Document Type : Original Article


1 PhD Candidate, Department of Microbiology and Immunology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

2 Department of Microbiology and Immunology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran


Major histocompatibility complex (MHC) represents an important genetic marker for manipulation to improve the health and productivity of cattle. It is closely associated with numerous disease susceptibilities and immune responses. Bovine MHC, also called bovine leukocyte antigen (BoLA), is considered as a suitable marker for genetic diversity studies. In cattle, most of the polymorphisms are located in exon 2 of BoLA-DRB3, which encodes the peptide-binding cleft. In this study, the polymorphism of the BoLA-DRB3.2 gene in Holstein's calves was studied using high resolution melting curve analysis (HRM). Observed HRM results were compared to PCR-RFLP and direct sequencing techniques. Eight different HRM and seven different RFLP profiles were identified among the population studied. By comparing to sequencing data, HRM could completely discriminate all genotypes (8 profiles), while the RFLP failed to distinguish between the genotypes *1101/*1001 and *1104/*1501. According to the results, the HRM analysis method gave more accurate results than RFLP by differentiating between the BoLA-DRB3.2 genotypes. Due to the Co-dominant nature of the MHC alleles, HRM technique could be used for investigating the polymorphisms of genotypes and their associations with immune responses.



    1. Radwan J, Biedrzycka A, Babik W. Does reduced MHC diversity decrease viability of vertebrate populations? Biol Conserv 2010; 143(3): 537-544.
    2. Gorer PA. The genetic and antigenic basis of tumour transplantation. J Pathol 1937; 44(3): 691-697.
    3. Bonner J. Major histocompatibility complex influences reproductive efficiency: Evolutionary implications. J Craniofac Genet Dev Biol Suppl 1986; 2: 5-11.
    4. Penn DJ, Potts WK. The evolution of mating preferences and major histocompatibility complex genes. Am Nat 1999; 153(2): 145-164.
    5. Graham RR, Ortmann W, Rodine P, et al. Specific combinations of HLA-DR2 and DR3 class II haplotypes contribute graded risk for disease susceptibility and autoantibodies in human SLE. Eur J Hum Genet 2007; 15(8): 823-830.
    6. Ovsyannikova IG, Pankratz VS, Vierkant RA, et al. Human leukocyte antigen haplotypes in the genetic control of immune response to measles-mumps-rubella vaccine. J Infect Dis 2006; 193(5): 655-663.
    7. Glass EJ, Oliver RA, Russell GC. Duplicated DQ haplotypes increase the complexity of restriction element usage in cattle. J Immunol 2000; 165(1): 134-138.
    8. Juliarena M, Poli M, Sala L, et al. Association of BLV infection profiles with alleles of the BoLA-DRB3.2 gene. Anim Genet 2008; 39(4): 432-438.
    9. Maillard JC, Berthier D, Chantal I, et al. Selection assisted by a BoLA-DR/DQ haplotype against susceptibility to bovine dermatophilosis. Genet Sel Evol 2003; 35(Suppl 1): S193-S200.
    10. Nikbakht Brujeni G, Ghorbanpour R, Esmailnejad A. Association of BoLA-DRB3.2 alleles with BLV infection profiles (persistent lymphocytosis/lymphosarcoma) and lymphocyte subsets in Iranian Holstein cattle. Biochem Genet 2016; 54(2): 194-207.
    11. Brown JH, Jardetzky TS, Gorga JC, et al. Three-dimensional structure of the human class II histocompatibility antigen HLA-DR1. Nature 1993; 364(6432): 33-39.
    12. Rothschild M, Skow L, Lamont S. The major histo-compatibility complex and its role in disease resistance and immune responsiveness. In: Axford RFE, Bishop SC, Nicholas FW, et al. (Eds). Breeding for disease resistance in farm animals. 2nd ed. Wallingford, UK: CAB International 2000; 73-105.
    13. Mosafer J, Heydarpour M, Manshad E, et al. Distribution of BoLA-DRB3 allelic frequencies and identification of two new alleles in Iranian buffalo breed. Sci World J 2012; 863024. doi: 10.1100/2012/863024.
    14. Ellis SA, Hammond JA. The functional significance of cattle major histocompatibility complex class I genetic diversity. Annu Rev Anim Biosci 2014; 2(1): 285-306.
    15. McShane R, Gallagher D, Newkirk H, et al. Physical localization and order of genes in the class I region of the bovine MHC. Anim Genet 2001; 32(5): 235-239.
    16. Dukkipati V, Blair H, Garrick D, et al. Ovar-Mhc'-ovine major histocompatibility complex: Structure and gene polymorphisms. Genet Mol Res 2006; 5(4): 581-608.
    17. Ackerman AL, Cresswell P. Cellular mechanisms governing cross-presentation of exogenous antigens. Nat Immunol 2004; 5(7): 678-684.
    18. Yakubu A, Salako AE, De Donato M, et al. Genetic diversity in exon 2 of the major histocompatibility complex class II DQB1 locus in Nigerian goats. Biochem Genet 2013; 51(11-12): 954-966.
    19. Wu XX, Yang ZP, Wang XL, et al. Restriction fragment length polymorphism in the exon 2 of the BoLA-DRB3 gene in Chinese Holstein of the south China. J Biomed Sci Eng 2010; 3: 221-225.
    20. Da Mota AF, Martinez ML, Coutinho LL. Genotyping BoLA-DRB3 alleles in Brazilian Dairy Gir cattle (Bos indicus) by temperature-gradient gel electrophoresis (TGGE) and direct sequencing. Eur J Immunogenet 2004; 31(1): 31-35.
    21. Emery D, Puri N, Dufty J, et al. A functional and biochemical analysis of bovine class II MHC antigens using monoclonal antibodies. Vet Immunol Immunopathol 1987; 16(3-4): 215-234.
    22. van der Poel JJ, Groenen MA, Dijkhof RJ, et al. The nucleotide sequence of the bovine MHC class II alpha genes: DRA, DQA, and DYA. Immunogenetics 1990; 31(1): 29-36.
    23. Glass E, Oliver R, Williams J, et al. Alloreactive T-cell recognition of bovine major histocompatibility complex class II products defined by one-dimensional isoelectric focusing. Anim Genet 1992; 23(2): 97-111.
    24. Sitte K, East IJ, Lavin MF, et al. Identification and characterization of new BoLA-DRB3 alleles by heteroduplex analysis and direct sequencing. Anim Genet 1995; 26(6): 413-417.
    25. Aldridge BM, McGuirk SM, Clark RJ, et al. Denaturing gradient gel electrophoresis: A rapid method for differentiating BoLA-DRB3 alleles. Anim Genet 1998; 29(5): 389-394.
    26. van Eijk MJ, Stewart-Haynes JA, Lewin HA. Extensive polymorphism of the BoLA-DRB3 gene distinguished by PCR-RFLP. Anim Genet 1992; 23(6): 483-496.
    27. Takeshima S, Nakai Y, Ohta M, et al. Characterization of DRB3 alleles in the MHC of Japanese shorthorn cattle by polymerase chain reaction-sequence-based typing. J Dairy Sci 2002; 85(6): 1630-1632.
    28. Reed GH, Wittwer CT. Sensitivity and specificity of single-nucleotide polymorphism scanning by high-resolution melting analysis. Clin Chem 2004; 50(10): 1748-1754.
    29. Lin JH, Tseng CP, Chen YJ, et al. Rapid differentiation of influenza A virus subtypes and genetic screening for virus variants by high-resolution melting analysis. J Clin Microbiol 2008; 46(3): 1090-1097.
    30. Li JH, Yin YP, Zheng HP, et al. A high-resolution melting analysis for geno-typing urogenital Chlamydia trachomatis. Diagn Microbiol Infect Dis 2010; 68(4): 366-374.
    31. Robertson T, Bibby S, O’Rourke D, et al. Identification of chlamydial species in crocodiles and chickens by PCR-HRM curve analysis. Vet Microbiol 2010; 145(3-4): 373-379.
    32. Simenc J, Potocnik U. Rapid differentiation of bacterial species by high resolution melting curve analysis. Prikl Biokhim Mikrobiol 2011; 47(3): 283-290.
    33. Holterman MHM, Oggenfuss M, Frey JE, et al. Evaluation of high-resolution melting curve analysis as a new tool for root-knot nematode diagnostics. J Phytopathol 2012; 160(2): 59-66.
    34. Ricchi M, Barbieri G, Cammi G, et al. High-resolution melting for analysis of short sequence repeats in Mycobacterium avium subsp. paratuberculosis. FEMS Microbiol Lett 2011; 323(2): 151-154.
    35. Ganopoulos I, Argiriou A, Tsaftaris A. Adulterations in Basmati rice detected quantitatively by combined use of microsatellite and fragrance typing with high resolution melting (HRM) analysis. Food Chem 2011; 129(2): 652-659.
    36. Wittwer CT, Reed GH, Gundry CN, et al. High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem 2003; 49(6): 853-860.
    37. Lundgren A, Kim S, Stadnisky MD, et al. Rapid discrimination of MHC class I and killer cell lectin-like receptor allele variants by high-resolution melt analysis. Immunogenetics 2012; 64(8): 633-640.
    38. Gelhaus A, Schnittger L, Mehlitz D, et al. Sequence and PCR-RFLP analysis of 14 novel BoLA-DRB3 alleles. Anim Genet 1995; 26(3): 147-153.
    39. Maillard JC, Renard C, Chardon P, et al. Characterization of 18 new BoLA-DRB3 alleles. Anim Genet 1999; 30(3): 200-203.
    40. Paswan C, Bhushan B, Patra BN, et al. Characterization of MHC DRB3.2 alleles of crossbred cattle by polymerase chain reaction-restriction fragment length polymorphism. Asian-Aust J Anim Sci 2005; 18(9): 1226-1230.
    41. Nassiri MR, Shahroudi FE, Tahmoorespur M, et al. The diversity of BoLA-DRB3 gene in Iranian native cattle. Asian-Aust J Anim Sci 2008; 21(4): 465-470.
    42. Rupp R, Hernandez A, Mallard BA. Association of bovine leukocyte antigen (BoLA) DRB3.2 with immune response, mastitis, and production and type traits in Canadian Holsteins. J Dairy Sci 2007; 90(2): 1029-1038.
    43. Oprządek J, Urtnowski P, Sender G, et al. Frequency of BoLA-DRB3 alleles in Polish Holstein-Friesian cattle. Anim Sci Pap Rep 2012; 30(2): 91-101.
    44. Yoshida T, Furuta H, Kondo Y, et al. Association of BoLA-DRB3 alleles with mastitis resistance and susceptibility in Japanese Holstein cows. Anim Sci J 2012; 83(5): 359-366.
    45. Schwab AE, Geary TG, Baillargeon P, et al. Association of BoLA DRB3 and DQA1 alleles with susceptibly to Neospora caninum and reproductive outcome in Quebec Holstein cattle. Vet Parasitol 2009; 165(1-2): 136-140.
    46. Takeshima SN, Aida Y. Structure, function and disease susceptibility of the bovine major histocompatibility complex. Anim Sci J 2006; 77(2): 138-150.