Molecular and Pathological Characterization of Classical Swine Fever Virus Genotype 2 Strains Responsible for the 2013-2018 Outbreak in Colombia.

Classical swine fever (CSF) is a highly contagious transboundary viral disease of domestic and wild pigs. Despite mass vaccination and continuous eradication programs, CSF remains endemic in Asia, some countries in Europe, the Caribbean and South America. Since June 2013, Northern Colombia has reported 137 CSF outbreaks, mostly in backyard production systems with low vaccination coverage. The purpose of this study was to characterize the virus responsible for the outbreak. Phylogenetic analysis based on the full-length E2 sequence shows that the virus is closely related to CSF virus (CSFV) genotype 2.6 strains circulating in Southeast Asia. The pathotyping experiment suggests that the virus responsible is a moderately virulent strain. The 190 nucleotide stretch of the E2 hypervariable region of these isolates also shows high similarity to the CSFV isolates from Colombia in 2005 and 2006, suggesting a common origin for the CSF outbreaks caused by genotype 2.6 strains. The emergence of genotype 2.6 in Colombia suggests a potential transboundary spread of CSFV from Asia to the Americas, complicating the ongoing CSF eradication efforts in the Americas, and emphasizes the need for continuous surveillance in the region.

Research Alliance for Veterinary Science and Biodefense BSL-3 Network (RAV3N): Report on network origin and phase I activities.

Recent disease events have heightened awareness for the need for collaboration between the nation's public health and veterinary infectious disease communities to improve preparedness for current and future biological threats. To address this need, the U.S. Department of Agriculture's National Bio- and Agro-Defense Facility (USDA NBAF) has partnered with Texas A&M University through its Global Health Research Complex (TAMGHRC) to establish the Research Alliance for Veterinary Science and Biodefense BSL-3 Network (RAV3N). As a collaborative network of U.S. university and federal BSL-3Ag/BSL-3/BSL-4 laboratory research facilities, the objective of RAV3N is to establish strategic and coordinated approaches for harnessing collective large-animal biocontainment infrastructure and research capacity to improve bio-surveillance, diagnostics, and countermeasure development against high-consequence pathogens of veterinary and zoonotic importance. Here, we describe the origin and development of RAV3N, detail phase I activities, and summarize the proceedings of its first membership meeting held in August 2022.

Meat Exudate for Detection of African Swine Fever Virus Genomic Material and Anti-ASFV Antibodies.

African swine fever (ASF) is one of the most important viral diseases of pigs caused by the ASF virus (ASFV). The virus is highly stable over a wide range of temperatures and pH and can survive in meat and meat products for several months, leading to long-distance transmission of ASF. Whole blood, serum, and organs from infected pigs are used routinely as approved sample types in the laboratory diagnosis of ASF. However, these sample types may not always be available. Here, we investigated meat exudate as an alternative sample type for the detection of ASFV-specific nucleic acids and antibodies. Pigs were infected with various ASFV strains: the highly virulent ASFV Malawi LIL 18/2 strain, the moderately-virulent ASFV Estonia 2014 strain, or the low-virulent ASFV OURT/88/3 strain. The animals were euthanized on different days post-infection (dpi), and meat exudates were collected and tested for the presence of ASFV-specific nucleic acids and antibodies. Animals infected with the ASFV Malawi LIL 18/2 developed severe clinical signs and succumbed to the infection within seven dpi, while pigs infected with ASFV Estonia 2014 also developed clinical signs but survived longer, with a few animals seroconverting before succumbing to the ASFV infection or being euthanized as they reached humane endpoints. Pigs infected with ASFV OURT/88/3 developed transient fever and seroconverted without mortality. ASFV genomic material was detected in meat exudate from pigs infected with ASFV Malawi LIL 18/2 and ASFV Estonia 2014 at the onset of viremia but at a lower amount when compared to the corresponding whole blood samples. Low levels of ASFV genomic material were detected in the whole blood of ASFV OURT/88/3-infected pigs, and no ASFV genomic material was detected in the meat exudate of these animals. Anti-ASFV antibodies were detected in the serum and meat exudate derived from ASFV OURT/88/3-infected pigs and in some of the samples derived from the ASFV Estonia 2014-infected pigs. These results indicate that ASFV genomic material and anti-ASFV antibodies can be detected in meat exudate, indicating that this sample can be used as an alternative sample type for ASF surveillance when routine sample types are unavailable or are not easily accessible.

Evaluation of oral fluid as an aggregate sample for early detection of African swine fever virus using four independent pen-based experimental studies.

The sustained spread of African swine fever (ASF) virus throughout much of the world has made ASF a global animal health priority, with an increased emphasis on enhancing preparedness to prevent, detect and respond to a potential outbreak of ASF virus (ASFV). In the event of ASFV entry to the North American swine population, enhanced surveillance and diagnostic testing strategies will be critical to facilitate progressive response and eradication of the disease. Compared to individual animal sampling, pen-based oral fluid collection for active surveillance is a non-invasive alternative that is less resource and time-intensive. To evaluate the feasibility of using rope-based oral fluid for early detection of ASFV, four independent animal experiments were conducted in weaned pigs housed in numbers that mimic the industry settings, utilising either highly virulent ASFV Georgia 2007/1 strain or moderately virulent ASFV Malta'78 strain. Pen-based oral fluid and individual oropharyngeal swabs were collected daily and blood samples from each animal were collected every other day. All samples were subsequently tested for ASFV by real-time PCR. ASFV genome was detected in individual blood samples as early as one day post-infection and detected in oral fluids at low-to-moderate levels as early as 3-5 days post-infection in all four independent experiments. These results suggest that pen-based oral fluid samples may be used to supplement the use of traditional samples for rapid detection of ASFV during ASF surveillance.

Rapid and highly sensitive portable detection of African swine fever virus.

African swine fever (ASF) continues to spread across Asia, devastating pig populations. The disease is nearly 100% fatal in pigs, and currently, there is no effective vaccine available. Therefore, early detection of ASF is critical for effective disease control. The testing process usually requires samples to be shipped to a central laboratory, which may take many hours of travel or shipping time, delaying the results needed for a rapid response. The ability to confirm ASFV-infected animals on-site or in a regional laboratory that has limited technical capacity and/or infrastructure should eliminate these issues. This study describes the successful transfer of a highly sensitive and specific laboratory-validated real-time PCR assay to a portable pen-side thermocycler, which can be operated in the field for rapid detection of ASFV following a quick manual nucleic acid extraction from a wide array of clinical samples including aggregate samples such as oral fluids. The performance of the portable assay was comparable to the laboratory-based assay. The true portability of the assay was evaluated in seven ASF-suspected farms in Vietnam by testing eighty-nine freshly collected whole blood samples on-site. The results obtained on-site were in agreement with the laboratory data obtained the following day. Availability of this field-deployable molecular assay would eliminate the need to ship samples to a central laboratory, when rapid laboratory results are required, ultimately improving the response time.

Development and evaluation of swine vesicular disease isotype-specific antibody ELISAs based on recombinant virus-like particles.

Swine vesicular disease (SVD) is a contagious viral disease of pigs. The clinical signs of SVD are indistinguishable from other vesicular diseases, such as senecavirus A infection (SVA) and foot-and-mouth disease (FMD). Rapid and accurate diagnostic tests of SVD are considered essential in countries free of vesicular diseases. Competitive ELISA (cELISA) is the serological test used routinely. However, although cELISA is the standard test for SVD antibody testing, this test produces a small number of false-positive results, which caused problems in international trade. The current project developed a SVD isotype antibody ELISA using recombinant SVD virus-like particles (VLP) and an SVD-specific monoclonal antibody (mAb) to reduce the percentage of false positives. The diagnostic specificities of SVD-VLP isotype ELISAs were 98.7% and 99.6% for IgM and IgG. The SVD isotype ELISAs were SVD-specific, without cross-reactivity to other vesicular diseases. A panel of 16 SVD-positive reference sera was evaluated using the SVD-VLP isotype ELISAs. All sera were correctly identified as positive by the two combined SVD-VLP isotype ELISAs. Comparison of the test results showed a high level of correlation between the SVDV antigen isotype ELISAs and SVD-VLP isotype ELISAs. 303 sera from animals lacking clinical signs and history of SVDV exposure were identified positive using SVD cELISA. These samples were examined using SVD-VLP isotype ELISAs. Of the 303 serum samples, five were positive for IgM, and five of 303 were positive for IgG. Comparable to virus neutralization test results, SVD isotype ELISAs significantly reduced the false-positive samples. Based on above test results, the combined use of cELISA and isotype ELISAs can reduce the number of false-positive samples and the use of time-consuming virus neutralization tests, with benefit for international trade in swine and related products.

Preliminary evaluation of diagnostic accuracy and precision of a competitive ELISA for detection of antibodies to Rift Valley fever virus in cattle and sheep sera.

Rift Valley fever virus (RVFV), a mosquito-borne, zoonotic pathogen, is endemic to sub-Saharan Africa and has spread beyond the continent to the Arabian Peninsula. The high likelihood of RVFV's spread to other non-endemic countries spurs the need for development and implementation of rapid diagnostic tests and surveillance programs. In this preliminary evaluation, we assessed the diagnostic accuracy and precision of a recombinant RVFV nucleoprotein based competitive ELISA (cELISA) assay to detect RVFV antibodies compared to a plaque reduction neutralization test (PRNT80), using sera of cattle and sheep that were experimentally infected with either the MP-12 RVFV vaccine or a wild-type RVFV strain, as well as using known RVFV negative sera. With the manufacturer recommended 60% inhibition for the cELISA and a 1:40 titer for the PRNT80, the sensitivity and specificity of the cELISA assay was determined to be 95.1% (95% CI = 83.5-99.4%) and 91.8% (95% CI = 85.0-96.2%), respectively. Antibodies to RVFV were first detected at 5 days post inoculation (dpi) in both species' sera. The prototype cELISA is an easy to implement, sensitive, specific, and safe test for the detection of antibodies to RVFV that can be used in surveillance programs.

An improved, rapid competitive ELISA using a novel conserved 3B epitope for the detection of serum antibodies to foot-and-mouth disease virus.

The highly contagious foot-and-mouth disease virus (FMDV) afflicts cloven-hoofed animals, resulting in significant costs because of loss of trade and recovery from disease. We developed a sensitive, specific, and rapid competitive ELISA (cELISA) to detect serum antibodies to FMDV. The cELISA utilized a monoclonal blocking antibody specific for a highly conserved FMDV nonstructural 3B epitope, a recombinant mutant FMDV 3ABC coating protein, and optimized format variables including serum incubation for 90 min at 20-25°C. Samples from 16 animals experimentally infected with one FMDV serotype (A, O, Asia, or SAT-1) demonstrated early detection capacity beginning 7 d post-inoculation. All samples from 55 vesicular stomatitis virus antibody-positive cattle and 44 samples from cloven-hoofed animals affected by non-FMD vesicular diseases were negative in the cELISA, demonstrating 100% analytical specificity. The diagnostic sensitivity was 100% against sera from 128 cattle infected with isolates of all FMDV serotypes, emphasizing serotype-agnostic results. Diagnostic specificities of U.S. cattle ( n = 1135) and swine ( n = 207) sera were 99.4% and 100%, respectively. High repeatability and reproducibility were demonstrated with 3.1% coefficient of variation in percent inhibition data and 100% agreement using 2 kit lots and 400 negative control serum samples, with no difference between bench and biosafety cabinet operation. Negative results from vaccinated, uninfected cattle, pig, and sheep sera confirmed the DIVA (differentiate infected from vaccinated animals) capability. This rapid (<3 h), select agent-free assay with high sensitivity and specificity, DIVA capability, and room temperature processing capability will serve as a useful tool in FMDV surveillance, emergency preparedness, response, and outbreak recovery programs.

An inter- laboratory proficiency testing exercise for rabies diagnosis in Latin America and the Caribbean.

The direct fluorescent antibody test (DFA), is performed in all rabies reference laboratories across Latin America and the Caribbean (LAC). Despite DFA being a critical capacity in the control of rabies, there is not a standardized protocol in the region. We describe the results of the first inter-laboratory proficiency exercise of national rabies laboratories in LAC countries as part of the regional efforts towards dog-maintained rabies elimination in the American region. Twenty three laboratories affiliated to the Ministries of Health and Ministries of Agriculture participated in this exercise. In addition, the laboratories completed an online questionnaire to assess laboratory practices. Answers to the online questionnaire indicated large variability in the laboratories throughput, equipment used, protocols availability, quality control standards and biosafety requirements. Our results will inform actions to improve and harmonize laboratory rabies capacities across LAC in support for the regional efforts towards elimination of dog-maintained rabies.

Safety and immunogenicity of mammalian cell derived and Modified Vaccinia Ankara vectored African swine fever subunit antigens in swine.

A reverse vaccinology system, Vaxign, was used to identify and select a subset of five African Swine Fever (ASF) antigens that were successfully purified from human embryonic kidney 293 (HEK) cells and produced in Modified vaccinia virus Ankara (MVA) viral vectors. Three HEK-purified antigens [B646L (p72), E183L (p54), and O61R (p12)], and three MVA-vectored antigens [B646L, EP153R, and EP402R (CD2v)] were evaluated using a prime-boost immunization regimen swine safety and immunogenicity study. Antibody responses were detected in pigs following prime-boost immunization four weeks apart with the HEK-293-purified p72, p54, and p12 antigens. Notably, sera from the vaccinees were positive by immunofluorescence on ASFV (Georgia 2007/1)-infected primary macrophages. Although MVA-vectored p72, CD2v, and EP153R failed to induce antibody responses, interferon-gamma (IFN-γ+) spot forming cell responses against all three antigens were detected one week post-boost. The highest IFN-γ+ spot forming cell responses were detected against p72 in pigs primed with MVA-p72 and boosted with the recombinant p72. Antigen-specific (p12, p72, CD2v, and EP153R) T-cell proliferative responses were also detected post-boost. Collectively, these results are the first demonstration that ASFV subunit antigens purified from mammalian cells or expressed in MVA vectors are safe and can induce ASFV-specific antibody and T-cell responses following a prime-boost immunization regimen in swine.

Construction and characterization of a full-length cDNA infectious clone of emerging porcine Senecavirus A.

A full-length cDNA infectious clone, pKS15-01-Clone, was constructed from an emerging Senecavirus A (SVA; strain KS15-01). To explore the potential use as a viral backbone for expressing marker genes, the enhanced green fluorescent protein (EGFP)-tagged reporter virus (vKS15-01-EGFP) was generated using reverse genetics. Compared to the parental virus, the pKS15-01-Clone derived virus (vKS15-01-Clone) replicated efficiently in vitro and in vivo, and induced similar levels of neutralizing antibody and cytokine responses in infected animals. In contrast, the vKS15-01-EGFP virus showed impaired growth ability and induced lower level of immune response in infected animals. Lesions on the dorsal snout and coronary bands were observed in all pigs infected by parental virus KS15-01, but not in pigs infected with vKS15-01-Clone or vKS15-01-EGFP viruses. These results demonstrated that the infectious clone and EGFP reporter virus could be used as important tools in further elucidating the SVA pathogenesis and development of control measures.

Development of a competitive enzyme-linked immunosorbent assay for detection of antibodies against the 3B protein of foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is one of the most highly contagious and economically devastating diseases, and it severely constrains the international trade of animals. Vaccination against FMD is a key element in the control of FMD. However, vaccination of susceptible animals raises critical issues, such as the differentiation of infected animals from vaccinated animals. The current study developed a reliable and rapid test to detect antibodies against the conserved, nonstructural proteins (NSPs) of the FMD virus (FMDV) to distinguish infected animals from vaccinated animals. A monoclonal antibody (MAb) against the FMDV NSP 3B was produced. A competitive enzyme-linked immunosorbent assay (cELISA) for FMDV/NSP antibody detection was developed using a recombinant 3ABC protein as the antigen and the 3B-specific MAb. Sera collected from naive, FMDV experimentally infected, vaccinated carrier, and noncarrier animals were tested using the 3B cELISA. The diagnostic specificity was 99.4% for naive animals (cattle, pigs, and sheep) and 99.7% for vaccinated noncarrier animals. The diagnostic sensitivity was 100% for experimentally inoculated animals and 64% for vaccinated carrier animals. The performance of this 3B cELISA was compared to that of four commercial ELISA kits using a panel of serum samples established by the World Reference Laboratory for FMD at The Pirbright Institute, Pirbright, United Kingdom. The diagnostic sensitivity of the 3B cELISA for the panel of FMDV/NSP-positive bovine serum samples was 94%, which was comparable to or better than that of the commercially available NSP antibody detection kits. This 3B cELISA is a simple, reliable test to detect antibodies against FMDV nonstructural proteins.

Testicular lesions and antler abnormalities in Colorado, USA mule deer (Odocoileus hemionus): a possible role for epizootic hemorrhagic disease virus.

Antler abnormalities of deer and other cervids often result from testicular lesions and decreased levels of testosterone, inhibiting normal cycles of antler growth. Affected males have antlers with retained velvet, numerous short, misshapen points ("cactus bucks"), and failure to shed these abnormal antlers annually. In Colorado, US, we observed a high occurrence of "cactus bucks" in mule deer (Odocoileus hemionus) populations after management efforts to increase the number of mature male deer in the state. Affected males consistently had antibody to epizootic hemorrhagic disease virus serotype 2 (EHDV-2), and examination of the testes of these animals demonstrated nonspecific end-stage lesions of chronic inflammation, fibrosis, and mineralization. To examine more acute stages of testicular lesions, and to screen for EHDV specifically within the testes, we sampled 16 male mule deer from affected herds, but with essentially normal antlers (n = 14) or retained velvet only (n = 2). Testicular and epididymal lesions identified from these samples included necrotizing vasculitis (n = 2), hemorrhage (n = 6), edema (n = 2), seminiferous tubular necrosis (n = 5), orchitis (n = 5), epididymitis (n = 10), hypospermia (n = 6), and end-stage lesions of seminiferous tubular loss (n = 2), fibrosis (n = 2), and mineralization (n = 2). Each of the 16 cases was blindly scored on the basis of number of histologic lesions, with a median score of two. Five of seven (71%) testes that were PCR positive for EHDV had lesion scores above the median, whereas none of the nine (0%) EHDV PCR-negative testes had lesion scores above the median, suggesting an association between testicular lesions and detection of EHDV RNA in the testes (P = 0.003). Although the role of EHDV infection remains unconfirmed, the association between testicular and epididymal lesions and presence of EHDV RNA in the affected tissues suggests that cactus buck antlers may be a sequela of EHDV infection.

Molecular typing of epizootic hemorrhagic disease virus serotypes by one-step multiplex RT-PCR.

Epizootic hemorrhagic disease virus (EHDV) causes a highly infectious noncontagious hemorrhagic disease in wild and captive deer (Cervidae) populations in the US. Although rapid and accurate identification of the disease is important, identification of the serotype is equally important for understanding the epidemiology of the disease in white-tailed deer (Odocoileus virginianus) populations. We developed a one-step multiplex reverse transcriptase PCR assay for rapid differentiation and identification of EHDV serotypes 1, 2, and 6 in cell culture and clinical samples by targeting the viral gene segment 2 (L2) that encodes for the structural protein VP2. From 2009 to 2012, 427 clinical samples including tissue and blood (in ethylenediaminetetraacetic acid) from white-tailed deer, found EHDV positive by real-time PCR, were used to evaluate this subtyping assay. Eighteen percent of the positive samples tested were EHDV-1, 59% were EHDV-2, and 21% were EHDV-6; 2% of the samples were positive for more than one subtype, indicating mixed infection. This assay provides a rapid, sensitive, specific diagnostic tool for differentiation and identification of EHDV serotypes in field samples and virus isolates.

Validation of an improved competitive enzyme-linked immunosorbent assay to detect Equine arteritis virus antibody.

The objective of the present study was to validate a previously described competitive enzyme-linked immunosorbent assay (cELISA) to detect antibody to Equine arteritis virus (EAV) based on GP5-specific nonneutralizing monoclonal antibody (mAb) 17B7(9) using the World Organization for Animal Health (OIE)-recommended protocol, which includes the following 5 in-house analyses. 1) The assay was calibrated with the OIE-designated reference serum panel for EAV; 2) repeatability was evaluated within and between assay runs; 3) analytical specificity was evaluated using sera specific to related viruses; 4) analytical sensitivity was evaluated with sera from horses vaccinated with an EAV modified live virus (MLV) vaccine; and 5) the duration of cELISA antibody detection following EAV vaccination was determined. The positive cELISA cutoff of ≥35% inhibition (%I) was confirmed by receiver operating characteristic plot analysis. Analytical sensitivity of the cELISA was comparable to the serum neutralization (SN) assay in that it detected EAV-specific antibody as early as 8 days postvaccination. The duration of EAV-specific antibody detected by cELISA was over 5 years after the last vaccination. This cELISA could detect EAV-specific antibody in serum samples collected from horses infected with various EAV strains. In the field trial performed by American Association of Veterinary Laboratory Diagnosticians-accredited state laboratories and OIE laboratory, the diagnostic specificity of the cELISA was 99.5% and the diagnostic sensitivity was 98.2%. The data using various serum panels also had consistently significant positive correlation between SN titers and cELISA %I results. The results further confirm that the EAV antibody cELISA is a reliable, simple alternative to the SN assay for detecting EAV-specific antibodies in equine sera.

Development and performance evaluation of a streamlined method for nucleic acid purification, denaturation, and multiplex detection of Bluetongue virus and Epizootic hemorrhagic disease virus.

Bluetongue virus (BTV) and Epizootic hemorrhagic disease virus (EHDV) possess similar structural and molecular features, are transmitted by biting midges (genus Culicoides), and cause similar diseases in some susceptible ruminants. Generally, BTV causes subclinical disease in cattle, characterized by a prolonged viremia. EHDV-associated disease in cattle is less prominent; however, it has emerged as a major economic threat to the white-tailed deer (Odocoileus virginianus) industry in many areas of the United States. The recent emergence of multiple BTV and EHDV serotypes previously undetected in the United States demonstrates the need for robust detection of all known serotypes and differential diagnosis. For this purpose, a streamlined workflow consisting of an automated nucleic acid purification and denaturation method and a multiplex one-step reverse transcription quantitative polymerase chain reaction for the simultaneous detection of BTV serotypes 1-24 and EHDV serotypes 1-7 was developed using previously published BTV and EHDV assays. The denaturation of double-stranded (ds) BTV and EHDV RNA was incorporated into the automated nucleic acid purification process thus eliminating the commonly used separate step of dsRNA denaturation. The performance of this workflow was compared with the World Organization of Animal Health BTV reference laboratory (National Veterinary Services Laboratory, Ames, Iowa) workflow for BTV and EHDV detection, and high agreement was observed. Implementation of the workflow in routine diagnostic testing enables the detection of, and differentiation between, BTV and EHDV, and coinfections in bovine blood and cervine tissues, offering significant benefits in terms of differential disease diagnosis, herd health monitoring, and regulated testing.

Progress towards eliminating canine rabies: policies and perspectives from Latin America and the Caribbean.

Human rabies transmitted by dogs is considered a neglected disease that can be eliminated in Latin America and the Caribbean (LAC) by 2015. The aim of this paper is to discuss canine rabies policies and projections for LAC regarding current strategies for achieving this target and to critically review the political, economic and geographical factors related to the successful elimination of this deadly disease in the context of the difficulties and challenges of the region. The strong political and technical commitment to control rabies in LAC in the 1980s, started with the regional programme coordinated by the Pan American Health Organization. National and subnational programmes involve a range of strategies including mass canine vaccination with more than 51 million doses of canine vaccine produced annually, pre- and post-exposure prophylaxis, improvements in disease diagnosis and intensive surveillance. Rabies incidence in LAC has dramatically declined over the last few decades, with laboratory confirmed dog rabies cases decreasing from approximately 25 000 in 1980 to less than 300 in 2010. Dog-transmitted human rabies cases also decreased from 350 to less than 10 during the same period. Several countries have been declared free of human cases of dog-transmitted rabies, and from the 35 countries in the Americas, there is now only notification of human rabies transmitted by dogs in seven countries (Bolivia, Peru, Honduras, Haiti, Dominican Republic, Guatemala and some states in north and northeast Brazil). Here, we emphasize the importance of the political commitment in the final progression towards disease elimination. The availability of strategies for rabies control, the experience of most countries in the region and the historical ties of solidarity between countries with the support of the scientific community are evidence to affirm that the elimination of dog-transmitted rabies can be achieved in the short term. The final efforts to confront the remaining obstacles, like achieving and sustaining high vaccination coverage in communities that are most impoverished or in remote locations, are faced by countries that struggle to allocate sufficient financial and human resources for rabies control. Continent-wide cooperation is therefore required in the final efforts to secure the free status of remaining countries in the Americas, which is key to the regional elimination of human rabies transmitted by dogs.

Development of a quick and simple detection methodology for foot-and-mouth disease virus serotypes O, A and Asia 1 using a generic RapidAssay Device.

BACKGROUND: Outbreaks of Foot-and-mouth disease (FMD) have resulted in tremendous economic losses. Thus, the development of a rapid and easily performed test for FMD detection is important for controlling a FMD outbreak and containing its spread. The purpose of this project is to develop a lateral flow immunochromatographic (LFI) strip test for rapid detection of FMD virus serotypes O, A and Asia 1. METHODS: Specific monoclonal antibodies (mAbs) against each serotype were produced and used as the capture mAbs. A serotype independent mAb was selected and used as the detection mAb with the aim of subsequently developing a multi-serotype strip test. A new generation of the generic RapidAssay Device (gRAD) was used for the test. RESULT: Each strip test can specifically detect the FMDV O, A or Asia 1 viruses, but not other vesicular disease viruses. The LFI strip tests for serotypes A and Asia 1 were able to identify all tested serotype A (n= 39) and Asia 1 field isolates (n=17). Whereas the test for serotype O detected 45 out of 46 field isolates. The sensitivity of this strip test was comparable with the double antibody sandwich ELISA for viral antigen detection. All vesicular fluid and epithelium samples collected from experimentally infected animals with serotype O, A and Asia 1 were identified as positive by the LFI strip test. Swab samples (n=11) collected over the lesion area from experimentally inoculated animals (serotype A) were examined. All of them demonstrated positive results using the LFI serotype A strip test and double antibody sandwich (DAS) ELISA. CONCLUSIONS: The ability of strip tests to produce rapid results and high specificity makes it a valuable tool for early detection of FMDV O, A and Asia 1 in the field.