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The infectious bronchitis virus is a causative agent of avian infectious bronchitis (AIB), and is is an important disease that produces severe economic losses to the poultry industry worldwide. Recent AIB outbreaks in India have been associated with poor growth in broilers, drop in egg production, and thin egg shells in layers. The complete spike gene of Indian AIB vaccine strain was amplified and sequenced using a conventional reverse transcription polymerase chain reaction and is submitted to the GenBank (accession no KF188436). Phylogenetic analysis revealed that the vaccine strain currently used belongs to H120 genotype, an attenuated strain of Massachusetts (Mass) serotype. Nucleotide and amino acid sequence comparisons have shown that the reported spike gene from Indian isolates have 71.8%-99% and 71.4%-96.9% genetic similarity with the sequenced H120 strain. The study identifies live attenuated IBV vaccine strain, which is routinely used for vaccination, for the first time. Based on nucleotide and amino acid relatedness studies of the vaccine strain with reported IBV sequences from India, it is shown that the current vaccine strain is efficient in controlling the IBV infection. Continuous monitoring of IBV outbreaks by sequencing for genotyping and in vivo cross protection studies for serotyping is not only important for epidemiological investigation but also for evaluation of efficacy of the current vaccine.

Duck virus enteritis, also known as duck plague, is an acute herpes viral infection of ducks caused by duck enteritis virus (DEV). The method of repeated immunization with a live attenuated vaccine has been used for the prevention and control of duck enteritis virus (DEV). However, the incidence of the disease in vaccinated flocks and latency reactivation are the major constraints in the present vaccination programme. The immunogenicity and protective efficacy afforded by intramuscular inoculation of plasmid DNA encoding DEV glycoprotein D (pCDNA-gD) followed by DEV gD expressed in Saccharomyces cerevisia (rgD) was assessed in a murine model. Compared with mice inoculated with DNA (pCDNA-gD) or protein (rgD) only, mice inoculated with the combination of gD DNA and protein had enhanced ELISA antibody titers to DEV and had accelerated clearance of virus following challenge infection. Furthermore, the highest levels of lymphocyte proliferation response, IL-4, IL-12 and IFN-γ production were induced following priming with the DNA vaccine and boosting with the rgD protein. For instance, the specially designed recombinant DEV vector vaccine would be the best choice to use in ducks. It offers an excellent solution to the low vaccination coverage rate in ducks. We expect that the application of this novel vaccine in the near future will greatly decrease the virus load in the environment and reduce outbreaks of DEV in ducks.

Toll-like receptors (TLRs) are one of the types of pattern recognition receptors (PRRs) that recognize conserved pathogen molecules. TLRs link innate and adaptive arms of immune system and are implicated in the development of defense against invading pathogens. Lipopolysaccharide (LPS) and flagellin are recognized by TLR4 and TLR5, respectively. In this study, the effect of flagellin and lipopolysaccharide alone and in combination on chicken peripheral blood mononuclear cells (PBMCs) was investigated. The FliC gene of Salmonella typhimurium was expressed in a prokaryotic expression system and the recombinant flagellin was used to stimulate the chicken PBMCs. A combination of recombinant flagellin and LPS synergistically upregulated nitric oxide production, IL-12 and IL-6 expression but antagonistically down regulated IL-4 expression in comparison to recombinant flagellin alone. The results indicate that these agonists synergistically interact and enhance macrophage function and promote Th1 immune response in chicken PBMCs.

The routine technique for detecting antibodies specific to infectious bursal disease virus is a serological evaluation by enzyme linked immunosorbent assay (ELISA) with preparations of whole virions as antigens. To avoid the use of complete virus in the standard technique, in-house VP2 and VP3 based ELISAs were developed. Accordingly, four types of indirect ELISAs viz., a commercial IDEXX-ELISA kit, VP2 and or VP3 antigen based ELISAs and a whole virus ELISA were compared with the virus neutralization test. It was concluded that the sensitivity and specificity at receiver-operating characteristics (ROC) optimized cut-off of four ELISAs viz., IDEXX-ELISA, VP2-ELISA and VP3-ELISA indicated similar performance whereas whole virus antigen based ELISA showed poor performance in comparison to other ELISAs. Similarly the positive and negative likelihood ratio of four ELISAs at an optimized cut-off indicated IDEXX-ELISA to be the best among all the four ELISAs while the performance of rVP3-ELISA and rVP2-ELISA is good as compared to the whole virus ELISA. Finally, the area under the ROC curve (AUC) of four ELISAs which represented a summary statistics of the overall diagnostic performance of the test also indicated that the IDEXX-ELISA, VP3-ELISA and VP2-ELISA had similar and relatively better performance when compared to whole virus antigen-ELISA.

A recombinant haemagglutinin neuraminidase (HN) antigen-based single serum dilution enzyme linked immuno-sorbent assay (ELISA) was developed to measure the specific antibody in sera of chickens against Newcastle disease virus. A linear relationship was found between the predicted antibody titres at a single working dilution of 1:1000 and the corresponding observed serum titres as determined by the standard serial dilution method. Regression analysis was used to determine a standard curve from which an equation was derived that allowed the demonstration of this correlation. The equation was then used to convert the corrected absorbance readings of the single working dilution directly into the predicted ELISA antibody titres. The assay proved to be sensitive, specific and accurate as compared to the standard haemagglutination inhibition (HI) test.

A velogenic Newcastle disease virus isolate typed to belong to group C1 by monoclonal antibody typing was adapted 50 times in chicken embryo fibroblast cell culture and 60 times in Vero cells. At every 10th passage the virus was characterized on the basis of mean death time, intracerebral pathogenicity indices and viral titration studies. A gradual reduction in the virulence of the virus was noted as the passage number increased. RT-PCR of a 254 bp region of the fusion gene encompassing the fusion protein cleavage site was carried out for the virulent as well as cell culture-adapted viruses at every 10th passage level. The amplicons were subsequently digested with three restriction enzymes, viz. AluI, HaeIII and PstI. It was found out that there was difference in banding patterns between the virulent and adapted viruses, indicating nucleotide substitutions in the virulent virus when it was sequentially passaged onto cell culture systems.

Leptospirosis, a zoonosis with a worldwide distribution is an acute febrile illness caused by spirochaetes of the pathogenic Leptospira interrogans. Microscopic agglutination test (MAT), the reference method for diagnosis was successively done to evaluate the modified ELISA which was developed with the recombinant LipL32 antigen for the detection of anti-leptospiral antibodies in human serum samples.The recombinant LipL32 antigen was developed from the serovar Pomona strain Pomona of the pathogenic L. interrogans species. The predicted titre at a single working dilution was plotted against the observed antiserum titre. Subsequently, predicted antibody activity titres were determined directly from the standard curve by solving the regression line equation. The relative sensitivity, specificity and accuracy of the single dilution ELISA for the detection of anti-leptospiral antibodies were determined in comparison to the MAT.A linear relationship was found between the predicted antibody titres at a single working dilution of 1:250 and the corresponding observed serum titres by the standard serial-dilution method. Regression analysis was used to determine a standard curve from which an equation was derived that allowed demonstration of the mentioned correlation. The equation was then used to convert the corrected absorbance readings of the single working dilution directly into the predicted ELISA antibody titres. A high level of sensitivity of 96 per cent and specificity of 91 per cent between ELISA and MAT titres was found. The kappa value was almost 1.0 indicating perfect agreement.The r LipL32 ELISA was proved to be sensitive, specific and accurate as compared to the standard MAT and the test could be efficiently utilized as a screening test for a large number of human serum samples for the detection of leptospiral antibodies.