Comparative analysis of innate immune response following in vitro stimulation of sheep and goat peripheral blood mononuclear cells with bluetongue virus - serotype 23.

Bluetongue is an infectious disease caused by bluetongue virus (BTV), which affects sheep, goat, cattle and certain wild ruminants. However severe clinical signs are usually seen with significant mortality in sheep than cattle and goat. To date, comparative studies on innate immune responses of sheep and goat infected with BTV is lacking. In this study, we compared the innate immune response of sheep and goat by infecting the peripheral blood mononuclear cells (PBMCs) with BTV serotype 23. In our study, we observed that sheep PBMCs supports higher virus replication than goat PBMCs. To delineate the role of innate immune response in differential viral replication observed in this study, we examined TLR3 (Receptor for dsRNA virus) mRNA expression and cytokine profiles (IL-1ß, Il-6, IL-8, Il-10, IL-12p40, TNF-α, IFN-γ and IFN-α) following Poly I:C (TLR3 ligand) stimulation and BTV 23 infection. In our present study, sheep PBMCs had significantly higher TLR3 mRNA levels, TLR3 specific ligand (Poly I:C) stimulation resulted in increased levels of IFN-γ at transcriptional and translational levels along with IL-8 and IL-10 at transcriptional levels. Whereas, the levels of TNF-α was higher in goat PBMCs at transcriptional levels. BTV infected sheep PBMCs expressed significantly higher levels of IFN-γ at transcriptional and translational levels along with IL-6, IL-8 and IL-10 at transcriptional levels. Whereas the expression levels of TNF-α and IFN-α at transcriptional and translational levels were significantly high in goat PBMCs. To examine the potential factor for consistent increase in the expression of TNF-α, we sequenced the promoter region of TNF-α and identified a total of five single nucleotide polymorphisms (SNP) and one indel in goat TNF-α promoter region. Luciferase assay for transcriptional activity of the promoter showed that goat TNF-α has significantly enhanced transcriptional activity in comparison with sheep TNF-α promoter. Altogether, our data suggests that the expression levels of TNF-α and IFN-α and/or IL-10 plays crucial role in replication of BTV 23.

Comparative in vitro toll-like receptor ligand induced cytokine profiles of Toda and Murrah buffaloes-Identification of tumour necrosis factor alpha promoter polymorphism.

The objective of this study was to assess cytokine production upon activation of pattern recognition receptors responsible for sensing bacterial and viral pathogen associated molecular patterns in two genetically diverse buffalo breeds, Toda and Murrah. A very limited molecular-epidemiological analysis showed a higher prevalence of Anaplasma and Theileria in Murrah than Toda buffaloes. Toda buffalo peripheral blood mononuclear cells (PBMC) produced significantly higher levels of IFN γ and/or TNF α mRNAs in response to peptidoglycan, poly I:C, lipopolysaccharide, imiquimod and CpG. Flagellin stimulation did not result in any significant differences in the expression levels of the cytokines tested between these breeds. The levels of ligand induced IFN γ and TNF α mRNA and proteins also correlated except when induced with CpG. The proximal promoter region of TNF α across these two breeds were also sequenced to detect SNPs and promoter assay performed to determine their role in altering the transcriptional activity. Two polymorphisms were identified at -737 (T/A) and -1092 (G/T) positions in Toda buffalo TNF α promoter and promoter assay revealed higher transcription activity in Toda buffalos than in Murrah. This suggests that disease tolerance of these buffalo breeds could be due to the differences in their cytokine transcription levels in response to the respective PAMPs that may be at least in part determined by polymorphisms in the cytokine promoter regions.

Transcript profiling of pattern recognition receptors in a semi domesticated breed of buffalo, Toda, of India.

The primary objective of this study was to assess the expression profile and levels of toll-like receptor (TLR) mRNAs in the spleen, lung, mediastinal lymph node (MLN), jejunum, rectum, skin and peripheral blood mononuclear cells (PBMC) of Toda and Murrah buffalos. Spleen and PBMC had increased expression of TLR mRNAs 2, 4, 5, 6, 8, 9 and 10; lung had increased expression of TLR mRNAs 2, 4, 5, 6 and 8, MLN TLR mRNA 6, 9, 10 and decrease in TLR 3 and 7 mRNAs in skin. No significant differences were observed in the expression levels of any of the TLR mRNA in jejunum and rectum. Toda buffaloes showed significantly higher expression levels of TLR 9 mRNA in MLN, TLR mRNAs 1, 5, 6, 9 and 10 in skin and TLR mRNAs 2, 4, 7 and 9 in PBMC than Murrah buffaloes living in the vicinity. Toda and Murrah buffaloes were inoculated with TLR5 (flagellin) and TLR9 (CpG ODN) ligands in vivo and expression levels of the respective TLRs analyzed 12h later. Following CpG inoculation, Toda buffaloes had significantly higher levels of TLR 9 mRNA expression but not in Murrah. However, flagellin induction did not increase TLR 5 mRNA expression in both these breeds. Histological sections of the skin were made and infiltrating cell clusters were graded and quantified. Following CpG inoculation, Toda buffaloes showed higher numbers of infiltrating grade 1 and grade 3 cell clusters while Murrah showed lower numbers of infiltrating grade 1 cells as compared to mock-inoculated skin sections. Flagellin treatment revealed no significant differences in infiltrating cell clusters in both the breeds. The results have shown differential expression of TLR mRNAs in various tissues between two divergent buffalo breeds with the highest difference in TLR expression profile seen in the skin, the largest portal of entry of pathogens, of Toda.

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.