Molecular characterisation of lineage IV peste des petits ruminants virus using multi gene sequence data.

Peste des petits ruminants is responsible for an economically important plague of small ruminants that is endemic across much of the developing world. Here we describe the detection and characterisation of a PPR virus from a recent outbreak in Tamil Nadu, India. We demonstrate the isolation of PPR virus from rectal swab and highlight the potential spread of disease to in-contact animals through faecal materials and use of faecal material as non-invasive method of sampling for susceptible wild ruminants. Finally we have performed a comprehensive 'multi-gene' assessment of lineage IV isolates of PPRV utilising sequence data from our study and publically available partial N, partial F and partial H gene data. We describe the effects of grouping PPRV isolates utilising different gene loci and conclude that the variable part of N gene at C terminus gives the best phylogenetic assessment of PPRV isolates with isolates generally clustering according to geographical isolation. This assessment highlights the importance of careful gene targeting with RT-PCR to enable thorough phylogenetic analysis.

Evaluation of immune responses by live infectious bursal disease vaccines to avoid vaccination failures.

Infectious Bursal Disease (IBD) is a viral, contagious immunosuppressive disease posing an important threat to the commercial poultry industry. Evolution of highly virulent strains of IBD virus warranted the need for detailed characterization of the immune responses offered by the currently available vaccines. Two extensively used live vaccines of varied attenuation levels - intermediate and intermediate plus - strains were analyzed for the induction of immune responses. Both the vaccines induced protective antibody titers with the onset, quicker and higher with the intermediate plus vaccine. The intermediate plus strain vaccinate was observed to induce higher levels of IFN-γ in the birds. These results were supported by immunophenotype analyses with an increase in CD8+ and simultaneous decrease in CD4+ cell population. Both vaccine strains conferred protective immunity against virulent challenge. The study warrants the use of intermediate plus vaccines in disease endemic regions and intermediate vaccines in non-endemic regions to prevent IBD infection.

Evaluation of rt-PCR assay for routine laboratory diagnosis of rabies in post mortem brain samples from different species of animals.

Rabies in domestic and wild animals continues to be a major public health threat in India. Rapid and accurate diagnosis of rabies in animals is therefore of utmost importance as the individuals who were in contact with the rabid animals are at a greater risk. A significant amount of diagnostic tissue samples submitted to our laboratory are often autolysed and the WHO recommended direct fluorescent antibody test (FAT) for rabies diagnosis cannot be used in such samples. In this pilot study we have evaluated three different diagnostic primer sets for rapid sensitive and specific detection of rabies genome from the brain samples of different species of animals. We have validated a sensitive RT-PCR assay using brain tissue samples from different species of animals such as cat, cattle, dog, mouse and human, for routine diagnosis of rabies. Our results show the potential of this assay as a confirmatory test when the FAT results are unreliable and also as an alternative diagnostic test in circumstances when the diagnostic samples are unsuitable for use in FAT. Furthermore the nucleotide sequence of nucleoprotein gene amplified using this assay can also be used for the molecular epidemiological study of the rabies viruses in India.

Rabies in South Indian Cows: An evidence of Sri Lankan Rabies virus Variant Infection Based on the Analysis of Partial Nucleoprotein Gene.

Rabies is a highly fatal non-suppurative encephalomyelitis, caused by the Rabies virus. Dogs are the major reservoir of rabies in India and are the source of infection to other domestic animals. In this report, laboratory investigation and molecular characterization of isolates from two cows with paralytic rabies is described. Necropsy brain samples from the two cows were tested for the presence of rabies antigen using a fluorescent antibody test and the results were confirmed using RT-PCR. Rabies virus was successfully isolated from both the brain samples in a murine neuroblastoma cell line. The phylogenetic analysis of partial nucleoprotein gene sequences of these isolates showed them to be of a variant of Rabies virus which is closely related to the Sri Lankan Rabies virus lineage as previously reported. In addition, partial nucleoprotein genes of 19 more Rabies virus isolates from southern India were sequenced and of these 11 isolates were found to be closely related to the Sri Lankan lineage. The deduced amino acid sequences of the partial nucleoprotein of the Indian isolates were 96-99% identical to the Sri Lankan isolates. This investigation re-confirms the previous speculations that the Sri Lankan variant of the virus may still be actively transmitted by animals in India.

Nucleotide sequence analysis of the L1 loop variable region of hexon gene of fowl adenovirus 4 isolates from India.

Three fowl adenovirus 4 (FAV4) isolates from chicken and one from quail, all from Tamil Nadu, India were analyzed. The L1 loop variable region of hexon gene of these isolates was amplified by PCR and sequenced. The nucleotide sequences (442 bp) and deduced amino acid sequences of the four isolates were compared with those of other isolates of FAV4. The nucleotide sequences of the four isolates had a 98% homology with other Indian isolates and a 96% homology with Belgian and Russian isolates. The amino acid sequences of the four Indian isolates had a more than 98% homology with other Indian isolates and a more than 92% homology with Belgian and Russian isolates. Hence, the variable of L1 loop region of hexon gene was found to be highly homologous in all the FAV4 isolates tested both at nucleotide and amino acid level.

Rapid serological profiling by an immunocomb-based dot-enzyme-linked immunosorbent test for three major poultry diseases.

An immunocomb-based dot-ELISA, employing specially designed apparatus, was used to measure the antibody status for the three major poultry diseases--Newcastle disease, infectious bursal disease and infectious bronchitis--in single test sera. Positive samples could be classified into strong, moderate and weak positives by comparison with the colour reaction given by known strong and weak positive serum controls. The simultaneous dot-immunobinding assay gave reproducible results and allowed considerable savings on the cost of reagents compared to liquid ELISA. The antigen-coated immunocomb can be stored under refrigeration and the test can be performed rapidly under field conditions by trained personnel.