New approaches for post-vaccination surveillance of foot-and-mouth disease.

Foot-and-mouth disease (FMD) is a contagious and important transboundary disease of cloven-hoofed animals and ruminants. In ruminants, an animal is considered as a foot-and-mouth disease virus (FMDV) carrier if a live FMDV/FMDV RNA is obtained from the oro-pharyngeal fluid (OPF) beyond 28 days after infection. These carrier animals may pose a risk for causing outbreaks in healthy animals. Moreover, it is important to conduct serosurveillance to know the virus circulation. In the present study, an ELISA was developed using field samples to detect FMDV specific secretory IgA antibodies. These samples were also tested for the presence of FMDV RNA using quantitative real-time PCR (qRT-PCR). It was found that more carrier animals were detected by IgA ELISA in comparison to qRT-PCR. Thus, IgA ELISA is an important tool to detect FMD carriers. An ELISA based on detection of antibodies against FMDV 2B non-structural protein (NSP) was also used to confirm the results obtained from screening of 3AB3 NSP ELISA. These two new approaches (IgA ELISA and 2B ELISA) form important tools for detection of carriers and virus circulation, respectively, during FMD eradication program. Keywords: foot-and-mouth disease virus; carriers; IgA; 2B non-structural protein; 3AB3 non-structural protein.

Reverse transcription loop-mediated isothermal amplification assays for rapid identification of eastern and western strains of bluetongue virus in India.

Bluetongue virus (BTV) infects all ruminants, including cattle, goats and camelids, causing bluetongue disease (BT) that is often severe in naïve deer and sheep. Reverse-transcription-loop-mediated-isothermal-amplification (RT-LAMP) assays were developed to detect eastern or western topotype of BTV strains circulating in India. Each assay uses four primers recognizing six distinct sequences of BTV genome-segment 1 (Seg-1). The eastern (e)RT-LAMP and western (w)RT-LAMP assay detected BTV RNA in all positive isolates that were tested (n=52, including Indian BTV-1, -2, -3, -5, -9, -10, -16, -21 -23, and -24 strains) with high specificity and efficiency. The analytical sensitivity of the RT-LAMP assays is comparable to real-time RT-PCR, but higher than conventional RT-PCR. The accelerated eRT-LAMP and wRT-LAMP assays generated detectable levels of amplified DNA, down to 0.216 fg of BTV RNA template or 108 fg of BTV RNA template within 60-90min respectively. The assays gave negative results with RNA from foot-and-mouth-disease virus (FMDV), peste des petits ruminants virus (PPRV), or DNA from Capripox viruses and Orf virus (n=10), all of which can cause clinical signs similar to BT. Both RT-LAMP assays did not show any cross-reaction among themselves. The assays are rapid, easy to perform, could be adapted as a 'penside' test making them suitable for 'front-line' diagnosis, helping to identify and contain field outbreaks of BTV.