A proteomics informed by transcriptomics insight into the proteome of Ornithodoros erraticus adult tick saliva.

BACKGROUND: The argasid tick Ornithodoros erraticus is the main vector of tick-borne human relapsing fever (TBRF) and African swine fever (ASF) in the Mediterranean Basin. The prevention and control of these diseases would greatly benefit from the elimination of O. erraticus populations, and anti-tick vaccines are envisaged as an effective and sustainable alternative to chemical acaricide usage for tick control. Ornithodoros erraticus saliva contains bioactive proteins that play essential functions in tick feeding and host defence modulation, which may contribute to host infection by tick-borne pathogens. Hence, these proteins could be candidate antigen targets for the development of vaccines aimed at the control and prevention of O. erraticus infestations and the diseases this tick transmits. The objective of the present work was to obtain and characterise the proteome of the saliva of O. erraticus adult ticks as a means to identify and select novel salivary antigen targets. METHODS: A proteomics informed by transcriptomics (PIT) approach was applied to analyse samples of female and male saliva separately using the previously obtained O. erraticus sialotranscriptome as a reference database and two different mass spectrometry techniques, namely liquid chromatography-tandem mass spectrometry (LC-MS/MS) in data-dependent acquisition mode and sequential window acquisition of all theoretical fragment ion spectra MS (SWATH-MS). RESULTS: Up to 264 and 263 proteins were identified by LC-MS/MS in the saliva of O. erraticus female and male ticks, respectively, totalling 387 non-redundant proteins. Of these, 224 were further quantified by SWATH-MS in the saliva of both male and female ticks. Quantified proteins were classified into 23 functional categories and their abundance compared between sexes. Heme/iron-binding proteins, protease inhibitors, proteases, lipocalins and immune-related proteins were the categories most abundantly expressed in females, while glycolytic enzymes, protease inhibitors and lipocalins were the most abundantly expressed in males. Ninety-seven proteins were differentially expressed between the sexes, of which 37 and 60 were overexpressed in females and males, respectively. CONCLUSIONS: The PIT approach demonstrated its usefulness for proteomics studies of O. erraticus, a non-model organism without genomic sequences available, allowing the publication of the first comprehensive proteome of the saliva of O. erraticus reported to date. These findings confirm important quantitative differences between sexes in the O. erraticus saliva proteome, unveil novel salivary proteins and functions at the tick-host feeding interface and improve our understanding of the physiology of feeding in O. erraticus ticks. The integration of O. erraticus sialoproteomic and sialotranscriptomic data will drive a more rational selection of salivary candidates as antigen targets for the development of vaccines aimed at the control of O. erraticus infestations and the diseases it transmits.

Prevention and Control Strategies of African Swine Fever and Progress on Pig Farm Repopulation in China.

African swine fever (ASF) is a devastating disease in domestic and wild pigs. Since the first outbreak of ASF in August 2018 in China, the disease has spread throughout the country with an unprecedented speed, causing heavy losses to the pig and related industries. As a result, strategies for managing the disease are urgently needed. This paper summarizes the important aspects of three key elements about African swine fever virus (ASFV) transmission, including the sources of infection, transmission routes, and susceptible animals. It overviews the relevant prevention and control strategies, focusing on the research progress of ASFV vaccines, anti-ASFV drugs, ASFV-resistant pigs, efficient disinfection, and pig farm biosecurity. We then reviewed the key technical points concerning pig farm repopulation, which is critical to the pork industry. We hope to not only provide a theoretical basis but also practical strategies for effective dealing with the ASF epidemic and restoration of pig production.

Genotype I African swine fever viruses emerged in domestic pigs in China and caused chronic infection.

The Georgia-07-like genotype II African swine fever virus (ASFV) with high virulence has been prevalent in China since 2018. Here, we report that genotype I ASFVs have now also emerged in China. Two non-haemadsorbing genotype I ASFVs, HeN/ZZ-P1/21 and SD/DY-I/21, were isolated from pig farms in Henan and Shandong province, respectively. Phylogenetic analysis of the whole genome sequences suggested that both isolates share high similarity with NH/P68 and OURT88/3, two genotype I ASFVs isolated in Portugal in the last century. Animal challenge testing revealed that SD/DY-I/21 shows low virulence and efficient transmissibility in pigs, and causes mild onset of infection and chronic disease. SD/DY-I/21 was found to cause necrotic skin lesions and joint swelling. The emergence of genotype I ASFVs will present more problems and challenges for the control and prevention of African swine fever in China.

Evaluation of the Presence of ASFV in Wolf Feces Collected from Areas in Poland with ASFV Persistence.

African swine fever (ASF), caused by a DNA virus (ASFV) belonging to genus Asfivirus of the Asfarviridae family, is one of the most threatening diseases of suids. During last few years, it has spread among populations of wild boars and pigs in countries of Eastern and Central Europe, causing huge economical losses. While local ASF occurrence is positively correlated with wild boar density, ecology of this species (social structure, movement behavior) constrains long-range disease transmission. Thus, it has been speculated that carnivores known for high daily movement and long-range dispersal ability, such as the wolf (Canis lupus), may be indirect ASFV vectors. To test this, we analyzed 62 wolf fecal samples for the presence of ASFV DNA, collected mostly in parts of Poland declared as ASF zones. This dataset included 20 samples confirmed to contain wild boar remains, 13 of which were collected near places where GPS-collared wolves fed on dead wild boars. All analyzed fecal samples were ASFV-negative. On the other hand, eight out of nine wild boar carcasses that were fed on by telemetrically studied wolves were positive. Thus, our results suggest that when wolves consume meat of ASFV-positive wild boars, the virus does not survive the passage through intestinal tract. Additionally, wolves may limit ASFV transmission by removing infectious carrion. We speculate that in areas where telemetric studies on large carnivores are performed, data from GPS collars could be used to enhance efficiency of carcass search, which is one of the main preventive measures to constrain ASF spread.

Disinfection to control African swine fever virus: a UK perspective.

A review of African swine fever (ASF) was conducted, including manifestations of disease, its transmission and environmental persistence of ASF virus. Findings on infectious doses of contemporary highly-pathogenic strains isolated from outbreaks in Eastern Europe were included. Published data on disinfectant susceptibility of ASF virus were then compared with similar findings for selected other infectious agents, principally those used in the UK disinfectant approvals tests relating to relevant Disease Orders for the control of notifiable and zoonotic diseases of livestock. These are: swine vesicular disease virus, foot and mouth disease virus, Newcastle disease virus and Salmonella enterica serovar Enteritidis. The comparative data thus obtained, presented in a series of charts, facilitated estimates of efficacy against ASF virus for some UK approved disinfectants when applied at their respective General Orders concentrations. Substantial data gaps were encountered for several disinfectant agents or classes, including peracetic acid, quaternary ammonium compounds and products based on phenols and cresols.

Evaluating the distribution of African swine fever virus within a feed mill environment following manufacture of inoculated feed.

It is critical to understand the role feed manufacturing may have regarding potential African swine fever virus (ASFV) transmission, especially given the evidence that feed and/or ingredients may be potential vectors. The objective of the study was to evaluate the distribution of ASFV in a feed mill following manufacture of contaminated feed. To accomplish this, a pilot-scale feed mill consisting of a mixer, bucket elevator, and spouting was constructed in a BSL-3Ag facility. First, a batch of ASFV-free feed was manufactured, followed by a batch of feed that had an ASFV-contaminated ingredient added to feed, which was then mixed and discharged from the equipment. Subsequently, four additional ASFV-free batches of feed were manufactured using the same equipment. Environmental swabs from 18 locations within the BSL-3Ag room were collected after each batch of feed was discharged. The locations of the swabs were categorized into four zones: 1) feed contact surface, 2) non-feed contact surface < 1 meter away from feed, 3) non-feed contact surface > 1 meter from feed, and 4) transient surfaces. Environmental swabs were analyzed using a qPCR specific for the ASFV p72 gene and reported as genomic copy number (CN)/mL of environmental swab processing buffer. Genomic copies were transformed with a log10 function for statistical analysis. There was no evidence of a zone × batch interaction for log10 genomic CN/mL (P = 0.625) or cycle threshold (Ct) value (P = 0.608). Sampling zone impacted the log10 p72 genomic CN/mL (P < 0.0001) and Ct values (P < 0.0001), with a greater amount of viral genome detected on transient surfaces compared to other surfaces (P < 0.05). This study illustrates that once ASFV enters the feed mill environment it becomes widespread and movement of people can significantly contribute to the spread of ASFV in a feed mill environment.

African Swine Fever Virus as a Difficult Opponent in the Fight for a Vaccine-Current Data.

Prevention and control of African swine fever virus (ASFV) in Europe, Asia, and Africa seem to be extremely difficult in view of the ease with which it spreads, its high resistance to environmental conditions, and the many obstacles related to the introduction of effective specific immunoprophylaxis. Biological properties of ASFV indicate that the African swine fever (ASF) pandemic will continue to develop and that only the implementation of an effective and safe vaccine will ensure a reduction in the spread of ASFV. At present, vaccines against ASF are not available. The latest approaches to the ASFV vaccine's design concentrate on the development of either modified live vaccines by targeted gene deletion from different isolates or subunit vaccines. The construction of an effective vaccine is hindered by the complex structure of the virus, the lack of an effective continuous cell line for the isolation and propagation of ASFV, unpredictable and stain-specific phenotypes after the genetic modification of ASFV, a risk of reversion to virulence, and our current inability to differentiate infected animals from vaccinated ones. Moreover, the design of vaccines intended for wild boars and oral administration is desirable. Despite several obstacles, the design of a safe and effective vaccine against ASFV seems to be achievable.

Thoughts on African Swine Fever Vaccines.

African swine fever (ASF) is an acute viral hemorrhagic disease of domestic swine with mortality rates approaching 100%. Devastating ASF outbreaks and continuing epidemics starting in the Caucasus region and now in the Russian Federation, Europe, China, and other parts of Southeast Asia (2007 to date) highlight its significance. ASF strain Georgia-07 and its derivatives are now endemic in extensive regions of Europe and Asia and are "out of Africa" forever, a situation that poses a grave if not an existential threat to the swine industry worldwide. While our current concern is Georgia-07, other emerging ASFV strains will threaten for the indefinite future. Economic analysis indicates that an ASF outbreak in the U.S. would result in approximately $15 billion USD in losses, assuming the disease is rapidly controlled and the U.S. is able to reenter export markets within two years. ASF's potential to spread and become endemic in new regions, its rapid and efficient transmission among pigs, and the relative stability of the causative agent ASF virus (ASFV) in the environment all provide significant challenges for disease control. Effective and robust methods, including vaccines for ASF response and recovery, are needed immediately.

Preliminary analysis of whether mosquitoes can carry and transmit African swine fever.

BACKGROUND: Mosquitoes are important insect vectors, but whether they can carry and transmit African swine fever virus (ASFV) in large-scale pig farms in China is unknown. RESULTS: In this study, probe-based qPCR analysis was performed on mosquitoes from five pig farms with ASF virus (ASFV). Analysis of ASFV in 463 mosquitoes yielded negative cycle threshold (CT) value), and detection remained negative after mixing samples from all five pig farms. CONCLUSIONS: Therefore, mosquitoes appear unlikely to transmit ASFV, and pose little threat to large-scale pig farms. Thus, farms should continue to follow normal mosquito control procedures when formulating strategies for the prevention and control of ASF.

A stochastic network-based model to simulate farm-level transmission of African swine fever virus in Vietnam.

African swine fever virus is highly contagious, and mortality rates reach up to 100% depending on the host, virus dose, and the transmission routes. The main objective of this study was to develop a network-based simulation model for the farm-level transmission of ASF virus to evaluate the impact of changes in farm connectivity on ASF spread in Vietnam. A hypothetical population of 1,000 pig farms was created and used for the network-based simulation, where each farm represented a node, and the connection between farms represented an edge. The three scenarios modelled in this way (baseline, low, and high) evaluated the impact of connectivity on disease transmission. The median number of infected farms was higher as the connectivity increased (low: 659, baseline: 968 and high: 993). In addition, we evaluated the impact of the culling strategy on the number of infected farms. A total of four scenarios were simulated depending on the timing of culling after a farm was infected. We found that the timing of culling at 16, 12, 8, and 6 weeks had resulted in a reduction of the number of median infected farms by 81.92%, 91.63%, 100%, and 100%, respectively. Finally, our evaluation of the implication of stability of ties between farms indicated that if the farms were to have the same trading partners for at least six months could significantly reduce the median number of infected farms to two (95th percentile: 413) than in the basic model. Our study showed that pig movements among farms had a significant influence on the transmission dynamics of ASF virus. In addition, we found that the either timing of culling, reduction in the number of trading partners each farm had, or decreased mean contact rate during the outbreaks were essential to prevent or stop further outbreaks.

Spatiotemporal clustering and Random Forest models to identify risk factors of African swine fever outbreak in Romania in 2018-2019.

African swine fever (ASF) has affected Romania since July 2017, with considerable economic and social consequences, despite the implementation of control measures mainly based on stamping out of infected pig populations. On the basis of the 2973 cumulative recorded cases up to September 2019 among wild boars and domestic pigs, analysis of the epidemiological characteristics could help to identify the factors favoring the persistence and spread of ASF. A statistical framework, based on a random forest methodology, was therefore developed to assess the spatiotemporal features of the epidemics and their relationships with environmental, human, and agricultural factors. The landscape of Romania was associated with the infection dynamics, particularly concerning forested and wetland areas. Waterways were also identified as a pivotal factor, raising questions about possible waterborne transmission since these waterways are often used as a water supply for backyard holdings. However, human activity was clearly identified as the main risk factor for the spread of ASF. Although the situation in Romania cannot be directly transposed to intensive pig farming countries, the findings of this study highlight the need for strict biosecurity measures on farms, and during transportation, to avoid ASF transmission at large geographic and temporal scales.

The Role of Male Reproductive Organs in the Transmission of African Swine Fever-Implications for Transmission.

African swine fever (ASF) has evolved from an exotic animal disease to a threat to global pig production. An important avenue for the wide-spread transmission of animal diseases is their dissemination through boar semen used for artificial insemination. In this context, we investigated the role of male reproductive organs in the transmission of ASF. Mature domestic boars and adolescent wild boars, inoculated with different ASF virus strains, were investigated by means of virological and pathological methods. Additionally, electron microscopy was employed to investigate in vitro inoculated sperm. The viral genome, antigens and the infectious virus could be found in all gonadal tissues and accessory sex glands. The viral antigen and viral mRNAs were mainly found in mononuclear cells of the respective tissues. However, some other cell types, including Leydig, endothelial and stromal cells, were also found positive. Using RNAScope, p72 mRNA could be found in scattered halo cells of the epididymal duct epithelium, which could point to the disruption of the barrier. No direct infection of spermatozoa was observed by immunohistochemistry, or electron microscopy. Taken together, our results strengthen the assumption that ASFV can be transmitted via boar semen. Future studies are needed to explore the excretion dynamics and transmission efficiency.

Change in the ASF entry risk into Japan as a result of the COVID-19 pandemic.

Using a model developed previously by the authors, a risk assessment was conducted to predict the change in the risk of ASF entering Japan as a result of the coronavirus pandemic in humans. The monthly probability of ASF entering Japan through illegal importation of pig products from China was calculated to be 4.2% (90% prediction interval: 0.0%-24.9%) in January, 0.45% (0%-2.5%) in February, 0.03% (0%-0.2%) in March and 0.0002% (0%-0.001%) in April, 0.00005% (0%-0.0003%) in May and 0.0009% (0%-0.005%) in June 2020 indicating a significant decline in the risk of ASF entry into Japan from China. The decline was attributed to a decrease in the number of air travellers from China and amount of restaurant food waste.

Quantitative risk assessment of the African swine fever introduction into the Republic of Korea via legal import of live pigs and pig products.

There are several routes of African swine fever (ASF) introduction into a country. Among the possible routes of entry, quarantine policies determine the possibility of introduction by legal import of live pigs and pig products. This study aimed at assessing the probability of ASF introduction through legal import of live pigs and pig products during the high-risk period (HRP) using a quantitative stochastic approach during 2009-2018. The result indicates that the mean annual probability of ASF introduction by legal import of live pig was 1.58 × 10-7 (1.52~1.67 × 10-7 95% CI). The mean annual probability by legal import of pig products was 1.59 × 10-10 (1.55~1.64 × 10-10 95% CI), of which Poland assumed 87.9% of the mean annual risk. The current import quarantine policy of Korean government may be enough to block the release of the virus via legal import of live pigs and pig products, and it should be continually enforced. This result can help to elucidate source of infection and to minimize the catastrophic consequences of the potential ASF reintroduction into South Korea by designing risk mitigation strategies such as risk-based selection of routes to be assessed and prevented and decreased exposure possibility by increased control of food waste and swill feeding practices.

Predicting spread and effective control measures for African swine fever-Should we blame the boars?

An ongoing, continually spreading, outbreak of African swine fever (ASF), following its identification in Georgia in 2007, has resulted in 17 European and 12 Asian countries reporting cases by April 2020, with cases occurring in both wild boar and domestic pigs. Curtailing further spread of ASF requires understanding of the transmission pathways of the disease. ASF is self-sustaining in the wild boar population, and they have been implicated as one of the main drivers of transmission within Europe. We developed a spatially explicit model to estimate the risk of infection with ASF in wild boar and pigs due to natural movement of wild boar that is applicable across the whole of Europe. We demonstrate the model by using it to predict the probability that early cases of ASF in Poland were caused by wild boar dispersion. The risk of infection in 2015 is computed due to wild boar cases in Poland in 2014, compared against reported cases in 2015, and then the procedure is repeated for 2015-2016. We find that long- and medium-distance spread of ASF (i.e. >30 km) is unlikely to have occurred due to wild boar dispersal, due in part to the generally short distances wild boar will travel (<20 km on average). We also predict the relative success of different control strategies in 2015, if they were implemented in 2014. Results suggest that hunting of wild boar reduces the number of new cases, but a larger region is at risk of ASF compared with no control measure. Alternatively, introducing wild boar-proof fencing reduces the size of the region at risk in 2015, but not the total number of cases. Overall, our model suggests wild boar movement is only responsible for local transmission of disease; thus, other pathways are more dominant in medium- and long-distance spread of the disease.

An expert opinion assessment of blood-feeding arthropods based on their capacity to transmit African swine fever virus in Metropolitan France.

To deal with the limited literature data on the vectorial capacity of blood-feeding arthropods (BFAs) and their role in the transmission of African swine fever virus (ASFV) in Metropolitan France, a dedicated working group of the French Agency for Food, Environmental and Occupational Health & Safety performed an expert knowledge elicitation. In total, 15 different BFAs were selected as potential vectors by the ad hoc working group involved. Ten criteria were considered to define the vectorial capacity: vectorial competence, current abundance, expected temporal abundance, spatial distribution, longevity, biting rate, active dispersal capacity, trophic preferences for Suidae, probability of contact with domestic pigs and probability of contact with wild boar. Fourteen experts participated to the elicitation. For each BFA, experts proposed a score (between 0 and 3) for each of the above criteria with an index of uncertainty (between 1 and 4). Overall, all experts gave a weight for all criteria (by distributing 100 marbles). A global weighted sum of score per BFA was calculated permitting to rank the different BFAs in decreasing order. Finally, a regression tree analysis was used to group those BFAs with comparable likelihood to play a role in ASF transmission. Out of the ten considered criteria, the experts indicated vectorial competence, abundance and biting rate as the most important criteria. In the context of Metropolitan France, the stable fly (Stomoxys calcitrans) was ranked as the most probable BFA to be a vector of ASFV, followed by lice (Haematopinus suis), mosquitoes (Aedes, Culex and Anopheles), Culicoides and Tabanidea. Since scientific knowledge on their vectorial competence for ASF is scarce and associated uncertainty on expert elicitation moderate to high, more studies are however requested to investigate the potential vector role of these BFAs could have in ASFV spread, starting with Stomoxys calcitrans.

Mechanical transmission of African swine fever virus by Stomoxys calcitrans: Insights from a mechanistic model.

African swine fever (ASF) represents a global threat with huge economic consequences for the swine industry. Even though direct contact is likely to be the main transmission route from infected to susceptible hosts, recent epidemiological investigations have raised questions regarding the role of haematophagous arthropods, in particular the stable fly (Stomoxys calcitrans). In this study, we developed a mechanistic vector-borne transmission model for ASF virus (ASFV) within an outdoor domestic pig farm in order to assess the relative contribution of stable flies to the spread of the virus. The model was fitted to the ecology of the vector, its blood-feeding behaviour and pig-to-pig transmission dynamic. Model outputs suggested that in a context of low abundance (<5 flies per pig), stable flies would play a minor role in the spread of ASFV, as they are expected to be responsible for around 10% of transmission events. However, with abundances of 20 and 50 stable flies per pig, the vector-borne transmission would likely be responsible for almost 30% and 50% of transmission events, respectively. In these situations, time to reach a pig mortality of 10% would be reduced by around 26% and 40%, respectively. The sensitivity analysis emphasized that the expected relative contribution of stable flies was strongly dependent on the volume of blood they regurgitated and the infectious dose for pigs. This study identified crucial knowledge gaps that need to be filled in order to assess more precisely the potential contribution of stable flies to the spread of ASFV, including a quantitative description of the populations of haematophagous arthropods that could be found in pig farms, a better understanding of blood-feeding behaviours of stable flies and the quantification of the probability that stable flies partially fed with infectious blood transmit the virus to a susceptible pig during a subsequent blood-feeding attempt.

Could wild boar be the Trans-Siberian transmitter of African swine fever?

China has experienced a sudden multi-focal and multi-round of African swine fever (ASF) outbreaks during 2018. The subsequent epidemiological survey resulted in a debate including the possibility of a transboundary spread from European Russia to China through wild boar. We contribute to the debate by assessing a hypothetical overland Euro-Siberian transmission path and its associated ASF arrival dates. We selected the maximum entropy algorithm for spatial modelling of ASF-infected wild boar and the Spatial Distribution Modeller in ArcGIS to plot Least Cost Paths (LCPs) between Eastern Europe and NE China. The arrival dates of ASF-infected wild boar have been predicted by cumulative maximum transmission distances per season and cover with their associated minimum time intervals along the LCPs. Our results show high costs for wild boar to cross Kazakhstan, Xinjiang (NW China) and/or Mongolia to reach NE China. Instead, the Paths lead almost straight eastward along the 59.5° northern latitude through Siberia and would have taken a minimum of 219 or 260 days. Therefore, infected wild boar moving all the way along the LCP could not have been the source of the ASF infection in NE China on 2 August 2018.

Transmission of African swine fever in China Through Legal Trade of Live Pigs.

The African swine fever virus (ASFV) was first reported in China on 3 August 2018, which subsequently triggered a severe epidemic that spreads across the country. While the ASFV has numerous underlying transmission paths in China, this study primarily assessed the possibility of ASFV transmission through the legal animal husbandry trade. The reason for this is that, historically, this transmission path is one of the critical contacts for exotic diseases to access disease-free areas. This study employed a stochastic model to assess the monthly possibility for ASFV entering respective Chinese provinces. The results of this model suggest that the risk of ASFV transmission though the legal live-pig trade is highest in the southeastern regions of China. Vulnerable regions centred around Zhejiang, Jiangsu and Anhui provinces, especially throughout the months of January and December. Liaoning province contributes most to transmission risk with 46.7% of the overall annual risk. This study quantified the risk of ASFV spread in China related to the legal trade of pigs and provides detailed and new information for the development of ASFV monitoring and control plans in China and other countries who also face the challenge of ASFV.

A stochastic simulation model of African swine fever transmission in domestic pig farms in the Red River Delta region in Vietnam.

The main objectives of this study were to model various scenarios of African swine fever (ASF) virus transmission among farms in Vietnam and to evaluate the impact of control strategies using North American Animal Disease Spread Model (NAADSM). A total of 7,882 pig farms in the Red River Delta (RRD) region were obtained from the General Statistics Office, and then, random points corresponding to the number of farms in each province were generated as exact farm locations were not available. A total of 10 models were developed, including movement control scenarios. In addition, we conducted sensitivity analysis to assess the impact of indirect contact transmission probability (TP). Overall, the indirect contact exhibited an important role in transmitting the ASF virus. In order to minimize ASF transmission between farms, we found that movement restriction needed to reach a certain level (approximately between 50% and 75%) and that the restriction had to be applied in a timely manner. This study offers valuable insight into how ASF virus can be transmitted via direct and indirect contact and controlled among farms under the various simulation scenarios. Our results suggest that the enforcement of movement restriction was an effective control measure as soon as the outbreaks were reported. In addition, this study provided evidence that high standards of biosecurity can contribute to the reduction of disease spread.