Trichinella T9 in wild bears in Japan: Prevalence, species/genotype identification, and public health implications

In Japan, the recent series of sporadic outbreaks of human trichinellosis caused by Trichinella (Nematoda: Trichocephalida) has occurred owing to the consumption of raw or insufficiently cooked meat from wild bears. However, the infection status and molecular characteristics of Trichinella larvae in Japanese wild bears remain poorly understood. This study investigated the prevalence of Trichinella spp. in brown bears (Ursus arctos) from Hokkaido, and Japanese black bears (Ursus thibetanus japonicus) from three prefectures (Aomori, Akita, and Iwate) in northern Japan, between April 2019 and August 2022. Trichinella larvae were detected in 2.5% (6/236) of the brown bears and 0.9% (1/117) of the Japanese black bears. Sequence analysis using two genetic loci, the internal transcribed spacer region of nuclear ribosomal DNA and the mitochondrial cytochrome c oxidase subunit I gene, revealed that the larvae collected from the seven infected bears were identical to one of the two haplotypes of Trichinella T9. The prevalence of Trichinella T9 is low but is maintained in bears in the Hokkaido and Iwate prefectures suggesting that undercooked meat from these animals could cause human infection. Thus, continued health education campaigns are needed to raise awareness of the potential risk of trichinellosis among hunters, meat suppliers, consumers, and local governmental health agencies.


Introduction
Trichinellosis is a zoonotic disease caused by nematodes of the genus Trichinella (Enoplea: Trichocephalida: Trichinellidae). Trichinella is transmitted by predation and carrion consumption and circulates between carnivores and omnivores. Therefore, human infection is mainly associated with cultural factors such as consuming raw or undercooked meat from Trichinella-infected domestic and wild animals (Pozio and Murrell, 2006).
In this study, we investigated the prevalence and molecular characteristics of Trichinella larvae in wild bears in Japan. The role of wild bears in the life cycle of Trichinella in Japan is also discussed. Furthermore, we examined whether the recent series of human outbreaks were due to changes in the infection status of Trichinella in bears or the current popularity of wild meat dishes.

Ethical statement
All samples were collected from wild bears legally hunted for pest control or as game. This study did not involve deliberately killing animals; thus, ethical approval was not deemed necessary.

Sample collection
Licensed hunters legally hunted 353 wild bears, including 236 brown bears from 13 subprefectures of Hokkaido, and 117 Japanese black bears from Aomori (n = 15), Akita (n = 100), and Iwate (n = 2) prefectures, Japan, for pest control or as game between April 2019 and August 2022. Fig. 1 illustrates the details of the capture sites. Age was determined by counting cementum annual layers of the fourth premolar tooth (Craighead et al., 1970). Tongue samples were collected from all animals by hunters, sent to the co-authors' offices, and stored frozen at − 18 • C until shipping. The samples were then transported while frozen to the National Institute of Infectious Diseases, Tokyo, Japan, twice a year for the detection of Trichinella larvae. We requested the shipping companies to accept the samples as clinical specimens such as patient samples from medical settings, and the samples that arrived at this institute were all in good shape, showing no decomposition.

Detection of Trichinella larvae
A total of 20 g of the tongue muscle from each bear sample was excised, minced in a food processor, and artificially digested using a PrioCHECK Trichinella Alternative Artificial Digestion Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol. Trichinella larvae isolated from each sample in the digestants were mixed thoroughly with physiological saline in a plastic tube up to 50 mL. Subsequently, 5 mL of the solution was transferred to a 9 cm diameter plastic Petri dish. The number of larvae in each dish was counted thrice under a stereoscopic microscope, and the mean number of larvae per gram of tissue (LPG) was calculated. Differences in prevalence between bear species and sexes were separately examined using the chi-squared test.

Molecular identification of Trichinella larvae
DNA samples were extracted from a pool of larvae collected from each animal (4-100 larvae/head) using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Two sets of primer pairs (5′- CACCCAGAAGTATACATCC-3′ and 5′-GTAATAATAGGTCTAGGGAGG-3′ for cytochrome c oxidase subunit I gene, cox1, and 5′-CAATT-GAAAACCGGTGAG-3′ and 5′-ATCACTCAACATTAACCG-3′ for the nuclear internal transcribed spacer 2 region, ITS2) were used to identify the Trichinella species following the methods described in a previous report (Kanai et al., 2006). The PCR products were visualised using electrophoresis on a 1.5% agarose gel and subjected to direct sequencing at Eurofins Genomics, Inc. (Tokyo, Japan). Multiple sequence alignments were performed using MAFFT ver. 7.505 with the option Q-INS-I (Katoh and Standley, 2016). The sequences obtained were compared to those available in the International Nucleotide Sequence Database at NCBI using the BLASTN program (http://blast.ncbi.nlm.nih.gov/Blast. cgi).

Prevalence of Trichinella larvae in wild bears
Trichinella larvae were detected in 7 of the 353 bears examined (2.0%, Table 1). The infection rate of brown bears in Hokkaido was 2.5%; at the subprefecture level, it was 18.2% in Sorachi, 5.3% in Shiribeshi, and 2.3% in Hiyama (Table 2). Among the 117 Japanese black bears examined, Trichinella larvae were detected in one bear (0.9%, Table 2) which was captured in Iwate Prefecture. All positive bears were adults, with LPG ranging from 0.3 to 180.0 (Table 3).
A partial cox1 sequence (345 bp) was determined from the same positive samples, and these sequences were 100% identical. BLAST analysis revealed 100% sequence identity to that of Trichinella T9 from Japanese black bears from Iwate Prefecture (accession no. LC546041) and brown bears from Hokkaido (accession no. LC361217) and 99.7% of the sequences from Japanese black bears in Iwate Prefecture (accession no. LC546040) and raccoons from Hokkaido (accession no. AB267879). Two haplotypes have been reported in Japanese black bears, tentatively named h1 and h2, for descriptive purposes. However, in this study, only h1 was detected from two bear species in Hokkaido and Iwate. The other species with query coverage of >95% was T. murrelli with <95.4%.
The representative sequences determined in this study have been deposited in the DNA Data Bank of Japan under accession nos. LC764456 (ITS2) and LC764591 (cox1).

Discussion
In this study, we identified the larvae detected in tongue muscle samples from six brown bears in Hokkaido and one Japanese black bear in Iwate as Trichinella T9, which is indigenous to Japan where the sylvatic cycle takes place. It has been reported that Trichinella T9 belongs to the same phylogenetic group as T6 and T. nativa; however, unlike them, T9 does not resist freezing (Pozio and Murrell, 2006). Notably, the Trichinella sequence registered as T. britovi from the Japanese black bear (accession no. AB091477) was 100% identical to the ITS2 sequence of Trichinella T9 obtained in this study (345/345 bp). Additionally, all other available sequences of T. britovi had <92.4% identity, implying that the sequence in the previous study was misregistered (Pozio and Murrell, 2006). Two lineages of Trichinella T9 have been reported, which we tentatively named h1 and h2 haplotypes based on the differences in partial cox1 sequences. Haplotype h1 was found in Japanese black bears in Iwate (accession no. LC546041, Tominaga et al., 2021) and brown bears in Hokkaido (accession no. LC361217, Tada et al., 2018), whereas haplotype h2 was detected in Japanese black bears in Iwate (accession no. LC546040, Tominaga et al., 2021) and raccoons from Hokkaido (accession no. AB267879, Kobayashi et al., 2011). In this study, neither h2 nor T. nativa was detected in bears in Hokkaido. Interestingly, a common haplotype-h1-was observed in both bear species in Iwate and Hokkaido, which are geographically separated by the Tsugaru Strait. This is the location of the Blakiston Line, a faunal and floral boundary between Hokkaido and Honshu Islands, Japan . Clarifying the effect of the Blakiston Line on the genetic divergence of Trichinella T9 would have a significant meaning for biogeography.   Two species of bears exist in Japan: brown bears on the island of Hokkaido and Japanese black bears on the islands of Honshu and Shikoku, with their habitats separated by the Tsugaru Strait (Sato, 2010;Yamazaki, 2010). Both bear species are omnivores, feeding primarily on plant material, but occasionally hunt small animals or scavenge carcasses (Sato, 2010;Yamazaki, 2010;Matsubayashi et al., 2015), which transmit Trichinella in a predator-prey relationship, possibly regardless of their sex. The prevalence of Trichinella T9 in brown and Japanese black bears was 2.5% and 0.9%, respectively in this study. Given the low infection rates, wild bears in Japan may have limited contact with infected animals. Bears appear to be the dead-end hosts in the life cycle of Trichinella, as they are the highest-ranking species in natural ecosystems, especially in Japan. However, Trichinella larvae have a long lifespan in nurse cells within the muscle tissue, and they can survive for over 20 years in polar bears (Kumar et al., 1990). Since consuming bear carrion can transmit Trichinella to other animals, it has been suggested that wild bears play a role as long-term reservoirs of Trichinella in the natural environment. On the other hand, medium-sized carrion feeders, such as foxes, may be better suited as efficient sources of transmission in terms of the life cycle of Trichinella, probably including T9 (Campbell, 2018). In fact, red foxes had a relatively high prevalence of 13.8% (44/319, Kanai et al., 2007).
In recent years, the population and distribution of wild ungulates, particularly sika deer and wild boar, have increased throughout Japan. Furthermore, this trend has also affected wild bears, according to the Ministry of Agriculture, Forestry and Fisheries website (https://www. maff.go.jp/j/tokei/kouhyou/jibie/index.html).
Consequently, the number of slaughtered and processed bears has almost doubled from 160 in the fiscal year 2016 to 306 in 2021 for human consumption and as food for other animals. The Guidelines for the Hygienic Management of Meat from Wild Animals in Japan state that wild game should not be eaten or served raw (Takai, 2022) because wild game, including bears, are prepared in slaughtering and processing facilities where veterinarians do not conduct inspections. Nevertheless, people still consume insufficiently cooked bear meat in restaurants, causing trichinellosis in Japan (Tada et al., 2018). Trichinella larvae were detected in wild bear meat in this study, albeit at low levels. Therefore, we believe sporadic trichinellosis outbreaks will likely continue if no further robust countermeasures are taken.
In conclusion, similar to the previous prevalence of 1.2-3.2% (Yamaguchi, 1991;Kanai et al., 2007), this study shows that Trichinella T9 is maintained at low levels in wild bears in the northern part of Japan (2.0%, 7/353). The findings imply that the recent human trichinellosis outbreaks, especially those that have occurred after 2016, might not be due to increased infection rates among wild bears, but due to increasing consumption and preference for wild game. To frame a clear and lucid answer to this matter, extensive surveys covering larger areas of Hokkaido and Honshu Islands would help obtain a comprehensive picture of Trichinella prevalence among wild bears and other wild animals. Owing to the growing popularity of wild meat dishes, the risk of Trichinella infection in the general population through undercooked or underprocessed bear meat consumption is increasing (Takai, 2022). Ongoing health education campaigns are highly necessary to raise awareness of the potential risk of trichinellosis among hunters, meat suppliers, consumers, and local government health agencies responsible for overseeing the public health of local populations.

Funding
This study was financially supported partly by a grant from the Ministry of Health, Labour and Welfare, Japan (MHLW; 21KA1003, awarded to HS). The funders had no role in the study design, data collection, and interpretation, or in the decision to submit the work for publication.

Declaration of competing interest
The authors declare that they have no conflicts of interest concerning this study. A-D: Location on the map (Fig. 1). a LPG, larvae per gram. b NE, not examined.