TaqMan real-time RT-PCR assay for detecting Japanese encephalitis virus in swine blood samples and mosquitoes.

Japanese encephalitis (JE) is an emerging mosquito-borne zoonotic flaviviral disease. The present study was undertaken with the objective to develop TaqMan real-time reverse-transcription polymerase chain reaction (RT-PCR) assay for rapid detection and quantification of Japanese encephalitis virus (JEV) in swine blood and mosquito vectors. The amplification of envelope (E) gene was targeted by designing gene-specific MGB TaqMan fluorescent probe along with the primers. The best performance in terms of sensitivity was achieved by standardized TaqMan real-time RT-PCR with a detection limit of 2.8 copies/reaction and it was found to be 4-log more sensitive than conventional RT-PCR. The applicability of the standardized TaqMan assay was evaluated by screening representative sets of field swine blood samples and mosquito pools for JEV. The viral load ranged between 3.32 × 107-4.2 × 102 copies/ml of swine blood samples, and 5.7 × 109-1.3 × 102 copies/pool of mosquitoes. The standardized assay which is highly sensitive, specific and rapid would aid in screening sentinel swine and mosquitoes under JEV surveillance programs for effective prevention and control of disease in human beings.

Identification and evaluation of reference genes for accurate gene expression normalization of fresh and frozen-thawed spermatozoa of water buffalo (Bubalus bubalis).

The quantitative real time PCR (qRT-PCR) has become an important tool for gene-expression analysis for a selected number of genes in life science. Although large dynamic range, sensitivity and reproducibility of qRT-PCR is good, the reliability majorly depend on the selection of proper reference genes (RGs) employed for normalization. Although, RGs expression has been reported to vary considerably within same cell type with different experimental treatments. No systematic study has been conducted to identify and evaluate the appropriate RGs in spermatozoa of domestic animals. Therefore, this study was conducted to analyze suitable stable RGs in fresh and frozen-thawed spermatozoa. We have assessed 13 candidate RGs (BACT, RPS18s, RPS15A, ATP5F1, HMBS, ATP2B4, RPL13, EEF2, TBP, EIF2B2, MDH1, B2M and GLUT5) of different functions and pathways using five algorithms. Regardless of the approach, the ranking of the most and the least candidate RGs remained almost same. The comprehensive ranking by RefFinder showed GLUT5, ATP2B4 and B2M, MDH1 as the top two stable and least stable RGs, respectively. The expression levels of four heat shock proteins (HSP) were employed as a target gene to evaluate RGs efficiency for normalization. The results demonstrated an exponential difference in expression levels of the four HSP genes upon normalization of the data with the most stable and the least stable RGs. Our study, provides a convenient RGs for normalization of gene-expression of key metabolic pathways effected during freezing and thawing of spermatozoa of buffalo and other closely related bovines.

Expression Profiling of Activin type IIB Receptor During Ontogeny in Broiler and Indigenous Chicken.

Augmenting the meat production is among the primary breeding objective of genetic selection programs in poultry production. However, the knowledge about the expression of genes regulating muscle growth at the molecular level is inadequate. Activin type IIB receptor (ACTRIIB) has been reported to play vital role in the negative regulation of muscle growth by binding to multiple members of transforming growth factor-ß superfamily. The present investigation was carried out to comprehend the trend of ACTRIIB messenger RNA in pectoralis major muscle during embryonic (E5-20) and post embryonic age (days 1, 14, 28, and 42) in both Control Broiler (CB) and Aseel by using Real-time PCR. The expression profile of ACTRIIB gene displayed a similar trend in CB and Aseel, however Aseel showed significantly (P < 0.001) higher transcription throughout the period. The fold change in expression of ACTRIIB in Aseel relative to CB varied from 3.94 to 14.72 folds and 3.28 to 7.14 folds during embryonic and post embryonic age, respectively. ACTRIIB exhibited its peak on E7, E11, and E16 during embryonic age, which coincides with the formation of primary and secondary muscle fibers in both lines. While at the time of post-embryonic age, ACTRIIB showed highest transcription on day 1 and lowest transcription on day 28 in both CB and Aseel. Within each line, the expression of ACTRIIB differed significantly (P < 0.001) between days in the course of embryonic and post-embryonic period. ACTRIIB gene expression had significant (P < 0.05) effect on all carcass traits except neck weight. Our results suggest that Aseel expressed higher levels of ACTRIIB transcript than CB. The study inferred that expression pattern of ACTRIIB was analogous in both CB and Aseel, which might imply that molecular mechanisms underlying muscle development and regulation are comparable in nature.

Phylogenetic analysis of Indian rabies virus isolates targeting the complete glycoprotein gene.

Rabies a fatal viral zoonosis is endemic in India. There is no report on phylogenetic study of Indian rabies virus isolates based on the complete G gene. In the present study, a total of 25 rabies positive brain samples collected during 2001-2014 from North India (UP, MP, Delhi, Rajasthan), South India (Kerala and Karnataka) and Gujarat states belonging to six different host species were subjected to G gene amplification by RT-PCR as three overlapping fragments of 881 bp, 991 bp and 618 bp. Phylogenetic analysis revealed that all Indian rabies virus isolates are genetically closely related with Arctic-like 1a lineage viruses. However, two distinct clusters were identified namely, India South and India North. All the Indian rabies isolates had 95.5-100% homology related to geography, but not to host species. Deduced amino acids on comparison revealed two amino acid changes, aa 356 in ECTO; N→K and aa 458; M→I, which were found to distinguish between the India South and India North isolates.

Comparative evaluation of nucleic acid-based assays for detection of Japanese encephalitis virus in swine blood samples.

Japanese encephalitis is an emerging mosquito-borne flaviviral zoonotic disease. The present study was undertaken with the objective of developing rapid and sensitive nucleic-acid-based assays for detection of Japanese encephalitis virus (JEV) in swine blood samples. Three nucleic-acid-based assays, viz., reverse transcription polymerase chain reaction (RT-PCR), reverse transcription loop-mediated isothermal amplification (RT-LAMP), and real-time RT-PCR, were developed and compared in terms of their diagnostic efficacy. All three assays were found to be 100 per cent specific. The minimum detection limit of RT-LAMP and real-time RT-PCR was 12 copies/µl, while RT-PCR could detect 1.2 × 10(5) copies/µl. On comparison, RT-LAMP and real-time RT-PCR were 4-log more sensitive than RT-PCR. The applicability of the assays was evaluated by screening 135 field swine blood samples, of which 24 (17.77 %) were positive by RT-LAMP and real-time RT-PCR and only six (4.44 %) were positive by RT-PCR. The viral load in swine blood samples ranged between 2 × 10(6) and 4.8 × 10(9) copies per ml of blood by real-time RT-PCR. The comparative diagnostic sensitivity and specificity of RT-LAMP vis-à-vis real-time RT-PCR was found to be 100 %, while the sensitivity and specificity of RT-PCR vis-à-vis real-time RT-PCR was found to be 25 % and 100 %, respectively. Thus, the use of RT-PCR may cause the incidence of JEV in the swine population to be underestimated, while the real-time RT-PCR reported here is the test of choice for reference laboratories, and the newly developed one-step RT-LAMP assay will be suitable for field-level testing.

Differential Expression of Th1- and Th2- Type Cytokines in Peripheral Blood Mononuclear Cells of Murrah Buffalo (Bubalus Bubalis) on TLR2 Induction by B. Subtilis Peptidoglycan.

Peripheral blood mononuclear cells (PBMCs) discriminate microbial pathogens and induce T-cell responses of appropriate effector phenotype accordingly. Toll-like receptors (TLRs), in part, mediate this microbial recognition and differentiation while the development of T-cell effector functions critically depends on the release of Th1- or Th2- type cytokines. In the present study, buffalo PBMCs were stimulated under in vitro culture conditions by Bacillus subtilis cell wall petidoglycan, a TLR2 ligand, in a dose-and time- dependent manner. The expression of TLR2 as well as the subsequent differential induction of the Th1 and Th2 type cytokines was measured. Stimulation was analyzed across five doses of peptidoglycan (10 µ/ml, 20 µg/ml, 30 µg/ml, 40 µg/ml and 50 µg/ml) for 3 h, 12 h, 24 h and 36 h incubation periods. We observed the induction of TLR2 expression in a dose- and time-dependent manner and the peptidoglycan induced tolerance beyond 30 µg/ml dose at all incubation periods. The correlation between peptidoglycan stimulation and TLR2 induction was found positive at all doses and for all incubation periods. Increased production of all the cytokines was observed at low doses for 3 h incubation, but the expression of IL-4 was relatively higher than IL-12 at the higher antigen doses, indicating tailoring towards Th2 response. At 12 h incubation, there was a pronounced decrease in IL-4 and IL-10 expression relative to IL-12 in a dose- dependent manner, indicating skewing to Th1 polarization. The expression of IL-12 was highest for all doses across all the incubation intervals at 24 h incubation, indicating Th1 polarization. The relative expression of TNF-α and IFN-γ was also higher while that of IL-4 and IL-10 showed a decrease. For 36 h incubation, at low doses, relative increase in the expression of IL-4 and IL-10 was observed which decreased at higher doses, as did the expression of all other cytokines. The exhaustion of cytokine production at 36 h indicated that PBMCs became refractory to further stimulation. It can be concluded from this study that the cytokine response to sPGN initially was of Th2 type which skews, more pronouncedly, to Th1 type with time till the cells become refractory to further stimulation.

Molecular epidemiology of Mycobacterium avium subspecies paratuberculosis: IS900 PCR identification and IS1311 polymorphism analysis from ruminants in the Punjab region of India.

Johne's disease is chronic granulomatous infectious enteritis of animals caused by Mycobacterium avium subspecies paratuberculosis. A total of 153 animals from 19 dairy farms, 2 gaushalas (unproductive-animal rehabilitation centers), 2 goat and 2 sheep farms from different districts of the Punjab region were selected on the basis of clinical signs of disease. All samples from cattle (n=86), buffalo (n=34), goat (n=25) and sheep (n=26) were subjected to Ziehl-Neelsen staining and DNA extraction by a freeze and thaw method. Ziehl-Neelsen staining detected 71% samples positive for acid-fast bacilli whereas IS900 PCR detected 55% positive for Map DNA. IS1311 PCR-REA analysis of IS900 positive samples revealed 'Bison' type as the most prevalent (82%) genotype of Map, infecting all domestic ruminants. 'Cattle' type was present in a minority of cases (15%) from cattle, buffaloes and goats. This is the first report of 'Cattle' type Map from buffalo and goat species in India.

Sequencing, characterization and genetic variation of the Bos indicus glucose-6-phosphate-dehydrogenase gene.

The coding sequence of the bovine (Bos indicus) Glucose-6-phosphate-dehydrogenase (G6PD) gene was amplified by Reverse Transcriptase-PCR (RT-PCR), cloned, sequenced and characterized. The deduced amino acid sequence clustered the bovine G6PD sequence with the other mammalian G6PD proteins into a monophyletic group. The bovids (B. indicus and B. taurus) clustered clearly from the rodent (rat, mouse and hamster) subcluster and from humans. The multiple sequence alignment of the bovine G6PD with the mammalian species clearly revealed conservation of the substrate, coenzyme, catalytic and the dimer binding sites with the solved X-ray crystallographic structure of Homo sapiens. Also, four fragments of bovine (Bos indicus) G6PD gene viz. 118, 319, 683 and 408 bp were amplified and sequenced for the first time. A G/A and G/C single nucleotide polymorphisms in intron-9 and exon-10 were detected on PCR-RFLP of the 319 bp amplicon with Hae III and Pst I, respectively. This work is the first study on Bos indicus G6PD gene at the nucleotide level.