Evaluation of an in-house indirect immunoperoxidase test for detection of antibodies against African swine fever virus.

African swine fever (ASF) is a high-consequence transboundary animal disease caused by African swine fever virus (ASFV). Given that vaccines are not widely available, ASFV detection, including by molecular and serologic assays, is paramount to efficacious control and mitigation of ASF. ASFV-specific antibodies can be detected as early as 7-10 d postinfection in infected animals and may persist for several months or longer. Accurate detection of ASFV-specific antibody is critical for the identification of chronically infected, subclinically infected, or recovered animals. ELISAs are commonly used for the rapid screening of large numbers of animals for ASFV antibodies. The World Organisation for Animal Health recommends that ELISA-positive results should be confirmed with a second serologic method, such as an indirect immunofluorescent assay, indirect immunoperoxidase test (IPT), or immunoblot test. Commercial kits are not available for those tests. We developed and validated an in-house IPT by using a currently circulating genotype II ASFV strain as antigen. The sensitivity and specificity of the in-house IPT are comparable to the reference IPT developed by an international ASFV reference laboratory and superior to a commercial blocking ELISA.

Spleen Swabs for Sensitive and High-Throughput Detection of African Swine Fever Virus by Real-Time PCR.

African swine fever (ASF) continues to spread in Africa, Europe, Asia and the island of Hispaniola, increasing the need to develop more streamlined and highly efficient surveillance and diagnostic capabilities. One way to achieve this is by further optimization of already established standard operating procedures to remove bottlenecks for high-throughput screening. Real-time polymerase chain reaction (real-time PCR) is the most sensitive and specific assay available for the early detection of the ASF virus (ASFV) genome, but it requires high-quality nucleic acid extracted from the samples. Whole blood from live pigs and spleen tissue from dead pigs are the preferred samples for real-time PCR. Whole blood can be used as is in nucleic acid extractions, but spleen tissues require an additional homogenization step. In this study, we compared the homogenates and swabs prepared from 52 spleen samples collected from pigs experimentally inoculated with highly and moderately virulent ASF virus strains. The results show that not only are the spleen swabs more sensitive when executed with a low-cell-count nucleic acid extraction procedure followed by real-time PCR assays but they also increase the ability to isolate ASFV from positive spleen samples. Swabbing is a convenient, simpler and less time-consuming alternative to tissue homogenization. Hence, we recommend spleen swabs over tissue homogenates for high-throughput detection of ASFV by real-time PCR.

Further validation of 2 nonstructural protein-specific antibody tests for diagnosis and surveillance of foot-and-mouth disease in the United States.

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. FMD poses an economic threat to the livestock industry in the United States. Due to the potential use of vaccines composed of partially purified structural proteins of the FMD virus (FMDV), it is important to test samples from infected and vaccinated animals with a competitive ELISA that detects antibodies against nonstructural proteins (NSPs) of FMDV. Our study extends the diagnostic validation of the Prionics ELISA (Thermo Fisher) and the VMRD ELISA. We used diverse serum sample sets from bovine, porcine, and other cloven-hoofed animals to evaluate the analytical specificity and sensitivity, diagnostic specificity and sensitivity, and differentiation of infected from vaccinated animals (DIVA) per validation guidelines outlined by the World Organisation for Animal Health (WOAH). The 2 tests were analytically 100% accurate. The VMRD test was diagnostically more sensitive than Prionics, but Prionics was diagnostically more specific than the VMRD test. Both tests could tell if animals were infected or vaccinated. Considering these data, both VMRD and Prionics ELISAs can be used for serodetection of FMDV antibodies at the Foreign Animal Disease Diagnostic Laboratory and within the National Animal Health Laboratory Network laboratories.

Experimental Infection of Domestic Pigs with African Swine Fever Virus Isolated in 2019 in Mongolia.

African swine fever (ASF) is an infectious viral disease caused by African swine fever virus (ASFV), that causes high mortality in domestic swine and wild boar (Sus scrofa). Currently, outbreaks are mitigated through strict quarantine measures and the culling of affected herds, resulting in massive economic losses to the global pork industry. In 2019, an ASFV outbreak was reported in Mongolia, describing a rapidly progressing clinical disease and gross lesions consistent with the acute form of ASF; the virus was identified as a genotype II virus. Due to the limited information on clinical disease and viral dynamics within hosts available from field observations of the Mongolian isolates, we conducted the present study to further evaluate the progression of clinical disease, virulence, and pathology of an ASFV Mongolia/2019 field isolate (ASFV-MNG19), by experimental infection of domestic pigs. Intramuscular inoculation of domestic pigs with ASFV-MNG19 resulted in clinical signs and viremia at 3 days post challenge (DPC). Clinical disease rapidly progressed, resulting in the humane euthanasia of all pigs by 7 DPC. ASFV-MNG19 infected pigs had viremic titers of 108 TCID50/mL by 5 DPC and shed virus in oral secretions late in disease, as determined from oropharyngeal swabs. Whole-genome sequencing confirmed that the ASFV-MNG19 strain used in this study was a genotype II strain highly similar to other regional strains. In conclusion, we demonstrate that ASFV-MNG19 is a virulent genotype II ASFV strain that causes acute ASF in domestic swine.

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.

Generation and characterization of genetically stable heterohybridomas producing foot-and-mouth disease virus-specific porcine monoclonal antibodies.

This report covers the methodology for generation of stable heterohybridoma clones producing Foot-and-mouth disease virus (FMDV) reactive porcine monoclonal antibodies (mAbs). Swine received five inoculations of an inactivated O1 Manisa FMDV vaccine prior to the harvest of splenocytes. Due to the lack of a species-specific hybridoma fusion partner, the Sp2/0 murine myeloma cell line was utilized for the formation of porcine-murine heterohybridoma clones. Twenty-nine FMDV-reactive parental clones were generated. Following sub-cloning and monitoring of reactivity over 20 serial passages, eleven subclones derived from unique parental origins were characterized and are reported herein. This methodology demonstrated the production of porcine mAbs by fusion of porcine splenocytes from immunized pigs with murine myeloma cells to generate heterohybridomas. The porcine immune response may differ from the murine immune response in relation to recognized epitopes. Therefore, application of this methodology may provide valuable resources for swine immunology and enhance the understanding of the mechanisms for antibody based protection from diseases in swine.

Plant-made E2 glycoprotein single-dose vaccine protects pigs against classical swine fever.

Classical Swine Fever Virus (CSFV) causes classical swine fever, a highly contagious hemorrhagic fever affecting both feral and domesticated pigs. Outbreaks of CSF in Europe, Asia, Africa and South America had significant adverse impacts on animal health, food security and the pig industry. The disease is generally contained by prevention of exposure through import restrictions (e.g. banning import of live pigs and pork products), localized vaccination programmes and culling of infected or at-risk animals, often at very high cost. Current CSFV-modified live virus vaccines are protective, but do not allow differentiation of infected from vaccinated animals (DIVA), a critical aspect of disease surveillance programmes. Alternatively, first-generation subunit vaccines using the viral protein E2 allow for use of DIVA diagnostic tests, but are slow to induce a protective response, provide limited prevention of vertical transmission and may fail to block viral shedding. CSFV E2 subunit vaccines from a baculovirus/insect cell system have been developed for several vaccination campaigns in Europe and Asia. However, this expression system is considered expensive for a veterinary vaccine and is not ideal for wide-spread deployment. To address the issues of scalability, cost of production and immunogenicity, we have employed an Agrobacterium-mediated transient expression platform in Nicotiana benthamiana and formulated the purified antigen in novel oil-in-water emulsion adjuvants. We report the manufacturing of adjuvanted, plant-made CSFV E2 subunit vaccine. The vaccine provided complete protection in challenged pigs, even after single-dose vaccination, which was accompanied by strong virus neutralization antibody responses.

Pigs that recover from porcine reproduction and respiratory syndrome virus infection develop cytotoxic CD4+CD8+ and CD4+CD8- T-cells that kill virus infected cells.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection is difficult to control because the virus undergoes antigenic variation during infection and also modulates the protective host immune response. Although current vaccines do not provide full protection, they have provided insight into the mechanisms of protection. Live PRRSV vaccines induce partial protection before the appearance of neutralizing antibody, suggesting cell-mediated immunity or other mechanisms may be involved. Herein, we demonstrate recovery from infection is associated with development of cytotoxic T-lymphocytes (CTL) that can kill PRRSV-infected target cells. Initial experiments showed survival of PRRSV-infected monocyte derived macrophage (MDM) targets is reduced when overlaid with peripheral blood mononuclear cells (PBMC) from gilts that had recovered from PRRSV infection. Further studies with PBMC depleted of either CD4+ or CD8+ T-cells and positively selected subpopulations of CD4+ and CD8+ T-cells showed that both CD4+ and CD8+ T-cells were involved in killing. Examination of killing at different time points revealed killing was biphasic and mediated by CTL of different phenotypes. CD4+CD8+high were associated with killing target cells infected for 3-6 hours. CD4+CD8- CTL were associated with killing at 16-24 hours. Thus, all the anti-PRRSV CTL activity in pigs was attributed to two phenotypes of CD4+ cells which is different from the anti-viral CD4-CD8+ CTL phenotype found in most other animals. These findings will be useful for evaluating CTL responses induced by current and future vaccines, guiding to a novel direction for future vaccine development.

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.

A novel modified-indirect ELISA based on spherical body protein 4 for detecting antibody during acute and long-term infections with diverse Babesia bovis strains.

BACKGROUND: Cattle persistently infected with Babesia bovis are reservoirs for intra- and inter-herd transmission. Since B. bovis is considered a persistent infection, developing a reliable, high-throughput assay that detects antibody during all stages of the infection could be pivotal for establishing better control protocols. METHODS: A modified indirect enzyme-linked immunosorbent assay (MI-ELISA) was developed using the spherical body protein-4 (SBP4) of B. bovis to detect antibody against diverse strains through all infection stages in cattle. This SBP4 MI-ELISA was evaluated for sensitivity and specificity against field sera from regions with endemic and non-endemic B. bovis. Sera were also evaluated from cattle infected experimentally with various doses and strains during acute and persistent infection with parasitemia defined by nested PCR. RESULTS: The format variables for SBP4 MI-ELISA were optimized and the cutoff for positive and negative interpretation was determined based on receiver operating characteristic curve analysis using B. bovis positive and negative sera tested in the reference immunofluorescence assay (IFA). The diagnostic specificity of the SBP4 MI-ELISA using IFA-negative sera collected from Texas was 100%, significantly higher than the cELISA (90.4%) based on an epitope in the rhoptry-associated protein-1 (RAP-1 cELISA). The diagnostic sensitivity of the SBP4 MI-ELISA was 98.7% using the IFA-positive sera collected from several areas of Mexico, in contrast to that of the RAP-1 cELISA at 60% using these same sera. In cattle infected with low and high doses of three B. bovis strains, the SBP4 MI-ELISA remained antibody positive for 11 months or more after initial detection at 10 to 13 days post-inoculation. However, the RAP-1 cELISA did not reliably detect antibody after eight months post-inoculation despite the fact that parasitemia was occasionally detectable by PCR. Furthermore, initial antibody detection by RAP-1 cELISA in low-dose infected animals was delayed approximately nine and a half days compared to the SBP4 MI-ELISA. CONCLUSIONS: These results demonstrate excellent diagnostic sensitivity and specificity of the novel SBP4 MI-ELISA for cattle with acute and long-term carrier infections. It is posited that use of this assay in countries that have B. bovis-endemic herds may be pivotal in preventing the spread of this disease to non-endemic herds.

Recognition of Highly Diverse Type-1 and -2 Porcine Reproductive and Respiratory Syndrome Viruses (PRRSVs) by T-Lymphocytes Induced in Pigs after Experimental Infection with a Type-2 PRRSV Strain.

BACKGROUND/AIM: Live attenuated vaccines confer partial protection in pigs before the appearance of neutralizing antibodies, suggesting the contribution of cell-mediated immunity (CMI). However, PRRSV-specific T-lymphocyte responses and protective mechanisms need to be further defined. To this end, the hypothesis was tested that PRRSV-specific T-lymphocytes induced by exposure to type-2 PRRSV can recognize diverse isolates. METHODS: An IFN-gamma ELISpot assay was used to enumerate PRRSV-specific T-lymphocytes from PRRSVSD23983-infected gilts and piglets born after in utero infection against 12 serologically and genetically distinct type-1 and -2 PRRSV isolates. The IFN-gamma ELISpot assay using synthetic peptides spanning all open reading frames of PRRSVSD23983 was utilized to localize epitopes recognized by T-lymphocytes. Virus neutralization tests were carried out using the challenge strain (type-2 PRRSVSD23983) and another strain (type-2 PRRSVVR2332) with high genetic similarity to evaluate cross-reactivity of neutralizing antibodies in gilts after PRRSVSD23983 infection. RESULTS: At 72 days post infection, T-lymphocytes from one of three PRRSVSD23983-infected gilts recognized all 12 diverse PRRSV isolates, while T-lymphocytes from the other two gilts recognized all but one isolate. Furthermore, five of nine 14-day-old piglets infected in utero with PRRSVSD23983 had broadly reactive T-lymphocytes, including one piglet that recognized all 12 isolates. Overlapping peptides encompassing all open reading frames of PRRSVSD23983 were used to identify ≥28 peptides with T-lymphocyte epitopes from 10 viral proteins. This included one peptide from the M protein that was recognized by T-lymphocytes from all three gilts representing two completely mismatched MHC haplotypes. In contrast to the broadly reactive T-lymphocytes, neutralizing antibody responses were specific to the infecting PRRSVSD23983 isolate. CONCLUSION: These results demonstrated that T-lymphocytes recognizing antigenically and genetically diverse isolates were induced by infection with a type 2 PRRSV strain (SD23983). If these reponses have cytotoxic or other protective functions, they may help overcome the suboptimal heterologous protection conferred by conventional vaccines.

Enhanced sensitivity of an antibody competitive blocking enzyme-linked immunosorbent assay using Equine arteritis virus purified by anion-exchange membrane chromatography.

In an effort to improve a competitive blocking enzyme-linked immunosorbent assay (cELISA) for antibody detection to Equine arteritis virus (EAV), antigen purified by anion-exchange membrane chromatography capsule (AEC) was evaluated. Virus purification by the AEC method was rapid and easily scalable. A comparison was made between virus purified by the AEC method with that obtained by differential centrifugation based on the following: 1) the relative purity and quality of EAV glycoprotein 5 (GP5) containing the epitope defined by monoclonal antibody 17B7, and 2) the relative sensitivity of a commercial antibody cELISA with the only change being the 2 purified antigens. On evaluation by Western blot using GP5-specific monoclonal antibody 17B7, the AEC-purified EAV contained 86% GP5 monomer whereas the differentially centrifuged EAV contained <29% of the monomer. Improvement of analytical sensitivity without sacrifice of analytical specificity was clearly evident when cELISAs prepared with EAV antigen by each purification method were evaluated using 7 sensitivity and specificity check sets. Furthermore, the AEC-purified EAV-based cELISA had 30-40% higher agreement with the virus neutralization (VN) test than the cELISA prepared with differentially centrifuged EAV based on testing 40 borderline EAV-seropositive samples as defined by the VN test. In addition, the AEC-purified cELISA had highly significant (P = 0.001) robustness indicated by intra-laboratory repeatability and interlaboratory reproducibility when evaluated with the sensitivity check sets. Thus, use of AEC-purified EAV in the cELISA should lead to closer harmonization of the cELISA with the World Organization for Animal Health-prescribed VN test.