Development and Validation of an Indirect and Blocking ELISA for the Serological Diagnosis of African Swine Fever.

African swine fever (ASF) is an economically devastating viral disease of pigs caused by the ASF virus (ASFV). The rapid global spread of ASF has increased the demand for ASF diagnostics to be readily available and accessible. No commercial ASF enzyme-linked immunosorbent assay (ELISA) kits are manufactured and licensed in North America.

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.

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.

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.

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.

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.

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.

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 (PRNT), 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.

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.

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.

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.

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.

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.

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.

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.

Prioritization of capacities for the elimination of dog-mediated human rabies in the Americas: building the framework.

The region of the Americas pledged to eliminate dog-transmitted human rabies by 2015. After 30 years of sustained efforts, regional elimination appears possible as dog-mediated human rabies cases are at an all-time low, and a number of countries and territories have already eliminated the disease. In this setting, there is an opportunity to generate a framework to support countries strategies in the achievement and maintenance of rabies-free status (RFS).

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.

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.

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.

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.

A single electroporation delivery of a DNA vaccine containing the hemagglutinin gene of Asian H5N1 avian influenza virus generated a protective antibody response in chickens against a North American virus strain.

Protection against the avian influenza (AI) H5N1 virus is suspected to be mainly conferred by the presence of antibodies directed against the hemagglutinin (HA) protein of the virus. A single electroporation delivery of 100 or 250 μg of a DNA vaccine construct, pCAG-HA, carrying the HA gene of strain A/Hanoi/30408/2005 (H5N1), in chickens led to the development of anti-HA antibody response in 16 of 17 immunized birds, as measured by a hemagglutination inhibition (HI) test, competitive enzyme-linked immunosorbent assay (cELISA), and an indirect ELISA. Birds vaccinated by electroporation (n = 11) were protected from experimental AI challenge with strain A/chicken/Pennsylvania/1370/1/1983 (H5N2) as judged by low viral load, absence of clinical symptoms, and absence of mortality (n = 11).

Development and application of an indirect ELISA for detection of antibodies against avian hepatitis E virus.

An indirect enzyme-linked immunosorbent assay (iELISA) that could detect immunoglobulin G antibodies against avian hepatitis E virus (HEV) was developed. This assay employs a truncated C-terminal 268-amino acid recombinant ORF2 protein from an avian HEV genotype 3 strain isolated in China (CaHEV) as the coating antigen. The antigen concentration and serum dilution were optimized using a checkerboard titration.

Development and performance evaluation of calf diarrhea pathogen nucleic acid purification and detection workflow.

Calf diarrhea (scours) is a primary cause of illness and death in young calves. Significant economic losses associated with this disease include morbidity, mortality, and direct cost of treatment. Multiple pathogens are responsible for infectious diarrhea, including, but not limited to, Bovine coronavirus (BCV), bovine Rotavirus A (BRV), and Cryptosporidium spp.