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.

Antimicrobial resistance and molecular characterisation of E. coli from poultry in Eastern India.

In this study 252 poultry samples comprised of poultry meat (n = 228) and poultry eggs (n = 24) were screened for the isolation of Escherichia coli (E. coli). A total of 62 E. coli isolates were recovered from poultry meat. The E. coli isolates belonged to different serogroups based on ‘O’ serotyping of the isolates viz O29 (10.8%), O8 (7.7%), O40 (6.15%), O2 (4.61%), O60 (3.08%), O106 (3.08%), 42 (1.54%), O 87 (1.54%), and 01 serotypes of O1, O7, O30, O45, O59, O66, O105, O1116, O136, O141, O147, O148, O166, and O172. Sixteen (24.62%) of the isolates were UT (untypable) and 6 (9.23 %) were rough types. Molecular characterisation of the isolates was performed, targeting stx1 and stx2 virulence gene fragment. Out of 62 E. coli isolates, 10 (16.12%) were carrying virulence gene stx2, whereas none of the isolate was carrying stx1 gene. The E. coli isolates showed wide variation in resistance pattern against the antimicrobial agents that we used (9-90%). Among E. coli isolates, maximum resistance was observed against cefuroxime (89.1%) and penicillin (89.4%), followed by ampicillin (80.43%), vancomycin (74.1%), co-trimoxazole (73.1%), cephalothin (60.8%), ceftriaxone (28.2%), tetracycline (17.4%), gentamicin (13%), amikacin (13.04%), ofloxacin (13%), and ciprofloxacin (6.5%). A high degree of susceptibility was observed against amikacin (84.7%) and ciprofloxacin (76%) followed by gentamicin (71.73) and ofloxacin (60.86%). High multiple antibiotic resistances were observed and a total of 34 resistance patterns were identified.

Protection of mice against Brucella abortus 544 challenge by vaccination with recombinant OMP28 adjuvanted with CpG oligonucleotides.

Brucella abortus, a gram negative, facultative intracellular pathogen causes brucellosis in many animal species and humans. Although live, attenuated vaccines are available against this infection, they suffer from certain limitations. Therefore, the development of an effective subunit vaccine against brucellosis is an area of intense research. The outer membrane proteins (OMPs) of Brucella species have been extensively studied for its immunogenicity and protective ability. We have investigated the potential of CpG ODN to enhance the immunogenicity and protective efficacy of recombinant 28 kDa outer membrane protein (rOMP28) of Brucella melitensis. The study demonstrated vigorous immunoglobulin G (IgG) response of OMP28. The administration of rOMP28 with CpG caused increased cell mediated immune response in terms of induced IgG2a, T-cell proliferation and up-regulation of type I cytokine expression. In contrast, the free antigen suppressed the interferon gamma (type I cytokine) production on in-vitro stimulation of spleenocytes. The result indicates the role of OMP28 in the down regulation of IFN-gamma production. Moreover, the B. abortus S-19 vaccinated mice showed highest production of IL-4 and IFN-gamma. The protective ability of the antigen was evaluated by systemic bacterial clearance after challenging the mouse with B. abortus 544 pathogen. The level of protection was significant in rOMP28+CpG treated mice but was lower than the required level. The results of the present study indicate that rOMP28 could be an immunogen capable of inducing both humoral and cellular immune response. The humoral response was biased towards Th1 type when it was co-administered with CpG.

Genetic variants of beta-lactoglobulin gene and its association with milk composition traits in riverine buffalo.

A study was carried out to determine genetic variants of beta-lactoglobulin gene and to explore associations between these and milk composition traits in riverine buffalo. Single strand conformation polymorphism was employed to detect the genetic variants of the gene. Two fragments of this gene i.e. 119 bp of exon I and 400 bp spanning exon IV and intron IV were included in the study. For 119 bp fragment, three alleles namely, A, B and C were observed in all the buffalo breeds whereas four alleles (A, B, C and D) were detected for 400 bp fragment. The frequency distribution of alleles was different in different breeds of buffaloes for both the fragments. For exon I fragment, the milk composition traits such as total SNF, protein, solid, fat and whey protein yield were found to be significantly (P<0.05) associated with genotypes in Murrah and Bhadawari buffalo whereas in Mehsana breed genotypes were significantly (P<0.05) co-related with total SNF, solid and fat yield. Genotypes of 400 bp fragment, only total fat yield in Mehsana buffalo was found to be significantly (P<0.05) associated with genotypes.