Cluster of diffferention-14 (CD14) gene is involved in innate immunity and has been identified as one of the candidate gene for disease resistance. CD14 molecule works as cell surface receptor present on surface of inflammatory cells. Bovine CD14 gene spans over 2630 bp having 2 exons. Present study is based on identification of DNA polymorphism and to analyze the association between CD14 gene variants with incidence of mastitis in Sahiwal cattle. A total of 100 lactating Sahiwal cattle maintained at NDRI, Karnal (Haryana) were included in the present study. PCR-RFLP analysis of contig 4 using HinfI restriction enzyme showed three different genotypic patterns (CC, CD and DD). Remaining contig-enzyme combinations showed monomorphic pattern. Genotypic frequency of CC genotype was found highest (54%). Chi-square (2) analysis revealed that there is significant difference between these genotypes regarding mastitis incidence. CC genotyped animals were found to be less susceptible for mastitis.

Lactoferrin (Lf) gene promoter was screened for the presence of single nucleotide polymphism in indigenous and crossbred cattle from North India and to evaluate its association with Mastitis. Study revealed the presence of genetic variation in regulatory region of bovine Lactoferrin gene using PCR-RFLP technique. Three genotypes namely GG, GH and HH were identified. A single nucleotide change, from guanine to adenine at 25th position was found to be significantly associated (p<0.05) with clinical mastitis in indigenous Sahiwal and crossbred Karan Fries cattle maintained at organised herd of National Dairy Research Institute, Karnal. A non-significant association was observed between subclinical mastitis, somatic cell score (SCS), and GG genotype in Karan Fries cattle, however, a lower SCS was observed in animals having GG genotype. Overall a lower incidence of clinical mastitis was recorded in those animals having GG genotype of Lf in Sahiwal and Karan Fries (KF) cattle. The SNP identified in the promoter region may effect expression lactoferrin protein, which may lead to different levels of antibacterial and anti-inflammatory activity of Lf gene. Results from this study indicated the probable role played by Lactoferrin promoter to serve as candidate gene for mastitis susceptibility among indigenous and crossbred milch cattle.

Calcium channel, voltage-dependent, alpha-2/ delta subunit 1 (CACNA2D1) gene plays an important role in excitation-contraction coupling in muscle-cells, glial cells and neurons. The CACNA2D1 gene may be one of the candidate genes related with some phenotypic traits due to its location in QTLs associated with Somatic cell score (SCS ) and mastitis. A total of 120 Sahiwal cattle were selected to characterize exon 19 of CACNA2D1 gene to identify polymorphism and its association with mastitis susceptibility/resistance. A 249 bp PCR fragment of CACNA2D1 gene encompassing the exon 19 and partial intronic region was amplified and digested with Hae III to screen the SNP which was significantly associated with SCS. Genotype analysis using PCR-RFLP revealed a monomorphic banding pattern. Sequencing was also carried out to explore other SNPs which are deposited in dbSNP in the nucleotide sequence of a particular region. This study provides preliminary information that the targeted region of CACNA2D1 gene in indigenous cattle has no significant association with mastitis resistance which may be a breed specific characteristic.

Aim: The present study was undertaken with the objectives to characterize, identify DNA polymorphism in cluster of differentiation 14 (CD14) gene in Karan Fries (KF) cattle and to analyze association between genetic variants with incidence of clinical mastitis in National Dairy Research Institute (NDRI) herd, Karnal. Materials and Methods:Genomic DNA was extracted using blood of randomly selected hundred KF lactating cattle by phenol-chloroform method. After checking its quality and quantity, polymerase chain reaction (PCR) was carried out using reported primers to amplify 832 base pair region covering nucleotide base position number 1012 to 1843 (part of promoter, 5'UTR, exon 1, intron 1 and part of exon 2) of bovine CD14 gene. The PCR amplified target product was purified, sequenced and further ClustalW analysis was done to align edited sequence with reported Bos taurus sequence (EU148610.1). The restriction fragment length polymorphism (RFLP) analysis was performed for each KF cow using HinfI restriction enzyme (RE). Cows were assigned genotypes obtained by PCR-RFLP analysis and association study was done using Chi-square (χ 2 ) test.Results: After PCR amplification, DNA sequencing of amplicon confirmed the 832 bases covering 1012 to 1843 nucleotide base position of bovine CD14 gene. ClustalW multiple sequence alignment program for DNA revealed six nucleotide changes in KF cows at positions T1117D, T1239G, T1291C, G1359C, G1361A, and G1811A. Cows were also screened using PCR-RFLP with HinfI RE, which revealed three genotypes CC, CD and DD that differed significantly regarding mastitis incidence. Within CC genotype, 72.73% of cows were in a mastitis non-affected group whereas, those in CD and DD genotypes 69.44% and 60.38% respectively were mastitis affected. Conclusion:KF cows with allele C of CD14 gene were less susceptibility to mastitis compared with D allele.

Lactoferrin gene is one of the important candidate genes for mastitis resistance. The gene is located on chromosome BTA 22 and consists of 17 exons spanning over 34.5 kb of genomic DNA. The present study was undertaken with the objectives to identify allelic variants in exons 7 and 12 of lactoferrin gene and to analyze association between its genetic variants and incidence of clinical mastitis in Murrah buffalo. The amplification of exons 7 and 12 of lactoferrin gene yielded amplicons of 232- and 461-bp sizes. PCR-restriction fragment length polymorphism (RFLP) analysis of 232-bp amplicon using BccI restriction enzyme revealed three genotypes (AA, AB, and BB) with frequencies of 0.62, 0.22, and 0.16, respectively. The frequencies of two alleles, A and B, were estimated as 0.73 and 0.27. Hpy188I-RFLP for 461-bp amplicon revealed polymorphism with three genotypes, CC, CD, and DD, with respective frequencies of 0.06, 0.39, and 0.56, whereas frequencies for C and D alleles were 0.25 and 0.75. The chi-square (χ(2)) analysis revealed a significant association between incidence of clinical mastitis and genetic variants of exon 7, and animals of AA genotype of exon 7 were found to be least susceptible to mastitis. The findings indicate potential scope for incorporation of lactoferrin gene in selection and breeding of Murrah buffaloes for improved genetic resistance to mastitis.