Diagnosis Of African Swine Fever Research Articles

Evaluation of Selective Culling as a Containment Strategy for African Swine Fever at a Vietnamese Sow Farm.

Selective culling, also known as the "tooth extraction approach", is a strategy for controlling African swine fever (ASF) by removing only sick and suspect animals instead of the entire herd in Vietnam. This method prioritizes preserving healthy animals, particularly valuable breeding pigs. Despite its implementation in various forms, no standardized protocol based on scientific principles has been established. Farms typically adapt this strategy based on their understanding, which can vary significantly. In implementing of selective culling that is not based on scientific principles, there is a significant risk of spreading the disease. The aim of this study is to evaluate the consequences of selective culling as currently implemented in Vietnam. Our analysis on a large sow farm revealed that current practices rely heavily on clinical observations without laboratory confirmations. This approach allows ASF-infected animals to remain on the farm longer, potentially exacerbating the spread of the virus. Thus, selective culling poses a substantial risk by potentially exacerbating the spread of disease. Our findings emphasize that early diagnosis of ASF and systematic removal of infected pigs are critical components for the effective implementation of selective culling strategies and that a high level of fragmentation to minimize contact between animals plays a key role. The optimal approach is to test conspicuous animals and separate them. Under no circumstances should suspect animals be left in the herd for several days before they become severely ill and succumb to the disease.

Label-Free Detection of African Swine Fever and Classical Swine Fever in the Point-of-Care Setting Using Photonic Integrated Circuits Integrated in a Microfluidic Device.

Swine viral diseases have the capacity to cause significant losses and affect the sector's sustainability, a situation further exacerbated by the lack of antiviral drugs and the limited availability of effective vaccines. In this context, a novel point-of-care (POC) diagnostic device incorporating photonic integrated circuits (PICs), microfluidics and information, and communication technology into a single platform was developed for the field diagnosis of African swine fever (ASF) and classical swine fever (CSF). The device targets viral particles and has been validated using oral fluid and serum samples. Sensitivity, specificity, accuracy, precision, positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic odds ratio (DOR) were calculated to assess the performance of the device, and PCR was the reference method employed. Its sensitivities were 80.97% and 79%, specificities were 88.46% and 79.07%, and DOR values were 32.25 and 14.21 for ASF and CSF, respectively. The proposed POC device and PIC sensors can be employed for the pen-side detection of ASF and CSF, thus introducing novel technological advancements in the field of animal diagnostics. The need for proper validation studies of POC devices is highlighted to optimize animal biosecurity.

African Swine Fever Diagnosis in Africa: Challenges and Opportunities.

The global spread of African swine fever (ASF) in recent decades has led to the need for technological advances in sampling and diagnostic techniques. The impetus for these has been the need to enable sampling by lay persons and to obtain at least a preliminary diagnosis in the field for early control measures to be put in place before final laboratory confirmation. In rural Africa, rapid diagnosis is hampered by challenges that include lack of infrastructure as well as human and financial resources. Lack of animal health personnel, access to affordable means to transport field samples to a laboratory, and lack of laboratories with the capacity to make the diagnosis result in severe under-reporting of ASF, especially in endemic areas. This review summarizes the challenges identified in gap analyses relevant to low- and middle-income countries, with a focus on Africa, and explore the opportunities provided by recent research to improve field diagnosis and quality of diagnostic samples used. Sampling techniques include invasive sampling techniques requiring trained personnel and non-invasive sampling requiring minimal training, sampling of decomposed carcass material, and preservation of samples in situations where cold chain maintenance cannot be guaranteed. Availability and efficacy of point-of-care (POC) tests for ASF has improved considerably in recent years and their application, as well as advantages and limitations, are discussed. The adequacy of existing laboratory diagnostic capacity is evaluated and opportunities for networking amongst reference and other laboratories offering diagnostic services are discussed. Maintaining laboratory diagnostic efficiency in the absence of samples during periods of quiescence is another issue that requires attention, and the role of improved laboratory networking is emphasized. Early diagnosis of ASF is key to managing the disease spread. Therefore, the establishment of the Africa Chapter of the Global African Swine Fever Research Alliance (GARA) increases opportunities for collaboration and networking among the veterinary diagnostic laboratories in the region.

Overview of Modern Commercial Kits for Laboratory Diagnosis of African Swine Fever and Swine Influenza A Viruses.

Rapid and early detection of infectious diseases in pigs is important, especially for the implementation of control measures in suspected cases of African swine fever (ASF), as an effective and safe vaccine is not yet available in most of the affected countries. Additionally, analysis for swine influenza is of significance due to its high morbidity rate (up to 100%) despite a lower mortality rate compared to ASF. The wide distribution of swine influenza A virus (SwIAV) across various countries, the emergence of constantly new recombinant strains, and the danger of human infection underscore the need for rapid and accurate diagnosis. Several diagnostic approaches and commercial methods should be applied depending on the scenario, type of sample and the objective of the studies being implemented. At the early diagnosis of an outbreak, virus genome detection using a variety of PCR assays proves to be the most sensitive and specific technique. As the disease evolves, serology gains diagnostic value, as specific antibodies appear later in the course of the disease (after 7-10 days post-infection (DPI) for ASF and between 10-21 DPI for SwIAV). The ongoing development of commercial kits with enhanced sensitivity and specificity is evident. This review aims to analyse recent advances and current commercial kits utilised for the diagnosis of ASF and SwIAV.

Current detection methods of African swine fever virus.

African swine fever (ASF), caused by the African swine fever virus (ASFV), is a highly contagious and notifiable animal disease in domestic pigs and wild boars, as designated by the World Organization for Animal Health (WOAH). The effective diagnosis of ASF holds great importance in promptly controlling its spread due to its increasing prevalence and the continuous emergence of variant strains. This paper offers a comprehensive review of the most common and up-to-date methods established for various genes/proteins associated with ASFV. The discussed methods primarily focus on the detection of viral genomes or particles, as well as the detection of ASFV associated antibodies. It is anticipated that this paper will serve as a reference for choosing appropriate diagnostic methods in diverse application scenarios, while also provide direction for the development of innovative technologies in the future.

The emergence and spread of African Swine Fever in Western Europe

African swine fever (ASF) is the most dangerous viral disease of domestic pigs and wild boars, causing huge economic damage. It is characterized by acute, subacute, chronic course with high (up to 100%) mortality. The causative agent of ASF has been isolated into a separate family Asfarviridae, the genus Asfivirus. The disease is widespread in Africa, Europe, Asia. The purpose of the review article is to analyze the literature data on the occurrence and spread of epizootic foci of African swine fever in Western Europe. The materials of the article are based on the results of numerous epizootological and diagnostic studies by scientists from Spain, Portugal, Belgium, France, Italy, the Netherlands, Malta, Germany, Sweden.Due to the complex seroimmune structure of the pathogen and the impossibility of forming viral neutralizing antibodies, there are difficulties in creating effective vaccines against ASF. The main tools for preventing the spread of the disease are due to the use of strict quarantine measures. Early diagnosis is an important component of ASF control and eradication.Preventive and liquidation measures for ASF in Western European countries are considered. The recommendations of the OIE/WOAH-OIE on the diagnosis of ASF using a combination of tests are given: determination of the viral genome by PCR in national reference laboratories; detection of viral antigens and antibodies by ELISA or fluorescent test; detection of the pathogen by virus isolation. The explanation of the observed interspecific interaction during the watering of wild boars and free-range domestic pigs is given, which explains the endemicity of ASF on the island of Sardinia in Italy for more than forty years.Given the virulence of the ASF virus and the many ways of transmission throughout Europe, diagnostic and epizootological monitoring of this disease should be a priority in the fight against ASF.

Establishment of a Dual-Antigen Indirect ELISA Based on p30 and pB602L to Detect Antibodies against African Swine Fever Virus.

African swine fever (ASF) is an acute, virulent, and highly fatal infectious disease caused by the African swine fever virus (ASFV). There is no effective vaccine or diagnostic method to prevent and control this disease currently, which highlights the significance of ASF early detection. In this study, we chose an early antigen and a late-expressed antigen to co-detect the target antibody, which not only helps in early detection but also improves accuracy and sensitivity. CP204L and B602L were successfully expressed as soluble proteins in an Escherichia coli vector system. By optimizing various conditions, a dual-antigen indirect ELISA for ASFV antibodies was established. The assay was non-cross-reactive with antibodies against the porcine reproductive and respiratory syndrome virus, classical swine fever virus, porcine circovirus type 2, and pseudorabies virus. The maximum serum dilution for detection of ASFV-positive sera was 1:1600. The intra-batch reproducibility coefficient of variation was <5% and the inter-batch reproducibility coefficient of variation was <10%. Compared with commercial kits, the dual-antigen indirect ELISA had good detection performance. In conclusion, we established a detection method with low cost, streamlined production process, and fewer instruments. It provides a new method for the serological diagnosis of ASF.

A multi-state survey of farm-level preparedness towards African swine fever outbreak in Nigeria

Outbreaks of African Swine Fever (ASF) have severe economic implications for Nigeria and result in significant loss of livelihoods. The non-availability of vaccines makes biosecurity the reliable key to reducing ASF outbreaks. This study evaluated preparedness for ASF outbreaks at the farm level among 247 pig farmers randomly selected from Abia, Akwa-Ibom, Edo, Kwara, and Oyo states. We categorized each pig farmer's ASF preparedness rating (ASF – PR) as “poor”, “moderate”, and “satisfactory” based on their score on an 11-item scale. Finally, a multivariable logistic regression analysis was conducted to assess the association between the socio-demographic variables and farm-level ASF preparedness. The awareness of ASF among pig farmers was very high (87.9%, n = 217). Most farmers knew the clinical signs of the disease, the modes of transmission of ASF, and correctly identified the risk factors. They also considered the need for thorough cleaning and disinfection of piggeries (87.1%, n = 189), tightened biosecurity (85.7%, n = 186), culling all ASF-affected pigs (77.9%, n = 169) as well as the ban on the transport of pigs and their products (49.8%, n = 108) as very important in ASF control. Conversely, 27.6%, (n = 60) of the farmers thought ASF could affect humans, 12% (n = 27) of them openly discarded the carcasses of dead pigs, and there was a high antibiotic usage. Most of the pig farmers used antibiotics as prophylaxis (63.6%, n = 157), chemotherapeutics (66.4%, n = 164), growth promoters (15.4%, n = 38), and wrongly so, 13% (n = 32) of them thought that antibiotics could be used to prevent and treat ASF. At the farm level, two-thirds (68.8%, n = 170) of the farmers had strict movement restrictions, and 48.6% (n = 120) routinely quarantine new pigs before introduction into their herd. Across the five states, 36% (n = 89) of the farmers had witnessed sudden death with signs consistent with ASF amongst their pigs and only 10.1% (n = 27) had confirmatory ASF diagnosis. The mean score for the farm-level ASF-PR was 6.95 ± 2.7. Approximately one-quarter of the 247 pig farmers had satisfactory ASF – PR that might help to prevent the incursion of ASF into their farms. Most farmers had moderate ASF - PR (59.5%, n = 147) whereas 17% (n = 42) had very poor ASF-PR and were most prone to an ASF outbreak. Of the sociodemographic variables, only age was significantly associated with farm-level ASF preparedness as older pig farmers especially those aged between 50 and 59 years (OR: 4.83; 95% CI: 1.10, 21.22; p = 0.037) were more likely to have satisfactory ASF - PR than the others. Our findings showed pig farmers were not adequately prepared and the next ASF outbreak could pose more significant threat to pig populations across Nigeria. Government should urgently establish minimum biosecurity measures and improve its ASF surveillance mechanisms for commercial and backyard pig production.

A quadruple fluorescence quantitative PCR method for the identification of wild strains of african swine fever and gene-deficient strains

BackgroundOriginating in Africa, African swine fever (ASF) was introduced to China in 2018. This acute and highly virulent infectious disease affects domestic pigs. The World Organization for Animal Health has listed it as a statutory reportable disease, and China has listed it as a category A infectious disease.MethodsPrimers and probes were designed for four ASFV genes (B646L, EP402R, MGF505-3R, and A137R). The primers/probes were highly conserved compared with the gene sequences of 21 ASFV strains.ResultsAfter optimization, the calibration curve showed good linearity (R2 > 0.99), the minimum concentration of positive plasmids that could be detected was 50 copies/µL, and the minimum viral load detection limit was 102 HAD50/mL. Furthermore, quadruple quantitative polymerase chain reaction (qPCR) with nucleic acids from three porcine-derived DNA viruses and cDNAs from eight RNA viruses did not show amplification curves, indicating that the method was specific. In addition, 1 × 106, 1 × 105, and 1 × 104 copies/µL of mixed plasmids were used for the quadruple qPCR; the coefficient of variation for triplicate determination between groups was < 2%, indicating the method was reproducible.ConclusionsThe results obtained by testing clinical samples containing detectable EP402R, MGF505-3R, and A137R strains with different combinations of gene deletions were as expected. Therefore, the established quadruple qPCR method was validated for the molecular diagnosis of ASF using gene-deleted ASFV strains.

Improving African Swine Fever Surveillance Using Fluorescent Rapid Tests.

African swine fever (ASF) is a viral disease of swine with a huge impact due to its high mortality. Lately, the disease has actively spread around the world, affecting new areas from which it had been eradicated long ago. To date, ASF control is carried out by the implementation of strict biosecurity measures such as the early identification of infected animals. In this work, two fluorescent rapid tests were developed to improve the sensitivity of point-of-care diagnosis of ASF. For antigen (Ag) detection in blood, a double-antibody sandwich fluorescent lateral flow assay (LFA) was developed, employing a newly developed recombinant antibody to the VP72 of the virus. To complement the diagnosis, a double-recognition fluorescent LFA was developed using the VP72 for the detection of specific antibodies (Ab) in sera or blood. Both assays statistically improved the detection of the disease when compared to the commercial colorimetric assays INgezim® ASFV CROM Ag and INgezim® PPA CROM Anticuerpo, respectively, with higher statistical significance between 11 and 39 days post-infection. From the observation of results, it can be concluded that the combination of both Ag-LFA and Ab-LFA assays would facilitate the identification of infected animals, regardless of post-infection time.

Development of molecular and antigenic-based rapid tests for the identification of African swine fever virus in different tissues.

African swine fever (ASF) is a severe haemorrhagic infectious disease affecting suids, thus representing a great economic concern. Considering the importance of the early diagnosis, rapid point of care testing (POCT) for ASF is highly demanded. In this work, we developed two strategies for the rapid onsite diagnosis of ASF, based on Lateral Flow Immunoassay (LFIA) and Recombinase Polymerase Amplification (RPA) techniques. The LFIA was a sandwich-type immunoassay exploiting a monoclonal antibody directed towards the p30 protein of the virus (Mab). The Mab was anchored onto the LFIA membrane to capture the ASFV and was also labelled with gold nanoparticles for staining the antibody-p30 complex. However, the use of the same antibody for capturing and as detector ligand showed a significant competitive effect for antigen binding, so required an experimental design to minimize reciprocal interference and maximize the response. The RPA assay, employing primers to the capsid protein p72 gene and an exonuclease III probe, was performed at 39°C. The limit of detection of the method was assessed using a plasmid encoding the target gene and resulted in 5 copy/μL. The new LFIA and RPA were applied for ASFV detection in the animal tissues usually analysed by conventional assays (i.e., real-time PCR), such as kidney, spleen, and lymph nodes. A simple and universal virus extraction protocol was applied for sample preparation, followed by DNA extraction and purification for the RPA. The LFIA only required the addition of 3% H2O2 to limit matrix interference and prevent false positive results. The two rapid methods (25min and 15min were needed to complete the analysis for RPA and LFIA, respectively) showed high diagnostic specificity (100%) and sensitivity (93% and 87% for LFIA and RPA, respectively) for samples with high viral load (Ct<27). False negative results were observed for samples with low viral load (Ct>28) and/or also containing specific antibodies to ASFV, which decreased antigen availability and were indicative of a chronic, poorly transmissible infection. The simple and rapid sample preparation and the diagnostic performance of the LFIA suggested its large practical applicability for POC diagnosis of ASF.

Development of a Novel Indirect ELISA for the Serological Diagnosis of African Swine Fever Using p11.5 Protein as a Target Antigen.

African swine fever is a hemorrhagic viral disease with a mortality rate of nearly 100% in pigs. Hence, it is classified as a notifiable disease by the World Organization for Animal Health. Because no field-available vaccine exists, African swine fever virus (ASFV) control and eradication solely depend on good farm biosecurity management and rapid and accurate diagnosis. In this study, we developed a new indirect serological enzyme-linked immunosorbent assay (ELISA) using recombinant p11.5 protein from ASFV as a solid-phase target antigen. The cutoffs were determined by receiver operating curve analysis performed with serum samples obtained from naïve and infected pigs. Based on the results of a commercially available serological ELISA, the relative sensitivity and specificity of our assay were 93.4% and 94.4% (N = 166; area under the curve = 0.991; 95% confidence interval = 0.982-0.999), respectively. Furthermore, to compare the performance of the serological ELISAs, we conducted the assays on a panel of sera collected from pigs and boars experimentally infected with different ASFV isolates. The results indicated the greater sensitivity of the newly developed assay and its ability to detect anti-ASFV antibodies earlier after virus inoculation.

Establishment of an indirect immunofluorescence assay for the detection of African swine fever virus antibodies

African swine fever (ASF) continues to cause enormous economic loss to the global pig industry. Since there is no safe and effective vaccine, accurate and timely diagnosis of ASF is essential to implement control measures. Indirect immunofluorescence assay (IFA) is a gold standard serological method recommended by the World Organization for Animal Health (WOAH). In this study, we used primary fetal kidney cells to establish a wild boar cell line (BK2258) that supported the efficient replication of ASF virus (ASFV) SD/DY-I/21 and showed visible cytopathic effect (CPE). Moreover, using BK2258, we established a sensitive and specific IFA for ASFV antibody detection. To standardize and evaluate the performance of this assay, we used serum samples from pigs infected with the low virulent genotype I SD/DY-I/21 and genotype II HLJ/HRB1/20, and immunized with the vaccine candidate HLJ/18-7GD, field samples, and negative serum samples. The IFA reacted with the ASFV-positive sera and displayed bright fluorescence foci. There was no non-specific green fluorescence due to cellular senescence or other cell damage-causing factors. Compared to a commercial indirect enzyme-linked immunosorbent assay (iELISA), ASFV antibodies were detected 1–4 days earlier using our IFA. The detection limits of the IFA and iELISA for the same ASFV-antibody positive serum samples were 1:25,600 and 1:6,400, respectively, indicating that the IFA is more sensitive than iELISA. The newly established IFA was highly specific and did not cross-react with sera positive for six other important porcine pathogens (i.e., Classical swine fever virus (CSFV), Porcine reproductive and respiratory syndrome virus (PRRSV), Porcme circovirus type 2 (PCV2), Pseudorabies virus (PRV), Foot-and-Mouth disease virus type O (FMDV/O), and Porcine epidemic diarrhea virus (PEDV)). This study thus provides a sensitive, specific, and reliable detection method that is suitable for the serological diagnosis of ASF.

Novel Epitopes Mapping of African Swine Fever Virus CP312R Protein Using Monoclonal Antibodies.

African Swine Fever (ASF) is a highly contagious and lethal pig disease and poses a huge threat to the pig industry worldwide. ASF virus (ASFV) encodes more than 150 different proteins, but the biological properties of most viral proteins are still unknown. ASFV CP312R protein has been proven to be one of the most immunogenic proteins during ASFV infection in pigs; however, its specific epitopes have yet to be identified. In this study, we verified the immunogenicity of CP312R protein in the sera from attenuated ASFV-inoculated pigs. We generated seven anti-ASFV CP312R mouse monoclonal antibodies (mAbs) from mice immunized with recombinant CP312R protein (rCP312R). All seven mAbs are the IgG2b-Kappa isotype and specifically interacted with the CP312R protein expressed in various cells that were infected by ASFVs or transfected with plasmid CP312R. The epitope mapping was performed by using these characterized mAbs and the peptide scanning (Pepscan) method followed by Western blot. As a result, two antigenic determinant regions were identified: two of the seven mAbs recognized the 122KNEQGEEIYP131 amino acids, and the remaining five mAbs recognized the 78DEEVIRMNAE87 amino acids of the CP312R protein. These antigenic determinants of CP312R are conserved in different ASFV strains of seven genotypes. By using the characterized mAb, confocal microscopy observation revealed that the CP312R was mainly localized in the cytoplasm and, to some extent, in nuclei and on the nuclear membrane of infected host cells. In summary, our results benefit our understanding on the antigenic regions of ASFV CP312R and help to develop better serological diagnosis of ASF and vaccine research.

Multiepitope array as the key for African Swine Fever diagnosis

African Swine Fever (ASF) is an acute hemorrhagic fever affecting suids with high mortality and morbidity rate. The causal agent of ASF, the African Swine Fever Virus (ASFV), is an icosahedral virus of 200 nm diameter, composed of an outer envelope layer of host derivation and a linear 170–190 kb long dsDNA molecule. As of today, no efficient therapeutic intervention nor prophylactic measures exist to fight ASFV diffusion, underlining the importance of the early diagnosis and the need for efficient in-field screening of ASF. Recommended guidelines for the diagnosis of ASF are unpracticable in the desirable context of the rapid in-farm screening. In this view, the design of innovative diagnostics based on a panel of multiple ASFV epitopes would amend versatility and the analytical performances of the deliverable, ensuring high quality and accuracy standards worth of implementation in rapid in-field monitoring programs. Pursuing this view, we performed epitope prediction from the major AFSV structural proteins holding the potential to be targeted in innovative rapid diagnostic tests. Selected ASFV structural protein sequences were retrieved from data repositories and their tridimensional structure was computed. Linear and 3D protein structures were subjected to the prediction of the epitope sequences, that are likely to elicit antibody production, by independent bioinformatic tools, providing a list of candidate biomarkers whose batch employment held the potential suitability for the unbiased rapid in-field diagnosis and, in turn, might be implemented in screening programs, crowing the current monitoring and control campaigns that are currently running worldwide.

Hematological and serum biochemical profiles of a natural African swine fever virus infection in pigs.

African swine fever (ASF) is a contagious viral disease that affects pigs of all ages, inducing hemorrhagic fever with high mortality and severe threat to pig production. This study investigated the hematological and serum biochemical abnormalities associated with a natural ASF infection in pigs. A total of 100 serum samples of pigs from piggery suspected of ASFV infection were screened for antibodies by ELISA. Thirty‑two blood samples from serologically positive pigs and 32 negative pigs were undergo to hematological and serum biochemical analyses following standard procedures. The results showed that the mean values of the red blood cell (RBC) count, total white blood cell (TWBC) count, absolute lymphocyte count, absolute monocyte count, serum total protein (TP) and globulin were significantly (p < 0.05) lower while the mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), absolute neutrophil count and serum gamma glutamyl transferase (GGT) were significantly (p < 0.05) higher in the infected than the healthy pigs. There were no significant differences (p > 0.05) in the mean values of the packed cell volume (PCV), hemoglobin concentration, absolute eosinophil count, cholesterol, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities between the infected and healthy pigs. Hence, natural ASFV infection may have caused alterations in the hematological and serum biochemical parameters in the infected pigs. The generated data could complement the existing laboratory diagnostic techniques such as polymerase chain reaction, direct fluorescence antibody test, indirect fluorescent antibody test and ELISA in the diagnosis of ASF in pigs.

Indirect ELISA Using Multi-Antigenic Dominants of p30, p54 and p72 Recombinant Proteins to Detect Antibodies against African Swine Fever Virus in Pigs.

African swine fever (ASF) caused by ASF virus (ASFV) is a fatal disease in pigs and results in great economic losses. Due to the lack of available vaccines and treatments, serological diagnosis of ASF plays a key role in the surveillance program, but due to the lack of knowledge and the complexity of the ASFV genome, the candidate target viral proteins are still being researched. False negativity is still a big obstacle during the diagnostic process. In this study, the high antigenic viral proteins p30, p54 and p72 were screened to find the antigenic dominant domains and the tandem His-p30-54-72 was derived. An indirect enzyme-linked immunosorbent assay (iELISA) coated with His-p30-54-72 was developed with a cut-off value of 0.371. A total of 192 clinical samples were detected by His-p30-54-72-coated indirect ELISA (iELISA) and commercial ASFV antibody kits. The results showed that the positive rate of His-p30-54-72-coated iELISA was increased by 4.7% and 14.6% compared with a single viral protein-based commercial ASFV antibody kits. These results provide a platform for future ASFV clinical diagnosis and vaccine immune effect evaluation.

Alternative approaches to the diagnosis of African swine fever in the Russian Federation in 2017–2021

Prevention and control of African swine fever (ASF) transmission on the territory of the Russian Federation requires monitoring based on testing of samples from pigs and wild boars. Specific anti-ASFV antibodies are rarely detected in samples during routine serological diagnostics. Although, ASF isolates with weakened virulence were confirmed in Russia and neighboring countries.The aim of this work was to determine the possibility of using alternative samples for ASF diagnosis and evaluate the effectiveness of the diagnostic methods used on the territory of Russia. Biological materials obtained from experimentally infected animals and samples collected in the "field" conditions were used in this study. Complex testing (RT-PCR and ELISA) is a more effective approach to diagnose chronic and asymptomatic forms of ASF compared to the separate use of these techniques. The possibility and efficiency of using alternative samples in diagnostics are demonstrated. It was confirmed that IPT method overcomes ELISA by high diagnostic sensitivity and detection of antibodies on earlier stages in extended range of samples. Anti-ASFV antibodies were detected in domestic and wild pigs in five regions of Russia. Samples from infected pigs that are negative in RT-PCR can be positive for anti-ASFV antibodies. The detection of antibodies in samples from shot wild boars (negative or uncertain in RT-PCR test) suggests the existence of animals surviving ASF infection. The data obtained suggest a revision of the ASF surveillance strategy, by introducing complex diagnostic methods aimed at detection of both the virus genome and anti-ASFV antibodies simultaneously.

Identification of novel linear epitopes in P72 protein of African swine fever virus recognized by monoclonal antibodies.

African swine fever (ASF) is one of the highly contagious and lethal diseases among domestic pigs and wild boars. The capsid protein P72 of African swine fever virus (ASFV) is very important for the diagnosis and vaccine development. However, the epitope of the protein is not clear. In this study, capsid protein P72 was expressed in Sf9 cells along with its chaperone B602L. A total of ten monoclonal antibodies (mAbs) specific to P72 protein were developed by fusions between SP2/0 cells and spleen cells of mice immunized with the recombinant-P72&B602L proteins expressed in Sf9 cells. Four linear B cell epitopes 31SNIKNVNKSY40, 41GKPDP45, 56HLVHFNAH63 and 185ERLYE189 were identified. Biological information analysis illustrated that epitopes 31SNIKNVNKSY40, 41GKPDP45 and 185ERLYE189 were highly conserved within different ASFV strains. These findings may lead to a better understanding of the antibody-antigen interaction and provide new insights into the vaccine research and serological diagnosis of ASF.

Clinical Validation of DNA Extraction-Free qPCR, Visual LAMP, and Fluorescent LAMP Assays for the Rapid Detection of African Swine Fever Virus.

The global pig industry and food safety are seriously threatened by outbreaks of African swine fever (ASF). To permit early diagnosis of African swine fever virus (ASFV), prevent its spread, and limit its outbreaks, a highly sensitive diagnostic method that can be performed at pig farms is required. Herein, we established DNA extraction-free real-time PCR (qPCR), visual loop-mediated isothermal amplification (LAMP), and fluorescent LAMP assays, which were compared with the results of World Organization for Animal Health (OIE) qPCR to assess ASFV-infected clinical samples. Based on plasmid DNA, the limit of detection for the three assays and OIE qPCR were 5.8 copies/μL. All four assays had good ASFV specificity and showed no cross-reactivity with other tested viruses. These assays were used to diagnose 100 clinical samples. The assays showed good diagnostic consistency, with kappa values of 1.0, 0.84, and 0.88, respectively. Compared with OIE qPCR, the diagnostic specificity/sensitivity of DNA extraction-free qPCR, visual LAMP, and fluorescent LAMP assays were 100%/100%, 100%/87.1%, and 100%/90.32%, respectively. The assays eliminated the need for DNA extraction and are more suitable for ASF diagnosis by inexperienced farmers in low-resource environments, making them a good choice for on-site monitoring of pig farms.