Molecular characterization of CRBR2 fragment of TLR4 gene in association with mastitis in Vrindavani cattle.

The bovine TLR4 gene is an interesting candidate marker for mastitis resistance, since it is involved in neutrophil migration to and from the mammary gland during mastitis. TLR4 detects pathogen ligands, such as the Escherichia coli lipopolysaccharide (LPS) endotoxin and facilitates innate and adaptive immune responses. In the current study, a total of 130 crossbred cows (74 mastitis tolerant and 56 with clinical mastitis) kept at the Cattle and Buffalo Farm, IVRI, Izatnagar, were selected to explore the polymorphism in the co-receptor binding region 2 (CRBR2) fragment of the TLR4 gene. PCR-SSCP and sequence analysis showed two genotypes of the TLR4 gene's CRBR2 fragment, AA and AB, which were polymorphic in both the afflicted and tolerant groups. Sequencing revealed eight single nucleotide polymorphisms (SNPs) in allele A and ten SNPs in allele B. This genotype had no significant effect on the incidence of clinical mastitis according to the logistic regression model. Our study found insufficient evidence linking SNP variants in the CRBR2 region of the TLR4 gene to mastitis susceptibility in crossbred cattle.

Molecular Characterization of Mx1 Gene in Native Indian Breeds of Chicken.

The genetic polymorphism of Mx1 gene was explored in Indian chicken breeds. PCR-RFLP analysis in 102 bp fragment of partial intron 13 and partial exon 14 of Mx1 gene revealed two genotypes viz. RS and SS with two alleles viz. R and S both in Naked Neck and Tellicherry breeds of chicken. The homozygous genotype RR was not identified. When deduced amino acid sequences were compared, the asparagine amino acid was found to be substituted in "R" allele for serine in "S" allele. PCR-SSCP analysis of 284 bp fragment in 5'-UTR and partial promoter region revealed three genotypes viz. CC, CG, and CH with three different alleles viz. C, G, and H in Naked Neck breed of chicken and five genotypes viz. DI, JK, KK, KL, and KM with six different alleles viz. D, I, J, K, L, and M in Tellicherry breed of chicken. The homozygous genotypes viz. GG and HH in Naked Neck and DD, II, JJ, LL, and MM in Tellicherry chicken was not identified. The nucleotide substitution rate estimated to be in the range of 0.004-0.011. The identified genetic variation can be helpful for better insight to disease resistance property of the Mx1 gene.

Molecular characterization and expression profile of partial TLR4 gene in association to mastitis in crossbred cattle.

Crossbred cattle are more prone to mastitis in comparison to indigenous cattle. Toll-like receptor 4 (TLR4) recognizes pathogen ligands, for example, lipopolysaccharide (LPS) endotoxin from Escherichia coli and mediates signaling to initiate innate and adaptive immune responses. Mutations in TLR4 can compromise the host immune response to certain pathogens, so it may be a potential candidate for marker assisted selection to enhance mastitis resistance in dairy cattle. Hence, in this study role of bovine TLR4 gene in mastitis resistance was investigated by association as well as expression profiling analysis in crossbred cattle. The animals were divided into mastitis affected and unaffected groups on the basis of history of animals and California Mastitis Test (CMT). PCR-SSCP and Sequence analysis revealed three genotypes of coreceptor binding region 1 (CRBR1) fragment of TLR4 gene namely AA, AB, and BB in both groups of cattle. The logistic regression model did not show any significant effect of these genotypes on the occurrence of clinical mastitis. Moreover, in vitro challenge of peripheral blood mononuclear cells (PBMCs) with LPS failed to show any association of the genotypes with TLR4 gene expression. In a nutshell, in the present study enough evidence was not found for association of the SNP variants of CRBR1 fragment of TLR4 gene with mastitis susceptibility in crossbred cattle.

Genotypic to expression profiling of bovine calcium channel, voltage-dependent, alpha-2/delta subunit 1 gene, and their association with bovine mastitis among Frieswal (HFX Sahiwal) crossbred cattle of Indian origin.

Calcium channel, voltage-dependent, alpha-2/delta subunit 1 (CACNA2D1) gene is considered to be an important noncytokine candidate gene influencing mastitis. Scanty of reports are available until today regarding the role play of CACNA2D1 gene on the susceptibility of bovine mastitis. We interrogated the CACNA2D1 G519663A [A>G] SNP by PCR-RFLP among two hundreds Frieswal (HF X Sahiwal) crossbred cattle of Indian origin. Genotypic frequency of AA (51.5, n=101) was comparatively higher than AG (35, n=70) and GG (14.5, n=29). Association of Somatic cell score (SCS) with genotypes revealed that, GG genotypes showing lesser count (less susceptible to mastitis) compare to AA and AG. Relative expression of CACNA2D1 transcript (in milk samples) was significantly higher among GG than AG and AA. Further we have also isolated blood sample from the all groups and PBMCs were cultured from each blood sample as per the standard protocol. They were treated with Calcium channel blocker and the expression level of the CACNA2D1 gene was evaluated by Real Time PCR. Results show that expression level decline in each genotypic group after treatment and expression level of GG are again significantly higher than AA and AG. Thus, it may be concluded that GG genotypic animals are favorable for selecting disease resistant breeds.

Effect of heat stress on the expression profile of Hsp90 among Sahiwal (Bos indicus) and Frieswal (Bos indicus × Bos taurus) breed of cattle: a comparative study.

We evaluated the effect of thermal challenge on the expression profile of heat shock protein 90 (Hsp90) among Sahiwal (Bos indicus) and Frieswal (Bos indicus × Bos taurus) breeds of cattle. The present investigation was focused on the comparative studies on Hsp90 expression among Frieswal and Sahiwal under in vitro and environmental heat stress. Measured immediately after the in vitro heat shock to the peripheral blood mononuclear cells (PBMCs), the relative expression of Hsp90 mRNA was significantly (P<0.05) higher in Sahiwal compared to those in Frieswal. In later intervals of time, the differences in the expression levels between the two breeds become negligible coming down towards the basal level. A similar pattern was observed in the protein concentration showing significantly (P<0.05) higher levels in Sahiwal compared to those in Frieswal. The second sets of experiments were undertaken during summer months (March to May) when temperature peaked from 37 to 45 °C. During these months, Frieswal cows consistently recorded higher rectal temperatures than the Sahiwal breed. Further during this peak summer stress, Sahiwal showed significantly higher levels of mRNA transcripts as well as protein concentration compared to the Frieswal breed. Our findings also interestingly showed that, the cell viability of PBMC are significantly higher among the Sahiwal than Frieswal. Taken together, the experiments of both induced in vitro and environmental stress conditions indicate that, Sahiwal may express higher levels of Hsp90 then Frieswal to regulate their body temperature and increase cell survivality under heat stressed conditions.

Evaluation of three bovine Y specific microsatellite loci as an alternative biomarkers for semen quality traits in crossbred bull.

Although some of the studies earlier reported that bovine semen parameters are associated with some candidate markers genes, but scanty of reports available regarding the effect of allelic variation in Y specific microsatellite markers on semen quality parameters in bulls. In the present study we have targeted three Y specific microsatellite markers (INRA126, INRA 189 and BM861) for their association ship analysis with some semen quality parameters among Frieswal (HF × Sahiwal) crossbred bulls of Indian origin. The polymorphic loci of INRA 126, bulls with 182 and 184 alleles had significantly (P<0.01) higher semen volume as compared to 186 allele, however, 186 allele showed significantly (P<0.01) higher concentration per ml of semen compared to 182 and 184. Interestingly our study also revealed that number of sperm/ejaculate is also significantly (P<0.05) higher in 184 allele compared to 182 and 186. Similarly, association analysis of INRA 189 major three alleles also revealed a significant difference in semen volume and concentration. Allele 89 and 96 having significantly (P<0.01) higher volume compared to 86, whereas allele 86 having significantly (P<0.01) higher concentration per volume of semen than 89 and 96. Again after association of two major alleles (160 and 164) of BM861 loci with semen parameters revealed no significant difference with any of the semen quality parameters chosen here. Therefore the present study may be for the first time revealed that the Y chromosomal microsatellite alleles are important male reproductive biomarkers for improving semen quality traits in bulls.

Promoter variants at AP2 box region of Hsp70.1 affect thermal stress response and milk production traits in Frieswal cross bred cattle.

Heat shock proteins (Hsp) are known to play major role in protection of cells from thermal stress. Nucleotide polymorphisms within the promoter of Hsp affect degree of expression and inducibility of Hsp mRNA. The present study aimed to investigate the effect of polymorphism within promoter region on the cellular expression of Hsp70.1 mRNA and association of identified polymorphisms with the physiological parameters during summer stress and milk production traits in dairy cattle. Two hundred Frieswal cows were genotyped using double PCR-RFLP to identify deletion of cytosine within the Hsp70.1 promoter AP2 box at base position 895. Homozygous wild type genotypes (CC) were found in lower frequency (39.29, n=78) than heterozygous cytosine deletion mutant genotypes (C-) (60.71, n=122). In the observed physiological parameters (rectal temperature, respiration rate and heat tolerance coefficient), cows that were homozygous wild types had better significant (P<0.05) summer tolerance than the heterozygous deletion genotypes. Cytosine deletion mutation in the promoter region negatively affected (P<0.01) the expression of Hsp70.1 mRNA in peripheral bovine mononuclear cells (PBMC) subjected to in vitro heat stress. Further association of observed polymorphism with the milk production traits was significant as the heterozygous cytosine deletion cows had lower total milk yield, peak yield, yield at 300 days, protein% (P<0.01) and fat% (P<0.05) than the native wild type promoter cows. The results from the present study suggest that the promoter region of bovine hsp70.1 gene is polymorphic and may be useful in selection of dairy cows for relatively better thermotolerance and higher milk production.

Microsatellite (GT)n polymorphism at 3'UTR of SLC11A1 influences the expression of brucella LPS induced MCP1 mRNA in buffalo peripheral blood mononuclear cells.

A (GT)n microsatellite polymorphism at 3'UTR of SLC11A1(solute carrier family 11A1) is associated with the natural resistance to bovine brucellosis. A pleiotropic effect of SLC11A1 on other candidate genes influencing the host resistance including monocyte chemotactic/chemoattractant protein 1 (MCP1) is also hypothesized. In the present study, we report the cloning and characterization of the complete coding sequence of bubaline (bu) MCP1 and its tissue distribution at the transcript level. The buMCP1 exhibited as high as 99% and >80% of sequence identities with the bovine and other domestic animal species homologues. The buMCP1 mRNA was abundant across the different tissues: most abundant in liver and mammary gland, moderate in ovary, skeletal muscle and testis, and least in uterus. Further, quantitative real-time PCR (RTqPCR) analysis revealed that PBMCs carrying so called resistant GT13 allele produced more MCP1 mRNA endogenously as well as when induced with brucella LPS suggesting the pleiotropic roles of SLC11A1 in conferring resistance against the intracellular pathogens particularly against brucellosis. However, the underlying molecular mechanisms by which 3'UTR SLC11A1 concomitantly increases the production of chemokines like MCP1 are yet to be investigated.

Differential expression of protamine 1 and 2 genes in mature spermatozoa of normal and motility impaired semen producing crossbred Frieswal (HF×Sahiwal) bulls.

Mature spermatozoa contain thousands of mRNA transcripts. These untranslated mRNA may perhaps serve as a "footprint" of spermatogenesis since many of them might directly or indirectly be involved in fertilization, early embryo cleavage, poor semen quality and fertility. In this study, we tried to isolate high-quality RNA from mature spermatozoa and to monitor the expression profile of protamine 1 (PRM1) and protamine 2 (PRM2) gene in ejaculated spermatozoa of normal (good, % initial progressive motility: 57.61±1.41, n=9) and motility impaired (poor, % initial progressive motility: 18.45±1.61, n=8) crossbred Frieswal (HF×Sahiwal) bulls semen using real time quantitative PCR. Semen samples were subjected to discontinuous (45:90) Percoll gradient centrifugation, specifically to eliminate damaged spermatozoa and contaminating somatic cells. Total RNA was extracted from sperm pellets and cDNA was synthesized. Furthermore, the absence of contamination of germ cells, epithelial cells and leucocytes in all the RNA extractions was tested by RT-PCR targeting specific molecular markers like KIT, CDH1 and CD4, respectively. The presence of transcripts like PRM1, PRM2, DAZL, and PPIA were demonstrated in ejaculated spermatozoa using appropriate PCR primers without RNA amplification. Expression of PRM1 and PRM2 genes were evaluated by real time quantitative PCR using TaqMan chemistry, where PPIA was used as internal control. The cDNA synthesized from normal buffalo testicular tissue was served as positive control. The good quality semen producing group showed significantly higher level of PRM1 mRNAs expression as compared to the poor quality semen producers (P<0.05) indicating putative role of the gene and semen quality parameters especially initial progressive motility. However, PRM2 transcript levels were not significantly different between the groups (P>0.05).

Molecular characterization of MHC-DRB cDNA in water buffalo (Bubalus bubalis).

In the present study, water buffalo MHC (Bubu)-DRB cDNA was cloned and characterized. The 1022 base long-amplified cDNA product encompassed a single open reading frame of 801 bases that coded for 266 amino acids. The Bubu-DRB sequence showed maximum homology with the BoLA-DRB3*0101 allele of cattle. A total of seven amino acid residues were found to be unique for the Bubu-DRB sequence. The majority of amino acid substitutions was observed in the ß(1) domain. Residues associated with important functions were mostly conserved. Water buffalo DRB was phylogenetically closer to goat DRB*A.

Molecular characterization of MHC-DRB cDNA in water buffalo (Bubalus bubalis)

In the present study, water buffalo MHC (Bubu)-DRB cDNA was cloned and characterized. The 1022 base long-amplified cDNA product encompassed a single open reading frame of 801 bases that coded for 266 amino acids. The Bubu-DRB sequence showed maximum homology with the BoLA-DRB3*0101 allele of cattle. A total of seven amino acid residues were found to be unique for the Bubu-DRB sequence. The majority of amino acid substitutions was observed in the β1 domain. Residues associated with important functions were mostly conserved. Water buffalo DRB was phylogenetically closer to goat DRB*A.

Molecular Characterization and SNP Detection of CD14 Gene of Crossbred Cattle.

CD14 is an important molecule for innate immunity that can act against a wide range of pathogens. The present paper has characterized CD14 gene of crossbred (CB) cattle (Bos indicus×Bos taurus). Cloning and sequence analysis of CD14 cDNA revealed 1119 nucleotide long open reading frame encoding 373 amino acids protein and 20 amino acids signal peptide. CB cattle CD14 gene exhibited a high percentage of nucleotide identity (59.3-98.1%) with the corresponding mammalian homologs. Cattle and buffalo appear to have diverged from a common ancestor in phylogenetic analysis. 25 SNPs with 17 amino acid changes were newly reported and the site for mutational hot-spot was detected in CB cattle CD14 gene. Non-synonymous substitutions exceeding synonymous substitutions indicate the evolution of this protein through positive selection among domestic animals. Predicted protein structures obtained from deduced amino acid sequence indicated CB cattle CD14 molecule to be a receptor with horse shoe-shaped structure. The sites for LPS binding, LPS signalling, leucine-rich repeats, putative N-linked glycosylation, O-linked glycosylation, glycosyl phosphatidyl inositol anchor, disulphide bridges, alpha helix, beta strand, leucine rich nuclear export signal, leucine zipper and domain linker were predicted. Most of leucine and cysteine residues remain conserved across the species.

Allelic diversity at MHC class II DQ loci in buffalo (Bubalus bubalis): evidence for duplication.

The genetic diversity of MHC class II DQ genes was investigated in riverine buffalo (Bubalus bubalis) by PCR-RFLP and sequencing. Highly variable regions (exons 2-3) of DQ genes were amplified from 152 buffaloes and genotyped by PCR-RFLP. Alleles identified by differential restriction patterns were sequenced for the characterization. PCR-RFLP was a rapid method to discriminate between DQA1 and duplicated DQA2 genes in buffalo, however, the method appeared to be inadequate for determining the more complicated DQB genotypes. A total of 7 and 10 alleles were identified for DQA and DQB loci, respectively. Nucleotide as well as amino acid variations among DQ alleles particularly at peptide binding regions were high. Such variations were as expected higher in DQB than DQA alleles. The phylogenetic analysis for both genes revealed the grouping of alleles into two major sub-groups with higher genetic divergence. High divergence among DQ allelic families and the isolation of two diverse DQA and DQB sequences from individual samples indicated duplication of DQ loci was similar in buffalo to other ruminants.

Identification of novel allelic variants of integrin beta 2 (ITGB2) gene and screening for Bubaline leukocyte adhesion deficiency syndrome in Indian water buffaloes (Bubalus bubalis).

A fragment of 570 bp corresponding to exon 5 and 6 of integrin beta 2 (ITGB2) gene was amplified for screening D128G mutation in one hundred and fifty two buffaloes (Bubalus bubalis) which causes bovine leukocyte adhesion deficiency syndrome (BLAD) in cattle, as well as to ascertain polymorphism. TaqI PCR-RFLP revealed no such mutation thus indicating the absence of bubaline leukocyte adhesion deficiency (BuLAD) allele in animals under study. However, the polymorphism studies using MspI restriction enzyme revealed two genotypic patterns viz. AA pattern (bands of 293, 141, 105, and 31 bp) and BB pattern (bands of 293, 105, 77, 64, and 31 bp). The sequences of A and B alleles were submitted to the GenBank (EU853307 and AY821799).

Isolation of two cDNAs encoding MHC-DQA1 and -DQA2 from the water buffalo, Bubalus bubalis.

In the present study, we explored structural and functional variations and possible duplication of the major histocompatibility complex (MHC)-DQA gene in water buffalo (Bubalus bubalis). Two cDNA sequences, amplified from one individual water buffalo, were designated as Bubu-DQA1 (DQA*0101) and -DQA2 (DQA*2001). The percentage of nucleotide and amino acid similarity between Bubu-DQA1 and -DQA2 revealed that these sequences display more similarity to alleles of respective DQA1 and DQA2 genes from other ruminant species than to each other. The phylogenetic analysis also revealed a considerably larger genetic distance between these two genes than between homologous genes from other species. The larger genetic distance between DQA*0101 and DQA*2001, and the presence of different bovine DQA putative locus specific amino acid motifs, suggests these sequences are non-allelic. This finding is consistent with DQA gene duplication in other ruminants.

Cloning and characterization of DGAT1 gene of Riverine buffalo.

The present study was carried out to characterize the DGAT1 gene of Riverine buffalo. Total RNA was extracted from the mammary tissue of buffalo and DGAT1cDNA were synthesized by RT-PCR, then cloned using pDRIVE cloning vector and sequenced. The sequencing revealed that the size of DGAT1 gene was 1470 bp with GC content of 62.30%. The gene encoded for 489 amino acid precursors and that it possessed 32 amino acids signal peptide. The similarity of buffalo DGAT1 mRNA sequence with that of cattle, pig, monkey, human, mice and rat were determined as 98.4, 90.7, 85.4, 85.0, 77.4 and 77.1%, respectively. Phylogenetic tree constructed from the derived DGAT1 protein sequences of 15 different species illustrated a unique branches for mammals, fly, nematode and plants. Among mammals, cattle and buffalo grouped together, whereas swine formed another group in the same branch. Four motifs were predicted in buffalo DGAT1 peptide sequence, one N-linked glycosylation site (246th position), two putative tyrosine phosphorylation site (316 and 261), one putative diacylglycerol binding site (382-392 amino acid position) and a conserved domain MBOAT (membrane bound acyl transferase from 150 to 474 amino acids) with a histidine as an active residue.

Association of microsatellite (GT)n polymorphism at 3'UTR of NRAMP1 with the macrophage function following challenge with Brucella LPS in buffalo (Bubalus bubalis).

Brucella abortus is a facultative intracellular pathogen that survives and replicates in host macrophages. Hence, macrophage function plays an important role in influencing natural resistance/susceptibility to intracellular pathogen. The natural resistance associated macrophage protein 1 (NRAMP1; erstwhile referred as Ity/Lsh/Bcg), a transmembrane protein, regulates activity of macrophages against intracellular pathogens. In bovine, natural resistance to brucellosis is significantly associated with (GT)13 allelic variant of microsatellite locus at 3' untranslated region (3'UTR) of the NRAMP1 gene. In the present study we screened 65 Murrah breed of buffalo (Bubalus bubalis) to identify polymorphism at 3'UTR of NRAMP1 gene and evaluate the association of these polymorphisms with the macrophage function. Four allelic variants (viz., GT13, GT14, GT15 and GT16) were identified. Majority of the buffaloes were of either homozygous (GT)14/(GT)14 or heterozygous (GT)14/(GT)15 with (GT)14 allele occurring most frequently (62%). For association study, non-vaccinated and serologically negative animals were divided into three genotypic groups: group 1 (n=2) comprising animals of homozygous (GT)13 genotype, whereas, group 2 (n=4) and group 3 (n=6) consisted animals of heterozygous [(GT)13/(GT)n, where n not equal 13] and non-(GT)13 [(GT)n/(GT)n, where n not equal 13] genotype, respectively. Macrophages, after maturation, were challenged with Brucella LPS to assay the macrophage function in terms of H2O2 and NO production. The (GT)13 allele, either in homozygoous {(GT)13/(GT)13} or heterozygous {(GT)13/(GT)n, where n=14, 15 or 16}, was significantly (p<0.01) associated with increased production of H2O2 and NO. In this manuscript, for the first time, we have identified (GT)13 allelic variant and demonstrated its significant association with the improved macrophage function in buffalo.

Polymorphism of exon 2-3 of bovine major histocompatibility complex class I BoLa-A gene

The exon 2-3 region of bovine major histocompatibility complex (MHC) class I BoLa-A gene was investigated for polymorphisms in three breeds of cattle originated in the Indian subcontinent namely Sahiwal, Tharparkar, Hariana, as well as crossbred (Bos taurus x Bos indicus) cattle and Jersey, the exotic breed (Bos taurus). The PCR amplified fragment of 714 bp showed distinct DdeI-, TaqI- and HinfI- RFLP patterns, thus confirming a higher degree of polymorphism in this region. To our knowledge this is the first report of HinfI restriction patterns for BoLa-A exon 2-3. The sequencing results revealed a number of nucleotide substitutions in this region, which resulted in amino acid changes. The present investigation confirmed that MHC class I BoLa-A exon 2-3 is highly polymorphic in cattle.

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.