associated with bovine brucellosis in cattle. Mol Biol Rep 41(5):2815–2825Google Scholar
  44. Quesniaux V, Fremond C, Jacobs M, Parida S, Nicolle D, Yeremeev V, Bihl F, Erard F, Botha T, Drennan M, Soler M, Le Bert M, Schnyder B, Ryffel B (2004) Toll-like receptor pathways in the immune responses to mycobacteria. Microbes Infect 6:946–959Google Scholar
  45. Roupie V, Alonso-Velasco E, Van Der Heyden S, Holbert S, Duytschaever L, Berthon P, Van Dosselaer I, Van Campe W, Mostin L, Biet F, Roels S (2018) Evaluation of mycobacteria-specific gamma interferon and antibody responses before and after a single intradermal skin test in cattle naturally exposed to M. Avium subsp. paratuberculosis and experimentally infected with M. Bovis. Vet Immunol Immunopathol 196:35–47Google Scholar
  46. Roura-Mir C, Wang L, Cheng TY, Matsunaga I, Dascher CC, Peng SL, Fenton MJ, Kirschning C, Moody DB (2005) Mycobacterium tuberculosis regulates CD1 antigen presentation pathways through TLR-2. J Immunol 175:1758–1766Google Scholar
  47. Ruiz-Larranaga O, Garrido JM, Manzano C, Iriondo M, Molina E, Gil A, Koets AP, Rutten VPMG, Juste RA, Estonba A (2010) Identification of single nucleotide polymorphisms in the bovine solute carrier family 11 member 1 (SLC11A1) gene and their association with infection by Mycobacterium avium subspecies paratuberculosis. J Dairy Sci 93(4):1713–1721Google Scholar
  48. Ruiz-Larrañaga O, Manzano C, Iriondo M, Garrido JM, Molina E, Vazquez P, Juste RA, Estonba A (2011) Genetic variation of toll-like receptor genes and infection by Mycobacterium avium ssp. paratuberculosis in Holstein-Friesian cattle. J Dairy Sci 94(7):3635–3641Google Scholar
  49. Ruiz-Larrañaga O, Vázquez P, Iriondo M, Manzano C, Aguirre M, Garrido JM, Juste RA, Estonba A (2017) Evidence for gene-gene epistatic interactions between susceptibility genes for Mycobacterium avium subsp. paratuberculosis infection in cattle. Livest Sci 195:63–66Google Scholar
  50. Sadana T, Singh RV, Singh SV, Saxena VK, Sharma D, Singh PK, Kumar N, Gupta S, Chaubey KK, Jayaraman S, Tiwari R (2015) Single nucleotide polymorphism of SLC11A1, CARD15, IFNG and TLR2 genes and their association with Mycobacterium avium subspecies paratuberculosis infection in native Indian cattle population. Indian J Biotechnol 14:469–475Google Scholar
  51. Sambrook J, Russel DW (2001) Molecular cloning- a laboratory manual, 3rd edn. Cold Spring Harbor laboratory Press, Cold Spring HarborGoogle Scholar
  52. Schukken YH, Whitlock RH, Wolfgang D, Grphn Y, Beaver A, VanKessel J, Zurakowski M, Mitchell R (2015) Longitudinal data collection of Mycobacterium avium subspecies paratuberculosis infections in dairy herds: the value of precise feld data. Vet Res 46:65Google Scholar
  53. Schwartz DA (2002) The genetics of innate immunity. Chest 121(3):62S–68SGoogle Scholar
  54. Sergeant ESG, Nielsen SS, Toft N (2008) Evaluation of test-strategies for estimating probability of low prevalence of paratuberculosis in Danish dairy herds. Prev Vet Med 85(1-2):92–106Google Scholar
  55. Sharma BS, Abo-Ismail MK, Schenkel FS, You Q, Verschoor CP, Pant SD, Karrow NA (2015) Association of TLR4 polymorphisms with Mycobacterium avium subspecies paratuberculosis infection status in Canadian Holsteins. Anim Genet 46(5):560–565Google Scholar
  56. Singh SV, Singh AV, Singh R, Sharma S, Shukla N, Misra S, Singh PK, Sohal JS, Kumar H, Patil PK, Misra P (2008) Sero-prevalence of bovine 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(5):419–433Google Scholar
  57. Singh SV, Dhama K, Chaubey KK, Kumar N, Singh PK, Sohal JS, Mahima CS, Deb R (2013) Impact of host genetics on susceptibility and resistance to Mycobacterium avium subspecies paratuberculosis in domestic ruminants. Pak J Biol Sci 16:251–266Google Scholar
  58. Singh SV, Singh PK, Singh AV, Sohal JS, Kumar N, Chaubey KK, Gupta S, Rawat KD, Kumar A, Bhatia AK, Srivastav AK (2014) ‘Bio-Load’and bio-type profiles of Mycobacterium avium subspecies paratuberculosis infection in the domestic livestock population endemic for Johne's disease: a survey of 28 years (1985–2013) in India. Transbound Emerg Dis 61(s1):43–55Google Scholar
  59. Smith RL, Al-Mamun MA, Gröhn YT (2017) Economic consequences of paratuberculosis control in dairy cattle: a stochastic modeling study. Prev Vet Med 138:17–27Google Scholar
  60. Sun WW, Lv WF, Cong W, Meng QF, Wang CF, Shan XF, Qian AD (2015) Mycobacterium avium subspecies paratuberculosis and bovine leukemia virus seroprevalence and associated risk factors in commercial dairy and beef cattle in northern and northeastern China. Biomed Res Int 2015:1–7. Google Scholar
  61. Sweeney RW (2011) Pathogenesis of paratuberculosis. Vet Clin North Am Food Anim Pract 27:537–546Google Scholar
  62. Sweeney RW, Collins MT, Koets AP, McGuirk SM, Roussel AJ (2012) Paratuberculosis (Johne’s disease) in cattle and other susceptible species. J Vet Intern Med 26:1239–1250Google Scholar
  63. Tabeta K, Georgel P, Janssen E, Du X, Hoebe K, Crozat K, Mudd S, Shamel L, Sovath S, Goode J, Alexopoulou L, Flavell RA, Beutler B (2004) Toll-like receptors 9 and 3 as essential components of innate immune defense against mouse cytomegalovirus infection. Proc Natl Acad Sci U S A 101:3516–3521Google Scholar
  64. Thuong NTT, Hawn TR, Thwaites GE, Chau TTH, Lan NTN, Quy HT, Hieu NT, Aderem A, Hien TT, Farrar JJ, Dunstan SJ (2007) A polymorphism in human TLR2 is associated with increased susceptibility to tuberculous meningitis. Genes Immun 8(5):422–428Google Scholar
  65. Uehori J, Fukase K, Akazawa T, Uematsu S, Akira S, Funami K, Shingai M, Matsumoto M, Azuma I, Toyoshima K, Kusumoto S, Seya T (2005) Dendritic cell maturation induced by muramyl dipeptide (MDP) derivatives: monoacylated MDP confers TLR2/TLR4 activation. J Immunol 174:7096–7103Google Scholar
  66. Vazquez P, Ruíz-Larrañaga O, Garrido JM, Iriondo M, Manzano C, Agirre M, Estonba A, Juste RA (2014) Genetic Association Analysis of Paratuberculosis Forms in Holstein-Friesian Cattle. Vet Med Int 2014:321327Google Scholar
  67. Vincze T, Posfai J, Roberts RJ (2003) NEBcutter: a program to cleave DNA with restriction enzymes. Nucleic Acids Res 31(13):3688–3691Google Scholar
  68. Wadhwa A, Foote RS, Shaw RW, Eda S (2012) Bead-based microfluidic immunoassay for diagnosis of Johne’s disease. J Immunol Methods 382(2):196–202Google Scholar
  69. Weiss DJ, Souza CD, Evanson OA, Sanders M, Rutherford M (2008) Bovine monocyte TLR2 receptors differentially regulate the intracellular fate of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium. J Leukoc Biol 83(1):48–55Google Scholar
  70. Werling D, Hope JC, Howard CJ, Jungi TW (2004) Differential production of cytokines, reactive oxygen and nitrogen by bovine macrophages and dendritic cells stimulated with toll-like receptor agonists. Immunology 111:41–52Google Scholar
  71. Werling D, Piercy J, Coffey TJ (2006) Expression of toll-like receptors (TLR) by bovine antigen-presenting cells: potential role in pathogen discrimination? Vet Immunol Immunopathol 112:2–11Google Scholar
  72. White SN, Kata SR, Womack JE (2003) Comparative fine maps of bovine toll-like receptor 4 and toll-like receptor 2 regions. Mamm Genome 14(2):149–155Google Scholar
  73. Whitlock RH, Wells SJ, Sweeney RW, Van Tiem J (2000) ELISA and fecal culture for paratuberculosis (Johne’s disease): sensitivity and specificity of each method. Vet Microbiol 77:387–398Google Scholar
  74. Yadav R, Sharma AK, Singh R, Sonwane A, Kumar A, Chauhan A, Kumar S, Kumar T, Renjith R, Bhaladhare A, Prakash O (2014) An association study of SNPs with susceptibility to bovine paratuberculosis infection in cattle. Indian J Anim Sci 84(5):490–493Google Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Satish Kumar
    • 1
    • 2
  • Subodh Kumar
    • 1
  • Ran Vir Singh
    • 1
    Email author
  • Anuj Chauhan
    • 1
  • Amit Kumar
    • 1
  • Sourabh Sulabh
    • 1
  • Jaya Bharati
    • 2
  • Shoor Vir Singh
    • 3
  1. 1.Division of Animal Genetics and BreedingICAR- Indian Veterinary Research InstituteBareillyIndia
  2. 2.ICAR-National Research Centre on PigGuwahatiIndia
  3. 3.Animal Health DivisionICAR- Central Institute for Research on GoatsMakhdoomIndia

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