Rakesh Matura

Comparative evaluation of non-structural protein-antibody detecting ELISAs for foot-and-mouth disease sero-surveillance under intensive vaccination

Foot-and-mouth disease is a highly infectious and contagious disease of livestock animals with transboundary and economical importance. Animals in the endemic settings are regularly vaccinated in addition to intensive surveillance for control of the disease. Under intensive vaccination, detection of infected animals among the vaccinated population is essential to monitor the infection and to track down the virus movement. Sero-surveillance and retrospective disease diagnosis is performed primarily by detecting antibodies against non-structural proteins (NSPs) of FMD virus which are usually absent in the inactivated vaccine formulations. The study was conducted with an objective to compare simultaneously performance of six NSP ELISAs in detecting infected animals in the areas covered under intensive vaccination, and to assess their fit-for-purpose attribute for sero-surveillance of FMD in India. A panel of bovine serum samples consisting of samples collected from infected with FMDV, vaccinated and naive animals were constituted. In addition, samples collected at random from areas having varied FMD situation and vaccination coverage were tested simultaneously by the six NSP ELISAs to compare their performances. The four indigenous assays showed varying degrees of correlation with the two commercial kits. The study validated that, in all the groups of samples, the indigenous assays were equally sensitive and specific as the two commercial kits. Among all the six assays, PrioCheck and in-house 3ABC I-ELISAs showed maximum sensitivity for detection of infected animals, whereas 3AB3 I-ELISA and 3ABC C-ELISA showed maximum specificity. The study concluded that the in-house available assays are equally capable as the commercially available kits for differentiation of infected animals under intensive vaccination and identifies the 3AB3 I-ELISA with optimum sensitivity and specificity for the purpose of sero-surveillance in India.

Diagnostic assays developed for the control of foot-and-mouth disease in India.

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease of livestock, primarily affecting cattle, buffalo and pigs. FMD virus serotypes O, A and Asia1 are prevalent in India and systematic efforts are on to control and eventually eradicate the disease from the country. FMD epidemiology is complex due to factors like co-circulation, extinction, emergence and re-emergence of genotypes/lineages within the three serotypes, animal movement, diverse farm practices and large number of susceptible livestock in the country. Systematic vaccination, prompt diagnosis, strict biosecurity measures, and regular monitoring of vaccinal immunity and surveillance of virus circulation are indispensible features for the effective implementation of the control measures. Availability of suitable companion diagnostic tests is very important in this endeavour. In this review, the diagnostic assays developed and validated in India and their contribution in FMD control programme is presented.

Production and characterization of single-chain antibody (scFv) against 3ABC non-structural protein in Escherichia coli for sero-diagnosis of Foot and Mouth Disease virus.

Differentiation of Foot-and-Mouth Disease infected from vaccinated animals is essential for effective implementation of vaccination based control programme. Detection of antibodies against 3ABC non-structural protein of FMD virus by immunodiagnostic assays provides reliable indication of FMD infection. Sero-monitoring of FMD in the large country like India is a big task where thousands of serum samples are annually screened. Currently, monoclonal or polyclonal antibodies are widely used in these immunodiagnostic assays. Considering the large population of livestock in the country, an economical and replenishable alternative of these antibodies was required. In this study, specific short chain variable fragment (scFv) antibody against 3B region of 3ABC poly-protein was developed. High level of scFv expression in Escherichia coli system was obtained by careful optimization in four different strains. Two formats of enzyme immunoassays (sandwich and competitive ELISAs) were optimized using scFv with objective to differentiate FMD infected among the vaccinated population. The assays were statistically validated by testing 2150 serum samples. Diagnostic sensitivity/specificity of sandwich and competitive ELISAs were determined by ROC method as 92.2%/95.5% and 89.5%/93.5%, respectively. This study demonstrated that scFv is a suitable alternate for immunodiagnosis of FMD on large scale.

Isolation and characterization of novel microsatellite markers in endangered mahseer, Tor putitora (Hamilton 1822) (Family: Cyprinidae, Pisces)

A set of 12 microsatellites was developed for a freshwater fish golden mahseer, Tor putitora. Golden mahseer is an endangered and endemic in mid-hill regions of South-Asian countries. Constructing a partial genomic library, we obtained 44 clones containing putative microsatellite motifs and 12 microsatellite with enough flanking sequence for primer designing. The number of alleles per locus ranged from 3 to 8 (mean 5.41), with a mean expected and observed heterozygosity of 0.68 and 0.66, respectively. Most of them were in Hardy–Weinberg equilibrium except TPM20a. There was no significant evidence for linkage disequilibrium between any pair of loci. These microsatellites were highly polymorphic (mean PIC 0.68) and will be useful for genetic variability and conservation genetics studies.

Quantitative single dilution liquid phase blocking ELISA for sero-monitoring of foot-and-mouth disease in India

Three of the seven serotypes of foot-and-mouth disease (FMD) virus are prevailing in India. A massive vaccination campaign is on to control and eradicate the disease from the country. However, FMD vaccines provide short term immunity, hence regular assessment of antibody level in the vaccinated herds is indispensible for the success of the control programme. The antibodies are quantitatively estimated, either by virus neutralization test or by end-point dilution liquid-phase-blocking ELISA (LPBE). Millions of cattle and buffalo in the country are now systematically vaccinated, and thousands of serum samples are routinely screened in the country for estimation of herd immunity against FMDV serotypes O, A and Asia1. Testing such a large number of serum samples within limited a period of time by the conventional end point dilution method of LPBE requires lots of man power, and biological reagents. A more economical high throughput single dilution LPBE (SdLPBE) assay was optimized and validated for quantitative estimation of antibody levels against the three FMD virus serotypes. The assay was thoroughly validated against LPBE method before adopting it for country-wide use. The biological reagents used in the assay were prepared in thermo-stable form to enable transportation to the field level FMD diagnostic laboratories.

Development and characterization of microsatellite markers in Schizopyge niger (Family: Cyprinidae, Pisces) from Kashmir Valley

A set of 8 microsatellites was developed for a freshwater fish chush snowtrout, Schizopyge niger. Chush snowtrout is an endemic fish to Kashmir valley, India. Constructing a partial genomic library, we obtained 36 clones containing putative microsatellite motifs and 12 microsatellite with enough flanking sequence for primer designing. The number of alleles per locus ranged from 3 to 7 (mean 4.75), with a mean expected and observed heterozygosity of 0.71 and 0.68, respectively. All the loci were in Hardy–Weinberg equilibrium. There was no significant evidence for linkage disequilibrium between any pair of loci. These microsatellites were highly polymorphic (mean PIC 0.63) and will be useful for genetic variability and conservation genetics studies.