A. Raja

Differential expression of toll-like receptor mRNA in selected tissues of goat (Capra hircus)

Pattern recognition receptors (PRRs) expressed by various immune cells and tissues have been shown to play a pivotal role in the recognition of pathogens by the host. The present study was carried out to identify toll-like receptors (TLRs) 1-10 mRNA in goat peripheral blood mononuclear cells (PBMCs) and selected tissues including jejunum, lung, lymph node, skin, spleen and uterus using reverse transcriptase polymerase chain reaction (RT-PCR). Our results confirm earlier reports regarding the evolutionarily conserved nature of these receptors as successful amplification of the goat TLR mRNAs could be obtained with bovine TLR mRNA-specific primers. The partial sequences of the purified TLR PCR amplicons had 93.8-99.7% nucleotide identity with sheep TLR cDNA sequences available in the GenBank. Semi-quantification of the expression levels of the TLR mRNAs was done using densitometric analysis of band intensities. All the TLR mRNAs (1-10) were expressed in high amounts in the lymph node while spleen showed lower expression of TLR 6 and 10 mRNAs. PBMC and lung expressed all TLR mRNAs in high amounts except TLR 10 mRNA. In uterus and jejunum, lower expression of TLR 3, 4 and 10 mRNAs was seen. Skin had the lowest repertoire of TLR mRNA expression with lower or no expression of TLR 2, 3, 4, 8, 9 and 10 mRNAs. Another interesting observation was that tissues such as uterus, lung and skin that exhibited lower levels of TLR 2 had higher levels of TLR 6 mRNAs.

Expression profile of toll like receptors in a range of water buffalo tissues (Bubalus bubalis).

The present study was carried out to determine the expression profile of toll-like receptors (TLRs) 1-10 in buffalo peripheral blood mononuclear cells (PBMNC), neutrophils, spleen, liver, lung, heart, kidney, ovary and uterus using reverse transcriptase polymerase chain reaction (RT-PCR) with bovine TLR-specific primers The buffalo TLR partial nucleotide sequences had 95-98% nucleotide homology with bovine TLR sequences available in the GenBank. PBMNC expressed all TLRs except TLR1 and neutrophils expressed all TLRs except TLR3. Expression of all TLRs was observed in spleen, lung and liver tissues. Wide range of TLR mRNA expression was observed in heart, which lacked the expression of only TLR10. Among the tissues analyzed kidneys had the least repertoire of TLR expression. The kidney tissue revealed mRNA expression of only TLR2, TLR5, TLR7 and TLR9. Among the reproductive tissues analyzed, uterus expressed a wide range of TLRs such as 2, 5, 7, 8, 9 and 10 while ovary expressed all TLRs except TLR1 indicating their immuno competence.

Effect of siRNA mediated suppression of signaling lymphocyte activation molecule on replication of peste des petits ruminants virus in vitro.

Abstract Signaling lymphocyte activation molecule (SLAM) expression was inhibited in B95a cell line using siRNA and the effect of SLAM inhibition on peste des petits ruminants virus (PPRV) replication and infectivity titre was studied. SLAM suppression was assessed using real-time PCR and flow cytometry to confirm suppression at the m-RNA and protein levels, respectively. Three chemically synthesized siRNAs were transfected individually using oligofectamine into B95a cell line. This resulted in SLAM suppression from 48 to 454-folds, in comparison to the untransfected B95a cell line. When the SLAM suppressed B95a cell line was infected with PPRV, replication was reduced by 12–143-folds and virus titre was reduced from log10 1.09 to 2.28. siRNA 3 showed the most potent inhibition of SLAM expression both at m-RNA and protein levels. This also caused the maximum reduction of virus replication and virus titre. A 100-fold reduction in PPRV titres was seen in anti-SLAM antibody neutralized B95a cell line. This further confirms that SLAM is one of the (co) receptors for PPRV. However, the presence of other putative virus receptor(s) is/are not ruled out.

Rapid detection of Newcastle disease virus replication in embryonated chicken eggs using quantitative real time polymerase chain reaction.

Newcastle disease virus (NDV), an avian paramyxovirus, is an economically important disease of poultry globally. Rapid methods to detect and differentiate the virus are important to curtail the spread of this virus. Nucleic acid based detection methods are routinely employed for diagnosis that suffer from the disadvantage of failure to discriminate viable virus and non-infectious genome. However, virus isolation remains the gold standard for diagnosis of field outbreaks. The sensitivity of virus isolation was combined with nucleic acid based detection methods so that the time taken for confirmatory diagnosis could be considerably reduced while increasing sensitivity. Quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) and conventional RT-PCR techniques were compared for the detection of NDV genome replication in 9-11-day-old embryonated chicken eggs (ECE) using the nucleoprotein (NP) gene of the virus as a target. The results suggest that at least two to fourfold increase in cycle threshold (C(t)) values over the baseline C(t) value of samples lacking infectious virus, would indicate live NDV replication. The limit of detection of NDV replication using qRT-PCR was 1×10(4.0) mean embryo infective doses (EID(50)). The earliest time point when live virus replication was detectable by qRT-PCR or RT-PCR was 30h post-inoculation in ECE.

Toll-Like Receptor Responses to Peste des petits ruminants Virus in Goats and Water Buffalo

Ovine rinderpest or goat plague is an economically important and contagious viral disease of sheep and goats, caused by the Peste des petits ruminants virus (PPRV). Differences in susceptibility to goat plague among different breeds and water buffalo exist. The host innate immune system discriminates between pathogen associated molecular patterns and self antigens through surveillance receptors known as Toll like receptors (TLR). We investigated the role of TLR and cytokines in differential susceptibility of goat breeds and water buffalo to PPRV. We examined the replication of PPRV in peripheral blood mononuclear cells (PBMC) of Indian domestic goats and water buffalo and demonstrated that the levels of TLR3 and TLR7 and downstream signalling molecules correlation with susceptibility vs resistance. Naturally susceptible goat breeds, Barbari and Tellichery, had dampened innate immune responses to PPRV and increased viral loads with lower basal expression levels of TLR 3/7. Upon stimulation of PBMC with synthetic TLR3 and TLR7 agonists or PPRV, the levels of proinflammatory cytokines were found to be significantly higher while immunosuppressive interleukin (IL) 10 levels were lower in PPRV resistant Kanni and Salem Black breeds and water buffalo at transcriptional level, correlating with reduced viralloads in infected PBMC. Water buffalo produced higher levels of interferon (IFN) α in comparison with goats at transcriptional and translational levels. Pre-treatment of Vero cells with human IFNα resulted in reduction of PPRV replication, confirming the role of IFNα in limiting PPRV replication. Treatment with IRS66, a TLR7 antagonist, resulted in the reduction of IFNα levels, with increased PPRV replication confirming the role of TLR7. Single nucleotide polymorphism analysis of TLR7 of these goat breeds did not show any marked nucleotide differences that might account for susceptibility vs resistance to PPRV. Analyzing other host genetic factors might provide further insights on susceptibility to PPRV and genetic polymorphisms in the host.

Expression profile of toll-like receptor 2 mRNA in selected tissues of shark (Chiloscyllium sp.)

Sharks are a species of delight for immunologists from the evolutionary perspective since it is considered as the first species to have evolved the adaptive immune responses in addition to the innate immune system. One of the components of the highly conserved innate immune system is the toll-like receptors (TLR) which has a conserved overall protein structure throughout deuterostome evolution. There is no report that demonstrates the expression of these receptors in sharks. In this study we successfully amplified a 270 bp amplicon using a degenerate primer design strategy that corresponded to the Toll/IL-1 receptor (TIR) domain of TLR2 (GenBank ID: JF792813). BLAST analysis revealed a maximum nucleotide identity of 87% and 76% with the TLR2 of higher mammals and teleost fishes respectively. Domain prediction revealed a TIR structure between 1 and 87 amino acids that had a maximum identity of 58% and 76% with TLR2 - TIR protein of teleost fishes and higher mammals respectively. Phylogenetic analysis revealed a closer clustering of the shark TIR sequence with those from human, cattle, goat, sheep and chicken than with other fish species. Basal expression levels of the TLR2-TIR mRNA were found to be significantly higher in kidneys followed by fins, spleen and intestinal spiral valve (ISV). In tissues such as spleen and kidney the expression of the TLR2-TIR mRNA could be localized to lymphoid and macrophages like cells and tubular epithelial cells respectively. In-vivo exposure of sharks to peptidoglycan (TLR 2 ligand) resulted in 9 folds higher expression of TLR2-TIR mRNA in gills followed by 5 folds in the fins. However, when inoculated with a TLR ligand pool, the expression levels significantly increased to 12 fold in skin followed by epigonal, kidneys and ISV. These findings not only support the presence of the TLRs in sharks but also their induction upon exposure to specific ligands. Further studies are needed to identify their numbers, their ligand specificity and downstream cytokine responses.

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-1β, 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.

Isolation and molecular characterization of canine distemper virus from India

Ocular swabs from canine distemper virus (CDV) suspected live or brain tissue from dead dogs were tested for the presence of CDV nucleoprotein (N) gene using reverse transcriptase polymerase chain reaction (RT-PCR). Partial “N” gene sequencing of the RT-PCR-positive samples and the local vaccine virus revealed that the Ind/Andaman 01/07 virus was highly divergent from the rest of the CDV isolates and from the vaccine strain. Quantitative real-time PCR (qRT-PCR) using SYBR Green I chemistry for CDV haemagglutinin “H” gene quantification showed Ct values ranging from 29.76–30.67 in the RT-PCR-positive samples. Two of the positive samples, designated Ind/TN 01/07 and Ind/Andaman 01/07 were used for virus isolation in B95a cell line. Characteristic cytopathic changes such as rounding of cells, syncytia formation, and ballooning were seen from the first passage onwards. Specific cytoplasmic fluorescence was seen in infected cells with a commercial reference serum against CDV. To the best of our knowledge, this is the first report of CDV isolation from clinical cases in India.

Detection of Rabies Virus Antigen or Antibody Using Flow Cytometry

Rabies is invariably a fatal encephalomyelitis that is considered to be a serious public health problem. Rabies diagnosis must be rapid and conclusive. Detection and quantification of antirabies antibodies is used for assessment of the effectiveness of rabies vaccines. Hence, computer‐automated detection of fluorescence using flow cytometry was attempted to reduce the work time required to undertake the conventional rapid fluorescent focus inhibition test (RFFIT).

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.