Diagnostic and prophylactic potential of a stabilized foot-and-mouth disease serotype Asia1 virus like particles designed through a structure guided approach.

Intact capsids of foot-and-mouth disease virus (FMDV) play a vital role in eliciting a protective immune response. Any change in the physico-chemical environment of the capsids results in dissociation and poor immunogenicity. Structural bioinfomatics studies have been carried out to predict the amino acids at the interpentameric region that resulted in the identification of mutant virus-like particles(VLPs) of FMDV serotype Asia1/IND/63/1972. The insect cell expressed VLPs were evaluated for their stability by sandwich ELISA. Among 10 mutants, S93H showed maximum retention of antigenicity at different temperatures, indicating its higher thermal stability as revealed by the in-silico analysis and retained the antigenic sites of the virus demonstrated by Sandwich ELISA. The concordant results of the liquid phase blocking ELISA for estimation of antibody titre of known sera with stable mutant VLP as antigen in place of virus antigen demonstrate its diagnostic potential. The stable mutant VLP elicited a robust immune response with 85.6 % protection in guinea pigs against virus challenge. The stabilized VLP based antigen requires minimum biosafety and cold storage for production and transit besides, complying with differentiation of infected from vaccinated animals. It can effectively replace the conventional virus handling during antigen production for prophylactic and diagnostic use.

A species-independent indirect-ELISA for detection of antibodies to the non-structural protein 2B of foot-and-mouth disease virus.

Diagnostic assays that are able to detect foot-and-mouth disease (FMD) virus infection in the vaccinated population are essential tools in the progressive control pathway for the FMD. However, testing of serum samples using a single diagnostic assay may not completely substantiate freedom from the virus infection. Therefore, viral non-structural proteins (NSPs)-based various serological assays have been developed for the detection of FMD infection. Nevertheless, the NSPs-based ELISAs have been developed in the indirect-ELISA format, thereby necessitating the use of species-specific conjugated secondary-antibodies for the detection of anti-NSP antibodies in various FMD-susceptible species. Therefore, this study presents a novel recombinant 2B-NSP-based indirect ELISA, employing HRP-conjugated protein-A/G detection system which can detect anti-NSPs antibodies from multiple FMD-susceptible species in a single ELISA platform. Recombinant 2B (r2B) protein was expressed as His-SUMO tagged protein in the E. Coli cells and purified using NI-NTA affinity column chromatography. Using the r2B protein and HRP-conjugated protein A/G, an indirect ELISA was developed and validated for the detection of anti-2B antibodies in serum samples collected from multiple FMD-susceptible animal species with known FMD status. Further, a resampling based statistical technique has been reported for determination of optimal cut-off value for the diagnostic assay. Through this technique, the optimal cut-off of 44 percentage of positivity value was determined for the assay. At this optimal cut-off value, the developed diagnostic assay provided diagnostic sensitivity, specificity, and accuracy, positive and negative predictive values (PPV and NPV) of 92.35 %, 98.41 %, 95.21 %, 98.58 %, and 91.67 %, respectively. The assay was validated further by analyzing random serum samples collected across multi-locations in India. The assay can be used as a single platform for testing serum samples from different species of FMDV-susceptible animals and will be useful for NSP-based serosurveillance of FMDV.

Evaluation of specific chicken IgY antibody value developing diagnostic capture antibody ELISA kit against Foot and Mouth disease.

The most preferred method for the detection of foot-and-mouth disease (FMD) viral antigen and identification of viral serotype is the enzyme-linked immunosorbent assay (ELISA). Diagnostic tests with high sensitivity are necessary both to distinguish infected vaccinated animals and execute disease control programs for the identification of the carrier animals. The current strategies for the detection of FMD virus are mainly based on the capture antibody (sandwich) ELISA test. The usage of laying pullets as an animal bioreactor for the production of specific egg yolk antibodies (IgY) has increased in recent years due to its high yield, affinity, low price, and quick production turnover. The present study aimed to produce a concentrated and purified IgY polyclonal antibody to design a capture antibody ELISA kit against the FMD virus (FMDV) serotype A. At first, laying hens were immunized with inactivated FMDV serotype virus, and then, on days 14, 21, and 28 following vaccination, the eggs and sera were collected. Afterward, the IgY polyclonal antibodies were extracted and purified from the chicken egg yolk using a polyethylene glycol 6000-ethanol precipitation procedure. Extracts were filtered, purified by ion exchange chromatography, and dialyzed. The purified IgY concentration, estimated by Bradford assay, confirmed its presence by SDS-PAGE and Western blot and also its specific immune reaction by Ouchterlony double immunodiffusion and Dot blot tests. Moreover, for achieving the optimum concentration of antigen/antibody (sera) in sandwich ELISA, a checkerboard titration test was set up based on indirect ELISA results. Eventually, 119 previously confirmed samples (including 80 positive and 39 negative) by both real-time polymerase chain reaction (quantitative PCR, qPCR) and a commercial ELISA kit were used for evaluation of the sensitivity and accuracy of our developed Capture antibody ELISA kit. In this manner, the sensitivity and specificity of our designed kit were 100% and 98%, respectively. Accordingly, the present developed capture ELISA kit based on IgY had high sensitivity and specificity for FMD virus detection and it could be used in the future for both commercial detecting and serotyping applications.

Evaluation of commercial ELISA kits' diagnostic specificity for FAST diseases in wild animals.

Wild animals, sharing pathogens with domestic animals, play a crucial role in the epidemiology of infectious diseases. Sampling from wild animals poses significant challenges, yet it is vital for inclusion in disease surveillance and monitoring programmes. Often, mass surveillance involves serological screenings using enzyme-linked immunosorbent assay (ELISA) tests, typically validated only for domestic animals. This study assessed the diagnostic specificity of commercially available ELISA tests on 342 wild ruminant serum samples and 100 from wild boars. We evaluated three tests for foot-and-mouth disease: two for Peste des petits ruminants, two for Rift Valley fever and one for Capripox virus. Diagnostic specificity was calculated using the formula True Negative/(False Positive + True Negative). Cohen's kappa coefficient measured agreement between tests. Results showed high specificity and agreement across all tests. Specificity for foot-and-mouth disease (FMD) ranged from 93.89% for Prionics to 100% for IDEXX, with IDvet showing 99.6%. The highest agreement was between FMD IDvet and IDEXX at 97.1%. Rift Valley fever (RVF) tests, Ingezim and IDvet, achieved specificities of 100% and 98.83%, respectively. The optimal specificity was attained by retesting single reactors and inactivating the complement.Contribution: Commercially available ELISA kits are specific for foot-and-mouth disease and similar transboundary animal diseases and can be used for highly specific wild animal testing.

Establishment of the Foot-and-Mouth Disease Virus Type Asia1 Expressing the HiBiT Protein: A Useful Tool for a NanoBiT Split Luciferase Assay.

Foot-and-mouth disease virus (FMDV) is a highly contagious virus that affects cloven-hoofed animals and causes severe economic losses in the livestock industry. Given that this high-risk pathogen has to be handled in a biosafety level (BSL)-3 facility for safety reasons and the limited availability of BSL-3 laboratories, experiments on FMDV call for more attention. Therefore, we aimed to develop an FMDV experimental model that can be handled in BSL-2 laboratories. The NanoBiT luciferase (Nano-luc) assay is a well-known assay for studying protein-protein interactions. To apply the NanoBiT split luciferase assay to the diagnosis and evaluation of FMD, we developed an inactivated HiBiT-tagged Asia1 Shamir FMDV (AS-HiBiT), a recombinant Asia1 shamir FMDV with HiBiT attached to the VP1 region of Asia1 shamir FMDV. In addition, we established LgBiT-expressing LF-BK cell lines, termed LgBit-LF-BK cells. It was confirmed that inactivated AS-HiBiT infected LgBiT-LF-BK cells and produced a luminescence signal by binding to the intracellular LgBiT of LgBiT-LF-BK cells. In addition, the luminescence signal became stronger as the number of LgBiT-LF-BK cells increased or the concentration of inactivated AS-HiBiT increased. Moreover, we confirmed that inactivated AS-HiBiT can detect seroconversion in sera positive for FMDV-neutralizing antibodies. This NanoBiT split luciferase assay system can be used for the diagnosis and evaluation of FMD and expanded to FMD-like virus models to facilitate the evaluation of FMDV vaccines and antibodies.

Interfering factors in the diagnosis of Senecavirus A.

BACKGROUND: Senecavirus A (SV-A) is an RNA virus that belongs to the genus Senecavirus within the family Picornaviridae. This study aimed to analyze factors that can influence the molecular diagnosis of Senecavirus A, such as oligonucleotides, RNA extraction methods, and RT-qPCR kits. METHODS: Samples from suspected cases of vesicular disease in Brazilian pigs were analyzed for foot-and-mouth disease, swine vesicular disease, and vesicular stomatitis. All tested negative for these diseases but positive for SV-A. RT-qPCR tests were used, comparing different reagent kits and RNA extraction methods. Sensitivity and repeatability were evaluated, demonstrating efficacy in detecting SV-A in clinical samples. RESULTS: In RNA extraction, significant reduction in Cq values was observed with initial dilutions, particularly with larger supernatant volumes. Trizol and Maxwell showed greater sensitivity in automated equipment protocols, though results varied in tissue tests. RT-qPCR kit comparison revealed differences in amplification using viral RNA but minimal differences with plasmid DNA. Sensitivity among methods was comparable, with slight variations in non-amplified samples. Repeatability tests showed consistent results among RT-qPCRs, demonstrating similarity between methods despite minor discrepancies in Cq values. CONCLUSIONS: Trizol, silica columns, and semi-automated extraction were compared, as well as different RT-qPCR kits. The study found significant variations that could impact the final diagnosis.

Monoclonal antibody based solid phase competition ELISA to detect FMDV serotype A specific antibodies.

In Foot-and-mouth disease (FMD) enzootic countries, periodic vaccination is the key tool in controlling the disease incidence. Active seromonitoring of the vaccinated population is critical to assess the impact of vaccination. Virus neutralization test (VNT) and enzyme-linked immunosorbent assays (ELISA) are commonly used for antibody detection. Assays like liquid phase blocking ELISA (LPBE) or solid phase competition ELISA (SPCE) are preferred as they do not require handling of live FMDV and are routinely used for seromonitoring or for vaccine potency testing; however, false positives are high in LPBE. Here we report, a monoclonal antibody (mAb) based SPCE as a potential alternate assay for antibody titration. From a panel of 12 mAbs against FMDV serotype A, two mAbs were chosen for the development of SPCE. Based on a set of 453 sera, it was demonstrated that mAb 2C4G11, mAb 6E8D11and polyclonal antibody (pAb) based SPCE had a relative sensitivity of 86.1, 86.1 and 80.3 %; and specificity of 99.6, 99.1 and 99.1 %, respectively. The correlation, repeatability, and level of agreement of the assays were high demonstrating the potential use of mAb in large scale surveillance studies and regular vaccine potency testing.

Seroprevalence and molecular detection of foot and mouth disease virus in cattle in selected districts of Wolaita Zone, Southern Ethiopia.

Foot and mouth disease (FMD) is a highly contagious, endemic, and acute viral cattle ailment that causes major economic damage in Ethiopia. Although several serotypes of the FMD virus have been detected in Ethiopia, there is no documented information about the disease's current serostatus and serotypes circulating in the Wolaita zone. Thus, from March to December 2022, a cross-sectional study was conducted to evaluate FMDV seroprevalence, molecular detection, and serotype identification in three Wolaita Zone sites. A multistage sample procedure was used to choose three peasant associations from each study region, namely Wolaita Sodo, Offa district, and Boloso sore district. A systematic random sampling technique was employed to pick 384 cattle from the population for the seroprevalence research, and 10 epithelial tissue samples were purposefully taken from outbreak individuals for molecular detection of FMDV. The sera were examined using 3ABC FMD NSP Competition ELISA to find antibodies against FMDV non-structural proteins, whereas epithelial tissue samples were analyzed for molecular detection using real-time RT-PCR, and sandwich ELISA was used to determine the circulating serotypes. A multivariable logistic regression model was used to evaluate the associated risk variables. The total seroprevalence of FMD in cattle was 46.88% (95% CI 41.86-51.88), with Wolaita Sodo Town having the highest seroprevalence (63.28%). As a consequence, multivariable logistic regression analysis revealed that animal age, herd size, and interaction with wildlife were all substantially related to FMD seroprevalence (p < 0.05). During molecular detection, only SAT-2 serotypes were found in 10 tissue samples. Thus, investigating FMD outbreaks and identifying serotypes and risk factors for seropositivity are critical steps in developing effective control and prevention strategies based on the kind of circulating serotype. Moreover, further research for animal species other than cattle was encouraged.

In-process quality control in foot-and-mouth disease vaccine production by detection of viral non-structural proteins using chemiluminescence dot blot assay.

Foot-and-mouth disease (FMD) is a contagious viral disease of cloven-footed animals. Immunization with inactivated virus vaccine is effective to control the disease. Six-monthly vaccination regimen in endemic regions has proven to be effective. To enable the differentiation of infected animals from those vaccinated, non-structural proteins (NSPs) are excluded during vaccine production. While the antibodies to structural proteins (SPs) could be observed both in vaccinated and infected animals, NSP antibodies are detectable only in natural infection. Quality control assays that detect NSPs in vaccine antigen preparations, are thus vital in the FMD vaccine manufacturing process. In this study, we designed a chemiluminescence dot blot assay to detect the 3A and 3B NSPs of FMDV. It is sensitive enough to detect up to 20 ng of the NSP, and exhibited specificity as it does not react with the viral SPs. This cost-effective assay holds promise in quality control assessment in FMD vaccine manufacturing.

An innovative immunochromatographic assay employing Pt-Pd bimetallic nanoparticles as labels for the detection of foot-and-mouth disease virus serotype O.

OBJECTIVE: We created a novel, high sensitivity immunochromatographic assay that allows for clear and precise quantitative analysis by employing innovative bimetallic nanoparticles with peroxide-like activity as markers for the preparation of the test strip. METHODS: Initially, we synthesized Pt-Pd bimetallic nanoparticles through the reduction of K2PtCl4 and Na2PdCl4 using ascorbic acid (AA) in an ultrasonic water bath. These bimetallic nanoparticles were then utilized to label purified antigens from the foot-and-mouth disease virus (FMDV) type O (FMDV-146S), resulting in the creation of antigen-captured nanomarkers. Upon completion of the antigen-antibody reaction, we introduced a color-developing agent (3,3',5,5'-tetramethylbenzidine) for cascade amplification, significantly enhancing detection sensitivity while ensuring clear and accurate quantitative analysis. RESULTS: The quantitative detection sensitivity achieved was 1:28/test, with a linear range spanning from 1:26 âˆ¼ 1:29 /test. For FMDV type O positive serum, the detection sensitivity reached 96.7 %. Furthermore, this method exhibited a 95 % detection sensitivity for FMDV negative serum, FMDV type A and type AsiaⅠ positive sera, as well as sera positive for other common viral diseases in animals. In comparison to the OIE-recommended LPB-ELISA, this approach displayed higher correlation (correlation coefficient = 0.909). Innovation was at the core of establishing this immunochromatographic assay based on Pt-Pd bimetallic nanoparticles for the detection of FMDV antibodies. CONCLUSION: The findings revealed a striking 24-fold improvement in sensitivity when compared to colloidal gold, accompanied by a strong correlation coefficient (R2 > 0.9). This suggests a robust and consistent linear association in the results. This method represents a significant advancement in the field of rapid immunochromatographic assays, offering a promising alternative application for bimetallic nanoparticles.

Surveillance for lumpy skin disease and foot and mouth disease in the Kimberley, Western Australia.

We quantified the sensitivity of surveillance for lumpy skin disease (LSD) and foot and mouth disease (FMD) in cattle in the Kimberley region of Western Australia. We monitored producer and veterinary activity with cattle for 3 years commencing January 2020. Each year, ~274,000 cattle of 685,540 present on 92 pastoral leases (stations) were consigned to other stations, live export or slaughter. Veterinarians examined 103,000 cattle on the stations, 177,000 prior to live export, and 10,000 prior to slaughter. Detection probabilities for the disease prior to transport or during veterinary procedures and inspections were elicited by survey of 17 veterinarians working in Northern Australia. The veterinarians estimated the probabilities that they would notice, recognise, and submit samples from clinical cases of LSD and FMD, given a 5% prevalence of clinical signs in the herd. We used scenario tree methodology to estimate monthly surveillance sensitivity of observations made by producers and by veterinarians during herd management visits, pre-export inspections, and ante-mortem inspections. Average monthly combined sensitivities were 0.49 for FMD and 0.37 for LSD. Sensitivity was high for both diseases during the dry season and low in the wet season. We estimated the confidence in freedom from the estimated surveillance sensitivity given one hypothetically infected herd, estimated probability of introduction, and prior confidence in freedom. This study provided assurance that the Kimberley is free of these diseases and that routine producer and veterinary interactions with cattle are adequate for the timely detection of the disease should they be introduced.

Optimization of a foot-and-mouth disease virus Southern African Territories-specific solid-phase competitive ELISA for small ruminant serum samples.

We optimized and verified a single-spot solid-phase competitive ELISA (ss-SPCE) to detect antibodies against structural proteins of Southern African Territories (SAT) serotypes of foot-and-mouth disease virus (FMDV) in small ruminants. Sera from goats vaccinated and experimentally challenged with a SAT1 FMDV pool were tested in duplicate at 4 dilutions (1:10, 1:15, 1:22.5, 1:33.8) to optimize the assay. To assess the performance of the assay in naturally infected animals, we evaluated 316 goat and sheep field sera collected during active SAT2 outbreaks. Relative to results of the virus neutralization test, the optimal serum dilution and cutoff percentage inhibition (PI) were 1:15 and 50%, respectively. At these values, the Spearman rank correlation coefficient was 0.85 (p < 0.001), and the sensitivity and specificity (95% CI) were 80.3% (72.6, 87.2) and 91.1% (84.1, 95.9), respectively. Relative to the liquid-phase blocking ELISA and the nonstructural protein ELISA, the ss-SPCE exhibited divergent performance characteristics between the goat and sheep field sera. Repeatability was better for goats, but the correlation and agreement among all 3 assays were better for the sheep sera. The prevalence of SAT2 FMDV infection in the sampled sheep was 23.6%; sampled goats were seemingly FMDV-free. The ss-SPCE is an appropriate FMDV detection tool to investigate the role of small ruminants in the epidemiology of FMD in Africa.

Laboratory testing and on-site storage are successful at mitigating the risk of release of foot-and-mouth disease virus via production of bull semen in the USA.

Thousands of frozen bovine semen doses are produced daily in the US for domestic use. An incursion of foot-and-mouth disease (FMD) in the country would pose strong challenges to the movements of animals and animal products between premises. Secure supply plans require an estimation of the risk associated with target commodities and the effectiveness of mitigation measures. This study presents the results of a quantitative assessment of the risk of release of FMD virus from five of the largest commercial bull studs in the US via contaminated frozen processed semen. The methodology from a previous study was adapted to better fit the US production system and includes more recent data. Two models were combined, a deterministic compartmental model of FMD transmission and a stochastic risk assessment model. The compartmental model simulated an FMD outbreak within a collection facility, following the introduction of a latent-infected bull. The risk of release was defined as the annual likelihood of releasing at least one frozen semen batch, defined as the total amount of semen collected from a single bull on a given collection day, containing viable FMD virus. A scenario tree was built using nine steps leading from the collection to the release of a contaminated batch from a given facility. The first step, the annual probability of an FMD outbreak in a given facility, was modeled using an empirical distribution fitted to incidence data predicted by five models published between 2012 and 2022. An extra step was added to the previously published risk pathway, to account for routine serological or virological surveillance within facilities. The results showed that the mitigation measures included in the assessment were effective at reducing the risk of release. The median annual risk of release from the five facilities was estimated at less than 2 in 10 billion (1.5 x 10-10) in the scenario including a 30-day storage, routine genome detection assays performed every two weeks and RT-PCR testing of the semen. In this scenario, there was a 95% chance that the risk of release would be lower than 0.00041. This work provides strong support to the industry for improving their response plans to an incursion of FMD virus in the US.

A TaqMan-based RT-qPCR assay for serotyping of Southern African territories (SAT) 2 strains of Foot-and-Mouth disease virus (FMDV) in Southern Africa.

OBJECTIVE: Determining the serotype of circulating virus strains is important in implementing effective vaccination. In this study, Foot-and-Mouth Disease (FMD) Southern African territory 2 (SAT2) specific primers and TaqMan probe were designed towards rapid SAT2 detection and serotyping. The primers were tested by endpoint reverse transcription (RT) polymerase chain reaction (PCR) and quantitative PCR (RT-qPCR) using the vaccine strain SAT2035. The SAT2 serotype-specific RT-qPCR assay was compared with currently used ELISA and VP1 sequencing using Cohen's kappa statistics. RESULTS: The primers yielded amplicons of band size 190 bp during endpoint RT-PCR. When coupled with the probe, the primers reaction efficiency was determined to be 99% with an r2 value of 0.994. The results show that the SAT2 assay has comparable performance to VP1 sequencing (k = 1) and a moderate degree of agreement with ELISA (k = 0.571). The data shows that the newly designed assay could be considered for serotyping of SAT2 strains. However, for this assay to be complete there is a need to design effective SAT1 and SAT3 primers and probes that can be multiplexed to target other serotypes that co-circulate within relevant FMD endemic pools. For future implementation of the assay there is also a need to increase the number of field samples towards validation of the assay.

A reverse transcription-multiplex PCR strategy devised for concomitant detection and differentiation of foot and mouth disease virus serotypes O, A and Asia 1 in India.

Serotype identification occupies the central part of foot and mouth disease (FMD) diagnosis workflow and vaccination decision tree. In this study, a reverse transcription-multiplex PCR (RT-mPCR) strategy wherein three assays with unique combinations of serotype specific primers targeting the VP1 region was developed to differentiate FMD virus serotypes O, A and Asia 1 based on differential size of the PCR amplicons on agarose gel. Their diagnostic performance relative to the mPCR assay in use in India was evaluated on 169 clinical samples and 210 cell culture grown virus isolates. The relative diagnostic sensitivity was found to be 99.69%, 98.78% and 99.08% for primer combinations 1, 2 and 3, respectively. These assays proved their worth by detecting serotype in three FMD suspected specimens that went undiagnosed in the existing mPCR and also by identifying multiple serotypes in the same sample. Their detection limits varied from log10 2 to log10 4 viral RNA dilution and from 100 to 0.1 TCID50 virus depending on the serotype. The validated novel mPCR assays show promise to be included in the routine diagnostic tool-box to augment the efficiency of diagnosis of FMD virus serotypes that display extreme genetic diversity and a tendency of transboundary dispersal.

Development of TaqMan Probe-Based One-Step RT-qPCR Assay Targeting 2B-NSP Coding Region for Diagnosis of Foot-and-Mouth Disease in India.

A one-step TaqMan probe-based RT-qPCR assay in the duplex format simultaneously targeting FMD Virus (FMDV) 2B NSP-coding region and 18S rRNA housekeeping gene was developed and evaluated. The duplex RT-qPCR assay specifically detected FMDV genome in both infected cell culture suspensions and a variety of clinical samples such as FMD-affected tongue/feet epithelium, oral/nasal swabs, milk and oro-pharyngeal fluids. The RT-qPCR assay was found to be highly sensitive, since the assay was 105-fold more sensitive than the traditional FMDV detecting antigen-ELISA (Ag-ELISA) and 102-fold better sensitive than both virus isolation and agarose gel-based RT-multiplex PCR. In addition, the assay could detect up to 100 copies of FMDV genome per reaction. In the epithelial samples (n = 582) collected from the FMD-affected animals, the diagnostic sensitivity was 100% (95% CI 99-100%). Similarly, all the FMDV-negative samples (n = 65) tested were confirmed negative by the new RT-qPCR assay, corresponding to 100% diagnostic specificity (95% CI = 94-100%). Further, the duplex RT-qPCR assay proved to be robust, showing an inter-assay co-efficient of variations ranging from 1.4 to 3.56% for FMDV-2B gene target, and from 2 to 4.12% for 18S rRNA gene target. While analyzing FMDV-infected cell culture suspension, a fairly strong positive correlation (correlation coefficient = 0.85) was observed between 2B-based RT-qPCR and WOAH-approved 5'UTR RT-qPCR assays. Therefore, the one-step RT-qPCR assay developed here with an internal control could be used for rapid, effective, and reliable detection of FMDV in pan-serotypic manner, and has the potential for routine diagnosis of FMDV in high throughput manner.

Production and characterization of monoclonal antibodies against foot-and-mouth disease virus serotype O and development of a sandwich ELISA for virus antigen detection.

Foot-and-mouth disease (FMD) is endemic in India with a majority of outbreaks caused by FMD virus (FMDV) serotype O. In the present study a panel of eight (2F9, 2G10, 3B9, 3H5, 4C8, 4D6, 4G10 and 5B6) mouse monoclonal antibodies (MAbs) were developed against FMDV serotype O Indian vaccine strain, O/IND/R2/75 via hybridoma systems. The MAbs generated were FMDV/O specific without cross-reactivity against FMDV type A and Asia 1. All the MAbs were identified as IgG1/kappa type. Out of eight, three MAbs (3B9, 3H5 and 4G10) demonstrated virus neutralizing activity. The reactivity of all MAbs increased with heat treated (@560C) serotype O antigen compared to untreated antigen in sandwich ELISA indicating that their binding epitopes are linear. Six MAbs (except 2F9 and 4D6) reacted with recombinant P1 protein of homologous virus in an indirect ELISA among which only MAb 3B9 bound to VP1. MAb profiling of 37 serotype O field viruses isolated between the years 1962 and 2021 demonstrated antigenic similarity between field isolates and reference vaccine strain. MAbs 5B6 and 4C8 consistently reacted with all 37 isolates. In indirect immunofluorescence assay MAb 5B6 bound well with FMDV/O antigen. Finally, a sandwich ELISA was successfully developed using rabbit polyclonal anti-FMDV/O serum and MAb 5B6 for detection of FMDV/O antigen in clinical samples (n = 649). The new assay exhibited 100% and 98.89% diagnostic sensitivity and specificity respectively compared to traditional polyclonal antibody-based sandwich ELISA suggesting that the MAb-based ELISA developed here could be an effective method for detection of FMDV serotype O.

Diagnostic application of formalin fixed archived tissues for detection of foot-and-mouth disease.

Early and definitive disease diagnosis is critical for effective disease control. 50% buffered glycerine is commonly used viral transport medium, which is not always available and required cold chain. Tissues samples archived in 10% neutral buffered formalin (NBF) can preserve nucleic acid that can be used in molecular studies and disease diagnosis. The present study's goal was to detect the foot-and-mouth disease (FMD) viral genome in formalin-fixed archived tissue which may avoid cold chain during transportation. This study used FMD suspected samples preserved in 10% neutral buffered formalin from 0 to 730 days post fixation (DPF). All archived tissues were positive for FMD viral genome by multiplex RT-PCR and RT-qPCR up to 30 DPF, whereas archived epithelium tissues and thigh muscle were positive for FMD vial genome up to 120 DPF. FMD viral genome was detected in cardiac muscle up to 60 DPF and 120 DPF, respectively. The findings suggest that 10% neutral buffered formalin could be used for sample preservation and transportation for timely and accurate FMD diagnosis. More samples need to be tested before implementing the use of 10% neutral buffered formalin as a preservative and transportation medium. The technique may add value in ensuring biosafety measures for creation during disease free zone as well.

Development of a real-time RT-PCR system applicable for rapid and pen-side diagnosis of foot-and-mouth disease using a portable device, PicoGene® PCR1100.

Foot-and-mouth disease (FMD) is a highly contagious viral vesicular disease, causing devastating losses to the livestock industry. A diagnostic method that enables quick decisions is required to control the disease, especially in FMD-free countries. Although conventional real-time reverse transcription polymerase chain reaction (RT-PCR) is a highly sensitive method widely used for the diagnosis of FMD, a time lag caused by the transport of samples to a laboratory may allow the spread of FMD. Here, we evaluated a real-time RT-PCR system using a portable PicoGene PCR1100 device for FMD diagnosis. This system could detect the synthetic FMD viral RNA within 20 min with high sensitivity compared to a conventional real-time RT-PCR. Furthermore, the Lysis Buffer S for crude nucleic extraction improved the viral RNA detection of this system in a homogenate of vesicular epithelium samples collected from FMD virus-infected animals. Furthermore, this system could detect the viral RNA in crude extracts prepared using the Lysis Buffer S from the vesicular epithelium samples homogenized using a Finger Masher tube, which allows easy homogenization without any equipment, with a high correlation compared to the standard method. Thus, the PicoGene device system can be utilized for the rapid and pen-side diagnosis of FMD.

Evaluating serological tests for foot-and-mouth disease while accounting for different serotypes and uncertain vaccination status.

Controlling foot-and-mouth disease (FMD) by vaccination requires adequate population coverage and high vaccine efficacy under field conditions. To assure veterinary services that animals have acquired sufficient immunity, strategic post-vaccination surveys can be conducted to monitor the coverage and performance of the vaccine. Correct interpretation of these serological data and an ability to derive exact prevalence estimates of antibody responses requires an awareness of the performance of serological tests. Here, we used Bayesian latent class analysis to evaluate the diagnostic sensitivity and specificity of four tests. A non-structural protein (NSP) ELISA determines vaccine independent antibodies from environmental exposure to FMD virus (FMDV), and three assays measuring total antibodies derived from vaccine antigen or environmental exposure to two serotypes (A, O): the virus neutralisation test (VNT), a solid phase competitive ELISA (SPCE), and a liquid phase blocking ELISA (LPBE). Sera (n = 461) were collected by a strategic post-vaccination monitoring survey in two provinces of Southern Lao People's Democratic Republic (PDR) after a vaccination campaign in early 2017. Not all samples were tested by every assay and each serotype: VNT tested for serotype A and O, whereas SPCE and LPBE tested for serotype O, and only NSP-negative samples were tested by VNT, with 90 of them not tested (missing by study design). These data challenges required informed priors (based on expert opinion) for mitigating possible lack of model identifiability. The vaccination status of each animal, its environmental exposure to FMDV, and the indicator of successful vaccination were treated as latent (unobserved) variables. Posterior median for sensitivity and specificity of all tests were in the range of 92-99 %, except for the sensitivity of NSP (∼66%) and the specificity of LPBE (∼71 %). There was strong evidence that SPCE outperformed LPBE. In addition, the proportion of animals recorded as having been vaccinated that showed a serological immune response was estimated to be in the range of 67-86 %. The Bayesian latent class modelling framework can easily and appropriately impute missing data. It is important to use field study data as diagnostic tests are likely to perform differently on field survey samples compared to samples obtained under controlled conditions.