B. P. Mishra

DGAT1 and ABCG2 polymorphism in Indian cattle (Bos indicus) and buffalo (Bubalus bubalis) breeds

BackgroundIndian cattle (Bos indicus) and riverine buffalo (Bubalus bubalis) give a poor yield of milk but it has a high fat and protein percentage compared to taurine cattle. The identification of QTLs (Quantitative Trait Loci) on BTA14 and BTA6 and its subsequent fine mapping has led to identification of two non conservative mutations affecting milk production and composition. Our objective was to estimate the frequency of K232A (DGAT1 – diacylglycerol – acyltransferase 1) and Y581S (ABCG2 – ATP binding cassette sub family G member 2) polymorphisms in diverse cattle and buffalo breeds of India having large variation in terms of milk production.ResultsWe screened the reported missense mutations in six cattle and five buffalo breeds. The DGAT1K and ABCG2Y alleles were found to be fixed in Indian cattle and buffalo breeds studied.ConclusionThis study provides an indirect evidence that all the Indian cattle and buffalo breeds have fixed alleles with respect to DGAT1 and ABCG2 genes reported to be responsible for higher milk fat yield, higher fat and protein percent.

Establishment and characterization of a buffalo (Bubalus bubalis) mammary epithelial cell line.

Background The objective of this study was to establish the buffalo mammary epithelial cell line (BuMEC) and characterize its mammary specific functions. Methodology Buffalo mammary tissue collected from the slaughter house was processed enzymatically to obtain a heterogenous population of cells containing both epithelial and fibroblasts cells. Epithelial cells were purified by selective trypsinization and were grown in a plastic substratum. The purified mammary epithelial cells (MECs) after several passages were characterized for mammary specific functions by immunocytochemistry, RT-PCR and western blot. Principal Findings The established buffalo mammary epithelial cell line (BuMEC) exhibited epithelial cell characteristics by immunostaining positively with cytokeratin 18 and negatively with vimentin. The BuMEC maintained the characteristics of its functional differentiation by expression of β-casein, κ-casein, butyrophilin and lactoferrin. BuMEC had normal growth properties and maintained diploid chromosome number (2n = 50) before and after cryopreservation. A spontaneously immortalized buffalo mammary epithelial cell line was established after 20 passages and was continuously subcultured for more than 60 passages without senescence. Conclusions We have established a buffalo mammary epithelial cell line that can be used as a model system for studying mammary gland functions.

Population structure in Indian sheep ascertained using microsatellite information.

This study attempts to provide a comprehensive insight into the prevailing genetic status of Indian sheep breeds using microsatellite markers. Seventeen Indian sheep breeds from 3 agroecological zones were analysed using a panel of 25 microsatellite markers. All of the sheep breeds investigated were genetically diverse, as evident from the high allele (>6) and gene (>0.6) diversity values. The gene diversity values for all breeds ranged from 0.621 to 0.780. The within-population heterozygote deficit (F(IS)) varied from -0.098 to 0.234, reflecting significant levels for 12 of the 17 breeds investigated. The average genetic differentiation between all breeds (F(ST)) was 11.1%, revealing moderate discrimination between the indigenous sheep breeds. The genetic distance and principal component analysis revealed a separation of sheep breeds based on geographical propinquity. The Bayesian clustering approach suggested poor breed differentiation in the north-western arid and semi-arid region when compared to the breeds from the eastern and southern peninsular regions. The observed results mirror the divergent management strategies in the different agroecological regions, lack of specific selection policies, and intermixing of breeds in close proximity. Immediate steps to curb the intermixing and erosion of breed purity for some of these breeds need to be implemented, for example, by introducing measures like making proven rams available and ensuring their frequent exchange between flocks. The data generated here provides valuable information about the genetic structure of the 17 Indian sheep breeds and this can be used for designating priorities for their conservation.

Sequence analysis of Toll-like receptor genes 1–10 of goat (Capra hircus)

This study involved cloning and sequencing of the coding regions of all 10 Toll-like receptor (TLR) genes of goat. Goat TLR 1-10 gene sequences revealed a high degree of nucleotide identity with sheep and cattle sequences (>90%) and 75-85% with pig, mouse and human sequences. At the amino acid level, 85-99% similarity was observed with sheep and cattle and 60-85% with pig, mouse and human. TLR9c DNA of goat showed the highest amino acid identity to that of sheep (99%) while TLR8 cDNA showed the lowest identity of 88.7% to that of sheep. Variations were seen in the number of leucine rich repeats (LRRs) of goat TLRs as compared to other ruminant species with maximum differences in the TLR3 gene. Phylogenetic analysis through molecular evolution and genetic analysis (MEGA) software and multi dimensional scaling revealed a high degree of conservation of goat TLRs with those from other species. However when the TIR domain of all the TLRs were compared, goat TLR7 TIR alone showed a high divergence of 19.3 as compared to sheep sequences. This is the first report of the full-length cDNA sequences of all the 10 TLR genes of goats which would be a useful tool for the study of evolutionary lineages and for phylogenetic analysis.

Selection of suitable reference genes for quantitative gene expression studies in milk somatic cells of lactating cows (Bos indicus)

We assessed the suitability of 9 internal control genes (ICG) in milk somatic cells of lactating cows to find suitable reference genes for use in quantitative PCR (qPCR). Eighteen multiparous lactating Sahiwal cows were used, 6 in each of 3 lactation stages: early (25 ± 5 d in milk), mid (160 ± 15 d in milk), and late (275 ± 25 d in milk) lactation. Nine candidate reference genes [glyceraldehyde 3-phosphate dehydrogenase (GAPDH), protein phosphatase 1 regulatory subunit 11 (PPP1R11), β-actin (ACTB), β-2 microglobulin (B2M), 40S ribosomal protein S15a (RPS15A), ubiquitously expressed transcript (UXT), mitochondrial GTPase 1 (MTG1), 18S rRNA (RN18S1), and ubiquitin (UBC)] were evaluated. Three genes, β-casein (CSN2), lactoferrin (LTF), and cathelicidin (CAMP) were chosen as target genes. Very high amplification was observed in 7 ICG and very low level amplification was observed in 2 ICG (UXT and MTG1). Thus, UXT and MTG1 were excluded from further analysis. The qPCR data were analyzed by 2 software packages, geNorm and NormFinder, to determine suitable reference genes, based on their stability and expression. Overall, PPP1R11, ACTB, UBC, and GAPDH were stably expressed among all candidate reference genes. Therefore, these genes could be used as ICG for normalization of qPCR data in milk somatic cells through lactation.

Riverine status and genetic structure of Chilika buffalo of eastern India as inferred from cytogenetic and molecular marker‐based analysis

The present study aimed at assessing the status of the Chilika buffalo population of eastern India employing cytogenetic and molecular markers. The Chilika buffaloes investigated cytogenetically possess a somatic chromosome count of 50, identical to that of typical riverine buffaloes. Various diversity estimates, viz. observed number of alleles (4.68), effective number of alleles (2.79), and observed (0.487) and expected (0.602) heterozygosity across 25 heterologous microsatellite markers indicated the presence of a moderate level of genetic diversity in Chilika buffaloes, comparable with three other prominent Indian riverine buffalo breeds (Murrah, Nagpuri and Toda) included in this study. Across the four buffalo populations, mean estimates of F-statistics from Jackknifing over loci were significantly different from zero (p < 0.05), with F(IT) (total inbreeding estimate) = 0.315 +/- 0.038, F(IS) (within-population inbreeding estimate) = 0.178 +/- 0.038, and F(ST) (population differentiation) = 0.166 +/- 0.025. Inter-breed analysis reflected Chilika buffaloes to be genetically close to Nagpuri followed by Murrah and Toda buffaloes. Factorial correspondence analysis (FCA) revealed low breed-specific clustering of Chilika and Nagpuri buffaloes. Additionally, the neighbour-joining tree structure of mitochondrial DNA D-loop haplotypes indicated clear grouping of the Chilika haplotypes with the riverine buffalo. Thus the cytogenetic, microsatellite and mitochondrial data analysed in the present study classify Chilika buffalo of eastern India to be of the riverine type and not swamp-type buffalo.

Proteome analysis of functionally differentiated bovine (Bos indicus) mammary epithelial cells isolated from milk

Mammary gland is made up of a branching network of ducts that end in alveoli. Terminally differentiated mammary epithelial cells (MECs) constitute the innermost layer of aveoli. They are milk‐secreting cuboidal cells that secrete milk proteins during lactation. Little is known about the expression profile of proteins in the metabolically active MECs during lactation or their functional role in the lactation process. In the present investigation, we have reported the proteome map of MECs in lactating cows using 2DE MALDI‐TOF/TOF MS and 1D‐Gel‐LC‐MS/MS. MECs were isolated from milk using immunomagnetic beads and confirmed by RT‐PCR and Western blotting. The 1D‐Gel‐LC‐MS/MS and 2DE‐MS/MS based approaches led to identification of 431 and 134 proteins, respectively, with a total of 497 unique proteins. Proteins identified in this study were clustered into functional groups using bioinformatics tools. Pathway analysis of the identified proteins revealed 28 pathways (p < 0.05) providing evidence for involvement of various proteins in lactation function. This study further provides experimental evidence for the presence of many proteins that have been predicted in annotated bovine genome. The data generated further provide a set of bovine MEC‐specific proteins that will help the researchers to understand the molecular events taking place during lactation.

Genomic analysis of host - Peste des petits ruminants vaccine viral transcriptome uncovers transcription factors modulating immune regulatory pathways

Peste des petits ruminants (PPR), is an acute transboundary viral disease of economic importance, affecting goats and sheep. Mass vaccination programs around the world resulted in the decline of PPR outbreaks. Sungri 96 is a live attenuated vaccine, widely used in Northern India against PPR. This vaccine virus, isolated from goat works efficiently both in sheep and goat. Global gene expression changes under PPR vaccine virus infection are not yet well defined. Therefore, in this study we investigated the host-vaccine virus interactions by infecting the peripheral blood mononuclear cells isolated from goat with PPRV (Sungri 96 vaccine virus), to quantify the global changes in the transcriptomic signature by RNA-sequencing. Viral genome of Sungri 96 vaccine virus was assembled from the PPRV infected transcriptome confirming the infection and demonstrating the feasibility of building a complete non-host genome from the blood transcriptome. Comparison of infected transcriptome with control transcriptome revealed 985 differentially expressed genes. Functional analysis showed enrichment of immune regulatory pathways under PPRV infection. Key genes involved in immune system regulation, spliceosomal and apoptotic pathways were identified to be dysregulated. Network analysis revealed that the protein - protein interaction network among differentially expressed genes is significantly disrupted in infected state. Several genes encoding TFs that govern immune regulatory pathways were identified to co-regulate the differentially expressed genes. These data provide insights into the host - PPRV vaccine virus interactome for the first time. Our findings suggested dysregulation of immune regulatory pathways and genes encoding Transcription Factors (TFs) that govern these pathways in response to viral infection.

Whole-Genome Sequence of Sungri/96 Vaccine Strain of Peste des Petits Ruminants Virus

ABSTRACT We report the complete genome sequence of the Sungri/96 vaccine strain of peste des petits ruminants virus (PPRV). The whole-genome nucleotide sequence has 89 to 99% identity with the available PPRV genome sequences in the NCBI database. This study helps to understand the epidemiological and molecular characteristics of the Sungri/96 strain.

Identification of suitable housekeeping genes for normalization of quantitative real‐time PCR data during different physiological stages of mammary gland in riverine buffaloes (Bubalus bubalis)

Gene expression analysis unravels the complex changes or relations at transcriptomic level. To nullify all type of errors that can be incorporated during any stage of RNA extraction into cDNA synthesis and for reliable results, the data obtained from qPCR have to be normalized using the appropriate/suitable housekeeping genes (HKGs). Unfortunately, till date, no such HKG has been reported for bubaline mammary gland. The objective of the present study was thus to identify and validate the potential HKGs for the gene expression studies in buffalo mammary gland. Mammary tissues from twelve buffaloes during different physiological stages: pre-pubertal (heifer), lactation and involution were obtained for the present study. A total of 16 potential HKGs (GAPDH, β-actin, UXT, β2M, A2M, RPl4, RPS9, RPS15A, RPS18, RPS23, HMBS, HPRT1, GTP, EEF1A1, UB1 and RPL22) from different functional classes were evaluated. The analysis revealed that the expression of EEF1A1, RPl4, β2M and RPS15A was most consistent across different physiological stages of buffalo mammary gland. On the other hand, β-actin, A2M, RPL22 and GAPDH were the least stable genes making them unsuitable as HKGs. Based on our analysis, we recommend the use of EEF1A1, RPl4, β2M and RPS15A genes as suitable HKGs for accurate normalization of gene expression data in bubaline mammary gland.