No abstract

Bovine herpesvirus-1 (BHV-1) causes infectious bovine rhinotracheitis (IBR) and infectious pustular vulvovaginitis / balanoposthitis (IPV/IPB) in cattle and buffaloes. BHV-1 is closely related to bovine herpesvirus-5 (BHV-5), which has been recovered from both genital and respiratory tracts of cattle and buffaloes. Perusal of literature reveals paucity of information on genetic characteristics of BHV subtypes circulating in India. In the present study, 25 isolates originated from five different states of India viz. Gujarat, Uttar Pradesh, Maharashtra, Karantaka and Andhra Pradesh, during the period 1983-2010, were genetically characterized by restriction endonuclease analysis (REA) and partial sequencing of unique long (UL27, UL44) and unique short regions (US1.67) of the viral genome. The REA patterns (Hind III) of these isolates indicated that they were indistinguishable from BHV-1.1 subtype. The phylogenetic analysis based on nucleotide sequences of the unique long (UL) and unique short (US) regions revealed the high sequence homology (>99%) within the isolates and their close relatedness to the BHV-1.1 subtype. The present study established the prevalence of BHV-1.1 as the predominant circulating subtype of BHV in India. The current study also confirmed that the genomic fingerprinting based on HindIII cleavage, direct sequencing of gC (UL44) and gB (UL27) gene-derived PCR amplicons were useful tools for genetic characterization of BHV strains.

The extended frozen semen (EFS) batches produced from infectious bovine rhinotracheitis (IBR) sero-positive cattle and buffalo bulls housed in various semen stations in India are transported to the testing laboratory in liquid nitrogen (LN) for screening bovine herpesvirus-1 (BoHV-1). This procedure is laborious and poses LN related hazards. An alternative logistics for transportation of samples was investigated. Use of Flinders Technology Associates (FTA) elute card was evaluated for transportation of extended bovine semen to screen BoHV-1 DNA by real-time PCR targeting gB gene and the method was compared with the OIE approved Chelex resin based method. A protocol for extraction of BoHV-1 DNA from FTA card spotted with extended semen was optimized. The viral DNA was found to be stable on FTA card for at least 28 days when the cards are stored at 4°-37 °C. The analytical sensitivity for the assay was determined using variable dilutions of BoHV-1 spiked semen and positive plasmid harbouring gB gene (97bp) spotted onto FTA card and it was found to be 10 TCID/ml or 100 copies respectively in real-time PCR. The test could detect as low as 10 TCID/ml or 1 copy of positive plasmid when more number of replicates (n = 6) of the same sample were tested. This sensitivity was found to be comparable to Chelex method and both the methods demonstrated a very strong correlation (r = 0.9774; 95% CI: 0.9620-0.9860) in terms of Ct value (p < 0.0001). The diagnostic sensitivity and specificity of the FTA method in comparison to the Chelex method was 83.08% (95% CI: 71.73%-91.24%) and 93.23% (95% CI: 89.38%-96.01%) respectively when 316 samples were screened by both the methods. The degree of agreement between these two tests was good (Kappa value: 0.738; 95% CI: 0.646-0.829). The method was found to be robust and highly repeatable in inter-assay and intra-assay precision testing. The result suggests that the FTA card holds promise as an alternative system for transportation of EFS for downstream screening of BoHV-1 DNA.

| A diagnostic Real time PCR (qPCR) for detection of Mycobacterium avium subspecies paratuberculosis (MAP) was optimized using the ISMav2 sequence specific for MAP. The analytical sensitivity of the assay was 500 fg, and it was able to detect approximately 7 copies of the positive control plasmid construct. The qPCR detected up to 1x10 3 , 1x10 4 and 1x 10 5 MAP cells per reaction from spiked bovine semen, milk and feces respectively. The assay was reliable, reproducible and could be completed in 87 minutes. Comparative quantification for MAP copy number was established by utilizing normalized Cq values. The ISMav2 qPCR specifically detected 8 MAP strains, but was unable to amplify DNA from any other strains of Mycobacterium (n=5) or non-Mycobacterium strains (n=10). Several factors were tested to study their impact on the validation of the assay under field conditions. It was observed that sample size, number of sampling time points and test methods adopted for accepting the infection status were strongly correlated. The best return of validation estimates were obtained when sample-wise results of the qPCR were compared to the combined status of culture and acid fast bacilli (AFB) staining. The diagnostic sensitivity and specificity, positive and negative predictive values were estimated to be 100% (95% CI |63.06 -100.0|) and 99.9% (95% CI |99.44 100.0|), 88.89% (CI |51.75 -99.72|) and 100% (95%CI |99.75 -100.0|), respectively. Also, the qPCR and the MAP identification test conditions were strongly associated (κ = 0.941 (95% CI |0.825 -1.00|). The validation estimates were true only when the sample size was large (n=1008) and sample collection was based on a single time point sampling strategy. Thus it appears that the assay could be considered as a reliable and rapid diagnostic test for field application. Keywords