A case-control study of the infection risk of H5N8 highly pathogenic avian influenza in Japan during the winter of 2020-2021.

In Japan, outbreaks of H5N8 highly pathogenic avian influenza (HPAI) were reported between November 2020 and March 2021 in 52 poultry farms. Understanding HPAI epidemiology would help poultry industries improve their awareness of the disease and enhance the immediate implementation of biosecurity measures. This study was a simulation-based matched case-control study to elucidate the risk factors associated with HPAI outbreaks in chicken farms in Japan. Data were collected from 42 HPAI-affected farms and 463 control farms that were within a 5-km radius of each case farm but remained uninfected. When infected farms were detected as clusters, one farm was randomly selected from each cluster, considering the possibility that the cluster was formed by farm-to-farm transmission within an epidemic area. For each case farm, up to three control farms were selected within a 5-km radius. Overall, 26 case farms (16 layer and 10 broiler farms) and 75 control farms (45 layer and 30 broiler farms) were resampled 1000 times for the conditional logistic regression model with explanatory variables comprising geographical factors and farm flock size. A larger flock size and shorter distance to water bodies from the farm were found to increase infection risk in layer farms. Similarly, in broiler farms, a shorter distance to water bodies increased infection risk. On larger farms, frequent access of farm staff and instrument carriages to premises could lead to increased infection risk. Waterfowl visiting water bodies around farms may also be associated with infection risk.

Assessing population structure and migration patterns of wild boar (Sus scrofa) in Japan.

Geographical wildlife patterns reflect historical range expansion and connectivity and provide insights into wildlife population management. In our large-scale phylogeographic population analysis of wild boars (Sus scrofa leucomystax) in Japan, we identified 15 clusters using 29 microsatellite markers, each structured within a range of approximately 200 km. This suggests that evolution was essentially driven by isolation by distance, and that the range of gene flow was limited. One cluster contained subpopulations located approximately 900 km apart, indicating the occurrence of past anthropogenic introductions. Moreover, we estimated effective migration to visualize the geographic genetic population diversity. This analysis identified six potential barriers, one of which involved large plains and mountainous areas in the Kanto region of eastern Japan. This barrier likely persisted in the two eastern clusters for an extended period, restricting migration to the neighboring areas. Overall, our study sheds light on the demographic history of wild boar in Japan, provides evidence of past anthropogenic introductions from distant areas, and highlights the importance of geographic barriers in shaping genetic diversity and population dynamics. This knowledge will be beneficial for forming informed wildlife management strategies toward the conservation of genetic integrity and ecological balance of wild boar populations in Japan.

Analysis of effective spatial range of oral vaccination against classical swine fever for wild boar.

Classical swine fever (CSF) re-emerged in Gifu Prefecture, central Japan, in September 2018 and is currently widespread in wild boar populations. Due to its widespread in wild boars, an oral mass vaccination strategy was initiated in March 2019, employing a commercial bait vaccine that is a live attenuated vaccine. To enhance the effectiveness of oral vaccination, it is crucial to determine the vaccine's effective spatial range. This understanding is essential for devising a comprehensive vaccination strategy, which should also include a preliminary investigation of wild boar habitats before vaccination. This study aimed to estimate the effective range of oral vaccination for wild boars against CSF by analyzing the geographical relationship between immune wild boars and vaccination points within the vaccination areas in Gifu Prefecture. This study utilized oral vaccination data from April 2021 to March 2022. The prevalence of CSF infections in wild boars remained below 5% in this period, suggesting limited disease transmission and immune wild boars were considered to be induced by the effect of vaccination. Two vaccination campaigns were conducted during this period, with almost 2000 vaccination points each. To investigate the factors associated with the intensity (i.e., density) of immune wild boar, the nearest distances to a vaccination point and to a susceptible wild boar were evaluated as explanatory variables. The Rhohat procedure and point process model were utilized to analyze the relationship between the intensity of immune wild boars and the explanatory variables. The point process model revealed a significant decrease in the intensity of immune wild boars when the distance from the nearest vaccination point exceeded 500 m, indicating that the effective spatial range of bait vaccination is within 500 m of the vaccination point. Although the distance to the nearest susceptible animal did not show significance in the model, Rhohat plots indicated that the intensity of immune wild boars decreased at distances greater than 1200 m from the nearest susceptible wild boar. This finding highlights the importance of investigating susceptible wild boar populations within a range of at least 1200 m from a vaccination point before implementation. The present study revealed the effective range of oral vaccination for wild boars against CSF and indicated the importance of investigating susceptible wild boar habitats around vaccination points before the implementation of vaccination. These findings may help improve the effectiveness of oral vaccinations.

The probability distributions of the movement of dairy and beef cattle in Japan: a data note.

OBJECTIVES: Animal movement is an important factor in the transmission of infectious diseases among livestock. A better understanding of animal movement characteristics provides a more reliable estimation of disease spread and promotes modeling studies. In Japan, all the cattle movement information is recorded in a national database called the Individual Cattle Identification Register (ICIR)." Our previous studies using this information demonstrated heterogeneity in the movement of dairy and beef cows according to location, season, and age. The present study describes the probability distributions of the movement of Japanese dairy and beef cows in the following month on a regional basis. DATA DESCRIPTION: This publication contains four probability distribution datasets for the predicted locations of dairy and beef cows in Japan in the following month, which were developed using individual cattle movement information obtained from the ICIR. These datasets provide information on cattle movement in the following month on a regional basis, given properties such as birth region, location, time, and age.

Unique genomic sequences in a novel Mycobacterium avium subsp. hominissuis lineage enable fine scale transmission route tracing during pig movement.

Mycobacterium avium subsp. hominissuis (MAH) is one of the most prevalent mycobacteria causing non-tuberculous mycobacterial disease in humans and animals. Of note, MAH is a major cause of mycobacterial granulomatous mesenteric lymphadenitis outbreaks in pig populations. To determine the precise source of infection of MAH in a pig farm and to clarify the epidemiological relationship among pig, human and environmental MAH lineages, we collected 50 MAH isolates from pigs reared in Japan and determined draft genome sequences of 30 isolates. A variable number of tandem repeat analysis revealed that most pig MAH isolates in Japan were closely related to North American, European and Russian human isolates but not to those from East Asian human and their residential environments. Historical recombination analysis revealed that most pig isolates could be classified into SC2/4 and SC3, which contain MAH isolated from pig, European human and environmental isolates. Half of the isolates in SC2/4 had many recombination events with MAH lineages isolated from humans in East Asia. To our surprise, four isolates belonged to a new lineage (SC5) in the global MAH population. Members of SC5 had few footprints of inter-lineage recombination in the genome, and carried 80 unique genes, most of which were located on lineage specific-genomic islands. Using unique genetic features, we were able to trace the putative transmission route via their host pigs. Together, we clarify the possibility of species-specificity of MAH in addition to local adaptation. Our results highlight two transmission routes of MAH, one exposure on pig farms from the environment and the other via pig movement. Moreover, our study also warns that the evolution of MAH in pigs is influenced by MAH from patients and their residential environments, even if the MAH are genetically distinct.

Region-wise analysis of beef cow movements in Japan.

Animal movement is an important factor in the transmission of animal infectious diseases. A better understanding of movement patterns is therefore necessary for developing effective control measures against disease spread. In Japan, a cattle tracing system was established in 2003, following a bovine spongiform encephalopathy epidemic, and the information on all cattle movements has been stored in a national database maintained by the National Livestock Breeding Center. Using these data, we previously analyzed the movement of dairy cows, concluding that heterogeneities in cattle movement are associated with regional and seasonal factors. In the present study, we aimed to identify specific factors affecting the regional and seasonal movement patterns of beef cows in Japan. From April 2012 to March 2017, 797,553 farm-to-farm movement events were recorded. We analyzed movements by month and by cattle age and looked at the frequency of movement within and between seven regions spanning the national territory. Our results show that calf movement peaked at 9-10 months old; these movements were considered to be via the market and were frequent within and between regions. For inter-regional movements, Kyushu region was the top producer of calves for calf trading markets throughout Japan. With regard to intra-regional movements, round-trip movements for summer grazing were observed in May and October for cattle of various ages in the northern regions, especially Hokkaido and Tohoku. Moreover, the movements of Japanese Shorthorn breeds in Tohoku region exhibited consistent annual peaks in May and October/November, in accordance with their seasonal breeding practice. In the areas with high relative densities of dairy cows, such as Hokkaido, the shipping of newborn beef calves produced via embryo transfer to dairy cows was also observed. Overall, understanding the patterns of beef cow movement will help develop effective disease surveillance measures, such as pre-movement inspections focused on specific regions and types of movement.

Current research and future directions for realizing the ideal One-Health approach: A summary of key-informant interviews in Japan and a literature review.

The COVID-19 pandemic has highlighted the importance of the One Health (OH) approach, which considers the health of humans, animals, and the environment in preventing future pandemics. A wide range of sustainable interdisciplinary collaborations are required to truly fulfill the purpose of the OH approach. It is well-recognized, however, that such collaborations are challenging. In this study, we undertook key-informant interviews with a panel of stakeholders from Japan to identify their perceived needs and challenges related to OH research. This panel included scientists, government officials, journalists, and industry stakeholders. By combining a thematic analysis of these interviews and a literature review, we summarized two key themes pertinent to the effective implementation of OH research: types of required research and systems to support that research. As a technological issue, interviewees suggested the importance of research and development of methodologies that can promote the integration and collaboration of research fields that are currently fragmented. An example of such a methodology would allow researchers to obtain high-resolution metadata (e.g. ecological and wildlife data) with high throughput and then maximize the use of the obtained metadata in research, such as in environmental DNA analysis, database construction, or the use of computational algorithms to find novel viral genomes. In terms of systems surrounding OH research, some interviewees stressed the importance of creating a sustainable research system, such as one that has continuous budget support and allows researchers to pursue their academic careers and interests. These perceptions and challenges held by Japanese stakeholders may be common to others around the world. We hope this review will encourage more researchers and others to work together to create a resilient society against future pandemics.

Estimation of introduction time window of highly pathogenic avian influenza virus into broiler chicken farms during the 2020 - 2021 winter season outbreak in Japan.

When an infectious disease occurs in an area, early detection of infected farms is important to respond quickly and contain the outbreak on a small scale. Estimating the time window for the introduction of the infection is important for its prevention and control. The aim of this study was to estimate the farm-specific time window from the introduction of the highly pathogenic avian influenza (HPAI) virus into poultry farms using field data from the HPAI H5N8 outbreak in the 2020-2021 winter season in Japan. Daily mortality data from 12 broiler chicken farms during the outbreak were used for the analysis. A mathematical model (Susceptible-Exposed-Infectious-Removed, SEIR model) was applied to generate the within-flock transmission of HPAI. The model-predicted mortality was fitted to the observed excess mortality data induced by HPAI to estimate the farm-specific transmission rate and the time of virus introduction. The estimated value of the transmission rate in each farm was 1.449 day-1 in median (min: 0.661 day-1, max: 3.387 day-1). The time window from the introduction of the virus to notification in each farm was estimated at 14.0 days in median (min: 8.6 days, max: 24.1 days) in the deterministic model. In addition, in the stochastic model considering the randomness of transmission in the early phase of the outbreak, the upper value of 95 % credible interval of the time window ranged from 12 to 34 days, with a median of 21 days. The results suggest that although one to three weeks had elapsed on most farms until notification after the virus introduction, the time window could exceed three weeks considering the stochasticity of disease transmission. As for the potential farm characteristics affecting within-flock transmission, the transmission rate was smaller (p-value=0.02) and the estimated time window from introduction to notification was longer (p-value=0.02) when birds were older. This study provides reliable information for setting up a tracing period for a potential source farm and enhancing the efforts for early detection.

Subgrouping and analysis of relationships between classical swine fever virus identified during the 2018-2020 epidemic in Japan by a novel approach using shared genomic variants.

Classical swine fever (CSF) is a worldwide devastating disease of the pig industry caused by classical swine fever virus (CSFV). In September 2018, an outbreak of CSF occurred in Japan where the disease had been eradicated and was officially designated a CSF-free country since 2015. Following the detection of the first 2018 case on a farm in Gifu Prefecture, the disease spread among both farm pigs and wild boars and still continues. Epigenome analysis using whole-genome information is helpful in identifying the infection route, but the current approaches provide an insufficient resolution. In this study, a novel method of using single-nucleotide variants (SNVs) was employed to identify the associations among 158 isolates (65 from farms and 93 from wild boars). The identified groups of CSFV strains were plotted in different colours on a map, identifying the location where each strain was collected. The lack of an SNV set shared between the index case and the other strains suggested the first infection in Japan during the outbreak occurred in wild boars, not at the index farm. For the Atsumi Peninsula outbreaks, where nine farms were found infected within a 10-km radius area, the farm strains were assembled into three groups, suggesting these outbreaks resulted from at least three different infection events in this area. For the infections in the area around Saitama Prefecture, an area remote from the epicentre, strains from both the farms and wild boars were identified as being in the same group, suggesting they resulted from one viral introduction. Likewise, seven infected farms in Okinawa Prefecture, almost 1,500 km from Gifu Prefecture, were identified as being in a common, but separate group. By demonstrating the variety of transmission routes and possibility of long-distance infection, these results will help improve disease control measures.

Phylogenetic and phylodynamic analysis of a classical swine fever virus outbreak in Japan (2018-2020).

After 26 years, another classical swine fever virus (CSFV) outbreak in domestic pigs and wild boars occurred in Japan 2018. Herein, we investigated the entry and the spatial dynamics of the CSFV outbreak in Japan using the nearly complete genomes of strains isolated from both wild boars and domestic pigs during this epidemic. Phylogenetic analysis showed that the most recent common ancestor (MRCA) of the Japanese lineage emerged 146 days (95% highest posterior density (HPD): 85-216 days) before the index case was detected. Based on epidemiological analysis, the period for the 95% HPD was 1 month earlier than the time of virus introduction into the index farm. The disease mainly spreads to the adjoining regions during the epidemic, with no spread to the nonadjacent regions. This result indicates that human activities, such as the movement of vehicles, contributed to the infection spread. As cases occurred in nonadjacent regions, the MRCA for the epidemic in the Saitama prefecture was estimated to have emerged 93 days before the date of detection in the initial farm in this region. Similarly, the MRCA for the epidemic in Okinawa prefecture, more than 1,300 km away from the other infected regions, was estimated to have emerged 34 days before the date of detection in the region's primary farm. Therefore, our results indicate that if exotic diseases emerge after a long period of absence or in a disease-free country, a longer period of time will elapse before detection, resulting in further spread. Additionally, subsequent infections occurring in regions distant from the original infected region will require less time for detection than in the original region. This study provides valuable insights into a CSFV outbreak that occurred in a previously CSFV-free country and thus beneficial in enhancing producers' awareness and allow for better preparation for infections.

Pig farm vaccination against classical swine fever reduces the risk of transmission from wild boar.

In 2018, classical swine fever (CSF) re-emerged in the Gifu Prefecture, central Japan, causing an on-going outbreak among wild boars and domestic pigs in the country. Consequently, oral vaccination for wild boar and compulsory vaccination for pig farms started in 2019. We have previously shown that, before vaccination in the Gifu Prefecture, the presence of CSF-infected wild boar near pig farms increased the risk of CSF transmission. This study aimed to re-evaluate the transmission risk from wild boars to pig farms under a vaccination program. The effectiveness of vaccination was evaluated by comparing the transmission risk estimated before and after the implementation of vaccinations. In this study, we focused on two affected areas, the Kanto (eastern Japan) and Kinki (west-central Japan) regions, in which eight of 11 infected farms were detected between the start of pig farm vaccinations and April 2021. Wild boar surveillance data from an area within a 50-km radius from the infected farms were used for analysis, consisting of 18,870 1-km grid cells (207 infected cells) in the Kanto region, and 15,677 cells (417 infected cells) in the Kinki region. The transmission rates in the post-vaccination period in the Kanto and Kinki regions were much lower than that in the pre-vaccination period in the Gifu Prefecture. The values of transmission kernels (h0, transmission rate at 0 km) in the Kanto and Kinki regions decreased to 1% of the transmission kernel in the pre-vaccination period. In the pre-vaccination period, the risk of infection within 300 days was almost 95 % when one infected grid cell was detected within 1 km of a pig farm. Meanwhile, in the post-vaccination period, the risk of infection within 300 days was approximately 5% when several infected cells were detected within 1 km of a pig farm. Considering the limited effect of oral vaccination for wild boar due to distribution limitations in the Kanto and Kinki regions, vaccination on pig farms may seems to have mainly reduced the transmission risk from wild boar. However, despite the implementation of vaccination, the risk of infection on pig farms remains present due to the immunity gap of weaning pigs. Therefore, strict biosecurity measures on pig farms and an appropriate vaccination program are required to prevent and control CSF spread.

Additive Bayesian network analysis of the relationship between bovine respiratory disease and management practices in dairy heifer calves at pre-weaning stage.

BACKGROUND: Nursery farms that accept nursing and growing pre-weaned heifer calves from private dairy farms must work to prevent bovine respiratory disease (BRD). Knowledge of the BRD-associated risk factors related to calf management and calves' condition will help to develop appropriate neonatal management practices at original farms and to identify calves at higher risk for BRD at nursery farms. In this study, the relationship between BRD and calf management practices (colostrum feeding, dam parity, serum total protein concentration at introduction (TP), body weight at introduction, introduction season, and daily average growth) was investigated using observational data from pre-weaned dairy calves introduced into a nursery farm in Hokkaido, Japan between 2014 and 2018 (n = 3185). Using additive Bayesian network (ABN) analysis, which is a multivariate statistical modelling approach, the direct and indirect associations between these factors were assessed. RESULTS: Colostrum feeding contributed to an increase in TP (correlation 1.02 [95 % CI, 0.94;1.10]), which was negatively associated with BRD directly (log odds ratio - 0.38 [- 0.46;-0.31]) and indirectly through increasing daily growth (correlation 0.12 [0.09;0.16]). Calves of multiparous dams had higher body weight at introduction (correlation 0.82 [0.74;0.89]), which indirectly reduced BRD risk through the increasing daily growth (correlation 0.17 [0.14;0.21]). Calves introduced during winter had the highest risk for BRD (log odds ratio 0.29 [0.15;0.44]), while those introduced in summer had the lowest risk (log odds ratio - 0.91 [- 1.06;-0.75]). The introduction season was also associated with BRD indirectly through dam parity, body weight at introduction, and daily growth. CONCLUSIONS: The following calf management practices are recommended for preventing BRD in pre-weaned calves at nursery farms: (1) encouraging colostrum feeding to neonatal calves at their original farms; and (2) identifying calves with higher BRD risk, i.e., those without feeding colostrum, born to primiparous cattle, with low body weight at introduction, and/or introduced in winter, and paying intensive attention to the calves for rapid detection of BRD. ABN analysis applied enabled us to understand the complex inter-relationships between BRD incidence and the risk factors, which will help to reduce BRD incidence and to rear healthy calves at nursery farms.

Region-wise analysis of dairy cow movements in Japan.

BACKGROUND: Animal movement is considered the most significant factor in the transmission of infectious diseases in livestock. A better understanding of its effects would help provide a more reliable estimation of the disease spread and help develop effective control measures. If the movement pattern is heterogeneous, its characteristics should be considered in epidemiological analyses, such as when using simulation models to obtain reliable outputs. In Japan, following the bovine spongiform encephalopathy epidemic, a traceability system for cattle was established in 2003, and the registration of all cattle movements in the national database began. This study is the first to analyze cattle movements in Japan. We examined regional and seasonal heterogeneity in dairy cow movements, which accounted for most Japanese breeding cattle. RESULTS: In the 14 years from April 2005 to March 2018, 4,577,709 between-farm movements of dairy cows were recorded, and the number of movements was counted by month and age for both inter- and intra-regional movements. As a result, two characteristic round-trip movements were observed: one was non-seasonal and inter-regional movements related to cattle-breeding ranches in Hokkaido (the northern region of Japan), which consists of the movement of cows around ages 6 to 8 and 21 to 23 months old. In addition, the seasonal movement of heifers for summer grazing within Hokkaido occurred in May and October at the peak ages of 13 to 14 and 19 to 20 months old, respectively. The observed heterogeneity seemed to reflect the suitability of raising the Holstein breed in Hokkaido and the shortage of supply of replacement heifers and available farming areas outside Hokkaido. CONCLUSIONS: Understanding the patterns of dairy cow movements will help develop reliable infectious disease models and be beneficial for developing effective control measures against these diseases.

Genome variability of classical swine fever virus during the 2018-2020 epidemic in Japan.

Although RNA viruses exhibit extensive sequence diversity, the mutation rate must be limited to ensure protein functions that maintain the viral life cycle. Here, we compared the whole genome sequences of 150 isolates of classical swine fever virus (CSFV), obtained from a single epidemic that occurred in Japan during 2018-2020. After the detection of the first case, the disease spread among both farm pigs and wild boars and caused severe impact on the pig industry. To evaluate the diversification of the CSFV genome that eliminated mutations negatively affecting viral transmission, the substitution sets inherited by at least two isolates were separately evaluated as shared single nucleotide variants (SNVs) or shared single amino acid variants (SAVs). Comparisons of 12 protein-coding regions indicated that the percentages of SNVs and SAVs in the multifunctional nonstructural protein NS3 were the lowest, and shared SAVs were not detected in another nonstructural protein, NS4A. This demonstrated purifying negative selection suppressing changes in the protein sequences of NS3 and NS4A during virus transmission in the field. In contrast, a high possibility of nonsynonymous substitution among shared SNVs was detected only in genes encoding the secreted protein Erns and the nonstructural protein NS2, suggesting positive selection during the epidemic. Mapping of shared SAVs to the three-dimensional structure of Erns revealed that shared SAVs were not present in the substrate-binding sites but were instead localized to the peripheral region of the protein. These data will support efforts toward the development of diagnostic methods, recombinant vaccines, and antiviral agents targeting conserved and indispensable viral genes.

Epidemiological analysis of classical swine fever in wild boars in Japan.

BACKGROUND: Classical swine fever (CSF) is a contagious disease of pigs and wild boars that is transmitted through direct/indirect contact between animals or CSF virus-contaminated fomites. When the disease re-emerged in 2018 in Japan, a CSF-infected wild boar was reported shortly after the initial pig farm outbreak; subsequently, the disease spread widely. To control the disease spread among wild boars, intensive capturing, fencing, and oral bait vaccination were implemented with concomitant virological and serological surveillance. This study aimed to describe the disease spread in the wild boar population in Japan from September 2018, when the first case was reported, to March 2020, based on the surveillance data. We conducted statistical analyses using a generalized linear mixed model to identify factors associated with CSF infection among wild boars. Moreover, we descriptively assessed the effect of oral bait vaccination, which started in March 2019 in some municipalities in the affected areas. RESULTS: We observed a faster CSF infection spread in the wild boar population in Japan compared with the CSF epidemics in European countries. The infection probability was significantly higher in dead and adult animals. The influence of the multiple rounds of oral bait vaccination was not elucidated by the statistical modeling analyses. There was a decrease and increase in the proportion of infected and immune animals, respectively; however, the immunization in piglets remained insufficient after vaccination for 1 year. CONCLUSIONS: Conditions regarding the wild boar habitat, including forest continuity, higher wild boar population density, and a larger proportion of susceptible piglets, were addressed to increase the infection risk in the wild boar population. These findings could improve the national control strategy against the CSF epidemic among wild boars.

Genomic features of Mycobacterium avium subsp. hominissuis isolated from pigs in Japan.

Mycobacterium avium subsp. hominissuis (MAH) is one of the most important agents causing non-tuberculosis mycobacterial infection in humans and pigs. There have been advances in genome analysis of MAH from human isolates, but studies of isolates from pigs are limited despite its potential source of infection to human. Here, we obtained 30 draft genome sequences of MAH from pigs reared in Japan. The 30 draft genomes were 4,848,678-5,620,788 bp in length, comprising 4652-5388 coding genes and 46-75 (median: 47) tRNAs. All isolates had restriction modification-associated genes and 185-222 predicted virulence genes. Two isolates had tRNA arrays and one isolate had a clustered regularly interspaced short palindromic repeat (CRISPR) region. Our results will be useful for evaluation of the ecology of MAH by providing a foundation for genome-based epidemiological studies.

Epidemiology of Classical Swine Fever in Japan-A Descriptive Analysis of the Outbreaks in 2018-2019.

This study describes the epidemiological characteristics of classical swine fever (CSF) outbreaks in Japan. The first case was confirmed in September 2018, 26 years after the last known case. Outbreaks occurred on 39 farms, 34 commercial farms, and 5 non-commercial farms, between September 2018 and August 2019. In this study, a descriptive analysis was conducted of the epidemiological data on the characteristics of the affected farms, clinical manifestations, intra-farm transmission, association with infected wild boars, and control measures implemented on the farms. Twenty-eight of the 34 affected commercial farms were farrow-to-finish farms. It was assumed that the major risk factors were frequent human-pig interactions and the movement of pigs between farms. Fever and leukopenia were commonly observed in infected pigs. In 12 out of 18 farms where clinical manifestations among fattening pigs was the reason for notification, death was the most frequent clinical manifestation, but the proportion of dead animals did not exceed 0.5% of the total number of animals at most of the affected farms. Therefore, the clinical form of CSF in Japan was considered to be sub-acute. Twenty-three of the 29 farms (79%) with pigs at multiple stages (i.e., piglets, fattening pigs, and sows), had infection across the multiple stages. Many of these farms were within 5 km of the site where the first infected wild boars had been discovered, suggesting that infected wild boars were the source of infection. Infections still occurred at farms that had implemented measures at their farm boundaries to prevent the introduction of the virus into their farms, such as disinfection of vehicles and people, changing boots of the workers, and installation of perimeter fences. It is necessary to continue to strengthen biosecurity measures for farms located in areas with infected wild boars and to continue monitoring the distribution of infected wild boars so that any abnormalities can be reported and inspected at an early stage.

Evaluation of sampling methods for effective detection of infected pig farms during a disease outbreak.

Emergency surveillance following an outbreak of transboundary animal diseases such as classical swine fever (CSF), is conducted to find another new infection as early as possible. Although larger sample sizes can help achieve higher disease surveillance sensitivity, the sample size is limited by the availability of resources in an emergency situation. Moreover, the recent CSF outbreak reported in Japan was associated with fewer clinical signs; this emphasizes the importance of detecting infected farms by surveillance. In this study, we aimed to identify effective and labor-efficient sampling methods showing high probabilities of detecting infection, by simulating infection and sampling in pigsties. We found that impartial sampling, which involves selection of pigs to be sampled from the four corners and the center of the pigsty, and random sampling showed comparable probabilities of detection. Impartial sampling involves sample collection without pig identification and random selection. Owing to its simplicity, impartial sampling is labor-efficient and thus a possible substitute for random sampling. In a group-housing pigsty, testing five pigs from five pens showed a higher detection probability than testing five pigs from one pen. These results suggest preferable surveillance methods for conducting emergency surveillance of infectious diseases.

ISOLATION AND ANTIMICROBIAL SUSCEPTIBILITIES OF NONTUBERCULOUS MYCOBACTERIA FROM WILDLIFE IN JAPAN.

Nontuberculous mycobacteria (NTM) are opportunistic pathogens of humans and animals and are transmitted among the environment, wildlife, livestock, and humans. The aim of this study was to investigate the rate of isolation and antimicrobial susceptibility of NTM in wildlife. In total, 178 samples of feces (n=131) and tissues (n=47) were collected from 11 wildlife species in Gifu Prefecture and Mie Prefecture, Japan, between June 2016 and October 2018. We isolated NTM from 15.3% (20/ 131) of fecal samples using Ogawa medium, and isolates were identified by sequencing the rpoB and hsp65 genes. The rpoB sequences were compared with those from other strains of human and environmental origin. The NTM isolates were obtained from sika deer (Cervus nippon), wild boar (Sus scrofa), Japanese monkey (Macaca fuscata), raccoon dog (Nyctereutes procyonoides), masked palm civet (Paguma larvata), and Japanese weasel (Mustela itatsi) and were classified as rapidly growing mycobacteria (RGM) and slowly growing mycobacteria (SGM). The 12 RGM identified were Mycolicibacterium peregrinum (n=5), Mycolicibacterium fortuitum (n=3), Mycolicibacterium septicum (n=3), and Mycolicibacterium thermoresistibile (n=1), and the eight SGM were Mycobacterium paraense (n=4), Mycolicibacter arupensis (n=2), Mycolicibacter virginiensis (n=1), and Mycobacterium nebraskense (n=1). The NTM from wildlife showed ≥99% similarity with strains from different sources including humans. The RGM were susceptible to the antimicrobial agents tested except for M. fortuitum, which was resistant to azithromycin and clarithromycin. The SGM showed multiple drug resistance qualities but were susceptible to amikacin, clarithromycin, and rifabutin. These results indicate that wildlife may be reservoir hosts of NTM in Japan. The presence of antimicrobial-resistant NTM in wildlife suggests that the trends of NTM antimicrobial susceptibility in wildlife should be monitored.

Comparative Analysis of Fecal Microbiota in Grasscutter (Thryonomys swinderianus) and Other Herbivorous Livestock in Ghana.

The grasscutter (also known as the greater cane rat; Thryonomys swinderianus) is a large rodent native to West Africa that is currently under domestication process for meat production. However, little is known about the physiology of this species. In the present study, aiming to provide information about gut microbiota of the grasscutter and better understand its physiology, we investigated the intestinal microbiota of grasscutters and compared it with that of other livestock (cattle, goat, rabbit, and sheep) using 16S rRNA metagenomics analysis. Similar to the other herbivorous animals, bacteria classified as Bacteroidales, Clostridiales, Ruminococcaceae, and Lachnospiraceae were abundant in the microbiome of grasscutters. However, Prevotella and Treponema bacteria, which have fiber fermentation ability, were especially abundant in grasscutters, where the relative abundance of these genera was higher than that in the other animals. The presence of these genera might confer grasscutters the ability to easily breakdown dietary fibers. Diets for grasscutters should be made from ingredients not consumed by humans to avoid competition for resources and the ability to digest fibers may allow the use of fiber-rich feed materials not used by humans. Our findings serve as reference and support future studies on changes in the gut microbiota of the grasscutter as domestication progresses in order to establish appropriate feeding methods and captivity conditions.