Generation of single stranded DNA with selective affinity to bovine spermatozoa.

OBJECTIVE: This study was conducted to generate single stranded DNA oligonucleotides with selective affinity to bovine spermatozoa, assess its binding potential and explore its potential utility in trapping spermatozoa from suspensions. METHODS: A combinatorial library of 94 mer long oligonucleotide was used for systematic evolution of ligands by exponential enrichment (SELEX) with bovine spermatozoa. The amplicons from sixth and seventh rounds of SELEX were sequenced, and the reads were clustered employing cluster database at high identity with tolerance (CD-HIT) and FASTAptamer. The enriched nucleotides were predicted for secondary structures by Mfold, motifs by Multiple Em for Motif Elicitation and 5' labelled with biotin/6-FAM to determine the binding potential and binding pattern. RESULTS: We generated 14.1 and 17.7 million reads from sixth and seventh rounds of SELEX respectively to bovine spermatozoa. The CD-HIT clustered 78,098 and 21,196 reads in the top ten clusters and FASTAptamer identified 2,195 and 4,405 unique sequences in the top three clusters from the sixth and seventh rounds, respectively. The identified oligonucleotides formed secondary structures with delta G values between -1.17 to -26.18 kcal/mol indicating varied stability. Confocal imaging with the oligonucleotides from the seventh round revealed different patterns of binding to bovine spermatozoa (fluorescence of the whole head, spot of fluorescence in head and mid- piece and tail). Use of a 5'-biotin tagged oligonucleotide from the sixth round at 100 pmol with 4×106 spermatozoa could trap almost 80% from the suspension. CONCLUSION: The binding patterns and ability of the identified oligonucleotides confirms successful optimization of the SELEX process and generation of aptamers to bovine spermatozoa. These oligonucleotides provide a quick approach for selective capture of spermatozoa from complex samples. Future SELEX rounds with X- or Y- enriched sperm suspension will be used to generate oligonucleotides that bind to spermatozoa of a specific sex type.

RNAseq Reveals the Contribution of Interferon Stimulated Genes to the Increased Host Defense and Decreased PPR Viral Replication in Cattle.

Peste des petits ruminants virus (PPRV) is known to replicate in a wide variety of ruminants causing very species-specific clinical symptoms. Small ruminants (goats and sheep) are susceptible to disease while domesticated cattle and buffalo are dead-end hosts and do not display clinical symptoms. Understanding the host factors that influence differential pathogenesis and disease susceptibility could help the development of better diagnostics and control measures. To study this, we generated transcriptome data from goat and cattle peripheral blood mononuclear cells (PBMC) experimentally infected with PPRV in-vitro. After identifying differentially expressed genes, we further analyzed these immune related pathway genes using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and selected candidate genes were validated using in-vitro experiments. Upon PPRV infection, we identified 12 and 22 immune related genes that were differentially expressed in goat and cattle respectively. In both species, this included the interferon stimulated genes (ISGs) IFI44, IFI6, IFIT1, IFIT2, IFIT3, ISG15, Mx1, Mx2, OAS1X, RSAD2, IRF7, DDX58 and DHX58 that were transcribed significantly higher in cattle. PPRV replication in goat PBMCs significantly increased the expression of phosphodiesterase 12 (PDE12), a 2',5'-oligoadenylate degrading enzyme that contributes to the reduced modulation of interferon-regulated gene targets. Finally, a model is proposed for the differential susceptibility between large and small ruminants based on the expression levels of type-I interferons, ISGs and effector molecules.

Complete Genome Sequence of a Newcastle Disease Virus from a Coturnix coturnix japonica (Japanese Quail) Covey in India.

The genome of a Newcastle disease virus isolated from a Japanese quail in 2003 is reported here. The genome is 15,192 nucleotides (nt) long, as found in the recent genotypes, and grouped as genotype VIIb, with a 6-nt insertion. This is the first report on the sequence of a genotype VII Newcastle disease virus (NDV) from India.

Homology modeling and structural comparison of leucine rich repeats of Toll like receptors 1-10 of ruminants.

Toll-like receptors (TLRs) are transmembrane receptors composed of extra cellular leucine rich repeats (LRRs) that identify specific pathogen associated molecular patterns triggering a innate immune cascade. The LRR regions of TLR 1-10 proteins of goat (Capra hircus), sheep (Ovis aries), buffalo (Bubalus bubalis) and bovine (Bos taurus) were modeled using MODELLER 9v7 tool and validated. The similarities and variations of these 10 TLRs extracellular regions of each species were compared using online servers like FATCAT, SSM and SSAP. It was evident that the LRRs of TLRs like 1, 2, 3 and 6 showed structural convergence with <1 % RMSD deviation while TLRs like 5, 7, 8 and 9 had high divergence. Docking analysis showed that TLR 2, 3 and 7 of all the selected four ruminant species were able to bind with their corresponding ligands like Peptidoglycan (PGN), Poly I:C, Resiquimod (R-848) and Imiquimod. However, there were variations in the active site regions, interacting residues and the number of bonded interactions. Variations seen among TLR structures and their ligand binding characteristics is likely to be responsible for species and breed specific genetic resistance observed among species or breeds.

Differential expression of toll-like receptor mRNA in corneal epithelium of ruminants.

PURPOSE: The present study was carried out to identify and assess the expression levels of toll-like receptors (TLRs) 1-10 mRNA in corneal epithelial cells of buffalo, goat, sheep and bull. MATERIALS AND METHODS: The globes from the respective species were collected and the corneal epithelium was denuded. The expression levels of the different TLR mRNAs were assessed by densitometry of the band intensities following reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: In all species TLR5 and TLR7 were highly expressed with lower levels of TLR10. The TLR6 mRNA levels were the lowest in the cornea of bull. The TLR2 expression was high in sheep and bulls, while TLR4 mRNA was higher in sheep alone. The level of the other TLRs varied across other species. CONCLUSION: Based on these observations it may be inferred that these animals would be resistant to flagellin (TLR5) and single-stranded RNA viruses (TLR7), while sheep alone may show more resistance to lipo-polysaccharide (TLR4) and peptidoglycan (TLR2)-mediated injury of the cornea. This work reports for the first time the expression of TLR mRNAs in the corneal epithelial cells of farm animals. The relationship of the TLR mRNA levels to corneal disease susceptibilities and pathogenicity remains to be explored further.