Lessons from CanSpotASF: Moving towards risk-based African Swine Fever surveillance with rule-out testing in Western Canada.

African swine fewer (ASF) is a serious disease present in Africa, Eurasia, and the Caribbean but not in continental North America. CanSpotASF describes the ASF surveillance in Canada as a phased in approach. The first enhancement to the passive surveillance was the risk-based early detection testing (rule-out testing) where eligible cases were tested for ASF virus (ASFv). The objective was to describe how the eligibility criteria were applied to cases in western Canada. In particular, to assess if cases tested for ASFv had eligible conditions and if pathology cases with eligible conditions were tested for ASFv based on the data collated by Canada West Swine Health Intelligence Network (CWSHIN) from British Columbia, Alberta, Saskatchewan, and Manitoba. The study period was August 2020 to December 2022 and the data included two study laboratories. We found that over 90% of cases tested for ASFv had eligible conditions as defined in CanSpotASF. The eligibility criteria were applied at three stages of the disease investigation process: 1) the clinical presentation in the herd; 2) at the initial laboratory assessment; and 3) the final pathology diagnosis. At the two study laboratories the proportion of all submitted cases (culture, serology, PCR, pathology) tested for ASFv was very low 1%. However, in the pathology cases specifically targeted in CanSpotASF, and the proportion of tested cases was 12%. In addition, for eligible pathology cases (eligible diagnosis or test) the proportion tested was higher 15%. These results indicated that CanSpotASF targeted herds with submissions for pathological examination and to some degree eligible conditions which would be herds with health issues (known or unknown). We interpret this as a first step towards risk-based surveillance with health as the defining factor.

Development of a novel sensitive single-tube nested PCR assay for the detection of African swine fever virus.

African swine fever (ASF) is a highly fatal and contagious viral disease caused by African swine fever virus (ASFV). It has caused significant economic losses to the swine industry and poses a serious threat to food security worldwide. Diagnostic tests with high sensitivity are essential for the effective management of ASF. Here, we describe a single-tube nested PCR (STN-PCR) assay for the detection of ASFV in which two consecutive amplification steps are carried out within a single tube. Two pairs of primers (outer and inner) were designed to target the p72 gene of ASFV. The primer concentrations, annealing temperatures, and number of amplification cycles were optimized to ensure the consecutive utilization of outer and inner primer pairs during amplification while minimizing the likelihood of amplicon contamination. In comparison with two conventional endpoint PCR assays (one of which is recommended by the World Organization for Animal Health), the newly developed STN-PCR assay demonstrated a 100-fold improvement in the limit of detection (LOD), detecting 100 copies of ASFV genomic DNA, whereas the endpoint PCR assays could detect no fewer than 10,000 copies. The clinical performance of the STN-PCR assay was validated using 95 tissue samples suspected of being positive for ASFV, and the assay showed 100% specificity. A Cohen's kappa value of 0.91 indicated perfect agreement between the assays. This new STN-PCR assay is a potentially valuable tool that will facilitate the control of ASF.

A highly efficient blocking ELISA based on p72 monoclonal antibody for the detection of African swine fever virus antibodies and identification of its linear B cell epitope.

Due to the absence of effective vaccine and treatment, African swine fever virus (ASFV) control is entirely dependent on accurate and early diagnosis, along with culling of infected pigs. The B646L/p72 is the major capsid protein of ASFV and is an important target for developing a diagnostic assays and vaccines. Herein, we generated a monoclonal antibody (mAb) (designated as 2F11) against the trimeric p72 protein, and a blocking ELISA (bELISA) was established for the detection of both genotype I and II ASFV antibodies. To evaluate the performance of the diagnostic test, a total of 506 porcine serum samples were tested. The average value of percent of inhibition (PI) of 133 negative pig serum was 8.4 % with standard deviation (SD) 6.5 %. Accordingly, the cut-off value of the newly established method was set at 28 % (mean + 3SD). Similarly, a receiver operating characteristic (ROC) was applied to determine the cut off value and the p72-bELISA exhibited a sensitivity of 100 % and a specificity of 99.33 % when the detection threshold was set at 28 %. The bELISA was also able to specifically recognize anti-ASFV sera without cross-reacting with other positive serums for other major swine pathogens. Moreover, by designing a series of overlapped p72 truncated proteins, the linear B cell epitope recognized by 2F11 mAb was defined to be 283NSHNIQ288. Amino acid sequence comparison revealed that the amino acid sequence 283NSHNIQ288 is highly conserved between different ASFV isolates. Our findings indicate that the newly established mAb based blocking ELISA may have a great potential in improving the detection of ASFV antibodies and provides solid foundation for further studies.

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.

KP177R-based visual assay integrating RPA and CRISPR/Cas12a for the detection of African swine fever virus.

Introduction: Early detection of the virus in the environment or in infected pigs is a critical step to stop African swine fever virus (ASFV) transmission. The p22 protein encoded by ASFV KP177R gene has been shown to have no effect on viral replication and virulence and can serve as a molecular marker for distinguishing field virus strains from future candidate KP177R deletion vaccine strains. Methods: This study established an ASFV detection assay specific for the highly conserved ASFV KP177R gene based on recombinase polymerase amplification (RPA) and the CRISPR/Cas12 reaction system. The KP177R gene served as the initial template for the RPA reaction to generate amplicons, which were recognized by guide RNA to activate the trans-cleavage activity of Cas12a protein, thereby leading to non-specific cleavage of single-stranded DNA as well as corresponding color reaction. The viral detection in this assay could be determined by visualizing the results of fluorescence or lateral flow dipstick (LFD) biotin blotting for color development, and was respectively referred to as fluorescein-labeled RPA-CRISPR/Cas12a and biotin-labeled LFD RPA-CRISPR/Cas12a. The clinical samples were simultaneously subjected to the aforementioned assay, while real-time quantitative PCR (RT-qPCR) was employed as a control for determining the diagnostic concordance rate between both assays. Results: The results showed that fluorescein- and biotin-labeled LFD KP177R RPA-CRISPR/Cas12a assays specifically detected ASFV, did not cross-react with other swine pathogens including PCV2, PEDV, PDCoV, and PRV. The detection assay established in this study had a limit of detection (LOD) of 6.8 copies/µL, and both assays were completed in 30 min. The KP177R RPA-CRISPR/Cas12a assay demonstrated a diagnostic coincidence rate of 100% and a kappa value of 1.000 (p < 0.001), with six out of ten clinical samples testing positive for ASFV using both KP177R RPA-CRISPR/Cas12a and RT-qPCR, while four samples tested negative in both assays. Discussion: The rapid, sensitive and visual detection assay for ASFV developed in this study is suitable for field application in swine farms, particularly for future differentiation of field virus strains from candidate KP177R gene-deleted ASFV vaccines, which may be a valuable screening tool for ASF eradication.

Development of visual detection of African swine fever virus using CRISPR/LwCas13a lateral flow strip based on structural protein gene D117L.

African swine fever virus (ASFV) is a large double stranded DNA arbovirus that is highly contagious and seriously endangers domestic and wild pigs. In the past decade, African swine fever (ASF) has spread in many countries in the Caucasus, Russian Federation, Eastern Europe and Asia, causing significant losses to the pig industry. At present, there is a lack of effective vaccine and treatment for ASF. Therefore, the rapid and accurate detection is crucial for ASF prevention and control. In this study, we have developed a portable lateral flow strip (LFS) detection mediated by recombinase polymerase amplification (RPA) and CRISPR/LwCas13a, which is performed at 37 ℃ and visualized by eyes without the need for complex instruments. This RPA-LwCas13a-LFS is based on the ASFV structural protein p17 gene (D117L), with a detection sensitivity up to 2 gene copies. This method is highly specific and has no cross reactivity to 7 other pig viruses. In the detection of two batches of 100 clinical samples, the p17 (D117L) RPA-LwCas13a-LFS had 100% coincidence with conventional quantitative PCR (qPCR). These findings demonstrate the potential of this simple, rapid, sensitive, and specific ASFV detection method for on-site ASFV detection.

Establishment of a simple, sensitive, and specific ASFV detection method based on Pyrococcus furiosus argonaute.

African swine fever (ASF), which is casued by African swine fever virus (ASFV), is a fatal infectious disease of pigs that results in significant losses to the breeding industry. Therefore, screening and detection are crucial for the control and prevention of the ASFV. Argonaute is a new detection tool that is being extensively used due to its high specificity and programmability. This study reports on a new nucleic acid assay method, termed REPD, which uses recombinase-aided amplification and restriction endonuclease-assisted Pyrococcus furiosus argonaute (PfAgo) detection. One-pot REPD was developed for the detection of ASFV. The one-pot REPD could detect a single copy of ASFV nucleic acid and showed no cross-reactivity with other pathogens. Detection in clinical samples was 100% consistent with the results of real-time PCR analysis. The results showed that the one-pot REPD assay is convenient, sensitive, specific, and potentially adaptable to the detection of ASFV. In summary, this study highlights a novel method that can be employed for the detection of pathogens.

Characterization of the monoclonal antibody and the immunodominant B-cell epitope of African swine fever virus pA104R by using mouse model.

The African swine fever virus (ASFV) structural protein pA104R is the only histone-like protein encoded by eukaryotic viruses. pA104R is an essential DNA-binding protein required for DNA replication and genome packaging of ASFV, which are vital for pathogen survival and proliferation. pA104R is an important target molecule for diagnosing, treating, and immune prevention of ASFV. This study characterized monoclonal antibodies (mAbs) against pA104R and found them to recognize natural pA104R in ASFV strains with different genotypes, showing high conservation. Confirmation analyses of pA104R epitopes using mAbs indicated the presence of immunodominant B-cell epitopes, and further characterization showed the high antigenic index and surface accessibility coefficients of the identified epitope. Furthermore, the pA104R protein functions through the polar interactions between the binding amino acid sites; however, these interactions may be blocked by the recognition of generated mAbs. Characterizing the immunodominant B-cell epitope of the ASFV critical proteins, such as pA104R, may contribute to developing sensitive diagnostic tools and vaccine candidate targets.IMPORTANCEAfrican swine fever (ASF) is a highly pathogenic, lethal, and contagious viral disease affecting domestic pigs and wild boars. As no effective vaccine or other treatments have been developed, the control of African swine fever virus (ASFV) relies heavily on virus detection and diagnosis. A potential serological target is the structural protein pA104R. However, the molecular basis of pA104R antigenicity remains unclear, and a specific monoclonal antibody (mAb) against this protein is still unavailable. In this study, mAbs against pA104R were characterized and found to recognize natural pA104R in ASFV strains with different genotypes. In addition, confirmation analyses of pA104R epitopes using mAbs indicated the presence of immunodominant B-cell epitopes, and further characterization showed the high antigenic index and surface accessibility coefficients of the identified epitope. Characteristics of the immunodominant B-cell epitope of ASFV proteins, such as pA104R, may contribute to developing sensitive diagnostic tools and identifying vaccine candidate targets.

The p30 protein of the African swine fever virus behaves as an RNase.

The African Swine Fever Virus (ASFV) is responsible for causing African Swine Fever (ASF), a severe contagious disease characterized by hemorrhagic symptoms. The p30 protein of ASFV is the most abundantly expressed viral protein. It is reported to be antigenic and has recognized phosphorylation, glycosylation, and membrane attachment sites, which also shows that the C-terminal region of p30 is more active than the N-terminal region. The present study reports the unique RNase activity of recombinant p30. The RNase activity of p30 was stable at an optimum temperature of 37 °C, and the maximum activity was recorded at pH 7-9 in the presence of monovalent salts. The mutant of p30 (p30m), where cysteine was mutated to alanine at position 109, showed a loss of RNase activity. Our understanding of ASFV biology is significantly less; until now, we have little knowledge about the functions of its proteins. The results of the present study will assist in exploring the biology of ASFV and the role of its protein in counteracting the host immune response.

Advanced Strategies for Developing Vaccines and Diagnostic Tools for African Swine Fever.

African swine fever (ASF) is one of the most lethal infectious diseases affecting domestic pigs and wild boars of all ages. Over a span of 100 years, ASF has continued to spread over continents and adversely affects the global pig industry. To date, no vaccine or treatment has been approved. The complex genome structure and diverse variants facilitate the immune evasion of the ASF virus (ASFV). Recently, advanced technologies have been used to design various potential vaccine candidates and effective diagnostic tools. This review updates vaccine platforms that are currently being used worldwide, with a focus on genetically modified live attenuated vaccines, including an understanding of their potential efficacy and limitations of safety and stability. Furthermore, advanced ASFV detection technologies are presented that discuss and incorporate the challenges that remain to be addressed for conventional detection methods. We also highlight a nano-bio-based system that enhances sensitivity and specificity. A combination of prophylactic vaccines and point-of-care diagnostics can help effectively control the spread of ASFV.

Establishment and characterization of a novel indirect ELISA method based on ASFV antigenic epitope-associated recombinant protein.

African Swine Fever (ASF) is an acute and highly lethal disease in pigs caused by African Swine Fever Virus (ASFV). Viral proteins have been commonly used as antigenic targets for the development of ASF diagnostic methods. However, the prokaryotic expression of viral proteins has deficiencies such as instability, insolubility, and high cost in eukaryotic situations. This study screened and verified ASFV-encoded p72, p54, and p30 protein antigenic epitopes. Subsequently, a novel antigenic epitope-associated recombinant protein was designed based on an ideal structural protein and expressed in Escherichia coli (E. coli). Western blot analysis indicated that the recombinant protein could specifically react with the monoclonal antibody (mAb) of p72 and polyclonal antibodies of p54 and p30, respectively. Next, an ASF indirect ELISA (iELISA) method was established based on the recombinant protein, which has no specific reaction with sera of other important pig viral diseases. Meanwhile, it shows a sensitivity to detecting dilutions of ASF-positive reference serum up to 1:6400. The clinical sample detection results showed a high coincidence rate of 98 % with a commercial competition ELISA kit. In conclusion, we established a novel specific, and sensitive ASF serologic detection method that opens new avenues for ASF serodiagnostic method development.

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.

Developing an Indirect ELISA for the Detection of African Swine Fever Virus Antibodies Using a Tag-Free p15 Protein Antigen.

African swine fever (ASF) is one of the most severe diseases caused by the ASF virus (ASFV), causing massive economic losses to the global pig industry. Serological tests are important in ASF epidemiological surveillance, and more antigen targets are needed to meet market demand for ASFV antibody detection. In the present study, ASFV p15 protein was fusion-expressed in Escherichia coli (E. coli) with elastin-like polypeptide (ELP), and the ELP-p15 protein was purified using a simple inverse transition cycling (ITC) process. The ELP tag was cleaved off using tobacco etch virus protease (TEVp), resulting in a tag-free p15 protein. Western blot analysis demonstrated that the p15 protein reacted strongly with ASFV-positive serum. The p15 protein was used as a coating antigen in an indirect ELISA (iELISA) for detecting ASFV antibodies. The p15-iELISA method demonstrated high specificity to ASFV-positive sera, with a maximum detection dilution of 1:1600. Moreover, the method exhibited good reproducibility, with less intra-assay and inter-assay CV values than 10%. Therefore, p15-iELISA offers a novel approach for accurately detecting ASFV antibodies with significant clinical application potential.

Accelerometer-based detection of African swine fever infection in wild boar.

Infectious wildlife diseases that circulate at the interface with domestic animals pose significant threats worldwide and require early detection and warning. Although animal tracking technologies are used to discern behavioural changes, they are rarely used to monitor wildlife diseases. Common disease-induced behavioural changes include reduced activity and lethargy ('sickness behaviour'). Here, we investigated whether accelerometer sensors could detect the onset of African swine fever (ASF), a viral infection that induces high mortality in suids for which no vaccine is currently available. Taking advantage of an experiment designed to test an oral ASF vaccine, we equipped 12 wild boars with an accelerometer tag and quantified how ASF affects their activity pattern and behavioural fingerprint, using overall dynamic body acceleration. Wild boars showed a daily reduction in activity of 10-20% from the healthy to the viremia phase. Using change point statistics and comparing healthy individuals living in semi-free and free-ranging conditions, we show how the onset of disease-induced sickness can be detected and how such early detection could work in natural settings. Timely detection of infection in animals is crucial for disease surveillance and control, and accelerometer technology on sentinel animals provides a viable complementary tool to existing disease management approaches.

Visual isothermal amplification detection of ASFV based on trimeric G-quadruplex cis-cleavage activity of Cas-12a.

African swine fever virus (ASFV) is a kind of DNA virus and can infect both domestic pigs and wild boars with fatality up to 100%. The contaminated meat products mainly led to the worldwide transmission of ASFV. The outbreak of ASF greatly affects the supply stability of meat products as well as the development of the global pig industry. In this study, a visual isothermal amplification detection assay for ASFV based on trimeric G-quadruplex cis-cleavage activity of Cas12a was developed. The introduction of Cas12a could discriminate the specific amplification from the non-specific amplification and improve the sensitivity. The detection limit was as low as 0.23 copies/µL. This assay had good potential in the detection of ASFV and would be helpful for the stability of meat production and supply.

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

BACKGROUND: Originating 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. METHODS: Primers 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. RESULTS: After 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. CONCLUSIONS: The 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.

I329L protein-based indirect ELISA for detecting antibodies specific to African swine fever virus.

African swine fever (ASF) is a disease that causes severe economic losses to the global porcine industry. As no vaccine or drug has been discovered for the prevention and control of ASF virus (ASFV), accurate diagnosis and timely eradication of infected animals are the primary measures, which necessitate accurate and effective detection methods. In this study, the truncated ASFV I329L (amino acids 70-237), was induced using IPTG and expressed in Escherichia coli cells. The highly antigenic viral protein I329L was used to develop an indirect enzyme-linked immunosorbent assay (iELISA), named I329L-ELISA, which cut-off value was 0.384. I329L-ELISA was used to detect 186 clinical pig serum samples, and the coincidence rate between the indirect ELISA developed here and the commercial kit was 96.77%. No cross-reactivity was observed with CSFV, PRRSV, PCV2, or PRV antibody-positive pig sera, indicating good specificity. Both intra- assay and inter-assay coefficients were below 10%, and the detection sensitivity of the iELISA reached 1:3200. In this study, an iELISA for ASFV antibody detection was developed based on the truncated ASFV I329L protein. Overall, the I329L-ELISA is a user-friendly detection tool that is suitable for ASFV antibody detection and epidemiological surveillance.

Identification and Characterization of Nanobodies from a Phage Display Library and Their Application in an Immunoassay for the Sensitive Detection of African Swine Fever Virus.

African swine fever (ASF) is one of the most lethal and devastating diseases of domestic and wild swine. The continual spread and frequent outbreaks of ASF have seriously threatened the pig and pig-related industries, causing great socioeconomic losses at unprecedented proportions. Although ASF has been documented for a century, no effective vaccine or antiviral treatment is currently available. Nanobodies (Nbs) derived from heavy-chain-only antibodies in camelids have been discovered to be effective as therapeutics and robust biosensors in imaging and diagnostic applications. In the present study, a high-quality phage display library containing specific Nbs raised against ASFV proteins was successfully constructed, and 19 nanobodies specific to ASFV p30 were preliminarily identified by phage display technology. After extensive evaluation, nanobodies Nb17 and Nb30 were employed as immunosensors and applied to develop a sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of ASFV in clinical specimens. This immunoassay showed a detection limit of approximately 1.1 ng/mL target protein and 102.5 hemadsorption (HAD50/mL) of ASFV and exhibited high specificity with no cross-reaction with the other porcine viruses tested. The performances of the newly developed assay and a commercial kit in testing 282 clinical swine samples were very similar (93.62% agreement). However, the novel sandwich Nb-ELISA showed higher sensitivity than the commercial kit when serial dilutions of ASFV-positive samples were tested. The present study describes a valuable alternative technique for the detection and surveillance of ASF in endemic regions. Furthermore, additional nanobodies specific to ASFV may be developed using the generated VHH library and employed in different biotechnology fields.

Enhanced passive surveillance for early detection of African and classical swine fevers.

African swine fever (ASF) and classical swine fever (CSF) are transboundary animal diseases (TADs) of pigs. Much effort and resources are regularly put into preventing these diseases' introduction in free areas. Passive surveillance activities bring the highest chances for the early detection of TAD incursions because they are routinely and widely conducted at farms, and because these activities focus on the time between introduction and when the first sample is sent for diagnostic testing. The authors proposed the implementation of an enhanced passive surveillance (EPS) protocol based on collecting data through participatory surveillance actions using an objective and adaptable scoring system to aid the early detection of ASF or CSF at the farm level. The protocol was applied in two commercial pig farms for ten weeks in the Dominican Republic, which is a CSF- and ASF-infected country. This study was a proof of concept, based on the EPS protocol to aid detection of substantial variations in the risk score triggering testing. One of the followed farms had score variation, which triggered testing of the animals, although the test results were negative. The study enables assessment of some of the weaknesses associated with passive surveillance and provides lessons applicable to the problem. Results demonstrate the potential for overcoming some issues preventing the broad application of EPS protocols and suggest that standardised approaches may contribute to the early detection of CSF and ASF introductions.

La peste porcine africaine (PPA) et la peste porcine classique (PPC) sont des maladies animales transfrontalières touchant les porcs. De nombreux efforts et ressources sont régulièrement alloués pour prévenir l'introduction de ces maladies dans des zones indemnes. Les activités de surveillance passive offrent les meilleures perspectives de détection précoce des incursions de maladies animales transfrontalières parce qu'elles sont menées de manière systématique et exhaustive dans les élevages, et parce qu'elles se concentrent sur la période entre l'introduction de la maladie et le moment où le premier échantillon est envoyé au laboratoire pour analyse. Les auteurs proposent la mise en oeuvre d'un protocole de surveillance passive renforcée fondé sur la collecte de données via des actions de surveillance participative utilisant un système de notation objectif et adaptable, en vue d'une détection précoce de la PPA et de la PPC dans les élevages. Ce protocole a été appliqué en République dominicaine, pays infecté par la PPA et la PPC, dans deux élevages porcins commerciaux pendant dix semaines. Cette étude était destinée à valider le principe de la méthode et se fondait sur le protocole de surveillance passive renforcée pour mieux détecter les variations substantielles de la note de risque qui conduisent à tester les animaux. L'un des élevages suivis a présenté une variation de cette note, ce qui a conduit à tester les animaux mais les tests se sont révélés négatifs. L'étude permet d'évaluer certaines des faiblesses associées à la surveillance passive et apporte des enseignements applicables à ce problème. Les résultats illustrent le potentiel de l'approche à surmonter certaines des problématiques empêchant l'application extensive des protocoles de surveillance passive renforcée. Ils suggèrent également que des approches normalisées pourraient contribuer à la détection précoce des cas d'introduction de la PPC et de la PPA.

La peste porcina africana (PPA) y la peste porcina clásica (PPC) son enfermedades animales transfronterizas que afectan al cerdo. Periódicamente se dedican grandes esfuerzos y cuantiosos recursos a evitar que estas enfermedades penetren en zonas que están exentas de ellas. Las actividades de vigilancia pasiva son las más eficaces para detectar con prontitud toda incursión de enfermedades animales transfronterizas, no solo por la regularidad y amplitud con que se llevan a cabo en las explotaciones, sino también porque inciden específicamente en el intervalo entre la penetración de una enfermedad y el momento en que se envía la primera muestra para que sea sometida a pruebas de diagnóstico. Los autores propusieron que se aplicara un protocolo de vigilancia pasiva reforzada que reposaba en la obtención de datos mediante actividades de vigilancia participativa, empleando para ello un sistema objetivo y adaptable de puntuación que ayudaba a detectar con prontitud la presencia en las explotaciones de PPA o PPC. Dicho protocolo fue aplicado a lo largo de diez semanas en dos explotaciones porcinas industriales de la República Dominicana, país en el que ambas infecciones están presentes. El estudio, que sirvió para poner a prueba la idea, pasaba por la aplicación del protocolo de vigilancia pasiva reforzada para ayudar a detectar variaciones sustanciales de la puntuación del nivel de riesgo que activa la realización de pruebas. En una de las explotaciones estudiadas se produjo una variación de la puntuación, cosa que activó la realización de pruebas en los animales, aunque estas arrojaron resultado negativo. El estudio aquí descrito permite evaluar algunos de los puntos débiles de la vigilancia pasiva y extraer enseñanzas aplicables al problema. Los resultados demuestran que es posible salvar algunas de las dificultades que impiden la aplicación generalizada de protocolos de vigilancia pasiva reforzada y dejan pensar que quizá el uso de planteamientos normalizados pueda ayudar a detectar con prontitud los casos de penetración de PPC o PPA.

A chemiluminescent magnetic microparticle immunoassay for the detection of antibody against African swine fever virus.

The p30 protein is abundantly expressed in the early stage of African swine fever virus (ASFV) infection. Thus, it is an ideal antigen candidate for serodiagnosis with the use of an immunoassay. In this study, a chemiluminescent magnetic microparticle immunoassay (CMIA) was developed for the detection of antibodies (Abs) against ASFV p30 protein in porcine serum. Purified p30 protein was coupled to magnetic beads, and the experimental conditions including concentration, temperature, incubation time, dilution ratio, buffers, and other relevant variables were evaluated and optimized. To evaluate the performance of the assay, a total of 178 pig serum samples (117 negative and 61 positive samples) were tested. According to receiver operator characteristic curve analysis, the cut-off value of the CMIA was 104,315 (area under the curve, 0.998; Youden's index, 0.974; 95% confidence interval: 99.45 to 100%). Sensitivity results showed that the dilution ratio of p30 Abs in ASFV-positive sera detected by the CMIA is much higher when compared to commercial blocking ELISA kit. Specificity testing showed that no cross-reactivity was observed with sera positive for other porcine disease viruses. The intraassay coefficient of variation (CV) was < 5%, and the interassay CV was < 10%. The p30-magnetic beads could be stored at 4 °C for more than 15 months without loss of activity. The kappa coefficient between CMIA and INGENASA blocking ELISA kit was 0.946, showing strong agreement. In conclusion, our method showed superiority with high sensitivity, specificity, reproducibility, and stability and potentialized its application in the development of a diagnostic kit for the detection of ASF in clinical samples. KEY POINTS: • ASFV tag-free p30 was successfully purified. • High sensitivity, specificity, relatively simple, and time-saving to detect antibody against ASFV were developed. • The development of CMIA will help the clinical diagnosis of ASFV and will be useful for large-scale serological test.