Profiling of Antimicrobial Resistance Genes and Integron from Escherichia coli Isolates Using Whole Genome Sequencing.

This study is designed to investigate Escherichia coli for the antibiotic resistance genes (ARGs) and integrons from healthy as well as diarrhoeic/diseased animals/birds' faecal samples. A total of eight samples were selected for the study; from each animal, two samples were taken, one from healthy animals/birds and one from diarrhoeic/diseased animals/birds. Antibiotic sensitivity testing (AST) and whole genome sequencing (WGS) was performed for selected isolates. The E. coli isolates showed resistance to moxifloxacin, followed by erythromycin, ciprofloxacin, pefloxacin, tetracycline, levofloxacin, ampicillin, amoxicillin, and sulfadiazine (4/8, 50.00% each). The E. coli isolates were 100% sensitive to amikacin, followed by chloramphenicol, cefixime, cefoperazone, and cephalothin. A total of 47 ARGs from 12 different antibiotic classes were detected among the eight isolates by WGS. The different classes of antibiotics included aminoglycoside, sulphonamide, tetracycline, trimethoprim, quinolone, fosfomycin, phenicol, macrolide, colistin, fosmidomycin, and multidrug efflux. The class 1 integrons were detected in 6/8 (75.00%) isolates with 14 different gene cassettes.

RNA-sequencing attest increased sperm motility in bovine spermatozoa treated with ethanolic extract of Putranjiva roxburghii.

In the course of time, scientific communities have a growing interest in understanding ethano medicines. The Putranjiva roxburghii, a native plant of the Indian Subcontinent is described as a "Child amulet tree" in Ayurveda. Based on the fact that this herbal medicine has an indispensable component of integrative medicine, the present study was planned to assess the effect of ethanolic dried extract of Putranjiva seeds on the motility of X and Y-bearing bovine spermatozoa. The in-vitro effect of seed extract diluted in S-TALP medium on bull semen has been evaluated by Computer Assisted Semen Analysis (CASA) shows a marked increase in the motility of spermatozoa. Motile and non-motile spermatozoa have been separated by glass wool column from the control as well as treated group. The X and Y-bearing sperm quantification have been carried out by droplet digital polymerase chain reaction (ddPCR). The extract didn't exert any differential effect on the motility and viability of X and Y chromosome-bearing spermatozoa. The transcriptome profiling (RNA-Seq) identified 93 differentially expressed genes between the extract treated and control group. It unveils the up-regulation of CATSPER, AKAP3, SPAG, ADAM1B, ADAM2 and ADAM32 genes that are involved in increasing sperm motility. Transcriptome profile also unveil the expression of ZAR1, CYP17A1, APPL2, HOXB4 and SP9 genes involved with embryonic development processes in Putranjiva extract-treated motile spermatozoa. The results envisaged the medicinal value of Putranjiva herb on increased fertility due to combinatory effect like increased sperm motility and favourableness on embryogenesis. The study ruled out the possibility of herbs having any biased effect on the selection of either male or female-bearing spermatozoa in the bull. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03452-4.

Understanding of molecular basis of histological graded horn cancer by transcriptome profiling.

Horn cancer is most devastating and prominent cancer in Indian zebu cattle that affects socio-economic condition of small-scale farmers who depends on their cattle for farm work. Development in the field for genomics through next generation sequencing and bioinformatics advancement have helped to identify genes which have a role in horn cancer development. Histopathological examination of cancerous tissues of horn revealed myxomatous changes, well, moderate and poorly differentiated squamous cell carcinoma. Differential gene expression analysis showed 40, 11, 66 and 29 upregulated genes and 10, 14, 08 and 07 down-regulated genes in myxomatous, well, moderate and poorly differentiated squamous cell carcinoma as compared to normal. Significant differentially expressed genes are related to cell development, cell proliferation, cell-cell communication, cell signaling and angiogenesis which are linked to Akt pathway, mTOR pathway and Wnt pathway. Activity of these genes and related pathways have already been established about their role in development of cancer. Among the candidate genes; keratin family, keratin family related gene, chemokine signaling and cytokines signaling associated genes could be a prominent target for the development of stage specific prognosis marker after further detailed study at large sample population level. CSTA, PTN, SPP1 genes have upregulation in all stages of cancer and they have enrolled as biomarkers for horn cancer.

Potential of camel rumen derived Bacillus subtilis and Bacillus velezensis strains for application in plant biomass hydrolysis.

Rumen inhabiting Bacillus species possesses a high genetic potential for plant biomass hydrolysis and conversion to value-added products. In view of the same, five camel rumen-derived Bacillus strains, namely B. subtilis CRN 1, B. velezensis CRN 2, B. subtilis CRN 7, B. subtilis CRN 11, and B. velezensis CRN 23 were initially assayed for diverse hydrolytic activities, followed by genome mining to unravel the potential applications. CRN 1 and CRN 7 showed the highest endoglucanase activity with 0.4 U/ml, while CRN 23 showed high ß-xylosidase activity of 0.36 U/ml. The comprehensive genomic insights of strains resolve taxonomic identity, clusters of an orthologous gene, pan-genome dynamics, and metabolic features. Annotation of Carbohydrate active enzymes (CAZymes) reveals the presence of diverse glycoside hydrolases (GH) GH1, GH5, GH43, and GH30, which are solely responsible for the effective breakdown of complex bonds in plant polysaccharides. Further, protein modeling and ligand docking of annotated endoglucanases showed an affinity for cellotrioside, cellobioside, and ß-glucoside. The finding indicates the flexibility of Bacillus-derived endoglucanase activity on diverse cellulosic substrates. The presence of the butyrate synthesis gene in the CRN 1 strain depicts its key role in the production of important short-chain fatty acids essential for healthy rumen development. Similarly, antimicrobial peptides such as bacilysin and non-ribosomal peptides (NRPS) synthesized by the Bacillus strains were also annotated in the genome. The findings clearly define the role of Bacillus sp. inside the camel rumen and its potential application in various plant biomass utilizing industry and animal health research sectors.

Exploration of rumen microbial and carbohydrate-active enzyme profiles in cattle fed coir a lignin-rich diet using a metagenomic approach.

Lignocellulosic biomass is a rich source of feed for cattle. Amongst them, coconut coir may be the potential source of feed supplements. To assess, the effect of various concentrations of coconut coir (0 %, 21 % and 40 %) as a feed supplement on the rumen microbiome of cattle (Kankrej breed), a metagenomic (16S rRNA gene amplicon and shotgun sequencing) study was performed. The Alpha diversity estimation from the amplicon study suggested that the group of cattle fed food without the coconut coir has a higher number of genera than the cattle fed with mixed ration. Within the liquid fraction, bacterial phyla Bacteroidetes were abundant followed by Firmicutes and Fibrobacteres, whereas the proportion of Tenericutes, TM7, SRI, Verrucomicrobia, Lentisphaerae, and Elusimicrobia had decreased with the rise in the coir concentration. While within the solid fractions, the proportion of Elusimicrobia increased, but the count of Bacteriodetes, Firmicutes, Fibrobacteres Tenericutes, TM7, SRI, Verrucomicrobia, and Lentisphaerae decreased with an increase in coir percentages. The results obtained from shotgun sequencing show similar results for bacterial diversity. The functions associated with carbohydrate metabolism were abundant in both the treatments as compared to the control. Functions related to glycoside hydrolases, glycosyltransferases and carbohydrate-binding modules were abundant in both the treatments as compared to control. Thus, the study indicates that the microbiome does alter after feeding coir as a supplement and may be used as feed for cattle.

Rumen and fecal microbial profiles in cattle fed high lignin diets using metagenome analysis.

Coconut coir (a lignin-rich, organic material) is widely used for its commercial and economic benefits. In this study, crossbred (exotic) and Kankrej (indigenous) breeds of cattle were fed diets containing 7 or 14% coconut coir. Metagenomic analyses (16S rRNA gene amplicon and shotgun sequencing) were used to characterize the microbial community in the rumen and fecal samples along with their functional capabilities. Both amplicon and shotgun analyses revealed the predominance of bacterial phyla, Bacteroidetes, Firmicutes, Actinobacteria and Fibrobacter in ruminal liquid, ruminal solid and fecal samples. 16S rRNA gene amplicon sequencing revealed a total of 18 different bacterial taxa were found to be enriched exclusively in the animals fed with 14% coir. The shotgun analysis revealed abundance of bacterial genera, Fibrobacter, Clostridium, Prevotella, Butyrivibrio, and Ruminococcus in both liquid and solid fractions of ruminal contents, while in the fecal sample, Bacteroides, Alistipes, Plaudibacter, Parabacteroides, Porphyromonas, and Victivallis and archaeal genus, Methanocorpusculum were abundant. The functional analysis based on dbCAN database suggested that among the Glycoside hydrolase family, genes that encode oligosaccharide degrading enzymes, GH3, GH13 (p-value < 0.05), and GH43 were abundant in the feces. In ruminal solid, cellulase encoding the GH5 family was abundant. Also, lignocellulosic binding modules encoded by the CBM family, including cellulose (CBM3) and hemicellulose binding modules (CBM32 and CBM67) were abundant. Thus, the study indicated the enrichment of lignocellulosic enzymes in ruminal contents in response to feeding the coconut coir, which could be mined for potential biofuel production and other biotechnological applications.

Isolation, purification and characterization of a novel esterase from camel rumen metagenome.

Bacterial esterases are gaining the importance in pharmaceuticals and agrochemical industries due to their excellent biocatalytic properties and a wide range of applications. In the present study, a novel gene encoding an esterase (designated as Est-CR) was identified from shotgun metagenomic sequencing data of camel rumen (Camelus dromedarius) liquor. The open reading frame consisted of 1,224bp, which showed 84.03% sequence identity to Bacteroidales bacterium, corresponding to a protein of 407 amino acids and has a catalytic domain belonging to an esterase. Est-CR belonged to family V with GLSMG domain. The purified enzyme with a molecular mass of 62.64 kDa was checked on SDS-PAGE, and its expression was confirmed by western blotting. The enzyme was active and stable over a broad range of temperature (35-65 °C), displayed the maximum activity at 50 °C and pH 7.0. Individually all metal ions inhibited the enzyme activity, while in combination, K2+, Ca2+, Mg2+ and Mn2+ metal ions enhanced the enzyme activity. The detergents strongly inhibited the activity, while EDTA (10 mM) increased the activity of the Est-CR enzyme. The enzyme showed specificity to short-chain substrates and displayed an optimum activity against butyrate ester. This novel enzyme might serve as a promising candidate to meet some harsh industrial processes enzymatic needs.

Characterizing rumen microbiota and CAZyme profile of Indian dromedary camel (Camelus dromedarius) in response to different roughages.

In dromedary camels, which are pseudo-ruminants, rumen or C1 section of stomach is the main compartment involved in fiber degradation, as in true ruminants. However, as camels are adapted to the harsh and scarce grazing conditions of desert, their ruminal microbiota makes an interesting target of study. The present study was undertaken to generate the rumen microbial profile of Indian camel using 16S rRNA amplicon and shotgun metagenomics. The camels were fed three diets differing in the source of roughage. The comparative metagenomic analysis revealed greater proportions of significant differences between two fractions of rumen content followed by diet associated differences. Significant differences were also observed in the rumen microbiota collected at different time-points of the feeding trial. However, fraction related differences were more highlighted as compared to diet dependent changes in microbial profile from shotgun metagenomics data. Further, 16 genera were identified as part of the core rumen microbiome of Indian camels. Moreover, glycoside hydrolases were observed to be the most abundant among all Carbohydrate-Active enzymes and were dominated by GH2, GH3, GH13 and GH43. In all, this study describes the camel rumen microbiota under different dietary conditions with focus on taxonomic, functional, and Carbohydrate-Active enzymes profiles.

Unraveling the camel rumen microbiome through metaculturomics approach for agriculture waste hydrolytic potential.

Cellulose is the most abundant natural polymer present on Earth in the form of agriculture waste. Hydrolysis of agriculture waste for simple fermentable reducing sugars is the bottleneck in the area of biofuel generation and other value-added products. The present study aims to utilize the camel rumen as a bioreactor for potent cellulolytic and hemicellulolytic bacteria by altering the feed types with varying cellulosic concentrations. A total of 6716 bacterial cultures were subjected to three layers of screening, where plate zymography and chromophoric substrate screening served as primary screening method for cellulolytic and hemicellulolytic potential. The potential isolates were genetically grouped using RAPD, and 51 representative isolates from each group were subjected to molecular identification through 16S rDNA sequencing, followed by quantification of various cellulolytic and hemicellulolytic enzymes. Out of 51 potent isolates, 5 isolates had high endoglucanase activity ranging from 0.3 to 0.48 U/ml. The selected five key isolates identified as Pseudomonas, Paenibacillus, Citrobacter, Bacillus subtilis, and Enterobacter were employed for hydrolyzing the various agriculture residues and resulted in approximately 0.4 mg/ml of reducing sugar. Furthermore, the metaculturomics approach was implemented to deduce the total cultured diversity through 16S rRNA amplicon library sequencing. The metaculturomics data revealed the dominance of proteobacteria and unidentified bacterial population in all four feed types, which indicates the possibility of culturing novel cellulose-deconstructing bacteria. Moreover, the presence of diverse hydrolytic enzymes in cultured isolates supports the usage of these bacteria in bio-processing of agriculture waste residues and obtaining the biofuels and other value-added products.

MetaRNAseq analysis of surti buffalo rumen content reveals that transcriptionally active microorganisms need not be abundant.

The present study describes rumen microbiota composition and their functional profiles in Indian Surti buffaloes by metagenomic (MG) and metatranscriptomic (MT) approaches. The study compares samples from buffaloes fed three different proportion of roughages; green and dry type of roughage; and different rumen liquor fractions. Irrespective of sample, Bacteroidetes and Firmicutes were the most predominant bacterial phyla, followed by Proteobacteria, Fibrobacteres and Actinobacteria while, Prevotella, Bacteroides, Ruminococcus and Clostridium were the most abundant genera. Different proportions of taxa were observed in both MG and MT approaches indicating the differences in organisms present and organisms active in the rumen. Higher proportions of fungal taxa were observed in MT while important organisms like Fibrobacter and Butyrivibrio and abundant organisms like Bacteroides and Prevotella were underrepresented in MT data. Functionally, higher proportions of genes involved in Carbohydrate metabolism, Amino acid metabolism and Translation were observed in both data. Genes involved in Metabolism were observed to be underrepresented in MT data while, those involved in Genetic information processing were overrepresented in MT data. Further, genes involved in Carbohydrate metabolism were overexpressed compared to genes involved in Amino acid metabolism in MT data compared to MG data which had higher proportion of genes involved in Amino acid metabolism than Carbohydrate metabolism. In all significant differences were observed between both approaches, different fractions of rumen liquor (liquid and solid) and different proportions of roughage in diet.

Exploring the eukaryotic diversity in rumen of Indian camel (Camelus dromedarius) using 18S rRNA amplicon sequencing.

In addition to a wide variety of anaerobic and facultative anaerobic bacteria, camel rumen also harbors a diverse of eukaryotic organisms. In the present study, the eukaryotic communities of camel rumen were characterized using 18S rRNA amplicon sequencing. Metagenomic DNA was isolated from rumen samples of fourteen adult Bikaneri and Kachchhi breeds of camel fed different diets containing Jowar, Bajra, Maize, and Guar. Illumina sequencing generated 27,161,904 number of reads corresponding to 1543 total operational taxonomic units (OTUs). Taxonomic classification of community metagenome sequences from all the samples revealed the presence of 92 genera belonging to 16 different divisions, out of which Ciliophora (73%), Fungi (13%) and Streptophyta (9%) were found to be the most dominant. Notably, the abundance of Ciliophora was significantly higher in the case of Guar feed, while Fungi was significantly higher in the case of Maize feed, indicating the influence of cellulose and hemicellulose content of feedstuff on the composition of eukaryotes. The results suggest that the camel rumen eukaryotes are highly dynamic and depend on the type of diet given to the animal. Pearson's correlation analysis suggested the ciliate protozoa and fungi were negatively correlated with each other. To the best of our knowledge, this is first systematic study to characterize camel rumen eukaryotes, which has provided newer information regarding eukaryotic diversity patterns amongst camel fed on different diets.

Transcriptomic analysis to affirm the regulatory role of long non-coding RNA in horn cancer of Indian zebu cattle breed Kankrej (Bos indicus).

Long non-coding RNA (lncRNA) was previously considered as a non-functional transcript, which now established as part of regulatory elements of biological events such as chromosome structure, remodeling, and regulation of gene expression. The study presented here showed the role of lncRNA through differential expression analysis on cancer-related coding genes in horn squamous cell carcinoma of Indian zebu cattle. A total of 10,360 candidate lncRNAs were identified and further analyzed for its coding potential ability using three tools (CPC, CPAT, and PLEK) that provide 8862 common lncRNAs. Pfam analysis of these common lncRNAs gave 8612 potential candidates for lncRNA differential expression analysis. Differential expression analysis showed a total of 59 significantly differentially expressed genes and 19 lncRNAs. Pearson's correlation analysis was used to identify co-expressed mRNA-lncRNAs to established relation of the regulatory role of lncRNAs in horn cancer. We established a positive relation of seven upregulated (XLOC_000016, XLOC_002198, XLOC_002851, XLOC_ 007383, XLOC_010701, XLOC_010272, and XLOC_011517) and one downregulated (XLOC_011302) lncRNAs with eleven genes that are related to keratin family protein, keratin-associated protein family, cornifelin, corneodesmosin, serpin family protein, and metallothionein that have well-established role in squamous cell carcinoma through cellular communication, cell growth, cell invasion, and cell migration. These biological events were found to be related to the MAPK pathway of cell cycle regulation indicating the role of lncRNAs in manipulating cell cycle regulation during horn squamous cell carcinomas that will be useful in identifying molecular portraits related to the development of horn cancer.

Metagenomic characterisation of ruminal bacterial diversity in buffaloes from birth to adulthood using 16S rRNA gene amplicon sequencing.

Microbial colonisation in the forestomach of a ruminant is one of the most crucial factors in determining many of its physiological developments and digestive capabilities. The present study attempts to identify establishment pattern of microbes in relation to food, age and rumen development in the buffalo calves at every fortnight interval from birth to 6 months of age, followed by every month till animals became 1 year of age. Diversity study based on 16S rRNA gene sequencing identified rapidly changing bacterial population during initial 60 days of life, which got assemblage as rumen became physiologically mature with increasing age of animals. A lactate fermenting aerobic to facultative anaerobic genera found during initial 30 days of life were expeditiously replaced by strict anaerobic cellulolytic bacterial population with increasing age. The study confirms that initial colonisation mainly depends on the oral cavity and skin of the mother, followed by the surrounding environment and feed offered, which is reversed in order once animal gets older. Some of the well-described genera based on culture-dependent studies like Ruminococcus spp. were found to be in lesser proportion suggesting an additional role of other microbes or niche in cellulose degradation. We report the presence of Porphyromonas spp. and Mannheimia glucosidal for the first time in bovine infants.

Illumina Next Generation Sequencing for the Analysis of Eimeria Populations in Commercial Broilers and Indigenous Chickens.

Eimeria species parasites can cause the enteric disease coccidiosis, most notably in chickens where the economic and welfare implications are significant. Seven Eimeria species are recognized to infect chickens, although understanding of their regional occurrence, abundance, and population structure remains limited. Reports of Eimeria circulating in chickens across much of the southern hemisphere with cryptic genotypes and the capacity to escape current anticoccidial vaccines have revealed unexpected levels of complexity. Consequently, it is important to supplement validated species-specific molecular diagnostics with new genus-level tools. Here, we report the application of Illumina MiSeq deep sequencing to partial 18S rDNA amplicons generated using Eimeria genus-specific primers from chicken caecal contents collected in India. Commercial Cobb400 broiler and indigenous Kadaknath type chickens were sampled under field conditions after co-rearing (mixed type farms, n = 150 chickens for each) or separate rearing (single type farms, n = 150 each). Comparison of MiSeq results with established Internal Transcribed Spacer (ITS) and Sequence Characterised Amplified Region (SCAR) quantitative PCR assays suggest greater sensitivity for the MiSeq approach. The caecal-dwelling Eimeria tenella and E. necatrix dominated each sample set, although all seven species which infect chickens were detected. Two of the three cryptic Eimeria genotypes were detected including OTU-X and OTU-Y, the most northern report for the latter to date. Low levels of DNA representing other Eimeria species were detected, possibly representing farm-level contamination with non-replicating oocysts or Eimeria DNA, or false positives, indicating a requirement for additional validation. Next generation deep amplicon sequencing offers a valuable resource for future Eimeria studies.

Microbial diversity and community composition of caecal microbiota in commercial and indigenous Indian chickens determined using 16s rDNA amplicon sequencing.

BACKGROUND: The caecal microbiota plays a key role in chicken health and performance, influencing digestion and absorption of nutrients, and contributing to defence against colonisation by invading pathogens. Measures of productivity and resistance to pathogen colonisation are directly influenced by chicken genotype, but host driven variation in microbiome structure is also likely to exert a considerable indirect influence. METHODS: Here, we define the caecal microbiome of indigenous Indian Aseel and Kadaknath chicken breeds and compare them with the global commercial broiler Cobb400 and Ross 308 lines using 16S rDNA V3-V4 hypervariable amplicon sequencing. RESULTS: Each caecal microbiome was dominated by the genera Bacteroides, unclassified bacteria, unclassified Clostridiales, Clostridium, Alistipes, Faecalibacterium, Eubacterium and Blautia. Geographic location (a measure recognised to include variation in environmental and climatic factors, but also likely to feature varied management practices) and chicken line/breed were both found to exert significant impacts (p < 0.05) on caecal microbiome composition. Linear discriminant analysis effect size (LEfSe) revealed 42 breed-specific biomarkers in the chicken lines reared under controlled conditions at two different locations. CONCLUSION: Chicken breed-specific variation in bacterial occurrence, correlation between genera and clustering of operational taxonomic units indicate scope for quantitative genetic analysis and the possibility of selective breeding of chickens for defined enteric microbiota.

Emergence of a genotype I variant of avian infectious bronchitis virus from Northern part of India.

Infectious bronchitis virus (IBV) is one of the foremost causes of a persistent economic burden to poultry industries worldwide. IBV belongs to the genus Gammacoronavirus within the family Coronaviridae. The IBV infection leads to respiratory and nephrogenic symptoms in broiler chickens. In addition, its infection leads to reduced fertility and hatchability in layer birds. We determined the first complete genome sequence of a variant IBV from an outbreak in Haryana state of the Northern part of India using next generation sequencing. On phylogenetic analysis of the IBV isolate, it clustered with genotype I lineage 1 (GI-1). The deduced amino acid sequence of S gene of IBV isolates showed a high level of identity with strains isolated from Tamil Nadu and the reference vaccine strains. Our result suggests that the IBV virus isolated from unvaccinated chicken flocks in North India might be a revertant strain originally evolved from the live attenuated vaccine strains used in the region. Determination of the complete genome sequence of additional IBV isolates from India is necessary to understand the epidemiology of IBV in India.

Metagenomic data of DNA viruses of poultry affected with respiratory tract infection.

The incidence and severity of respiratory diseases in commercial broiler chicken flocks have increased recently in India because of intensification of the broiler industry. Viral population are predominant in respiratory tract infections and they pose continuous economic burden to poultry industry by causing severe economic losses through decreased productivity [1], [2]. To understand viral metagenome of poultry associated with respiratory infections, we performed DNA virome sequencing and data analysis of broilers from 8 districts of Gujarat State in India. We report high quality sequencing reads and highly abundant DNA viral population present in the infected broiler birds. The raw sequencing data used to perform metagenomic analysis is available in the Sequence Read Archive (SRA) under the BioProject No. PRJNA322592 and Accession No. MAUZ00000000, MAVA00000000, MAVB00000000, MAVC00000000, MAVD00000000, MAVE00000000, MAVF00000000, MAVG00000000 (https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA322592).

Microbiota composition, gene pool and its expression in Gir cattle (Bos indicus) rumen under different forage diets using metagenomic and metatranscriptomic approaches.

Zebu (Bos indicus) is a domestic cattle species originating from the Indian subcontinent and now widely domesticated on several continents. In this study, we were particularly interested in understanding the functionally active rumen microbiota of an important Zebu breed, the Gir, under different dietary regimes. Metagenomic and metatranscriptomic data were compared at various taxonomic levels to elucidate the differential microbial population and its functional dynamics in Gir cattle rumen under different roughage dietary regimes. Different proportions of roughage rather than the type of roughage (dry or green) modulated microbiome composition and the expression of its gene pool. Fibre degrading bacteria (i.e. Clostridium, Ruminococcus, Eubacterium, Butyrivibrio, Bacillus and Roseburia) were higher in the solid fraction of rumen (P<0.01) compared to the liquid fraction, whereas bacteria considered to be utilizers of the degraded product (i.e. Prevotella, Bacteroides, Parabacteroides, Paludibacter and Victivallis) were dominant in the liquid fraction (P<0.05). Likewise, expression of fibre degrading enzymes and related carbohydrate binding modules (CBMs) occurred in the solid fraction. When metagenomic and metatranscriptomic data were compared, it was found that some genera and species were transcriptionally more active, although they were in low abundance, making an important contribution to fibre degradation and its further metabolism in the rumen. This study also identified some of the transcriptionally active genera, such as Caldicellulosiruptor and Paludibacter, whose potential has been less-explored in rumen. Overall, the comparison of metagenomic shotgun and metatranscriptomic sequencing appeared to be a much richer source of information compared to conventional metagenomic analysis.

Differential expression of microRNAs associated with thermal stress in Frieswal (Bos taurus x Bos indicus) crossbred dairy cattle.

Environmental temperature is one of the important abiotic factors that influence the normal physiological function and productive performance of dairy cattle. Temperature stress evokes complex responses that are essential for safeguarding of cellular integrity and animal health. Post-transcriptional regulation of gene expression by miRNA plays a key role cellular stress responses. The present study investigated the differential expression of miRNA in Frieswal (Holstein Friesian × Sahiwal) crossbred dairy cattle that are distinctly adapted to environmental temperature stress as they were evolved by using the temperate dairy breed Holstein Friesian. The results indicated that there was a significant variation in the physiological and biochemical indicators estimated under summer stress. The differential expression of miRNA was observed under heat stress when compared to the normal winter season. Out of the total 420 miRNAs, 65 were differentially expressed during peak summer temperatures. Most of these miRNAs were found to target heat shock responsive genes especially members of heat shock protein (HSP) family, and network analysis revealed most of them having stress-mediated effects on signaling mechanisms. Being greater in their expression profile during peak summer, bta-miR-2898 was chosen for reporter assay to identify its effect on the target HSPB8 (heat shock protein 22) gene in stressed bovine PBMC cell cultured model. Comprehensive understanding of the biological regulation of stress responsive mechanism is critical for developing approaches to reduce the production losses due to environmental heat stress in dairy cattle.

Molecular cloning, heterologous expression, and functional characterization of a cellulolytic enzyme (Cel PRII) from buffalo rumen metagenome.

A cellulase encoding gene, Cel PRII, was identified from Mehsani buffalo rumen metagenome, and cloned and expressed in Escherichia coli BL21(DE3)pLysS. The 1170 bp full length gene encodes a 389 residue polypeptide (Cel PRII) containing a catalytic domain belonging to glycosyl hydrolase (GH) 5 family. The fusion protein consisting of the Cel PRII, thioredoxin tag and 6x Histidine tag with predicted molecular weight of 63 kDa when recovered from inclusion bodies under denaturing conditions, exhibited cellulolytic activity against carboxymethyl cellulose (CMC). Recombinant Cel PRII was stable in the pH range 4.0-10.0 with pH optima 6.0. The optimal reaction temperature of Cel PRII was 30 °C with more than 50% of its activity retained at the temperatures ranging from 0 to 50 °C. Cel PRII exhibited enhanced enzymatic activity in the presence of Mn2+ ions and was inhibited in the presence of chelating agent EDTA. The K m and V max values for CMC were found to be 166 mg/mL and 1292 IU/mg, respectively. Cel PRII identified in the present study may act as an excellent candidate for industrial applications, and may aid in lignocellulosic biomass conversion because of its potential cellulolytic activity, thermostability, and excellent pH stability.