Anuj Chauhan

Single nucleotide polymorphisms in toll-like receptor genes and case-control association studies with bovine tuberculosis

Aim: Toll-like receptor 2 (TLR2) and TLR4 genes play critical roles in host recognition of Mycobacterium bovis infection and initiation of innate and adaptive immune response. The present study was aimed at exploring the association of seven single nucleotide polymorphisms (SNPs) in TLR2 and TLR4 genes with susceptibility/resistance against bovine tuberculosis (bTB) infection in cattle. Materials and Methods: A case-control resource population of 35 positive and 45 negative animals was developed after screening with single intradermal tuberculin test for bTB. Resource population was screened for SNPs in TLR2 and TLR4 genes using polymerase chain reaction-restriction fragment length polymorphism. The PROC LOGISTIC procedure of SAS 9.3 was used to find an association of allelic and genotypic frequencies with bTB. Results: In TLR2 gene, two of SNPs under study (rs55617172 and rs68268253) revealed polymorphism while in the case of TLR4 gene all four SNPs under investigation (rs8193041, rs207836014, rs8193060, and rs8193069) were found to be polymorphic in case-control population. SNP locus rs55617172 in TLR2 gene was found significantly (p<0.01) associated with susceptibility/resistance to TB in cattle. Conclusion: These findings indicate the presence of SNPs in TLR2 and TLR4 genes in our resource population. Upon validation in independent, large resource population and following biological characterization, SNP rs55617172 can be incorporated in marker panel for selection of animals with greater resistance to bTB.

Effect of heat shock protein 70 polymorphism on thermotolerance in Tharparkar cattle

Aim: Out of various members of heat shock protein (HSP) superfamily which act a molecular chaperon by binding to the denaturing protein thus stabilizing them and preserving their activity, HSP70 are of major importance in thermotolerance development. Thus, present investigation aimed at a screening of HSP70 gene for polymorphisms and possible differences in thermotolerance in Tharparkar breed of cattle. Materials and Methods: A 295 bp fragment of HSP70 gene was subjected to polymerase chain reaction-single-strand conformation polymorphism (SSCP) followed by sequencing of different SSCP patterns in 64 Tharparkar cattle. A comparative thermotolerance of identified genotypes was analyzed using heat tolerance coefficients (HTCs) of animals for different seasons. Results: Three SSCP patterns and consequently two alleles namely A and B were documented in one fragment of HSP70 gene. On sequencing, one single-nucleotide polymorphism with G > T substitution was found at a position that led to a change of amino acid aspartate to tyrosine in allele A. It was found that in maintaining near normal average rectal temperature, genotype AA was superior (p≤0.01). Genotype AA, thus, was found to be most thermotolerant genotype with the highest HTC (p≤0.01). Conclusion: The polymorphism at HSP70 is expected to be a potent determinant for heat tolerance in cattle, which may aid in selection for thermotolerance in cattle.

Association of ATP1A1 gene polymorphism with thermotolerance in Tharparkar and Vrindavani cattle.

Aim: One of the major biochemical aspects of thermoregulation is equilibrium of ion gradient across biological membranes. Na+/K+-ATPase, a member of P type-ATPase family, is a major contributor to the mechanism that actively controls cross-membrane ion gradient. Thus, we examined ATP1A1 gene that encodes alpha-1 chain of Na+/K+-ATPase, for genetic polymorphisms. Materials and Methods: A total of 100 Vrindavani (composite cross strain of Hariana x Holstein-Friesian/Brown Swiss/Jersey) and 64 Tharparkar (indigenous) cattle were screened for genetic polymorphism in ATP1A1 gene, using polymerase chain reaction single-strand conformation polymorphism and DNA sequencing. For association studies, rectal temperature (RT) and respiration rate (RR) of all animals were recorded twice daily for 3 seasons. Results: A SNP (C2789A) was identified in exon 17 of ATP1A1 gene. Three genotypes namely CC, CA, and AA were observed in both, Vrindavani and Tharparkar cattle. The gene frequencies in Tharparkar and Vrindavani for allele A were 0.51 and 0.48, and for allele C were 0.49 and 0.52, respectively, which remained at intermediate range. Association study of genotypes with RT and RR in both cattle population revealed that the animals with genotype CC exhibited significantly lower RT and higher heat tolerance coefficient than CA and AA genotypes. Conclusion: Differential thermoregulation between different genotypes of ATP1A1 gene indicate that the ATP1A1 gene could be potentially contributing to thermotolerance in both, Tharparkar, an indigenous breed and Vrindavani, a composite crossbred cattle.

INFLUENCE OF AMBIENT TEMPERATURE AND HUMIDITY ON ATP1A1 GENE EXPRESSION IN THARPARKAR AND VRINDAVANI CATTLE

The Na+/K+-ATPase is responsible for maintenance of the Na+ and K+gradients across the plasma membrane and responds to alteration in oxidative voltage and osmotic changes across membranes. ATP1A1 gene encodes for the a-1 chain of the Na+/K+-ATPase and there exists a positive linear correlation between Na+/K+-ATPase activity and mRNA level of a-1 isoforms. Present study aimed at exploring influence of temperature-humidity variation in winter, spring and summer test-periods on the expression of ATP1A1between Tharparkar and Vrindavani (cross of Holstein Frisian, Jersey and Brown Swiss withHariana) cattle. The value for THI were highest for summer (84.34), followed by spring (68.25) and lowest for winter (52.72),thus spring was consideredas reference season.Vrindavaniand Tharparkar both showed up-regulation of ATP1A1 expression in summer as well as inwinter.Higherup-regulationwas observed in summer. The up-regulation in Vrindavani was significantly higher than Tharparkar in both winter and summer seasons.

Association of polymorphisms in SLC11A1 gene with bovine tuberculosis trait among Indian cattle

ABSTRACT In the present study, polymorphisms at two single nucleotide polymorphisms (SNPs) and one microsatellite locus of SLC11A1 gene were investigated for finding their association with susceptibility to bovine tuberculosis trait (tuberculin reaction) in Indian cattle. A total of 245 animals were tested with single intradermal tuberculin test for screening positive animals for tuberculin reaction. At rs109915208 locus very low polymorphism information content of 0.0454 was observed, whereas it was moderate (0.352) at rs109453173 locus of SLC11A1. At rs109915208 locus the genotypic as well as allelic frequencies were differing significantly (p-value < .05) in case–control animals where the odds ratio (OR) of ‘CC’ verses ‘CT’ genotype and the OR of ‘C’ verses ‘T’ allele were approaching towards infinity, suggesting that animals having ‘CT’ genotype and ‘T’ allele were less susceptible for tuberculin reaction as compared to their contemporary genotype/allele. The significantly associated SNP was non-synonymous causing an amino acid change.

Exploring the molecular basis of resistance/ susceptibility to mixed natural infection of Haemonchus contortus in tropical Indian goat breed

The present investigation was carried out with the objective to identify putative candidate genes / Quantitative trait loci for resistance / susceptibility towards Haemonchus infestation in tropical goat breed (Rohilkhandi goat) of India. The mean faecal egg count (FEC) and packed cell volume (PCV) of the population were 142.78 ± 22.54 epg (eggs per gram) and 31.73% ± 0.49, respectively. Grouping of animals as per dot ELISA test showed 41.33% (n = 124) positive and 58.66% (n = 176) negative for Haemonchus infestation. The microsatellite loci DYA and ODRB1.2 were significantly associated (P ≤ 0.05) to parasite resistance. The locus DYA showed significant association with log FEC and dot ELISA and the locus ODRB1.2 showed significant association with log FEC, PCV and dot ELISA at P ≤ 0.05. Real time expression profiling revealed that the susceptible group (high FEC group) had 11.1-fold more expression of IFNγ mRNA (Th1 cytokine) and 0.11-fold lower expression of IL-10 mRNA (Th2 cytokine), which was found to be statistically significant (P ≤ 0.05).

Candidate Gene Polymorphism vis-a-vis Immune Response to Important Infectious Diseases in Bovines

Livestock sector play an important role in socio economic development and the national economy of the country. With an overall contribution of ~4% to GDP, livestock sector play an important role in national economy whilst contributing to the nutrition security, household income along with generating gainful employment in the rural areas, particularly among the landless, small and marginal farmers. India’s livestock sector is one of the largest in the world and boasts of a huge array of animal genetic resource. As per the 19 th Livestock Census-2012, India is home to a total of 512.05 million livestock population comprising cattle, buffalo, sheep, goat, pig, horse and ponies, mules, donkeys, camels, mithun and yak. India ranks first in milk production, accounting for 18.81 % of world production, achieving an annual output of 165.4 million tonnes during 2016-17 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 7 Number 08 (2018) Journal homepage: http://www.ijcmas.com

Nucleotide variability of protamine genes influencing bull sperm motility variables

Protamines (PRMs), important proteins of chromatin condensation in spermiogenesis, are promising candidate genes to explore markers of sperm motility. The coding and in-silico predicted promoter regions of these genes were investigated in 102 crossbred and 32 purebred cattle. Also, mRNA quantification was done to explore its possibility as diagnostic tool of infertility. The PCR-SSCP analysis indicated there were two band patterns only in fragment I of the PRM1 and fragment II of the PRM2 gene. The sequence analysis revealed A152G and G179A transitions in the PRM1 gene. Similarly, G35A, A49G and A64G transitions were identified in the PRM2 gene which resulted in altered amino acid sequences from arginine (R) to glutamine (Q), from arginine (R) to glycine (G) and from arginine (R) to glycine (G), respectively. This caused the reduction in molecular weight of PRM2 from 2157.66 to 1931.33 Da due to reduction in the number of basic amino acids. These altered properties of the PRM2 protein led to the reduction in Mass Motility (MM: P < 0.01), Initial Progressive Motility (IPM; P < 0.05) and Post Thaw Motility (PTM; P < 0.05) in crossbred bulls. The least squares analysis of variance indicated there was an effect of PRM2 haplotypes on MM (P = 0.0069), IPM (P = 0.0306) and PTM (P = 0.0500) in crossbred cattle and on PTM (P = 0.0408) in the overall cattle population. Based on the RT-qPCR analysis, however, there was not any significant variation of PRM1 and PRM2 gene expression among sperm of Vrindavani bulls with relatively lesser and greater sperm motility.

Single Nucleotide Polymorphisms in 5’ Upstream Region of Bovine TLR4 Gene Affecting Expression Profile and Transcription Factor Binding Sites

ABSTRACT The present study in the 5’ upstream region of TLR4 gene revealed four Single Nucleotide Polymorphisms (SNPs) in Vrindavani and Tharparkar cattle. The polymorphic information content (PIC), heterozygosity and allelic diversity values were low to moderate for these SNPs. In Vrindavani cattle, one SNP was found to be in Hardy-Weinberg Equilibrium (HWE) and the remaining three were found to be in linkage disequilibrium (LD) as indicated statistically (P > 0.05). In Tharparkar cattle, two SNPs were found to be in HWE and were not in LD as indicated statistically (P > 0.05). These SNPs were used for construction of haplotypes. In-silico analysis of these SNPs predicted abolition of eight transcription factor binding sites and creation of eight new sites. The quantitative real time PCR analysis did not show any significant variation of gene expression among haplotypes. However, gene expression between breed was found to be significant (P < 0.05) which suggested that upstream region of bovine TLR4 gene has a crucial role in its expression. These findings in TLR4 gene offer essential evidence that can be useful in future research exploring its role in immunity. TLR4 can be used as a marker for selection for disease resistance in bovines.

Peptidoglycan and Lipoteichoic Acid Induces Differential mRNA Response of Immune-Related Genes in PBMC of Crossbred, Tharparkar Cattle and Murrah Buffalo

ABSTRACT Subclinical mastitis, generally caused by Staphylococcus aureus, has a global economic impact all over the world. Hence, it needs to be resolved on higher priority which may be attained via. selection of mastitis resistant animals or inclusion of mastitis resistant trait into herd apart from management care. Diverse hosts with various genetic make-ups encounter pathogens in a diverse manner which in turn leads to contradicting outcome of the disease. Identification of species-wise or breed-wise differential expressed genes in response to S. aureus through relative evaluation of transcripts may be useful for judging the immuno-competency of a species or breed toward mastitis. The present study was undertaken to examine the stimulant effect of S. aureus peptidoglycan (PGN) and lipoteichoic acid (LTA) on Peripheral blood mononuclear cells (PBMC) harvested from blood samples of crossbred cattle, Tharparkar cattle, and Murrah buffaloes. After 6 h of in vitro stimulation qRT-PCR was used to measure the relative mRNA expression of TLR-2, TNF-α, IL-8, IFN-γ and IL-10 genes in stimulated and un-stimulated PBMC. The selected genes revealed significant differences in the pattern of immune response among crossbred cattle, Tharparkar cattle and Murrah buffalo in spite of the same stimulant dose.