Antiviral agents and disinfectants for foot­and­mouth disease (Review).

Fluorouracil, 5-azacytidine, 6-azauridine, ribavirin, favipiravir (T-705) and its derivative (T-1105) exhibit anti-foot-and-mouth disease virus (FMDV) effects. In particular, T-1105 exhibits promising results when administered to guinea pigs orally, and pigs in their feed. FMDV is excreted in the early stages of infection in aerosols and oral or nasal droplets from animals. T-1105 along with the FMDV vaccine can be used to combat foot-and-mouth disease (FMD) epidemics. Several studies have shown that sodium hypochlorous solutions are widely used to inactivate viruses, including FMDV. However, these solutions must be stored under cool and dark conditions to maintain their virucidal effects. Interestingly, a study indicated that the virucidal activity of a calcium bicarbonate solution with a mesoscopic structure (CAC-717) did not decrease after storage at room temperature for at least four years outside direct sunlight. Numerous lessons acquired from the 2010 FMD outbreak in Japan are relevant for the control of COVID-19. However, the widespread use of chlorite can cause environmental issues. Chlorite can be combined with nitrogen to produce chloramine or N-nitrosodimethylamine, which plays a role in carcinogenesis. Therefore, risk assessments should be conducted in aquatic environments. Moreover, there is a need to develop nonchlorine disinfectants that can be used during epidemics, including FMD. The approach of 'One Health' should be shared between the public health and veterinary fields to improve the management of viral outbreaks, including those due to FMD.

Ceramic absorbed with calcium bicarbonate mesoscopic crystals partially inactivate scrapie prions.

Prion diseases are fatal neurodegenerative disorders affecting both humans and animals. The causative agent, prion, is extremely resistant to common disinfection procedures. Thus, effective prion inactivation strategies using relatively safe and less corrosive disinfectants are required. A solution containing CAC-717, mesoscopic crystals of calcium bicarbonate, exerts both antimicrobial and virucidal activities without apparent harmful effects. This study demonstrated that combined treatment with CAC-717 absorbed on ceramic (CAC-717 ceramic) and sodium dodecyl sulfate (SDS) substantially reduced the protein misfolding cyclic amplification (PMCA) seeding activity of Chandler strain scrapie mouse-brain homogenates (ScBH). Additionally, bioassays demonstrated that ScBH-inoculated mice treated with CAC-717 ceramic in combination with sodium dodecyl sulfate (SDS) did not develop disease. Furthermore, this combination effectively inactivated PMCA seeding activity on ScBH-coated stainless-steel wires below the detection limit. Overall, the findings suggest that combined treatment with CAC-717 ceramic and SDS represents a promising and less damaging approach for prion inactivation.

Antimicrobial use and its association with the presence of methicillin-resistant staphylococci (MRS) and extended-spectrum beta-lactamases (ESBL)-producing coliforms in mastitic milk on dairy farms in the Chiba Prefecture, Japan.

Food-producing animals, including dairy cattle, are potential reservoirs of antimicrobial resistance. However, there is limited data on antimicrobial use and the selection of resistant bacteria. Therefore, we investigated the association between antimicrobial use and resistance to mastitis pathogens using 2016 data from milk samples collected from cows with mastitis in 134 dairy farms in Chiba Prefecture, one of the principal dairy production prefectures in Japan. We recorded the antimicrobial use and isolation of methicillin-resistant staphylococci (MRS) and extended-spectrum beta-lactamase (ESBL)-producing coliforms (E. coli and Klebsiella spp.), and used the antimicrobial treatment incidence (ATI; the theoretical number of animals per 1000 animal-days subjected to antimicrobial treatment) to indicate antimicrobial use on each farm. The farms in which MRS or ESBL-producing coliforms were isolated from at least one mastitic milk sample were classified as antimicrobial resistance (AMR)-positive, and those in which neither MRS nor ESBL-producing coliforms were isolated were classified as AMR-negative. The AMR-positive farms showed a significantly higher ATI (median 45.17) than AMR-negative farms (median 38.40). The results indicate that high antimicrobial usage is associated with AMR in staphylococci and coliforms isolated from mastitic milk on dairy farms in Chiba Prefecture.

Genetic Diversity and Epidemic Types of Porcine Reproductive and Respiratory Syndrome (PRRS) Virus in Japan from 2018 to 2020.

To clarify the genetic diversity of the porcine reproductive and respiratory syndrome virus (PRRSV) in Japan in recent years, we determined the nucleotide sequence of open reading frame 5 of 2482 PRRSV sequences obtained from samples collected from pigs between January 2018 and December 2020. As a result of molecular phylogenetic analysis, Cluster II represented the largest proportion (44.9−50.6%) throughout the study period, followed by Cluster IV (34.0−40.8%), Cluster III (7.8−12.1%), Cluster I (3.1−6.7%), and Cluster V (0.1−0.2%). The relative distributions between Clusters varied between geographic regions and between years: in 2018, Cluster II was the most prevalent in all regions. In 2019, Cluster II was dominant in the Hokkaido and Tohoku regions, while in other regions Cluster IV was dominant. In 2020, Cluster IV was dominant in the Kanto/Tosan and Kyushu/Okinawa regions, whilst in other regions Cluster II was predominant. Compared with a previous study, the proportions of genome sequences classified in Clusters II and IV significantly increased (p = 0.042 and 0.018, respectively) and those classified in Cluster III significantly decreased (p < 0.01). The widespread use of live attenuated vaccines using strains that belong to Cluster II might have accounted for these changes in the relative distribution between Clusters.

Evaluating the antimicrobial use on dairy farms in Chiba Prefecture in Japan using the antimicrobial treatment incidence, an indicator based on Japanese defined daily doses from 2014-2016.

The use of antimicrobial agents in food-producing animals may lead to the emergence and spread of antimicrobial resistance in bacteria of animal origin. However, there is a paucity of data on the quantity of antimicrobials use on dairy farms in Japan. This study describes antimicrobial use on dairy farms from 1 January 2014 to 31 December 2016 in five administrative districts (central, eastern, western, southern and northern) of Chiba Prefecture. The use of antimicrobial agents in dairy cattle over these three years was evaluated in terms of the antimicrobial treatment incidence (ATI; theoretical number of animals per 1,000 animal-days subjected to antimicrobial treatment) using data collected from a total of 442 dairy farms in that prefecture. Our results revealed that the average ATI on these farms for these years ranged from 38.7 to 39.4 with no significant difference between years and that the average ATI for these administrative districts varied between 32.9 and 43.2 with a significant variation between some of the districts. Approximately 84% of antimicrobials were administered intramammarily, 13-14% by injection and 1-2% orally. Scenario analyses were performed to assess the effect of changes in some of the defined daily dose (DDDjp) values used to calculate the ATI. Our results revealed that the calculated ATI is considerably affected by the changes in the long-acting factor used for assigning the DDDjp values of intramammary products for dry cows and the way in which DDD values are assigned for combination products.

Calcium bicarbonate as an antimicrobial, antiviral, and prion-inhibiting agent (Review).

Calcium bicarbonate does not act as a disinfectant at neutral pH; however, it exerts strong antimicrobial activity after it is placed in a high-voltage electric field, whereby it assumes an alkaline pH (12.4). Moreover, the microbicidal activity of the resulting solution (named CAC-717) is not influenced by the presence of organic material or resistance of the agent to inactivation. When sprayed on the skin surface, the pH of CAC-717 decreases rapidly to 8.84. CAC-717 comprises fine particles of 50-500 nm. When these mesoscopic crystals are dissolved in water, they destroy the genomes of bacteria or viruses and neutralize the infectious properties of abnormal prion proteins produced in ScN2a cells. The severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) pandemic has resulted in unprecedented international demand for disinfectants. A small titer of SARS-CoV-2 remains infectious even after 30 sec in growth medium at pH 12.4. CAC-717 has exhibited a strong virucidal effect (3.6 to 4.4 log10 decrease) against all examined SARS-CoV-2 isolates, including mutant forms. Similarly, human noroviruses also remain intact at pH 12.4; however, CAC-717 has been shown to cause a 3.25 log10 reduction in norovirus genomic RNA compared to untreated samples. Existing evidence suggests that an unidentified mechanism controls the virucidal activity of CAC-717.

Identifying the factors that affect the willingness of pig farmers to participate in the electronic prescription system.

Electronic data collection systems are being developed in countries around the world to monitor antimicrobial use at farm level. We conducted a questionnaire survey that is destined for pig farmers who are then able to communicate what factors influence their willingness to participate in an electronic prescription system. A principal component analysis was performed on the variables that were associated with the willingness to participate in the system. Using the principal components obtained from the principal component analysis and the attributes of the farmers as explanatory variables, we performed a logistic regression analysis. The results revealed that farmers with a high level of information technology (IT) literacy and a certain degree of active business management and farmers who are not currently familiar with business management practices but who are willing to use data were more willing to participate in the electronic prescription system than those who do not have a high level of IT literacy and/or who are not willing to use data for business management. Contrarily, farmers' intention to manage drug usage does not contribute to the willingness to participate in the system. These results show that the farmers' understanding of the benefits and ease of participation in the electronic prescription system is important for establishing the system, thus promoting the convenience of the system is the most effective way to gain cooperation of farmers when establishing the system.

Association between BoLA-DRB3 polymorphism and bovine leukemia virus proviral load in Vietnamese Holstein Friesian cattle.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis. Polymorphism in bovine leukocyte antigen (BoLA)-DRB3 allele can influence the host immune response to pathogens, including BLV. However, association between specific BoLA-DRB3 alleles and BLV proviral load (PVL), which is a useful index for estimating disease progression and transmission risk, in Vietnamese cattle are unknown. Here, association study of BoLA-DRB3 allele frequency between cattle with high or low PVL demonstrated BoLA-DRB3*12:01 associates with high PVL in Vietnamese Holstein Friesian (HF) crossbred cattle. This is the first study to demonstrate that BoLA-DRB3 polymorphism confers susceptibility to BLV high PVL in HF crossbred kept in Vietnam. Our results may be useful in disease control and eradiation for BLV through genetic selection.

Virucidal Effect of the Mesoscopic Structure of CAC-717 on Severe Acute Respiratory Syndrome Coronavirus-2.

Here, the virucidal effect of calcium bicarbonate with a mesoscopic structure (CAC-717) on severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was determined. Assays showed that CAC-717 had a strong virucidal effect on all examined SARS-CoV-2 isolates, including variant strains. The viral infectivity decreased within 15 s, and the virucidal activity of CAC-717 at a 1:49 ratio was similar to that of ethanol disinfectant. CAC-717 neutralization eliminated this virucidal effect, indicating that the alkaline condition of CAC-717 is important for virus inactivation and is an indicator of its mesoscopic structure and virucidal activity. The virucidal effect was observed in the presence of organic matter (bovine serum albumin). CAC-717 is a non-invasive and non-flammable substance with a low environmental burden, and its usefulness as a novel disinfectant has been confirmed.

Evaluation of antimicrobial use in dairy cattle, beef cattle and broilers in Japan using dosage-based indicators.

The sales amount of antimicrobials intended for use in dairy cattle, beef cattle and broilers from 2008 to 2019 was evaluated for each antimicrobial class and administration route using dosage-based indicators. Our results revealed that the antimicrobial sales amount sold for use in dairy cattle in 2019 in terms of total weight of active ingredient, the number of defined daily doses (DDDs) (theoretical amount of biomass subjected to antimicrobial treatment in a year) and the number of treatment days (TDs) (theoretical number of days of treatment that an animal is subjected to in a year) calculated using Japanese DDD values (DDDjp values) was 36,751 kg, 8,261,848,000 kg·days and 15.5 days, respectively. Likewise, the antimicrobial sales amount sold for use in beef cattle and broilers in 2019 in terms of these metrics was 33,403 kg, 3,928,248,000 kg·days and 3.61 days, and 69,773 kg, 2,947,848,000 kg·days and 10.66 days, respectively. There was a considerable difference between the number of DDDs calculated using DDDjp values and that calculated using European DDD values (DDDvet values) for antimicrobial products sold for use in dairy and beef cattle. Our results also revealed that the sales amount of some antimicrobials, such as cephalosporins and quinolones represented larger proportions when calculated using dosage-based indicators than when calculated using the weight of active ingredient. Considering that Japanese veterinarians and farmers are more likely to conform to the Japanese dosage recommendations rather than the European ones, these results indicate the need for using dosage-based metrics, in particular metrics based on Japanese dosages rather than European dosages.

Antimicrobial use on 74 Japanese pig farms in 2019: A comparison of Japanese and European defined daily doses in the field.

Defined daily doses (DDD) have been established in human medicine to standardize the measurement of treatment in a population. In veterinary medicine, the European Medicine Agency published defined daily dose (DDDvet) values for antimicrobial agents used in food-producing animals in 2016. National defined doses (DDDjp) for antimicrobials used for pigs in Japan have recently been determined. The aim of this study was to compare the results of calculated antimicrobial use in the field using the DDDjp and DDDvet values. Data from 74 pig farms in Japan relative to antimicrobial use in 2019 was collected. The numbers of DDDs (the weight of biomass treated in kg-days) using DDDjp and DDDvet values for each farm and for different antimicrobial classes were compared. Associations between calculated numbers of DDDjp and DDDvet on farm level were investigated. In addition, differences in antimicrobial use were investigated between different production types of farms (farrowing, finishing and farrow-to-finish farms). Using DDDjp and DDDvet values, the aggregated number of DDDs for 74 farms were 4,099,188 and 2,217,085 respectively, with the former being larger by 1.85 times than the latter. The most frequently used antimicrobial class was penicillin regardless of whether DDDjp or DDDvet was used. The absence of DDDvet values for certain antimicrobial agents used in Japan and the differences in the number of DDDjps/PCU and DDDvets/PCU indicated the need for Japanese DDDs. The number of DDDs per kg population correction unit (PCU) per farm tended to be higher in farrowing farms than in farrow-to-finish farms and finishing farms, with no significant difference (P = 0.19).

Comparison of prevalence of Felis catus papillomavirus type 2 in squamous cell carcinomas in cats between Taiwan and Japan.

Felis catus papillomavirus (FcaPV), especially type 2 (FcaPV2) is considered as one of the causative agents in squamous cell carcinoma (SCC) in cats. However, our previous study detected FcaPV3 and FcaPV4, but not FcaPV2 in feline SCCs collected in Japan, suggesting that the prevalence of FcaPV2 in SCC may vary depending on geographic locations. To evaluate this hypothesis, two conventional PCR reactions targeting E1 and E7 genes were performed to detect FcaPV2 in feline SCC samples collected in Taiwan and Japan. While 46.9% (23/49) of feline SCC cases from Taiwan were PCR positive for FcaPV2, only 8.6% (3/35) cases from Japan were positive. Our result suggests that the prevalence of FcaPV2 in feline SCCs may depend on the region.

Identifying causes of death of companion dogs in Japan using data from pet cemeteries.

This study aimed to identify the causes of death in dogs that were buried at pet cemeteries. Using data obtained from a questionnaire completed by 5,118 dog owners from 1 April 2016 to 31 March 2017, we estimated the life expectancy and the odds ratio for principal causes of death using multiple logistic regression analysis with potential risk factors as independent variables. The life expectancy determined at birth was 13.98 years. The commonest cause of death was senility. The odds ratios of dying of senility being significantly higher in Labrador Retrievers and Shiba breeds and aged dogs, and significantly lower in Chihuahuas, dogs living indoors and neutered dogs.

Establishing defined daily doses (DDDs) for antimicrobial agents used in pigs, cattle and poultry in Japan and comparing them with European DDD values.

Monitoring of antimicrobial use is essential in the management of the development and selection of antimicrobial resistance. A variety of indicators has become available to monitor antimicrobial use in human and animal medicine. One of them is an indicator based on defined daily dose (DDD). By using the number of DDDs administered and normalising it by the population at risk of being treated over a defined period, one can estimate the number of treatment days with antimicrobial agents in a population. For veterinary medicine, the European Medicines Agency (EMA) has published the European values of DDD (DDDvet) for food-producing animals. In this study, we defined Japanese defined daily doses for antimicrobial agents (DDDjp) using DDD values that we previously assigned for antimicrobial products approved for use in pigs, cattle and poultry in Japan and compared them with DDDvet values. For the comparison, the quotient of Japanese and European values (QDDD) was calculated and the effect of the administration route and the number of active substances contained in the preparation was investigated. A total of 59 DDDjp values were defined for 43 antimicrobial agents using the data of 276 products approved for use in pigs. Likewise, a total of 55 DDDjp values were defined for 32 antimicrobial agents using the data of 196 products for use in cattle, and a total of 27 DDDjps values were defined for 25 antimicrobial agents using the data of 131 products approved for use in poultry. A comparison was made for 42, 28 and 17 pairs of DDDjp and DDDvet values for antimicrobial agents used for pigs, cattle and poultry respectively. The comparison showed median QDDD value of 0.61 and 0.66 for antimicrobial agents used for pigs and cattle respectively (p<0.01), indicating that the Japanese daily doses are significantly lower than the corresponding EMA values in these species. For the antimicrobial agents used for poultry, no significant difference was observed between DDDjp and DDDvet values with a median QDDD value of 1.15. The difference between DDDvet and DDDjp values and absence of DDDvet values for some antimicrobial agents marketed in Japan indicate that DDDjp rather than DDDvet should be used as the basis for the calculation of antimicrobial use monitoring in farm animals in Japan.

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.

Genomic characterisation of bovine papillomavirus types 1 and 2 identified in equine sarcoids in Japan.

BACKGROUND: Bovine papillomavirus types 1 and 2 (BPV1/2) infection in horses has been associated with the development of equine sarcoids. Previous findings revealed the presence of sarcoid-associated BPV sequence variants that have been proposed as a key factor of cross-species infection in horses. To verify this hypothesis, sarcoid-associated BPV variants should be identified regardless of geographic location. OBJECTIVES: Sequence analyses of BPV1/2 derived from both horses and cattle were conducted to clarify the sarcoid-associated sequence variants. The aim of this study was to clarify the correlation between BPV phylogeny and the geographic origin/host species. STUDY DESIGN: Cross-sectional study. METHODS: Conventional PCR to detect BPV1/2 was performed with genomic DNA extracted from equine sarcoid (n = 10) and bovine papilloma (n = 10) samples collected in Japan. Direct sequencing results were compared between equine and bovine (equine/bovine)-derived BPV to identify sarcoid-associated variants of two early regions (E2, E5), one late region (L1) and the long control region (LCR). Phylogenetic and phylogeny-trait correlation were analysed using Bayesian Markov chain Monte Carlo (MCMC) method and Bayesian tip-association significance testing (BaTS). RESULTS: Seven BPV1 and three BPV2 were identified from equine sarcoids using PCR and direct sequencing. Sequence analysis of equine/bovine-derived samples showed no sarcoid-associated variants in four regions (E2, E5, L1 and LCR) of either BPV1 or BPV2. The phylogenetic tree of BPV1 E2, L1 and LCR tended to cluster within its geographic origins. BaTS analysis demonstrated that BPV1 sequence variability may be due to the geographic origin rather than host species difference. MAIN LIMITATIONS: There was a limitation in sample numbers. CONCLUSIONS: This study supports the geographic-specific hypothesis of sequence variability, suggesting that BPV1 is shared between local equids and bovids. However, more extensively collected sequences worldwide and functional evaluations are needed to verify the geographic-specific sequence variability of BPV1/2 between equine- and bovine-derived sequence.

Evaluation of the antimicrobial use in pigs in Japan using dosage-based indicators.

The use of antimicrobial agents in food-producing animals may lead to the emergence and spread of antimicrobial resistance in bacteria of animal origin. The use of antimicrobial agents in pigs in 2018 in Japan was evaluated in terms of the weight of active ingredient and number of defined daily doses (DDD), using annual sales data of veterinary antimicrobials sold for use in pigs. In addition, the use of antimicrobial agents in the Japanese pig sector in 2008 to 2017 was evaluated to determine whether or not there were any differences in temporal change pattern by use of different metrics. In 2018, 447 metric tons of active ingredient, corresponding to 77,379 × 106 kg-days (Japanese DDD) and 34,903 × 106 kg-days (European DDD) were sold. The proportion of the sales amount of sulfonamides, trimethoprim and lincosamides to the total sales amount was significantly different depending on the metric used. For most antimicrobial classes, the number of Japanese DDDs was greater than the number of European DDDs. These results indicate that the DDD-based metric, which is more reflective of the selective pressure of antimicrobials, is recommended for use in monitoring the antimicrobial use in pigs in Japan. The differences in the number of Japanese DDDs and European DDDs appear to confirm the need for Japanese DDDs.

Quantitative risk assessment of the introduction of rabies into Japan through animals accidentally placed in international freight containers.

Japan has been free from rabies since 1958 and various preventive measures are in place to protect the country from the introduction of the disease. With an increasing number of freight containers arriving in Japan every year, there is a concern that rabies might be reintroduced into Japan through animals arriving in international freight containers. A stochastic simulation model was built assuming the following entry and exposure pathway as being the most likely route of rabies entry: a rabies-infected animal is accidentally placed in a freight container in the country of origin; it survives transportation from the moment the container is sealed in the country of origin until it is opened at the destination in Japan; and it escapes from the container when it is opened at the destination in Japan. Input parameter values were based on surveys of container handling and warehouse agencies and scientific data from the literature. The annual probability of rabies introduction through this pathway worldwide was 5.47 × 10-6 (90 % PI: 9.72 × 10-7-1.33 × 10-5), or rabies would enter Japan every 368,864 (90 %PI: 75,267 - 1,027,568) years. Among sub-regions, the annual probability was highest for South-eastern Asia (4.54 × 10-6 (90 % PI: 8.04 × 10-7-1.11 × 10-5)), followed by Eastern Asia and Southern Asia. The rabies introduction risk from other sub-regions was negligible. The result of scenario analysis indicated that even if any of the main parameters changes, the risk of rabies introduction still remains very low, suggesting that unintentional movement of animals through international freight containers is not a very important pathway of rabies introduction into Japan.

Detection and genotyping of bovine leukemia virus (BLV) in Vietnamese cattle.

Bovine leukemia virus (BLV) belongs to the genus, Deltaretrovirus of the family, Retroviridae and it is the causative agent of enzootic bovine leukosis. The prevalence of BLV in three provinces in the Red River Delta Region in the North of Vietnam, Hanoi, Vinhphuc and Bacninh was studied from April 2017 to June 2018. A total of 275 blood samples collected from cattle were used for serum isolation and DNA extraction. Of these samples, 266 sera were subjected to ELISA test for detecting antibody against BLV gp51 protein and 152 DNA samples were used to detect the 444 bp fragment corresponding to a part of the gp51 region of the env by nested PCR. The results showed that 16.5% (n=44) and 21.1% (n=32) of samples were positive for BLV gp51 antibody and BLV proviral DNA, respectively. Phylogenetic analysis of the partial (423 bp) and complete (913 bp) BLV env-gp51 gene indicated that Vietnamese strains were clustered into genotypes 1, 6 and 10 (G1, G6 and G10). Of those genotypes, G1 genotype was dominant; G6 strains were designated as G6e and G6f subgenotypes; the existence of genotype 10 was confirmed for the first time in Vietnam. The present study provides important information regarding the prevalence of BLV infection and genetic characteristics of BLV strains identified in Vietnam, contributing to promote the establishment of disease control and eradication strategies in Vietnam.

Assessing the risk of ASFV entry into Japan through pork products illegally brought in by air passengers from China and fed to pigs in Japan.

A risk assessment was conducted to assess the risk of ASFV entry into Japan through pork products illegally brought in by air passengers from China and fed to pigs in Japan. Scenario tree modelling was used with the following entry and exposure pathway considered to be the most likely route of ASF entry: an ASFV infected pork product is illegally brought into Japan by air travellers from China; this pork product is then used in a restaurant where scrap waste is recycled for animal feed and subsequently fed to pigs without being heat-treated. Input parameter values were based on surveys conducted by the authors, scientific data gathered from the literature and official data published by government agencies. The annual probability of ASFV entry into Japan via this pathway was predicted to be 0.20 (90% prediction interval: 0.00-0.90). The wide prediction interval was mainly caused by the uncertainty regarding the dose response relation of ASFV, followed by the probability of an ASF infected pig dying on affected farms, the loading of ASFV in an infected pig and the probability of an illegally imported pork product being heat-treated in China and used in restaurants. The results of scenario analysis revealed that the annual probability of ASFV entry into Japan will increase with an increase in the number of ASF affected farms in China. The probability of ASFV entry will increase substantially even if only a small proportion of Ecofeed is not heat-treated during the production process. The probability will decrease if an increased proportion of farms that feed swill apply heat-treatment before feeding swill to their pigs. These findings indicate that stringent application of heat-treatment of Ecofeed and swill is key to protecting the Japanese pig industry from the introduction of ASFV.