Molecular Characterization and Seasonal Variation in Expression of HSP70.1 Gene in Gangatiri Cattle and Its Comparison with Buffalo.

Under tropical climate heat stress is a major challenge for livestock production. HSP70.1 is a ubiquitously expressed protein maintaining cellular machinery through proper folding of denatured proteins and prevents cellular apoptosis and protect cell from heat stress. Therefore, present investigation was undertaken to explore genetic variability in HSP70.1 gene in Gangatiri cattle, its comparison with buffalo sequences and differential expression in different season. The allelic variant was identified by sequencing amplified PCR product of HSP70.1 gene by primer walking. Season-wise total RNA samples was prepared for differential expression study. Brilliant SYBR Green QPCR technique was used to study the expression kinetics of this gene. DNA sequencing by primer walking identified four allelic variants in Gangatiri cattle. Sequence alignment study revealed four, six and one substitutions in the 5' untranslated region (5'UTR), coding and 3' untranslated region ((3'UTR) of HSP70.1 gene, respectively. Comparative analysis of HSP70.1 gene revealed that Cattle has shorter 5'UTR and 3' UTR than the buffalo. In Gangatiri cattle, summer season has significantly higher (P ≤ 0.05) expression of HSP70.1 than the spring and winter. The relative expression of HSP70.1 was increased by more than six folds in summer and nearly 1.5 folds higher in winter in comparison to the spring season. Therefore, HSP70.1 may be considered to have a critical role in the development of thermal tolerance in Gangatiri cattle.

Investigating genetic variability in Hsp70 gene-5'-fragment and its association with thermotolerance in Murrah buffalo (Bubalus bubalis) under sub-tropical climate of India.

The present study was undertaken to investigate genetic variability in a fragment comprising 5'UTR along with partial coding sequence of Hsp70 gene and its association with thermotolerance traits in Murrah buffalo at ICAR-Research Complex for Eastern Region, Patna (India). The allelic variants were identified from genomic DNA samples using SSCP technique. The PCR products were sequenced and analyzed. Data on different thermotolerance traits recorded in three seasons were analyzed by least squares ANOVA taking the SSCP genotypes as fixed effect. Two allelic variants (A and B), each of 503-bp in size, were documented with frequency of 0.59 and 0.41, respectively, and three genotypes (AA, AB and BB) with corresponding frequency of 0.30, 0.58 and 0.12. The allelic variants were due to single nucleotide substitution at 55th base position leading to a change of threonine (A) to methionine (B) in amino acid sequence. Both the allelic variants had 99.8% similarity in nucleotide sequence. In phylogenetic tree, allele A was in a cluster while allele B and Gangatiri cattle sequence formed a different cluster. The SSCP genotypes had significant effect on different thermotolerance traits in summer with thermo-humidity index of ≥ 84. Buffaloes with AA genotype had the highest (P Ë‚ 0.05) summer evening rectal temperature, respiration rate and pulse rate, inferring that the buffaloes carrying AA genotype had more stress in summer than those with AB and BB genotype. These SSCP genotypes might have differential role in heat shock protein response to induce thermotolerance of Murrah buffaloes in Gangetic plains.

Genetic profiling of Hsp70 gene in Murrah buffalo (Bubalus bubalis) under sub-tropical climate of India.

This study was aimed to genetic profiling of heat shock protein 70 (Hsp70) gene in Murrah buffalo investigating 50 unrelated adult animals at ICAR-Research Complex for Eastern Region, Patna (India) in winter, spring, and summer. PCR ready genomic DNA samples and season-wise total RNA samples were prepared. The PCR products of Hsp70 eluted from agarose gel were sequenced and analyzed. The first-strand cDNA was synthesized and concentration was equalized to 25 ng/µl. Expression kinetics of mRNA transcripts in different seasons was studied using Brilliant SYBR Green QPCR technique and the data retrieved was analyzed by least-squares ANOVA. DNA sequencing by primer walking revealed four allelic variants of Hsp70 gene. Alignment study revealed one substitution in 5'UTR, six substitutions in coding region, and one addition in 3'UTR. The highest percent identity and negligible phylogenetic distance were found among the alleles and reference bovine sequences. The relative mRNA expression was significantly higher in summer when THI ≥ 84 than the spring and winter; fold change increased by 4.5 times in summer than the spring whereas found nearly half in winter. These findings can be useful for heat stress management in buffaloes and help in understanding the mechanism of thermo-regulation well.

The effects of PPR on the reproductive health of Black Bengal goats and the possible role played by oxidative stress.

Outbreaks of Peste des petits ruminants (PPR) viral disease in Black Bengal goats were investigated from the middle Indo-Gangetic Plains of India. Clinical profile of PPR-affected flocks was recorded from four different outbreak sites of the region. The PPR outbreak was diagnosed serologically using commercially available sandwich ELISA kit. Relatively, low mortality rate (mean 26.75%) for PPR outbreak was recorded due to the endemic status of the disease. To understand the role of oxidative stress in PPR virus pathogenesis, various oxidant and antioxidant parameters in goats infected with PPR were estimated and compared with the uninfected/healthy goats of the same flock. The measured high level of pro-oxidant malondialdehyde (MDA) obtained from lipid peroxidation along with lower levels of anti-oxidants viz. superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) in PPR-affected Black Bengal goats suggests oxidative stress as one of the mechanism of pathogenesis of PPR virus. In addition, the correlation of oxidative stress due to PPR and the resulting reproductive disorders in the female goats were evaluated. The abortion in pregnant does observed during PPR outbreak was proportional to debility and oxidative stress manifested during PPR infection. The reproductive performance of recovered female goats in the period of 18 months of monitoring was significantly compromised in terms of kidding and twinning frequency. The mortality rate in kids born from PPR-recovered goats was significantly higher compared to those from health goats in the first 9 months post-recovery. From the present study, it may be concluded that together with the PPR virus, infection in goats and the resulting oxidative stress play a vital role for abortion and reduced post-reproductive performance in Black Bengal female goat.

Seroprevalence of Brucella abortus and Leptospira hardjo in cattle.

AIM: The aim was to assess the seroprevalence of B. abortus and Leptospira hardjo in the cattle population of Bihar, this work was carried out. MATERIALS AND METHODS: Randomly selected 450 cattle from nine districts of Bihar were serologically screened for antibodies against L. hardjo and B. abortus. DAS-ELISA for leptospira and AB-ELISA for brucella were carried out. Based on the results prevalence in each district and the state are reported herewith. RESULTS AND DISCUSSION: In this study, it was found that the seroprevalence of L. hardjo was 9.11% and that of B. abortus was 12.2% in Bihar. Indigenous cattle were found to be less susceptible to leptospirosis and brucellosis even though they accounted for 83.11% of the study population. CONCLUSION: Although there was no acute disease, antibodies detected against L. hardjo and B. abortus in the cattle population indicated the presence of chronic and subclinical infection, which could challenge the fertility of the animals.