Sonalika Mahajan

Immunodiagnosis of foot-and-mouth disease using mutated recombinant 3ABC polyprotein in a competitive ELISA

Differentiation of infected from vaccinated animals (DIVA) is essential for effective control of foot-and-mouth disease (FMD) by vaccination. The antibody response against FMD viral non-structural proteins (NSPs) has been used widely for this purpose. Among all the NSPs, the 3ABC polyprotein has been recognized as the most appropriate indicator for DIVA. In this study, mutated full-length 3ABC polyprotein was expressed in a prokaryotic system and monoclonal antibody against the recombinant protein was developed. A competitive ELISA (C-ELISA) for DIVA was standardized for different species of livestock animals using recombinant 3ABC and monoclonal antibodies. The diagnostic sensitivity and specificity of the assay were estimated by testing a panel of known serum samples consisting of sera from naive, vaccinated and infected animals as 86.9% with 66.4-97.2 (95%) confidence interval and 97% with 89.6-99.6 (95%) confidence interval respectively at 40% inhibition cut-off. The assay was validated further by testing sera from different livestock species collected at random from different parts of the country. The assay will provide a common method for testing sera from different species of livestock and wild animals. The C-ELISA is a sensitive and specific DIVA assay for FMD and can be used as a method for FMD control programme with vaccination.

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.

Truncated recombinant non-structural protein 2C-based indirect ELISA for FMD sero-surveillance.

Foot-and-mouth disease (FMD) is a transboundary animal disease caused by foot-and-mouth disease virus. In India, systematic preventive vaccination using inactivated trivalent (O, A and Asia 1) vaccine is the strategy being adopted to control FMD. The use of non-structural protein (NSP)-contaminated inactivated vaccine raises concerns over differentiation of infected and vaccinated animals (DIVA) by NSP based immunoassays. However, 2C being a membrane associated protein usually remain absent in vaccine formulations and thus, anti-2C response is one of the most reliable indicator of the FMDV infection. In this study, 34 amino acids from N-terminus of 2C protein were removed to eliminate membrane-binding amphipathic helicase activity for the expression of recombinant protein in soluble form. Truncated 2C (2Ct) was utilized for development of an indirect ELISA (I-ELISA) for bovine and the developed 2Ct I-ELISA was validated using a panel constituting of serum of naïve, vaccinated and infected animals. The assay was compared with the in-house r3AB3 I-ELISA and the overall concordance was 85.31%. The diagnostic sensitivity and specificity of the 2Ct I-ELISA were 92.9% and 94.0%, respectively. The apparent prevalence of anti-2C antibodies for random bovine samples tested by the developed assay was 23.7%. The developed ELISA will help in augmenting the sensitivity of detection if used in combination with r3AB3 I-ELISA for sero-surveillance.

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.

Indirect ELISA using recombinant nonstructural protein 3D to detect foot and mouth disease virus infection associated antibodies

Foot-and-mouth disease (FMD) is a highly infectious disease of transboundary importance. Routine biannual vaccination along with surveillance activities is seen as the principal to control FMD in India. Non-structural protein (NSP) based immunoassays are the test of choice for the differentiation between infected and vaccinated population. In this study, 3D protein of FMD virus was expressed in Escherichia coli and an indirect ELISA (I-ELISA) was developed to detect 3D-antibodies in the infected bovines. 3D I-ELISA demonstrated comparable diagnostic sensitivity (97.6%) but lower specificity (80.8%) as compared to the in-house r3AB3 I-ELISA. However, the specificity values varied significantly for naïve and vaccinated samples and were observed to be 98.42% and 76.93%, respectively. A moderate degree of concordance (88.5%) was observed between the overall results of two ELISAs. 3D I-ELISA displayed a considerably lower specificity in uninfected vaccinated samples, thereby suggesting against its application for serosurveillance in intensively vaccinated population. However by virtue of its high diagnostic sensitivity and longer duration of persistence of 3D-antibody post-infection, 3D I-ELISA could be adopted for seroepidemiological investigations in regions not practicing vaccination and could be extended to susceptible species which are generally not covered by vaccination.

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.

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.