Sero-epidemiology of porcine parvovirus, circovirus, and classical swine fever virus infections in India.

Reproductive problems in swine caused by porcine viruses pose a serious threat to the pig industry in developing countries like India. For evaluating the true extent of porcine infections, a total of 1308 representative sera samples were collected from 92 different pig farms covering 8 North-Eastern states and Punjab state of Northern India during a period of 2 years (2011-2013). Sera samples were tested for the presence of antibodies against porcine parvovirus (PPV), porcine circovirus-2 (PCV-2), and classical swine fever virus (CSFV) using commercial enzyme-linked immunosorbent assay (ELISA) kits. In the North-Eastern states, the seroprevalence of CSFV in non-vaccinated animals was 6.30% and that of PCV2 and PPV was 6.28% and 1.24%, respectively. In Punjab, the seroprevalence of CSFV in non-vaccinated animals was 44.44% and seroprevalence of PCV-2 and PPV was 34.07% and 39.10%, respectively. Detection of antibodies against more than one virus revealed that 4.66% animals had co-infection with PCV-2 and PPV, 1.75% with CSF and PPV, 1.98% with CSF and PCV-2, and 1.75% with all the three viruses. The receiver operator characteristics (ROC) curve analysis depicted that piglet mortality, parvovirus, and CSFV were the most important parameters with an AUC value of 0.997, 0.897, and 0.973, respectively. Incidence of single or co-infection with different viruses showed that the occurrence of single infection was significantly more prevalent than co-infection. This study provides useful information to set up future epidemiologic, flock management, and public animal health policies for the prevention and control of PCV-2, PPV, and CSF in India.

Prevalence of bovine herpes virus-1 among reproductive disorders in cattle and buffaloes in Punjab region of India.

Bovine Herpes Virus (BHV-1) is a virus prevalent among cattle and buffaloes which accounts for considerable reproductive failures. This study was undertaken with the objective of studying the prevalence of BHV-1 in reproductive tract infections of cattle and buffaloes in Punjab region in India. A total of 70 reproductive tract samples (like vaginal mucous, cervical mucous, uterine discharges, uterine pus and aborted materials like placenta, caruncles, foetal stomach contents, amniotic fluid and placental fluid) were taken from cattle and buffaloes from various areas of Punjab which were suffering from different reproductive disorders. The samples were screened for the presence of genome of BHV-1using PCR targeting gE gene. Out of 70 samples screened, only one sample was positive for the presence of BHV-1 genome which had an amplicon size of 468 bp, specific to the targeted gene. The study concluded that BHV-1 has very low prevalence among reproductive disorders in cattle and buffaloes in Punjab region, but has increased over last few years, particularly in female cattle and buffaloes.

In vitro and in silico study of eggplant extract on human cancer cell lines.

The aim of this research was to study the metabolite composition, antioxidative potential and cytotoxic activities of Solanum melongena fruit extracts. Phytochemical analyses of extracts were performed using LC-MS. Phenolic compounds were the major constituents present in the fruit extracts. Free radical scavenging activities were recorded and the highest activities were reported in methanolic extracts using DPPH (103.70 ± 2.75 EC50 µg/mL), ABTS (81.74 ± 3.64 EC50 µg/mL), and FRAP (22.39 ± 1.52 µmol TE/g) assays. Quantification has suggested the presence of delphinidin derivatives, and caffeic acid conjugates as major constituents of fruit extracts. The potential binding of these derivatives with human cell surface receptors was analysed using in silico analysis and validated for cytotoxic and apoptotic effects using in vitro studies on human cancer cell lines. The methanolic extract has shown the highest cytotoxic activity.

Development of recombinant subunit vaccine targeting InvH protein of Salmonella Typhimurium and evaluation of its immunoprotective efficacy against salmonellosis.

Salmonella Typhimurium is the most prevalent non-host specific Salmonella serovars and a major concern for both human and animal health systems worldwide contributing to significant economic loss. Type 3 secretion system (T3SS) of Salmonella plays an important role in bacterial adherence and entry into the host epithelial cells. The product of invH gene of Salmonella is an important component of the needle complex of the type 3 secretion system. Hence, the present study was undertaken to clone and express the 15 kDa InvH surface protein of Salmonella Typhimurium in an E. coli host and to evaluate its immune potency in mice. The purified recombinant InvH (r-InvH) protein provoked a significant (p < 0.01) rise in IgG in the inoculated mice. The immunized mice were completely (100%) protected against the challenge dose of 107.5 LD50, while protection against challenge with the same dose of heterologous serovars was 90%. The bacterin-vaccinated group showed homologous protection of 60% against all three serovars. Findings in this study suggest the potential of the r-InvH protein of S. Typhimurium as an effective vaccine candidate against Salmonella infections.

An open-label randomised pilot trial on safety of wheat variety C273 in patients with adult celiac disease.

BACKGROUND: The only effective treatment for celiac disease (CeD) is gluten free diet (GFD). However, GFD is restrictive and efforts are being made to explore alternative therapies including safer wheat varieties. Wheat variety C273 has been previously identified to have reduced load of intact T-cell stimulatory epitopes via in silico and in vitro analysis. METHODS: Adult patients diagnosed with CeD and recovered on GFD were included in the study. Patients were randomised into two groups in a 2:1 ratio. Patients in group I had graded introduction of C273 wheat in diet, maintained for 24 weeks; in Group II, wheat was restricted with continuation of GFD. Clinical symptoms, serology [anti-tissue transglutaminase (anti-tTG), anti-endomysial antibody (anti-EMA)], circulating inflammatory biomarkers [intestinal fatty-acid binding protein (I-FABP), plasma citrulline, interferon-γ (IFN-γ)] and histology were evaluated periodically. Final evaluation was performed at week 28. RESULTS: A total of 15 patients were enrolled (Group I: n = 10, Group II: n = 5). All patients except two in Group I remained compliant. None of the remaining eight patients in group I developed symptoms. No significant changes in serology (anti-tTG, anti-EMA) and histology were observed between the two groups at 28 weeks (p > 0.05). Significant changes in plasma citrulline(29.87 ± 8.98 versus 36.58 ± 3.09, p = 0.049) and IFN-γ (44.56 ± 9.74 versus 33.50 ± 3.68; p = 0.031) were observed in Group I. CONCLUSION: Consumption of C273 wheat did not result in development of symptoms or evident changes in serology and histology at 28 weeks. However, variations in circulating inflammatory markers were noted. Larger randomised trials are needed to corroborate these findings. CLINICAL TRIALS REGISTRY-INDIA: CTRI/2018/06/014521.

Variable Immunogenic Potential of Wheat: Prospective for Selection of Innocuous Varieties for Celiac Disease Patients via in vitro Approach.

Celiac Disease (CD) is a multifactorial, autoimmune enteropathy activated by cereal proteins in genetically predisposed individuals carrying HLA DQ2/8 genes. A heterogenous gene combination of the cereal prolamins is documented in different wheat genotypes, which is suggestive of their variable immunogenic potential. In the current study, four wheat varieties (C591, C273, 9D, and K78) identified via in silico analysis were analyzed for immunogenicity by measuring T-cell proliferation rate and levels of inflammatory cytokines (Interferon-γ and Tumor Necrosis Factor-α). Peripheral Blood Mononuclear Cells and biopsy derived T-cell lines isolated from four CD patients in complete remission and two controls were stimulated and cultured in the presence of tissue transglutaminase activated pepsin-trypsin (PT) digest of total gliadin extract from test varieties. The immunogenicity was compared with PBW 621, one of the widely cultivated wheat varieties. Phytohaemagglutinin-p was taken as positive control, along with unstimulated cells as negative control. Rate of cell proliferation (0.318, 0.482; 0.369, 0.337), concentration of IFN- γ (107.4, 99.2; 117.9, 99.7 pg/ml), and TNF- α (453.8, 514.2; 463.8, 514.2 pg/ml) was minimum in cultures supplemented with wheat antigen from C273, when compared with other test varieties and unstimulated cells. Significant difference in toxicity levels among different wheat genotypes to stimulate celiac mucosal T-cells and PBMC's was observed; where C273 manifested least immunogenic response amongst the test varieties analyzed.

Validation of immunomodulatory effects of lipopolysaccharide through expression profiling of Th1 and Th2 biased genes in Newcastle disease virus vaccinated indigenous chicken.

BACKGROUND AND AIM: Newcastle disease (ND) is considered one of the most important poultry diseases with chicken morbidity and mortality rates up to 100%. Current vaccination programs allow the use of live attenuated vaccines in the field to protect against the disease, which alone is inefficient and requires repeat booster doses. Toll-like receptor agonists (e.g., lipopolysaccharide [LPS]) as adjuvants are the ones, most extensively studied and have shown to be very promising in delivering a robust balanced immune response. In the present study, we have evaluated the potential of LPS to elicit a strong immune response with respect to the elicitation of both Th1 (cell-mediated) and Th2 (humoral) immune arms. MATERIALS AND METHODS: A total of 72 apparently healthy 1-day-old indigenous unvaccinated chicks were randomly divided into six experimental Groups A to F (n=12). At 8-week of age chicks in Group A, C, and E were vaccinated with live attenuated La Sota strain ND vaccine along with LPS, bovine serum albumin, and normal saline solution, respectively, and those in Group B, D, and E were kept separately without vaccination. Sampling was done on days 0, 1, 3, 7, 14, 21, 35, and 60 after vaccination. After vaccination and respective adjuvant application, Th1 and Th2 cytokine expression were measured in mRNA of both blood and tissue samples. RESULTS: The results were validated by, hemagglutination inhibition and enzyme-linked immunosorbent assay tests, to check for the humoral as well as cell-mediated immune response in blood serum levels. The results showed an increase in mRNA expression of the Th1 biased cytokines in Group A (LPS+NDV) as compared to the control groups. Similar mRNA expression pattern was seen in blood as well as tissue samples. Validation of results also indicates an increase in Cell-mediated Immunity as well as a humoral immune response in Group A (LPS+NDV). CONCLUSION: The results of the study provided enough evidence to consider LPS as a potential vaccine adjuvants candidate against ND in chicken.

Data on meq gene sequence analysis of Ludhiana MDV isolates.

The data described are related to the article entitled "Sequence Analysis of Meq oncogene among Indian isolates of Marek׳s Disease Herpesvirus" M. Gupta, D. Deka, Ramneek, 2016. Seven meq genes of Ludhiana Marek׳s disease virus (MDV) field isolates were PCR amplified by using proof reading Platinum Pfx DNA polymerase enzyme, sequenced and then analyzed for the distinct polymorphisms and point mutations. The sequences were named as LDH 1758, LDH 2003, LDH 2483, LDH 2614, LDH 2700, LDH 2929 and LDH 3262. At this point, their deduced Meq amino acid sequences were compared with GenBank available already sequenced meq genes worldwide in their deduced amino acid form to study their identity/similarity with each other.

Sequence analysis of Meq oncogene among Indian isolates of Marek's disease herpesvirus.

Marek's disease (MD), caused by Marek's disease virus (MDV), is a highly contagious neoplastic disease of chicken that can be prevented by vaccination. However, in recent years many cases of vaccine failure have been reported worldwide as chickens develop symptoms of MD in spite of proper vaccination. Distinct polymorphism and point mutations in Meq gene of MDV have been reported to be associated with virulence and oncogenicity. The present study was carried out with the objective to isolate and characterize field isolates of MDV on the basis of Meq gene. Twenty five samples of suspected cases of MD were collected and processed for virus isolation in duck embryo fibroblast (DEF) primary culture where 28% (7 of 25) samples showed characteristic cytopathic effects of MDV in the form of plaques and syncytia. Additional evidence of presence of MDV in these samples was confirmed by PCR. To analyze diversity in all seven isolates of MDV, a polymorphism study was carried out by cloning and sequencing of full length of Meq gene (1020 bp). Sequence homology of 7 isolates with 23 reference strains showed 98.10-99.40% similarity in nucleotide and 95.90-98.50% similarity in amino acid sequences. Six isolates revealed 5 repeat sequences of 4 prolines (PPPP) whereas, one isolate revealed only 4 repeats. In phylogenetic analysis, these isolates formed a separate cluster showing close relatedness to the Chinese isolates. The study indicates a high mutation rate in field isolates of MDV that may be probable cause of vaccination failure.

Minimization of apoptosis-like changes in cryopreserved buffalo bull sperm by supplementing extender with Bcl-2 protein.

AIM: This study was aimed at evaluating the anti-apoptotic effects of Bcl-2 protein in cryopreserved buffalo bull sperm. MATERIALS AND METHODS: A total 10 ejaculates from two buffalo bulls (5 each) were collected using artificial vagina method, and semen was evaluated using a standard protocol. Semen was extended by Tris egg yolk extender supplemented with Bcl-2 protein at 5, 10, and 15 µM. Semen was cryopreserved at ultra-low temperature using traditional vapor freezing method. Pre-freeze and post-thaw semen samples were evaluated for percent motility, viability, hypo-osmotic swelling test (HOST) reactive sperms; status of mitochondrial membrane activity and status of sperm phospholipase A1 and phospholipase A2 activity. RESULTS: There were no significant effects of Bcl-2 protein supplementation on pre-freeze sperm quality. Percent motility and active mitochondria in post-thaw Bcl-2 supplemented and control groups were also similar. However, viable sperms were significantly (p<0.05) higher (74.29±4.23%) in Bcl-2 supplemented group (5 µM) as compared to control (51.6±5.77%). The proportion of HOST reactive sperms was also higher (63.1±6.73%) in Bcl-2 supplemented (5 µM) group as compared to control (50.7±6.98%). The sperm with low PLA activity (non-apoptotic) was significantly (p<0.05) higher in all the supplemented doses of Bcl-2 protein, i.e., at 5 µM (73.42±5.79%), 10 µM (75.51±6.22%), and 15 µM (74.78±5.89%) as compared to control (60.23±4.45%). We found that Bcl-2 protein supplementation at 5 µM dose improved the post-thaw semen quality indicated by higher viability, HOST reactive sperms, and sperm with low PLA activity (non-apoptotic sperms). CONCLUSION: Bcl-2 protein supplementation exerts its protective effect on spermatozoa against apoptosis-like changes developed during cryopreservation.

Biocomputational analysis of evolutionary relationship between toll-like receptor and nucleotide-binding oligomerization domain-like receptors genes.

AIM: The active domains (TIR and NACHT) of the pattern recognition receptors (PRRs: Toll-like receptors [TLRs] and nucleotide-binding oligomerization domain [NOD]-like receptors [NLR], respectively) are the major hotspots of evolution as natural selection has crafted their final structure by substitution of residues over time. This paper addresses the evolutionary perspectives of the TLR and NLR genes with respect to the active domains in terms of their chronological fruition, functional diversification, and species-specific stipulation. MATERIALS AND METHODS: A total of 48 full-length cds (and corresponding peptide) of the domains were selected as representatives of each type of PRRs, belonging to divergent animal species, for the biocomputational analyses. The secondary and tertiary structure of the taurine TIR and NACHT domains was predicted to compare the relatedness among the domains under study. RESULTS: Multiple sequence alignment and phylogenetic tree results indicated that these host-specific PRRs formed entirely different clusters, with active domains of NLRs (NACHT) evolved earlier as compared to the active domains of TLRs (TIR). Each type of TLR or NLR shows comparatively less variation among the animal species due to the specificity of action against the type of microbes. CONCLUSION: It can be concluded from the study that there has been no positive selection acting on the domains associated with disease resistance which is a fitness trait indicating the extent of purifying pressure on the domains. Gene duplication could be a possible reason of genesis of similar kinds of TLRs (virus or bacteria specific).

Isolation and phylogenetic characterization of Canine distemper virus from India.

Canine distemper (CD), caused by canine distemper virus (CDV) is a highly contagious disease that infects a variety of carnivores. Sequence analysis of CDVs from different geographical areas has shown a lot of variation in the genome of the virus especially in haemagglutinin gene which might be one of the causes of vaccine failure. In this study, we isolated the virus (place: Ludhiana, Punjab; year: 2014) and further cloned, sequenced and analyzed partial haemagglutinin (H) gene and full length genes for fusion protein (F), phosphoprotein (P) and matrix protein (M) from an Indian wild-type CDV. Higher sequence homology was observed with the strains from Switzerland, Hungary, Germany; and lower with the vaccine strains like Ondersteport, CDV3, Convac for all the genes. The multiple sequence alignment showed more variation in partial H (45 nucleotide and 5 amino acid substitutions) and complete F (79 nucleotide and 30 amino acid substitutions) than in complete P (44 nucleotide and 22 amino acid substitutions) and complete M (22 nucleotide and 4 amino acid substitutions) gene/protein. Predicted potential N-linked glycosylation sites in H, F, M and P proteins were similar to the previously known wild-type CDVs but different from the vaccine strains. The Indian CDV formed a distinct clade in the phylogenetic tree clearly separated from the previously known wild-type and vaccine strains.

Proteomic Characterization of Lytic Bacteriophages of Staphylococcus aureus Isolated from Sewage Affluent of India.

Staphylococcus aureus is a Gram-positive bacterium that causes a variety of diseases, including bovine mastitis, which has severe economic consequences. Standard antibiotic treatment results in selection of resistant strains, leading to need for an alternative treatment such as bacteriophage therapy. Present study describes isolation and characterization of a staphylococcal phage from sewage samples. S. aureus isolates obtained from microbial type culture collection (MTCC), Chandigarh, India, were used to screen staphylococcal phages. A phage designated as ΦMSP was isolated from sewage samples by soft agar overlay method. It produced clear plaques on tryptone soya agar overlaid with S. aureus. Transmission electron microscopy revealed that the phage had an icosahedral symmetry. It had 5 major proteins and possessed a peptidoglycan hydrolase corresponding to 70 kDa. ΦMSP infection induced 26 proteins to be uniquely expressed in S. aureus. This phage can be proposed as a candidate phage to treat staphylococcal infections.