Anthelmintic Baiting of Foxes against Echinococcus multilocularis in Small Public Area, Japan.

We distributed anthelmintic baits on a university campus in Japan inhabited by foxes infected with Echinococcus multilocularis to design an effective baiting protocol for small public areas. High-density baiting can reduce the risk for human exposure to the parasite to near zero. However, monthly baiting is recommended to maintain this effect.

SPECIFIC MOLECULAR DETECTION OF PIROPLASMS AND CHARACTERIZATION OF ß-TUBULIN FOR A NOVEL BABESIA SPECIES IN SIKA DEER (CERVUS NIPPON YESOENSIS).

Piroplasms, which include Babesia spp. and Theileria spp., are protozoan parasites carried by ticks and commonly cause disease in animals and humans. Those caused by Babesia spp. manifest as fever, anemia, and hemoglobinuria, while Theileria spp. can lead to high fever, diarrhea, and lymphadenopathy. Recently, Theileria capreoli and an undescribed Babesia sp. were detected for the first time in sika deer (Cervus nippon yesoensis) from Hokkaido; however, there is limited information available on their epidemiology in Japan. Here, a touchdown polymerase chain reaction and reverse line blot hybridization were used to perform an epidemiological survey of T. capreoli and Babesia sp. using blood samples from 82 sika deer in Hokkaido, Japan. This was followed by partial sequencing and phylogenetic analysis of the 18S rRNA and ß-tubulin genes to characterize both piroplasm species. A total of 43 (52.4%) and 3 (3.7%) of the sika deer were positive for T. capreoli and Babesia sp., respectively. The ß-tubulin gene partial sequences for Babesia sp. were distinct from those of Babesia spp. in GenBank. Phylogenetic analysis showed that the unknown Babesia sp. is more closely related to B. bigemina and B. ovata than other Babesia spp. based on the ß-tubulin gene. Further studies are required to understand the ecology of these tick-borne pathogens in Japan.

Molecular detection of apicomplexan protozoa in Hokkaido brown bears (Ursus arctos yesoensis) and Japanese black bears (Ursus thibetanus japonicus).

Many tick-borne pathogens (TBPs) are present in wildlife. The objective of this study is to reveal the role of wild bears in maintaining TBPs. A total of 49 brown bears (Ursus arctos yesoensis) from Hokkaido, and 18 Japanese black bears (Ursus thibetanus japonicus) from Tochigi, and 66 Japanese black bears from Nagano were examined by two molecular methods, reverse line blot (RLB) hybridization, and nested PCR. A total of 5 TBPs (Hepatozoon ursi, Babesia sp. UR2-like group, Cytauxzoon sp. UR1, Babesia sp. UR1, and Babesia microti) were detected from bear blood DNA samples. B. microti was detected from blood DNA samples of Japanese black bear for the first time, with the prevalence of 6.0% (5/84). Out of detected pathogens, H. ursi, Babesia sp. UR2-like pathogens, and Cytauxzoon sp. UR1 were considered as three of the most prevalent TBPs in bears. The prevalence of H. ursi were significantly higher in Japanese black bear (0% vs 96.4%) while that of Babesia sp. UR2-like group was higher in Hokkaido brown bears (89.8% vs 40.5%). The prevalence of Babesia sp. UR1 were significantly higher in Japanese black bears from Tochigi (44.4%), comparing with those from Nagano (18.2%). The prevalence of the detected TBPs were significantly higher in adult bears, comparing with those in younger bears. The present study suggests that Japanese bear species contribute in the transmission of several TBPs in Japan. The expanding distribution of bears might cause the accidental transmission of TBPs to humans and domestic animals.

Utilizing attached hard ticks as pointers to the risk of infection by Babesia and Theileria species in sika deer (Cervus nippon yesoensis), in Japan.

Ticks are hematophagous ectoparasites that have a significant impact on their animal hosts. Along with mosquitoes, they are the main arthropod vectors of disease agents in domestic animals, wildlife and humans. To investigate the occurrence and prevalence of piroplasmids in ticks, DNA was extracted from 519 hard ticks collected from 116 hunted Hokkaido sika deer (Cervus nippon yesoensis). The success of the DNA extraction was confirmed by touchdown PCR targeting the mitochondrial 16S rDNA gene of ticks. Touchdown PCR and reverse line blot (RLB) hybridization targeting the 18S rRNA gene were used to detect 14 piroplasm species. All hard ticks parasitizing Hokkaido sika deer were identified as belonging to the genera Ixodes and Haemaphysalis. In total 163 samples (31.4%) were positive for Babesia and Theileria spp. among tick species according to RLB hybridization. Tick DNA hybridized to the oligonucleotide probes of Theileria sp. Thrivae (27.0% of ticks; 140/519), Theileria capreoli (10.6%; 55/519), Babesia divergens-like (1.7%; 9/519), Babesia sp. (Bab-SD) (0.6%; 3/519), Babesia microti U.S. (0.4%; 2/519), and B. microti Hobetsu (0.4%; 2/519). The partial sequencing and phylogenetic analyses of the 18S rRNA gene confirmed the RLB hybridization results. Further investigations are needed to reveal the epidemiology and respective vectors of these pathogens.

Mixed Mycobacterium tuberculosis Lineage Infection in 2 Elephants, Nepal.

Tuberculosis in elephants is primarily caused by Mycobacterium tuberculosis. We identified mixed M. tuberculosis lineage infection in 2 captive elephants in Nepal by using spoligotyping and large sequence polymorphism. One elephant was infected with Indo-Oceanic and East African-Indian (CAS-Delhi) lineages; the other was infected with Indo-Oceanic and East Asian (Beijing) lineages.

Comparison of feeding habits and habitat use between invasive raccoons and native raccoon dogs in Hokkaido, Japan.

BACKGROUND: In Japan, invasive raccoons cause severe ecological and social problems by transmitting pathogens to humans, livestock, and native species, causing substantial crop damage, and competing with native species. Possible competition between invasive raccoons and native raccoon dogs is of concern in Japan because Japanese raccoon dogs have a limited distribution and are native only to Japan and the two species have similar characteristics. We assessed potential competition between raccoons and raccoon dogs by comparing feeding habits and habitat use. RESULTS: Both species were captured in Hokkaido, Japan from 2004 to 2017. More raccoons were captured close to agricultural land at the forest periphery (70.1%, 358/511); conversely, more raccoon dogs were captured in the forest core (74.9%, 253/338). Feeding habits were then examined by fecal analysis and stable isotope analyses. Fecal analysis revealed both species to be opportunistic omnivores that consumed easily found food items. However, raccoon feces contained more crops, whereas raccoon dog feces contained more insects, reflecting the different locations in which the species were trapped. Moreover, stable isotope ratios were significantly higher in raccoons than raccoon dogs (Corn has the highest carbon stable isotope (δ13C) value, and amphibians and reptiles are high in nitrogen stable isotope (δ15N); forest resources such as insects and wild fruits are low in δ13C and δ15N). CONCLUSIONS: We conclude that both species ate similar food types, but their food preferences appeared to differ. Raccoon and raccoon dog habitat use also differed, possibly because the two species inhabited areas where they could easily obtain their preferred foods. Therefore, the current feeding habits and habitat use of raccoons do not appear to overlap sufficiently with those of raccoon dogs to impact the latter. The results of this study, particularly the stable isotope data, may provide a useful precedent for future studies of competition in medium-sized mammals, particularly canids.

Genetic Diversity and Population Structure of East Asian Raccoon Dog (Nyctereutes procyonoides): Genetic Features in Central and Marginal Populations.

The raccoon dog (Nyctereutes procyonoides) is endemic to East Asia but has been introduced in Europe. Its high adaptability enabled its rapid colonization of European countries, where population growth has been raising concerns regarding ecosystem disturbance and the spread of zoonotic diseases. The genetic diversity and structure of endemic, source, and introduced populations from seven locations across South Korea, China, Russian Far East, Finland (spread to Finland after introduction to European part of Russia from Russian Far East), Vietnam, and Japan (Honshu and Hokkaido) were examined based on 16 microsatellite loci. Two major and significantly different (FST = 0.236) genetic clusters were found: continental (South Korean, Chinese, Russian, Finnish, and Vietnamese) and island (Japanese) populations. The continental raccoon dog population comprises three subpopulations (Chinese_Russian_Finnish, South Korean, and Vietnamese) and the Japanese population consists of Honshu and Hokkaido subpopulations. The genetic diversity and geographic structure of raccoon dogs in East Asia has been influenced by natural barriers to gene flow and reveals a typical central-marginal trend in genetic diversity (continental vs. island, and central vs. marginal or source vs. introduced within continental populations). The detected differences between continental and island populations agree with those reported in previous studies that considered these populations as different species.

First molecular detection and characterization of Hepatozoon and Sarcocystis spp. in field mice and voles from Japan.

Sarcocystis and Hepatozoon species are protozoan parasites that are frequently detected in domestic and wild animals. Rodents are considered common intermediate and paratenic hosts for several Sarcocystis and Hepatozoon species. Here, blood DNA samples from a total of six rodents, including one Myodes rutilus, one Myodes rufocanus, and four Apodemus speciosus, collected from Hokkaido, Japan, were shown by conventional PCR of the 18S ribosomal RNA (rRNA) gene to contain Sarcocystis and Hepatozoon DNA. Sequencing of the DNA detected one Sarcocystis sp. in the M. rufocanus sample and two different Hepatozoon spp. in the M. rutilus and A. speciosus samples. Phylogenetic analysis showed that the detected Sarcocystis sp. sequence grouped with GenBank Sarcocystis sequences from rodents, snakes, and raccoons from Japan and China. The 18S rRNA partial gene sequences of both detected Hepatozoon spp. clustered with GenBank Hepatozoon sequences from snakes, geckos and voles in Europe, Africa, and Asia. This study provides evidence that wild rodents have a role in the maintenance of Sarcocystis and Hepatozoon species on the island of Hokkaido.

Pesticide Contamination Levels in the Stomach Contents of Wild Raccoons (Procyon lotor) and Masked Palm Civets (Paguma larvata) in Japan.

Pesticides, which are vital for agriculture, pose a significant threat to wildlife in transformed Japanese landscapes. Despite global reports of pesticide poisoning in animals, limited studies have examined current wildlife exposure in croplands or metropolitan areas in the region. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS), our study aimed to assess the contamination status of 368 commonly used pesticides. The stomach contents of raccoons living in croplands contained 13 pesticides, including six herbicides and 11 fungicides. Neonicotinoid insecticides, some fungicides, and previously banned insecticides (benzene hexachloride and dichlofenthion) were most frequently detected and found at the highest concentrations, suggesting direct soil-plant transfer and direct consumption by crop-eating species. In masked palm civets living in metropolitan areas, four insecticides and six fungicides were detected, indicating urban wildlife exposure from raided dustbins, urban gardens, and lumber from houses. Although the maximum measured concentrations of all pesticides were lower than the acceptable daily intake for humans, it remains unclear whether these concentrations may have toxic or adverse health effects on the species evaluated in these transformed landscapes. Our study is the first to examine recent pesticide exposures in wild mammals in Japan. Application of the method we developed will lay the foundation for the examination of pesticides in other wildlife species to assist conservation management efforts in the region. Environ Toxicol Chem 2024;43:943-951. © 2024 SETAC.

Timing of puberty and its relationship with body growth and season in male raccoons (Procyon lotor) in Hokkaido.

The raccoon (Procyon lotor), indigenous to North America, has naturalized in Japan as an invasive alien species, having been introduced into the country in the 1970s. In Hokkaido, the northernmost island of Japan, feral raccoons have been increasing in number and spreading throughout the island. The age at the onset of puberty for raccoons is important for estimating individual lifetime reproductive success and population growth. The present study investigated the timing of and potential factors affecting the onset of puberty in male raccoons in Hokkaido. External characteristics and histology of testes were studied in 151 male feral raccoons and in 1 captive juvenile. For the majority of feral yearling raccoons, prepubertal development began in May, and spermatozoa production began in October prior to their second mating season. However, some larger juveniles attained puberty during the juvenile period. The captive juvenile, which was fed throughout the winter, attained puberty only 11 months after birth. These results suggest that if male raccoons can achieve enough body growth before the first mating season, puberty can be attained early. In both juveniles and yearlings, spermatozoa production was only observed after autumn. This timing coincided with the recrudescence of seasonally active spermatogenesis in adult males. Therefore, attaining puberty in male raccoons appears to require both adequate body nutrient development and several environmental factors that control seasonal testicular changes.

Virological, pathological, and glycovirological investigations of an Ezo red fox and a tanuki naturally infected with H5N1 high pathogenicity avian influenza viruses in Hokkaido, Japan.

In winter/spring 2021-2022, high pathogenicity avian influenza viruses (HPAIVs) that are genetically closely related to each other were detected worldwide. In a public garden in Sapporo, Hokkaido, Japan, a crow die-off by HPAIV infection occurred from March 29 to May 18, 2022. During the event, H5N1 HPAIVs were isolated from an Ezo red fox (Vulpes vulpes schrencki) and a tanuki (Nyctereutes procyonoides albus) found in the same garden. The fox showed viral meningoencephalitis and moderate virus replication in the upper respiratory tract, whereas the tanuki showed viral conjunctivitis and secondary bacterial infection in the eyes accompanied with visceral larva migrans. Viruses isolated from the fox and the tanuki were genetically closely related to those isolated from crows in the same garden. Various α2-3 sialosides were found in the respiratory tracts of these canid mammals, consistent with HPAIV infections in these animals. This study highlighted the importance of monitoring HPAIV infections in wild carnivore mammals to detect the potential virus spreading in nature.

Age estimation based on blood DNA methylation levels in brown bears.

Age is an essential trait for understanding the ecology and management of wildlife. A conventional method of estimating age in wild animals is counting annuli formed in the cementum of teeth. This method has been used in bears despite some disadvantages, such as high invasiveness and the requirement for experienced observers. In this study, we established a novel age estimation method based on DNA methylation levels using blood collected from 49 brown bears of known ages living in both captivity and the wild. We performed bisulfite pyrosequencing and obtained methylation levels at 39 cytosine-phosphate-guanine (CpG) sites adjacent to 12 genes. The methylation levels of CpGs adjacent to four genes showed a significant correlation with age. The best model was based on DNA methylation levels at just four CpG sites adjacent to a single gene, SLC12A5, and it had high accuracy with a mean absolute error of 1.3 years and median absolute error of 1.0 year after leave-one-out cross-validation. This model represents the first epigenetic method of age estimation in brown bears, which provides benefits over tooth-based methods, including high accuracy, less invasiveness, and a simple procedure. Our model has the potential for application to other bear species, which will greatly improve ecological research, conservation, and management.

Habitat occupancy of sloth bear Melursus ursinus in Chitwan National Park, Nepal.

Mammals have experienced a massive decline in their populations and geographic ranges worldwide. The sloth bear, Melursus ursinus (Shaw, 1791), is one of many species facing conservation threats. Despite being endangered in Nepal, decades of inattention to the situation have hindered their conservation and management. We assessed the distribution and patterns of habitat use by sloth bears in Chitwan National Park (CNP), Nepal. We conducted sign surveys from March to June, 2020, in 4 × 4 km grids (n = 45). We collected detection/non-detection data along a 4-km trail that was divided into 20 continuous segments of 200 m each. We obtained environmental, ecological, and anthropogenic covariates to understand determinants of sloth bear habitat occupancy. The data were analyzed using the single-species single-season occupancy method, with a spatially correlated detection. Using repeated observations, these models accounted for the imperfect detectability of the species to provide robust estimates of habitat occupancy. The model-averaged occupancy estimate for the sloth bear was 69% and the detection probability was 0.25. The probability of habitat occupancy by sloth bears increased with the presence of termites and fruits and in rugged, dry, open, undisturbed habitats. Our results indicate that the sloth bear is elusive, functionally unique, and widespread in CNP. Future conservation interventions and action plans aimed at sloth bear management must adequately consider their habitat requirements.

Estimation of the Effects of Neonicotinoid Insecticides on Wild Raccoon, Procyon lotor, in Hokkaido, Japan: Urinary Concentrations and Hepatic Metabolic Capability of Neonicotinoids.

Toxicological effects of neonicotinoid insecticides (NNIs) have been reported for mammals, such as humans, rats, and mice. However, there are limited reports on their toxic effects on wild mammals. To predict NNI-induced toxic effects on wild mammals, it is necessary to determine the exposure levels and metabolic ability of these species. We considered that raccoons could be an animal model for evaluating NNI-induced toxicities on wildlife because they live near agricultural fields and eat crops treated with NNIs. The objective of the present study was to estimate the effects of NNI exposure on wild raccoons. Urinary concentrations of NNI compounds (n = 59) and cytochrome P450-dependent metabolism of NNIs (n = 3) were evaluated in wild raccoons captured in Hokkaido, Japan, in 2020. We detected either one of the six NNIs or one metabolite, including acetamiprid, imidacloprid, clothianidin, dinotefuran, thiacloprid, thiamethoxam, and desmethyl-acetamiprid in 90% of raccoons (53/59); the average cumulative concentration of the seven NNI compounds was 3.1 ng/ml. The urinary concentrations were not much different from those reported previously for humans. Furthermore, we performed an in vitro assessment of the ability of raccoons to metabolize NNIs using hepatic microsomes. The amounts of NNI metabolites were measured using liquid chromatography-electrospray ionization-tandem mass spectrometry and compared with those in rats. Raccoons showed much lower metabolic ability; the maximum velocity/Michaelis-Menten constant (Vmax /Km ) values for raccoons were one-tenth to one-third of those for rats. For the first time, we show that wild raccoons could be frequently exposed to NNIs in the environment, and that the cytochrome P450-dependent metabolism of NNIs in the livers of raccoons might be low. Our results contribute to a better understanding of the effects of NNIs on raccoons, leading to better conservation efforts for wild mammals. Environ Toxicol Chem 2022;41:1865-1874. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Diel and monthly activity pattern of brown bears and sika deer in the Shiretoko Peninsula, Hokkaido, Japan.

Mammals exhibit several types of diel activity pattern, including nocturnal, diurnal, crepuscular, and cathemeral. These patterns vary inter- and intra-specifically and are affected by environmental factors, individual status, and interactions with other individuals or species. Determining the factors that shape diel activity patterns is challenging but essential for understanding the behavioral ecology of animal species, and for wildlife conservation and management. Using camera-trap surveys, we investigated the species distributions and activity patterns of terrestrial mammals on the Shiretoko Peninsula, Hokkaido, Japan, with particular focus on brown bears and sika deer. From June to October 2019, a total of 7,530 observations were recorded by 65 camera-traps for eight species, including two alien species. The diel activity pattern of brown bears was diurnal/crepuscular, similar to that of bears in North America, but different from European populations. Bear observations were more frequent during the autumnal hyperphagia period, and adult females and sub-adults were more diurnal than adult males. In addition, bears inside the protected area were more diurnal than those outside it. These findings suggest that appetite motivation, competitive interactions between conspecifics, and human activities potentially affect bear activity patterns. Similar to other sika deer populations and other deer species, the diel activity patterns of sika deer were crepuscular. Deer showed less variation in activity patterns among months and sex-age classes, while adult males were observed more frequently during the autumn copulation period, suggesting that reproductive motivation affects their activity patterns.

Semen collection by urethral catheterization and electro-ejaculation with different voltages, and the effect of holding temperature and cooling rate before cryopreservation on semen quality in the Japanese macaque (Macaca fuscata).

In the Japanese macaque, semen has been collected by electro-ejaculation (EE), using the higher voltage stimuli compared to other species including genus Macaca. Semen coagulates immediately after ejaculation, which makes difficult to produce high-quality semen for artificial insemination. Recently, semen collection using urethral catheterization (UC) has been reported in carnivore and this technique may allow semen collection without coagulation in a less invasive manner. Further, the temporal preservation temperature and cooling rate of semen during cryopreservation affect post thawing sperm quality. In this study, to improve semen quality and quantity, as well as the animal welfare, semen collection was performed by EE with high (5-15 V) or low (3-6 V) voltage, UC and a combination of the two (EE-UC). It has been suggested that a high voltage is necessary for semen collection, but 10 V stimulation was effective enough and 15 V is for additional sperm collection. Also, liquid semen was collected by EE-UC and this could increase the total number of sperm. Further, to improve the post thawing sperm motility, semen was kept at four temperatures (4, 15, 25 and 37°C) for 60 min, and processed with two cooling procedures (slow cooling before second dilution and fast cooling after second dilution). Holding semen at 25°C and fast cooling after the second dilution maintained progressive motile sperm rate. The present results will contribute to the improvement of semen collection and animal welfare of Japanese macaques.

Detection of a Babesia sp. genotype closely related to marsupial-associated Babesia spp. in male Haemaphysalis shimoga from Sarawak, Malaysian Borneo.

In this study, Babesia screening was conducted in 55 rodents and 160 tick samples collected from primary forests and an oil palm plantation in Sarawak, Malaysian Borneo. PCR targeting the 18S ribosomal DNA revealed the presence of Babesia spp. DNA detected in two questing male Haemaphysalis shimoga ticks collected from the oil palm plantation. Sequence analysis revealed that both sequences were identical and had 98.6% identity to a Babesia macropus sequence obtained from Eastern grey kangaroos (Macropus giganteus) in Australia. Phylogenetic tree revealed clustering with marsupial-associated Babesia spp. in the Babesia sensu stricto clade. Whether or not H. shimoga is the competent vector and the importance of the Babesia sp. detected in this study warrants more investigation.

Highly pathogenic avian influenza virus infection in feral raccoons, Japan.

Although raccoons (Procyon lotor) are susceptible to influenza viruses, highly pathogenic avian influenza virus (H5N1) infection in these animals has not been reported. We performed a serosurvey of apparently healthy feral raccoons in Japan and found specific antibodies to subtype H5N1 viruses. Feral raccoons may pose a risk to farms and public health.

Changes in liver microRNA expression and their possible regulatory role in energy metabolism-related genes in hibernating black bears.

Hibernating bears survive up to 6 months without feeding while yet maintaining metabolic homeostasis. We previously reported expression changes in energy metabolism-related genes in the liver of hibernating Japanese black bears. The present study examined the role of microRNAs in the regulation of hepatic gene expression during hibernation. The quantitative analyses revealed significant increases in the expression of 4 microRNAs (miR-221-3p, miR-222-3p, miR-455-3p, and miR-195a-5p) and decreases of 2 microRNAs (miR-122-5p and miR-7a-1-5p) during hibernation. RNA sequencing and in silico target prediction regarding 3 upregulated microRNAs (miR-221-3p, miR-222-3p and miR-455-3p) found 13 target mRNAs with significantly decreased expression during hibernation. The transfection of microRNA mimics into cells showed that miR-222 and miR-455 reduced solute carrier family 16 member 4 (SLC16A4) and fatty acid synthase (FASN) mRNA expression, respectively. Our results suggest that the increased levels of hepatic miRNA during hibernation (miR-222-3p and miR-455-3p) negatively regulate the expression of targeted genes predicted to be involved in the transport of energy source and de novo fatty acid synthesis, is consistent with a regulatory role of these miRNAs in energy metabolism in hibernating black bears.

Dining from the coast to the summit: Salmon and pine nuts determine the summer body condition of female brown bears on the Shiretoko Peninsula.

Body condition in mammals fluctuates depending on energy intake and expenditure. For brown bears (Ursus arctos), high-protein foods facilitate efficient mass gain, while lipids and carbohydrates play important roles in adjusting dietary protein content to optimal levels to maximize energy intake. On the Shiretoko Peninsula, Hokkaido, Japan, brown bears have seasonal access to high-lipid pine nuts and high-protein salmon. To assess seasonal and annual fluctuation in the body condition of adult female brown bears in relation to diet and reproductive status, we conducted a longitudinal study in a special wildlife protection area on the Shiretoko Peninsula during 2012-2018. First, analyses of 2,079 bear scats revealed that pine nuts accounted for 39.8% of energy intake in August and salmon accounted for 46.1% in September and that their consumption by bears varied annually. Second, we calculated the ratio of torso height to torso length as an index of body condition from 1,226 photographs of 12 adult females. Results indicated that body condition continued to decline until late August and started to increase in September when salmon consumption increased. In addition, body condition began to recover earlier in years when consumption of both pine nuts and salmon was high. Furthermore, females with offspring had poorer body condition than solitary females, in particular in late August in years with low salmon consumption. Our findings suggest that coastal and subalpine foods, which are unique to the Shiretoko Peninsula, determine the summer body condition of female brown bears, as well as their survival and reproductive success.