Control Of African Swine Fever Research Articles

Rapid Visual Detection of African Swine Fever Virus with a CRISPR/Cas12a Lateral Flow Strip Based on Structural Protein Gene D117L

African swine fever virus (ASFV) is a large double-stranded DNA virus that is highly infectious and seriously affects domestic pigs and wild boars. African swine fever (ASF) has caused huge economic losses to endemic countries and regions. At present, there is still a lack of effective vaccines and therapeutics. Therefore, rapid and accurate detection is essential for the prevention and control of ASF. The portable DNA endonuclease (Cas12a)-mediated lateral flow strip detection method (Cas12a-LFS) combined with recombinant polymerase amplification (RPA) has been gradually recognized as effective for virus detection including ASFV. In this study, based on the ASFV structural protein p17 gene (D117L), an RPA-Cas12a-LFS detection method was established. The detection method exhibits a sensitivity of up to two gene copies and has no cross-reaction with nine other swine viruses. Thus, the method is highly sensitive and specific. In 68 clinical samples, the coincidence rate of the p17 strip was 100%, compared to the traditional quantitative PCR (qPCR). In conclusion, we have developed a simple, rapid, sensitive, and specific ASFV visual detection method and demonstrated the potential of on-site detection of ASFV.

Actions and perceived impact of African swine fever control measures along the smallholder pig value chain in Uganda

Pig production in Uganda is constrained by African swine fever (ASF) which is endemic in the country. Current measures taken by the Government of Uganda in controlling ASF outbreaks include trade and livestock movement restrictions, called “quarantine.” Little is known about the actions of, and impact of value chain actors in response to ASF quarantines. This study describes actions that different stakeholders in the smallholder pig value chain took, and the perceived economic impact, during ASF quarantines. Data was collected in ten focus group discussions (FGD) using participatory epidemiology tools and two key informants’ (KIs) interviews with District Veterinary Officers (DVOs) of Kisoro and Moyo districts in Uganda. The results show that during ASF quarantine, pig value chain actors shifted their activities from formal places such as livestock markets, slaughter slabs, pork butcheries and pork joints to informal places such as farmers’ homesteads. Farmers were perceived the most economically affected stakeholder group with forgone income due to unsold pigs, costs for implementing biosecurity measures and extra costs for feeding unsold pigs being the major perceived causes of the losses. The continued trade in pigs and pig products in informal marketplaces suggests that quarantine might not be effective for hindering activities that might spread ASF in these settings. The perceived economic losses provide an insight into the negative economic impact of the quarantine for the different stakeholders.

Preparation and epitope mapping of monoclonal antibodies against African swine fever virus p22 protein

African swine fever (ASF), caused by the African swine fever virus (ASFV), is now widespread in many countries and severely affects the commercial rearing of swine. Rapid and early diagnosis is crucial for the prevention of ASF. ASFV mature virions comprise the inner envelope protein, p22, making it an excellent candidate for the serological diagnosis and surveillance of ASF. In this study, the prokaryotic-expressed p22 recombinant protein was prepared and purified for immunization in mice. Four monoclonal antibodies (mAbs) were identified using hybridoma cell fusion, clone purification, and immunological assays. The epitopes of mAbs 14G1 and 22D8 were further defined by alanine-scanning mutagenesis. Our results showed that amino acids C39, K40, V41, D42, C45, G48, E49, and C51 directly bound to 14G1, while the key amino acid epitope for 22D8 included K161, Y162, G163, D165, H166, I167, and I168. Homologous and structural analysis revealed that these sites were highly conserved across Asian and European ASFV strains, and the amino acids identified were located on the surface of p22. Thus, our study contributes to a better understanding of the antigenicity of the ASFV p22 protein, and the results could facilitate the prevention and control of ASF.

A Robust Quadruple Protein-Based Indirect ELISA for Detection of Antibodies to African Swine Fever Virus in Pigs.

African swine fever (ASF) emerged in domestic pigs and wild boars in China in 2018 and rapidly spread to neighboring Asian countries. Currently, no effective vaccine or diagnostic tests are available to prevent its spread. We developed a robust quadruple recombinant-protein-based indirect enzyme-linked immunosorbent assay (QrP-iELISA) using four antigenic proteins (CD2v, CAP80, p54, and p22) to detect ASF virus (ASFV) antibodies and compared it with a commercial kit (IDvet) using ASFV-positive and -negative serum samples. The maximum positive/negative value was 24.033 at a single antigen concentration of 0.25 μg/mL and quadruple ASFV antigen combination of 1 μg/mL at a 1:100 serum dilution. Among 70 ASFV-positive samples, 65, 67, 65, 70, 70, and 14 were positive above the cut-offs of 0.121, 0.121, 0.183, 0.065, 0.201, and 0.122, for CD2v, CAP80, p54, p22-iELISA, QrP-iELISA, and IDvet, respectively, with sensitivities of 92.9%, 95.7%, 92.9%, 100%, 100%, and 20%, respectively, all with 100% specificity. The antibody responses in QrP-iELISA and IDvet were similar in pigs infected with ASFV I. QrP-iELISA was more sensitive than IDvet for early antibody detection in pigs infected with ASFV II. These data provide a foundation for developing advanced ASF antibody detection kits critical for ASF surveillance and control.

Stability of African swine fever virus genome under different environmental conditions.

African swine fever (ASF), a globally transmitted viral disease caused by ASF virus (ASFV), can severely damage the global trade economy. Laboratory diagnostic methods, including pathogen and serological detection techniques, are currently used to monitor and control ASF. Because the large double-stranded DNA genome of the mature virus particle is wrapped in a membrane, the stability of ASFV and its genome is maintained in most natural environments. This study aimed to investigate the stability of ASFV under different environmental conditions from both genomic and antibody perspectives, and to provide a theoretical basis for the prevention and elimination of ASFV. In this study, we used quantitative real-time polymerase chain reaction for pathogen assays and enzyme-linked immunosorbent assay for serological assays to examine the stability of the ASFV genome and antibody, respectively, under different environmental conditions. The stability of the ASFV genome and antibody under high-temperature conditions depended on the treatment time. In the pH test, the ASFV genome and antibody remained stable in both acidic and alkaline environments. Disinfection tests revealed that the ASFV genome and antibody were susceptible to standard disinfection methods. Collectively, the results demonstrated that the ASFV genome is highly stable in favorable environments but are also susceptible to standard disinfection methods. This study focuses on the stability of the ASFV genome under different conditions and provides various standard disinfection methods for the prevention and control of ASF.

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.

Remote Sensing Provides a Rapid Epidemiological Context for the Control of African Swine Fever in Germany.

Transboundary disease control, as for African swine fever (ASF), requires rapid understanding of the locally relevant potential risk factors. Here, we show how satellite remote sensing can be applied to the field of animal disease control by providing an epidemiological context for the implementation of measures against the occurrence of ASF in Germany. We find that remotely sensed observations are of the greatest value at a lower jurisdictional level, particularly in support of wild boar carcass search efforts.

What can we learn from the five-year African swine fever epidemic in Asia?

Today's global swine industry is exposed to the unprecedented threat of African swine fever (ASF). Asia, the site of the most recent epidemics, could serve as a huge viral reservoir for the rest of the world given the severity of the damage, the huge swine industry, and the high volume of trade with other countries around the world. As the majority of ASF notifications in Asia today originate from pig farms, the movement of live pigs and associated pork products are considered critical control points for disease management. Particularly, small-scale or backyard farms with low biosecurity levels are considered major risk factors. Meanwhile, wild boars account for most notified cases in some countries and regions, which makes the epidemiological scenario different from that in other Asian countries. As such, the current epidemic situation and higher risk factors differ widely between these countries. A variety of studies on ASF control have been conducted and many valuable insights have been obtained in Asia; nevertheless, the overall picture of the epidemic is still unclear. The purpose of this review is to provide an accurate picture of the epidemic situation across Asia, focusing on each subregion to comprehensively explain the disease outbreak. The knowledge gained from the ASF epidemics experienced in Asia over the past 5 years would be useful for disease control in areas that are already infected, such as Europe, as well as for non-affected areas to address preventive measures. To this end, the review includes two aspects: a descriptive analytical review based on publicly available databases showing overall epidemic trends, and an individualized review at the subregional level based on the available literature.

Development of a new effective African swine fever virus vaccine candidate by deletion of the H240R and MGF505-7R genes results in protective immunity against the Eurasia strain.

African swine fever (ASF) caused by ASF virus (ASFV) is a highly contagious and acute hemorrhagic viral disease in domestic pigs. Until now, no effective commercial vaccine and antiviral drugs are available for ASF control. Here, we generated a new live-attenuated vaccine candidate (ASFV-ΔH240R-Δ7R) by deleting H240R and MGF505-7R genes from the highly pathogenic ASFV HLJ/18 genome. Piglets immunized with ASFV-ΔH240R-Δ7R were safe without any ASF-related signs and produced specific antibodies against p30. Challenged with a virulent ASFV HLJ/18, the piglets immunized with high-dose group (105 HAD50) exhibited 100% protection without clinical symptoms, showing that low levels of virus replication with no observed pathogenicity by postmortem and histological analysis. Overall, our results provided a new strategy by designing live-attenuated vaccine candidate, resulting in protection against ASFV infection.

The diffusion metrics of African swine fever in wild boar

To control African swine fever (ASF) efficiently, easily interpretable metrics of the outbreak dynamics are needed to plan and adapt the required measures. We found that the spread pattern of African Swine Fever cases in wild boar follows the mechanics of a diffusion process, at least in the early phase, for the cases that occurred in Germany. Following incursion into a previously unaffected area, infection disseminates locally within a naive and abundant wild boar population. Using real case data for Germany, we derive statistics about the time differences and distances between consecutive case reports. With the use of these statistics, we generate an ensemble of random walkers (continuous time random walks, CTRW) that resemble the properties of the observed outbreak pattern as one possible realization of all possible disease dissemination patterns. The trained random walker ensemble yields the diffusion constant, the affected area, and the outbreak velocity of early ASF spread in wild boar. These methods are easy to interpret, robust, and may be adapted for different regions. Therefore, diffusion metrics can be useful descriptors of early disease dynamics and help facilitate efficient control of African Swine Fever.

African Swine Fever in Wild Boar: German Hunters' Perception of Surveillance and Control-A Questionnaire Study.

Since the first occurrence of African swine fever (ASF) in wild boar in Germany in 2020, the disease has primarily affected the wild boar population in the eastern part of the country close to the border with Poland. Local hunters play a crucial role in implementing surveillance and control. To evaluate their perceptions of existing control measures and analyze regional differences between hunters from ASF-affected and non-affected regions, a questionnaire study was conducted among the German hunting community. Hunters from non-affected areas held a more optimistic view regarding the effectiveness of control measures compared to hunters from affected areas. However, control measures that hinder hunting were generally perceived as ineffective. Measures that collided with hunters' understanding of fair hunting practices were regarded as controversial. Financial incentives and reducing bureaucracy were the most favored approaches to increase hunters' participation. Moreover, the possibility of eating or selling the meat of hunted wild boar and the provision of infrastructure for implementing ASF control were considered motivating. Thus, this study highlights the importance of compensating hunters and addressing their concerns to maintain their engagement in ASF control. To enhance compliance with controversial measures, thoughtful communication and raising awareness are essential.

Knowledge, Attitude, and Practices of Swine Farmers related to Livestock Biosecurity: A Case Study of African Swine Fever in Vietnam

Purpose: African Swine Fever (ASF), one of the world’s most contagious and deadly livestock infectious diseases, was introduced to Vietnam in 2019 and has since become one of the biggest threats to the country’s porcine industry. To this day, very little is known about the major drivers of ASF outbreaks – in particular, the generally low levels of biosecurity found on smallholder farms in Vietnam. The main objective of this paper is to assess the knowledge disparities, diverging attitudes, and different practices among smallholder pig farming households that influence the adoption, implementation, and continuation of ASF biosecurity measures in Vietnam. Research Method: Structured questionnaire surveys were conducted to 183 smallholder pig farming households in December 2020 in four districts of Dong Nai, the largest pig production province in Vietnam. The determinants of farmers’ knowledge and attitude toward ASF were estimated using an ordinal probit regression; a panel-data probit regression approximated the probability of farmers adopting biosecurity measures. Findings: It was found that, prior to the first outbreaks of ASF in Vietnam in 2019, surveyed pig farmers generally had an inadequate level of knowledge about ASF to be able to successfully prevent and control the disease’s spread. Rather, farmers only managed to significantly improve their understanding of ASF after outbreaks had begun. Model estimates indicate that a better understanding of ASF ultimately helped improve farmers’ attitudes toward ASF biosecurity measures. Additionally, farmers with previous experience in dealing with other livestock infectious disease outbreaks were, in most cases, found to be more willing to implement ASF biosecurity measures. Together, deeper understanding of ASF and more positive attitudes towards ASF prevention and control measures contributed to an overall increase in the likelihood of farmers adopting and implementing appropriate ASF biosecurity practices. Originality/ Value: This paper highlights the need for improved community education campaigns in efforts to prevent and contain ASF outbreaks, as well as the need for increased financial assistance and support for swine farmers to enable them to adopt effective biosecurity practices.

Factors Affecting the Spread, Diagnosis, and Control of African Swine Fever in the Philippines.

African Swine Fever (ASF) is a highly contagious disease that threatens the swine industry globally. Since its introduction into the Philippines in 2019, ASF has spread extensively in both commercial and backyard farms. Here, using a mix of qualitative and quantitative methods, including conjoint and SWOT analyses, world café discussions, and multivariable regression models, the most important factors that influence the spread, diagnosis, and control of ASF in the Philippines were identified. Research findings suggest that swill or contaminated feed, inadequate biosecurity protocols, and movement of personnel were the top risk factors favoring ASF spread among farms in general. For commercial farms, contaminated vehicles and personnel were also important, whereas for backyard farms, the introduction of new pigs, environmental contamination, and poor feeding quality were relevant risk factors. Notable clinical signs of ASF in pigs include reduced feed intake, huddled behavior, and reluctance to stand. This study highlights the need for timely reporting, trust-building initiatives, and enhanced biosecurity measures to effectively manage ASF outbreaks in the country. Results here contribute to the knowledge of factors affecting ASF spread in the Philippines and can help design prevention and control measures in ASF-infected countries while enhancing preparedness in countries free from the disease.

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.

B602L-Fc fusion protein enhances the immunogenicity of the B602L protein of the African swine fever virus.

African swine fever (ASF) is an acute, highly contagious, and deadly infectious disease caused by the African swine fever virus (ASFV) and has a huge impact on the pig industry. A lack of vaccines and effective therapeutic drugs has brought great challenges to the prevention and control of ASF. In this study, insect baculovirus expression system was used to express ASFV B602L protein (B602L) alone and the IgG FC-fused B602L protein (B602L-Fc), and evaluate the immune effect of B602L-Fc in mice model. To be specific, the ASFV B602L protein and B602L-Fc fusion protein were successfully expressed by the insect baculovirus expression system. Then, Functional analysis in vitro revealed that the B602L-Fc fusion protein bound and interacted with the FcRI receptor of antigen-presenting cells and significantly promoted the expression of proteins involved in antigen presentation and various cytokines at mRNA levels in porcine alveolar macrophages. Additionally, immunization using B602L-Fc fusion protein remarkably promoted the Th1-biased cellular immune response and humoral immune response in mice. In conclusion, The B602L-Fc fusion protein could up-regulate the expression of molecules involved in antigen presentation in APCs and enhance the humoral and cellular immune responses in mice. These results suggest that ASFV B602L-Fc recombinant fusion protein may be a promising candidate for subunit vaccine. This study provided useful data for the development of subunit vaccines for ASF.

Immunogenic response of recombinant pseudorabies virus carrying B646L and B602L genes of African swine fever virus in mice

African swine fever (ASF) is an acute infectious disease that poses a high lethality risk to domestic pigs and wild boars, causing substantial economic losses to the global pig industry. The prevention and control of ASF remain challenging, necessitating the urgent development of a safe and effective vaccine. This study focused on the essential structural protein p72 of ASFV (encoded by the B646L gene) and its chaperone protein pB602L (encoded by the B602L gene) as the target antigenic proteins. Based on CRISPR/Cas9 gene-editing technology, we constructed a live attenuated recombinant pseudorabies virus vector expressing the p72 and pB602L proteins (designated as rPRVXJ-EGFP/B602L/B646L), and assessed its immunization effect in mice. The recombinant virus rPRVXJ-EGFP/B602L/B646L successfully proliferated and demonstrated stable expression of the p72 and pB602L proteins in BHK-21 cells. Moreover, it exhibited excellent safety when used in mice and induced specific humoral and cellular immune responses targeting p72 and pB602L. In addition, it provided complete protection (100%) against the virulent PRV strain (PRV-XJ). These results indicate that the recombinant virus rPRVXJ-EGFP/B602L/B646L possesses robust immunogenicity and safety in mice. In conclusion, PRV represents a promising viral vector for expressing ASFV gene, and our study serves as an essential reference for the development of viral vector vaccines against ASFV.

Tools and opportunities for African swine fever control in wild boar and feral pigs: a review

Tools and opportunities for African swine fever control in wild boar and feral pigs: a review

Comparison of Attenuated and Virulent Strains of African Swine Fever Virus Genotype I and Serogroup 2.

African swine fever (ASF) is a contagious disease of pigs caused by the ASF virus (ASFV). The main problem in the field of ASF control is the lack of vaccines. Attempts to obtain vaccines by attenuating the ASFV on cultured cell lines led to the production of attenuated viruses, some of which provided protection against infection with a homologous virus. Here we report on the biological and genomic features of the attenuated Congo-a (KK262) virus compared to its virulent homologue Congo-v (K49). Our results showed differences in in vivo replication and virulence of Congo-a. However, the attenuation of the K49 virus did not affect its ability to replicate in vitro in the primary culture of pig macrophages. Complete genome sequencing of the attenuated KK262 strain revealed an 8,8 kb deletion in the left variable region of the genome compared to the virulent homologue K49. This deletion concerned five genes of MGF360 and three genes of MGF505. In addition, three inserts in the B602L gene, genetic changes in intergenic regions and missense mutations in eight genes were detected. The data obtained contribute to a better understanding of ASFV attenuation and identification of potential virulence genes for further development of effective vaccines.

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.

Recombinant porcine interferon cocktail delays the onset and lessens the severity of African swine fever

African swine fever (ASF) is a highly contagious and deadly disease that affects domestic and wild pigs. No commercial vaccine or antiviral is currently available against ASF. The control of ASF primarily relies on implementing effective biosecurity measures during the breeding process. Here, we evaluated the preventive and therapeutic potential of the interferon (IFN) cocktail (a mixture of recombinant porcine IFN α and γ) on ASF. The IFN cocktail treatment delayed the onset of ASF symptoms and ASF virus (ASFV) replication for approximately one week. However, IFN cocktail treatment could not prevent the death of the pigs. Further analysis showed that IFN cocktail treatment increased the expression of multiple IFN-stimulated genes (ISGs) in porcine peripheral blood mononuclear cells in vivo and in vitro. Additionally, IFN cocktail modulated the expression of pro- and anti-inflammatory cytokines and reduced tissue injury in the ASFV-infected pigs. Collectively, the results suggest that the IFN cocktail restricts the progression of acute ASF by inducing high levels of ISGs, contributing to the pre-establishment of antiviral status, and modulating the balance of pro- and anti-inflammatory mediators to lessen cytokine storm-mediated tissue damage.