Exploration of the rumen microbial diversity and carbohydrate active enzyme profile of black Bengal goat using metagenomic approach.

Black Bengal goats possess a rich source of rumen microbiota that helps them to adapt for the better utilization of plant biomaterial into energy and nutrients, a task largely performed by enzymes encoded by the rumen microbiota. Therefore the study was designed in order to explore the taxonomic profile of rumen microbial communities and potential biomass degradation enzymes present in the rumen of back Bengal goat using Illumina Nextseq-500 platform. A total of 83.18 million high-quality reads were generated and bioinformatics analysis was performed using various tools and subsequently, the predicted ORFs along with the rRNA containing contigs were then uploaded to MG-RAST to analyze taxonomic and functional profiling. The results highlighted that Bacteriodetes (41.38-59.74%) were the most abundant phyla followed by Firmicutes (30.59-39.96%), Proteobacteria (5.07-7.61%), Euryarcheaota (0.71-7.41%), Actinobacteria (2.05-2.75%). Genes that encode glycoside hydrolases (GHs) had the highest number of CAZymes, and accounted for (39.73-37.88%) of all CAZymes in goat rumen. The GT families were the second-most abundant in CAZymes (23.73-23.11%) and followed by Carbohydrate Binding module Domain (17.65-15.61%), Carbohydrate Esterase (12.90-11.95%). This study indicated that goat rumen had complex functional microorganisms produce numerous CAZymes, and that can be further effectively utilised for applied ruminant research and industry based applications.

A genome-wide approach to screen for genetic variants in broilers (Gallus gallus) with divergent feed conversion ratio.

Feed conversion ratio (FCR) is an economically important trait in broilers and feed accounts for a significant proportion of the costs involved in broiler production. To explore the contribution of functional variants to FCR trait, we analyzed coding and non-coding single-nucleotide variants (SNVs) across the genome by exome sequencing in seven pairs of full-sibs broilers with divergent FCR and with a sequence coverage at an average depth of fourfold. We identified 192,119 high-quality SNVs, including 30,380 coding SNVs (cSNVs) in the experimental population. We discovered missense SNVs in PGM2, NOX4, TGFBR3, and TMX4, and synonymous SNVs in TSNAX, ITA, HSP90B1, and COL18A1 associated with FCR. Haplotype analyses of genome-wide significant SNVs in PGM2, PHKG1, DGKZ, and SOD2 were also observed with suggestive evidence of haplotype association with FCR. Single-variant and FCR QTL-related genes-based association analyses of SNVs identified newly associated genes for FCR in the regions subjected to targeted exome sequencing. The top seven SNVs were next evaluated in independent replication data sets where SNV chr. 3: 13,990,160 (c. 961G>C) at TMX4 was replicated (p < 0.05). Collectively, we have detected SNVs associated with FCR in broiler as well as identification of SNVs in known FCR QTL region. These findings should facilitate the discovery of causative variants for FCR and contribute to marker-assisted selection.

Identification of putative SNPs in progressive retinal atrophy affected Canis lupus familiaris using exome sequencing.

Progressive retinal atrophy (PRA) is one of the major causes of retinal photoreceptor cell degeneration in canines. The inheritance pattern of PRA is autosomal recessive and genetically heterogeneous. Here, using targeted sequencing technology, we have performed exome sequencing of 10 PRA-affected (Spitz=7, Cocker Spaniel=1, Lhasa Aphso=1 and Spitz-Labrador cross breed=1) and 6 normal (Spitz=5, Cocker Spaniel=1) dogs. The high-throughput sequencing using 454-Roche Titanium sequencer generated about 2.16 Giga bases of raw data. Initially, we have successfully identified 25,619 single nucleotide polymorphisms (SNPs) that passed the stringent SNP calling parameters. Further, we performed association study on the cohort, and the highly significant (0.001) associations were short-listed and investigated in-depth. Out of the 171 significant SNPs, 113 were previously unreported. Interestingly, six among them were non-synonymous coding (NSC) SNPs, which includes CPPED1 A>G (p.M307V), PITRM1 T>G (p.S715A), APP G>A (p.T266M), RNF213 A>G (p.V1482A), C>A (p.V1456L), and SLC46A3 G>A (p.R168Q). On the other hand, 35 out of 113 unreported SNPs were falling in regulatory regions such as 3'-UTR, 5'-UTR, etc. In-depth bioinformatics analysis revealed that majority of NSC SNPs have damaging effect and alter protein stability. This study highlighted the genetic markers associated with PRA, which will help to develop genetic assay-based screening in effective breeding.

Whole-Genome Sequence of Listeria monocytogenes Strains from Clinical and Environmental Samples from Varanasi, India.

We present here the whole-genome sequences of Listeria monocytogenes from Ganges River water, agricultural soil, and human clinical samples from Varanasi, India, which will be used for a comparative analysis.

Potential functional gene diversity involved in methanogenesis and methanogenic community structure in Indian buffalo (Bubalus bubalis) rumen.

Understanding the methanogen community structure and methanogenesis from Bubalus bubalis in India may be beneficial to methane mitigation. Our current understanding of the microbial processes leading to methane production is incomplete, and further advancement in the knowledge of methanogenesis pathways would provide means to manipulate its emission in the future. In the present study, we evaluated the methanogenic community structure in the rumen as well as their potential genes involved in methanogenesis. The taxonomic and metabolic profiles of methanogens were assessed by shotgun sequencing of rumen metagenome by Ion Torrent semiconductor sequencing. The buffalo rumen contained representative genera of all the families of methanogens. Members of Methanobacteriaceae were found to be dominant, followed by Methanosarcinaceae, Methanococcaceae, Methanocorpusculaceae, and Thermococcaceae. A total of 60 methanogenic genera were detected in buffalo rumen. Methanogens related to the genera Methanobrevibacter, Methanosarcina, Methanococcus, Methanocorpusculum, Methanothermobacter, and Methanosphaera were predominant, representing >70 % of total archaeal sequences. The metagenomic dataset indicated the presence of genes involved in the methanogenesis and acetogenesis pathways, and the main functional genes were those of key enzymes in the methanogenesis. Sequences related to CoB--CoM heterodisulfide reductase, methyl coenzyme M reductase, f420-dependent methylenetetrahydromethanopterin reductase, and formylmethanofuran dehydrogenase were predominant in rumen. In addition, methenyltetrahydrofolate cyclohydrolase, methylenetetrahydrofolate dehydrogenase, 5,10-methylenetetrahydrofolate reductase, and acetyl-coenzyme A synthetase were also recovered.

De Novo Assembly and Transcriptome Characterization of Canine Retina Using High-Throughput Sequencing.

We performed transcriptome sequencing of canine retinal tissue by 454 GS-FLX and Ion Torrent PGM platforms. RNA-Seq analysis by CLC Genomics Workbench mapped expression of 10,360 genes. Gene ontology analysis of retinal transcriptome revealed abundance of transcripts known to be involved in vision associated processes. The de novo assembly of the sequences using CAP3 generated 29,683 contigs with mean length of 560.9 and N50 of 619 bases. Further analysis of contigs predicted 3,827 full-length cDNAs and 29,481 (99%) open reading frames (ORFs). In addition, 3,782 contigs were assigned to 316 KEGG pathways which included melanogenesis, phototransduction, and retinol metabolism with 33, 15, and 11 contigs, respectively. Among the identified microsatellites, dinucleotide repeats were 68.84%, followed by trinucleotides, tetranucleotides, pentanucleotides, and hexanucleotides in proportions of 25.76, 9.40, 2.52, and 0.96%, respectively. This study will serve as a valuable resource for understanding the biology and function of canine retina.

Insights into resistome and stress responses genes in Bubalus bubalis rumen through metagenomic analysis.

Buffalo rumen microbiota experience variety of diets and represents a huge reservoir of mobilome, resistome and stress responses. However, knowledge of metagenomic responses to such conditions is still rudimentary. We analyzed the metagenomes of buffalo rumen in the liquid and solid phase of the rumen biomaterial from river buffalo adapted to varying proportion of concentrate to green or dry roughages, using high-throughput sequencing to know the occurrence of antibiotics resistance genes, genetic exchange between bacterial population and environmental reservoirs. A total of 3914.94 MB data were generated from all three treatments group. The data were analysed with Metagenome rapid annotation system tools. At phyla level, Bacteroidetes were dominant in all the treatments followed by Firmicutes. Genes coding for functional responses to stress (oxidative stress and heat shock proteins) and resistome genes (resistance to antibiotics and toxic compounds, phages, transposable elements and pathogenicity islands) were prevalent in similar proportion in liquid and solid fraction of rumen metagenomes. The fluoroquinolone resistance, MDR efflux pumps and Methicillin resistance genes were broadly distributed across 11, 9, and 14 bacterial classes, respectively. Bacteria responsible for phages replication and prophages and phage packaging and rlt-like streptococcal phage genes were mostly assigned to phyla Bacteroides, Firmicutes and proteaobacteria. Also, more reads matching the sigma B genes were identified in the buffalo rumen. This study underscores the presence of diverse mechanisms of adaptation to different diet, antibiotics and other stresses in buffalo rumen, reflecting the proportional representation of major bacterial groups.

Use of real-time PCR technique in determination of major fibrolytic and non fibrolytic bacteria present in Indian Surti buffaloes (Bubalus bubalis).

In the milk industry in India, buffalo breeds are most commonly used for milk production. Efficiency of fiber digestion in ruminants is critical for animal productivity. Bacteria play an important role in fiber digestion and utilization. Absolute quantification real-time PCR was used to quantify ten bacterial species in rumen fluid of Surti buffalo fed green fodder, dry roughage and compound concentrate mixture. Abundance of each target taxon was calculated as a fraction of the total 16S rRNA gene copies in the samples, using taxon-specific primers. Bacterial populations showed a clear predominance of Ruminococcus albus, which comprised 5.66% of the bacterial rRNA gene copies in the samples. However, only 0.9% to 4.24% of the bacterial rRNA gene copies were represented by the ruminal Fibrobacter succinogenes, Ruminococcus flavefaciens and Prevotella species. The proportion of rRNA gene copies attributable to Selenomonas ruminantium, Streptococcus bovis, Ruminobacter amylophilus, Treponema bryantii and Anaerovibrio lipolytica was even less abundant, each comprising < 0.11% of the bacterial rRNA gene copies. The data suggest that the aggregate abundance of the most intensively studied ruminal bacterial species is relatively low and that a large fraction of the uncultured population represents a single bacterial genus.

Comparative evaluation of rumen metagenome community using qPCR and MG-RAST.

Microbial profiling of metagenome communities have been studied extensively using MG-RAST and other related metagenome annotation databases. Although, database based taxonomic profiling provides snapshots of the metagenome architecture, their reliability needs to be validated through more accurate methods. Here, we performed qPCR based absolute quantitation of selected rumen microbes in the liquid and solid fraction of the rumen fluid of river buffalo adapted to varying proportion of concentrate to green or dry roughages and compared with the MG-RAST based annotation of the metagenomes sequences of 16S r-DNA amplicons and high throughput shotgun sequencing. Animals were adapted to roughage-to-concentrate ratio in the proportion of 50:50, 75:25 and 100:00, respectively for six weeks. At the end of each treatment, rumen fluid was collected at 3 h post feeding. qPCR revealed that the relative abundance of Prevotella bryantii was higher, followed by the two cellulolytic bacteria Fibrobacter succinogens and Ruminococcus flavefaciens that accounted up to 1.33% and 0.78% of the total rumen bacteria, respectively. While, Selenomonas ruminantium and archaea Methanomicrobiales were lower in microbial population in the rumen of buffalo. There was no statistically significant difference between the enumerations shown by qPCR and analysis of the shotgun sequencing data by MG-RAST except for Prevotella. These results indicate the variations in abundance of different microbial species in buffalo rumen under varied feeding regimes as well as in different fractions of rumen liquor, i.e. solid and the liquid. The results also present the reliability of shotgun sequencing to describe metagenome and analysis/annotation by MG-RAST.

Metagenomic analysis of Surti buffalo (Bubalus bubalis) rumen: a preliminary study.

The complex microbiome of the rumen functions as an effective system for the conversion of plant cell wall biomass to microbial proteins, short chain fatty acids and gases. In this study, metagenomic approaches were used to study the microbial populations and metabolic potential of the microbial community. DNA was extracted from Surti Buffalo rumen samples (four treatments diet) and sequenced separately using a 454 GS FLX Titanium system. We used comparative metagenomics to examine metabolic potential and phylogenetic composition from pyrosequence data generated in four samples, considering phylogenetic composition and metabolic potentials in the rumen may remarkably be different with respect to nutrient utilization. Assignment of metagenomic sequences to SEED categories of the Metagenome Rapid Annotation using Subsystem Technology (MG-RAST) server revealed a genetic profile characteristic of fermentation of carbohydrates in a high roughage diet. The distribution of phylotypes and environmental gene tags (EGTs) detected within each rumen sample were dominated by Bacteroidetes/Chlorobi, Firmicutes and Proteobacteria in all the samples. The results of this study could help to determine the role of rumen microbes and their enzymes in plant polysaccharide breakdown is fundamental to understanding digestion and maximising productivity in ruminant animals.

Methanogenic diversity studies within the rumen of Surti buffaloes based on methyl coenzyme M reductase A (mcrA) genes point to Methanobacteriales.

Methane emissions from ruminant livestock are considered to be one of the more potent forms of greenhouse gases contributing to global warming. Many strategies to reduce emissions are targeting the methanogens that inhabit the rumen, but such an approach can only be successful if it targets all the major groups of ruminant methanogens. Therefore, basic knowledge of the diversity of these microbes in breeds of buffalo is required. Therefore, the methanogenic community in the rumen of Surti buffaloes was analyzed by PCR amplification, cloning, and sequencing of methyl coenzyme M reductase (mcrA) gene. A total of 76 clones were identified, revealing 14 different sequences (phylotypes). All 14 sequences were similar to methanogens belonging to the order Methanobacteriales. Within Methanobacteriales, 12 clones (6 OTUs) were similar to Methanosphaera stadtmanae and the remaining 8 phylotypes (64 clones) were similar to unclassified Methanobacteriales. Overall, members of the Methanobacteriales dominated the mcrA clone library in the rumen of Surti buffalo. Further studies and effective strategies can be made to inhibit the growth of Methanobacteriales to reduce methane emission from the rumen which would help in preventing global warming.

Low incidence of bovine leukocyte adhesion deficiency (BLAD) carriers in Indian cattle and buffalo breeds.

BLAD is an autosomal recessive genetic disease that affects Holstein-Friesian (HF) cattle worldwide. It is a disease characterized by a reduced expression of the adhesion molecules on neutrophils. The disease is caused by a mutation that replaces adenine at 383 with guanine, which causes an amino acid change from aspartic acid to glycine. Blood samples and a few semen samples were collected from 1250 phenotypically normal individuals, including HF (N=377), HF crossbred (N=334), Jersey (105), other breeds of cattle (N=160) and water buffalo Bubalus bubalis (N=274) belonging to various artificial insemination stations, bull mother farms (BMFs) and embryo transfer (ET) centres across the country. PCR-RFLP was performed to detect a point mutation in CD18, surface molecules of neutrophils. The results indicate that out of 1250 cattle and buffaloes tested for BLAD, 13 HF purebreds out of 377 and 10 HF crossbreds out of 334 appear to be BLAD carriers. In the HF and HF crossbred population, the percentage of BLAD carriers was estimated as 3.23%. The condition is alarming as the mutant gene has already entered the HF crossbred cattle population and therefore, the population of HF and its crossbreds needs regular screening to avoid the risk of spreading BLAD in the breeding cattle population of India.

Factor XI deficiency in Indian Bos taurus, Bos indicus, Bos taurus x Bos indicus crossbreds and Bubalus bubalis

We investigated the occurrence of Factor XI (FXI) deficiency syndrome in the following Indian dairy animals: Bos taurus Holstein-Friesian and Jersey cattle, Bos indicus Indian cattle breeds, B. taurus x B. indicus crossbreds and the river buffalo Bubalus bubalis. Factor XI deficiency is an autosomal recessive bleeding disorder known to affect Holstein cattle worldwide. A total of 1001 dairy animals, mainly bulls, were genotyped to detect the mutation within exon 12 of the gene encoding for factor XI. Two Holstein bulls were detected as heterozygous (carrier) for FXI deficiency, giving a carrier frequency of 0.6 percent in Indian Holstein cattle. None of the other cattle or buffalo breeds was found to be a carrier for FXI. Sequence comparison between normal and heterozygous animals revealed that there is a 77 base pair insertion fragment (AT (A)29 TAAAG (A)27 GAATTATTAATTCT) within exon 12 of the FXI gene. Both sequences were submitted to the National Center for Biotechnology Information (NCBI) GenBank and assigned the accession numbers DQ438908 for normal Holstein Friesian animals and DQ438909 for heterozygous Holstein Friesian animals.

Lack of carriers of citrullinaemia and DUMPS in Indian Holstein cattle.

The present study investigated the occurrence of 2 autosomal recessive genetic diseases, bovine citrullinaemia and deficiency of uridine monophosphate synthase (DUMPS), in Indian Holstein cattle. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was performed on a group of 642 animals, mainly HF and HF crossbred cattle, to identify carriers of these diseases. None of the animals were carriers of citrullinaemia or DUMPS. It is possible that with the mounting selection pressure, the international gene pool may diminish, and consequently the risk of dissemination of inherited defects will increase. It is therefore recommended to screen breeding bulls for their breed-specific genetic diseases before they are inducted in artificial insemination programmes, to minimize the risk.