Control Of African Swine Fever Research Articles

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.

Enhanced detection of African swine fever virus in samples with low viral load using digital PCR technology

Detection of low viral load samples has long been a challenge for African swine fever (ASF) prevention and control. This study aimed to compare the detection efficacy of droplet digital PCR(ddPCR) and quantitative PCR(qPCR) for African swine fever virus (ASFV) at different viral loads, with a focus on assessing the accuracy of ddPCR in detecting low viral load samples. The results revealed that ddPCR had a detection limit of 1.97 (95% CI 1.48 – 4.12) copies/reaction and was 18.99 times more sensitive than qPCR (detection limit: 37.42, 95% CI 29.56 – 69.87 copies/reaction). In the quantification of high, medium, and low viral load samples, ddPCR showed superior stability with lower intra- (2.06% – 7.58%) and inter-assay (3.83% – 7.50%) coefficients of variation than those of qPCR (intra-assay: 8.08%–29.86%; inter-assay: 9.27%–34.58%). Bland-Altman analysis indicated acceptable consistency between ddPCR and qPCR for high and medium viral load samples; however, discrepancies were observed for low viral load samples, where two samples (2/24, 8.33%) exhibited deviations beyond the acceptable range (−46.18 copies/reaction). Moreover, ddPCR demonstrated better performance in detecting ASFV in clinical samples from asymptomatic pigs and environmental samples, with qPCR showing false negative rates of 7.69% (2/26) and 27.27% (12/44), respectively. McNemar analysis revealed significant differences between the two methods (P = 0.000) for samples with a viral load <100 copies/reaction. The results of this study demonstrate that ddPCR has better detection limits and adaptability than qPCR, allowing for a more accurate detection of ASFV in early-stage infections and low-concentration environmental samples. These findings highlight the potential of ddPCR in the prevention and control of ASF.

Strategies for Transboundary Swine Disease Management in Asian Islands: Foot and Mouth Disease, Classical Swine Fever, and African Swine Fever in Taiwan, Japan, and the Philippines.

Swine transboundary diseases pose significant challenges in East and Southeast Asia, affecting Taiwan, Japan, and the Philippines. This review delves into strategies employed by these islands over the past two decades to prevent or manage foot and mouth disease (FMD), classical swine fever (CSF), and African swine fever (ASF) in domestic pigs and wild boars. Despite socio-economic differences, these islands share geographical and climatic commonalities, influencing their thriving swine industries. Focusing on FMD eradication, this study unveils Taiwan's success through mass vaccination, Japan's post-eradication surveillance, and the Philippines' zoning strategy. Insights into CSF in Japan emphasize the importance of wild boar control, whereas the ASF section highlights the multifaceted approach implemented through the Philippine National ASF Prevention and Control Program. This review underscores lessons learned from gained experiences, contributing to a comprehensive understanding of swine disease management in the region.

Understanding Smallholder Pigkeepers' Awareness and Perceptions of African Swine Fever and Its Control Measures in Ukraine.

African swine fever (ASF) has posed a significant threat to Ukrainian pig farming since its identification in 2012. In this study, recognising the pivotal role of pigkeepers in disease control, we conducted ten focus groups involving 52 smallholders across eight regions in Ukraine. Using participatory methods, we revealed their awareness of ASF signs, transmission routes, preventive measures, and the perceptions of stakeholders involved in ASF control. Furthermore, we identified the smallholders' acceptance of eradication and restriction measures, the perceived impact of zoning consequences, and their main sources of ASF information. Smallholders identified fever and skin haemorrhage as the most indicative signs of ASF and highlighted rodents as a primary transmission concern. Disinfection was seen as the most effective measure for preventing the introduction of ASF. Pigkeepers who perceived their stakeholder role in ASF control showed more trust in themselves and veterinarians than in central veterinary authorities. Farm-level ASF eradication measures were generally accepted; however, culling within the protection zone was least accepted, with economic losses listed as the most impactful consequence for pigkeepers. For ASF information, pigkeepers favour web searches and veterinarians, as well as traditional media and word-of-mouth news. This study provides valuable insights into refining the ASF communication strategies in Ukraine.

A vesicular stomatitis virus-based African swine fever vaccine prototype effectively induced robust immune responses in mice following a single-dose immunization.

African swine fever (ASF) is a highly contagious hemorrhagic fever disease in pigs caused by African swine fever virus (ASFV). It is very difficult to control and prevent ASF outbreaks due to the absence of safe and effective vaccines. In order to develop a safe and effective ASF vaccine for the control and prevention of ASF, two ASFV recombinant vesicular stomatitis virus (VSV) live vector vaccine prototypes, containing the gene of p72, and a chimera of p30 and p54, were developed based on the replication-competent VSV, and named VSV-p72 and VSV-p35. The immune potency of VSV-p72 or VSV-p35 alone and in combination was evaluated in BALB/c mice via intramuscular and intranasal vaccination. The results indicated that whether administered alone or in combination, the two vaccine prototypes showed acceptable safety in mice and, more importantly, induced high-level specific antibodies against p72, p30, and p54 of ASFV and a strong cellular immune response 28 days after vaccination. The sera from mice vaccinated with the vaccine prototypes significantly inhibited ASFV from infecting porcine alveolar macrophages (PAMs) in vitro. Most notably, the immunized sera from a mixture of VSV-p35 and VSV-p72 inhibited ASFV from infecting PAMs, with an inhibition rate of up to 78.58%. Overall, our findings suggest that ASFV recombinant VSV live vector vaccine prototypes may become a promising candidate vaccine for the control and prevention of ASF.

Identification of predilection sites for wild boar carcass search based on spatial analysis of Latvian ASF surveillance data

Targeted search for wild boar carcasses is essential for successful control of African swine fever (ASF) in wild boar populations. To examine whether landscape conditions influence the probability of finding ASF-positive carcasses, this study analyzed Global Positioning System (GPS) coordinates of Latvian wild boar carcasses and hunted wild boar, extracted from the CSF/ASF wild boar surveillance database of the European Union, and random coordinates in Latvia. Geographic information system (GIS) software was used to determine the landscape type and landscape composition of carcass detection sites and to measure distances from the carcasses to nearest waterbodies, forest edges, roads and settlements. The results of the automated measurements were validated by manually analyzing a smaller sample. Wild boar carcasses were found predominantly in forested areas and closer to waterbodies and forest edges than random GPS coordinates in Latvia. Carcasses of ASF-infected wild boar were found more frequently in transitional zones between forest and woodland shrub, and at greater distances from roads and settlements compared to ASF-negative carcasses and random points. This leads to the hypothesis, that ASF-infected animals seek shelter in quiet areas further away from human disturbance. A detailed collection of information on the environment surrounding carcass detection sites is needed to characterize predilection sites more accurately.

Tackling African swine fever in India

Abstract Globally, African swine fever (ASF) is the most feared swine viral disease, associated with a mortality rate of up to 100%. More than half of the world’s swine population is located in Asia. ASF outbreaks in Asia have a significant impact on the global pig industry. Pig industry’s economic losses have been huge because of ASF outbreaks. Swine husbandry in India is in a flourishing phase to meet food security demands, but the losses associated with ASF greatly hamper its expansion. ASF in India can be controlled and eradicated through strict biosecurity measures and vaccination. Development of penside point-of-care (POC) rapid diagnostics and effective indigenous vaccines are prerequisites to tackle ASF in India. Sensitization of veterinary professionals, researchers, and pig farmers about disease pathogenesis, transmission, and impact are crucial part of ASF control. This review presents the status of ASF in India in comparison to other parts of the world, and current approaches in India to deal with ASF. Further roadmap to combat this calamitous swine infection in India.

African Swine Fever and Its Control Measures in Wild Boar: A "De Iure Condito" Analysis in the European Union.

Recently, the African swine fever (ASF) epizootic has been reported in domestic pigs and wild boars in several European Union Member States (EU MS) and epidemiological evidence has accumulated which indicates that wild boar play a key role in maintaining and spreading the disease. Thanks to the experience gained when managing ASF outbreaks in Sardinia (Italy) and Eastern Europe, Directive 2002/60 CE was issued. This directive represented an important step forward in controlling the disease, particularly the risk of spreading the virus to wild animals. Since 2021, according to Regulation (EU) 2016/429, which is also called "Animal Health Law-AHL", when the MS competent authority suspects or confirms ASF (a cat. A listed disease) in wild animals, it is mandatory to conduct surveillance in the wild boar population and implement the necessary control measures. Within AHL, Regulations (EU) 2020/687 and 2023/594 established special ASF control measures in kept and wild porcine animals, and their products and by-products, focusing on and underlying old and new responsibilities that vets (both public and private ones) have to accomplish under the new regulations. The new change in the legal framework deals with specific measures to be applied in the wild and represents a great challenge for MS veterinary services. Some of these measures have been well established in the last two decades, particularly those related to application in the farming system, while other measures are still new to veterinary health management and require a holistic approach in terms of intensity, considering all geographical, ecological, productive, cultural and social features of the involved EU MS. In this contribution, the authors intend to focus on specific measures which have been issued in order to limit or stop the spread of ASF in a wild, "boundless" ecosystem. These measures expand the field of competence of the official veterinary service to wild areas in addition to farm activity.

Epidemiological Assessment of African Swine Fever Spread in the Dominican Republic.

Since African Swine Fever (ASF) was detected in the Dominican Republic in July 2021, it has negatively impacted the country's swine industry. Assessing the epidemiological situation is crucial to helping local authorities and industry stakeholders control the disease. Here, data on 155 reported outbreaks in the Dominican Republic from November 2022 to June 2023 were evaluated. Descriptive spatiotemporal analysis was performed to characterize disease distribution and spread, and between-herd R0 was calculated for the study period. The Knox test and a space-time permutation model were used to evaluate clustering. Data on clinical presentation, biosecurity measures, and suspected reasons for introduction were categorized and summarized. The majority (78%) of outbreaks occurred on backyard farms which generally had low biosecurity. Across farm types, the majority of pigs were still alive at the time of depopulation. Spatiotemporal findings and R0 estimates suggest an endemic pattern of disease geographically located centrally within the country. Clustering was detected even at small temporal and spatial distances due to outbreaks amongst neighboring backyard farms. These results provide critical information on the current state of the ASF epidemic in the Dominican Republic and will aid government officials and swine industry leaders in developing effective ASF control strategies.

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.