Sporadic endemicity of zoonotic Paragonimus in raccoon dogs and Japanese badgers from Miyazaki Prefecture, Japan.

Paragonimiasis is a zoonotic trematode infection caused by Paragonimus spp. To determine the recent status of Paragonimus infections in wild animals, this study investigated Paragonimus spp. in 39 raccoon dogs and 54 Japanese badgers from March 2019 to January 2021 in Miyazaki Prefecture, and examined metacercariae in freshwater crabs. Triploid P. westermani was found in one raccoon dog (2.6%), and metacercariae were recovered from Eriocheir japonica captured near the infected animal collected. One Japanese badger (1.9%) harbored P. skrjabini miyazakii; this prevalence was lower than the approximately 30% that was reported in the 1970s. Results indicated that zoonotic Paragonimus was sporadically prevalent in wild animals. Further investigation in various animals is awaited to elucidate current wildlife reservoirs for those Paragonimus.

Paragonimiasis is a parasitic infection caused by lung flukes of the genus Paragonimus. Approximately 50 annual cases of human paragonimiasis, caused by P. westermani and P. skrjabini miyazakii, have been reported in Japan [16, 23]. Several species of carnivorous/omnivorous animals have been recorded as being definitive hosts in nature. Raccoon dogs are capable of harboring both triploid and diploid P. westermani; however, epidemiological studies that evaluated P. westermani endemicity have been limited [18, 20].

Martens, weasels, and Japanese badgers are known to be suitable hosts for P. skrjabini miyazakii, and high prevalence of infection in those animals were reported in decades ago [2, 3, 5,6,7, 11, 21]. Moreover, although human paragonimiasis cases have been sporadically recognized, the current endemicity of zoonotic Paragonimus spp. in wild animals is poorly understood. In this study, prevalence of these parasites in two species of wild omnivores, raccoon dogs and Japanese badgers, were evaluated in Miyazaki Prefecture, which is a major endemic area of human paragonimiasis in Japan.

Carcasses of 39 raccoon dogs and 54 Japanese badgers that were found as roadkill or provided by the local government from vermin control in Miyazaki City and surrounding municipalities were collected from March 2019 to January 2021. A necropsy was conducted after determining body weight and length and checking the sex. Whole lungs were collected and macroscopically searched for Paragonimus cysts. Then, flukes were recovered from the detected cysts and stored at −30°C until molecular examination.

Genomic DNA was extracted from flukes collected from individual hosts using a QIAamp DNA mini kit (Qiagen GmbH, Hilden, Germany) in accordance with the manufacturer’s instructions. Species were molecularly identified by PCR sequencing of the second internal transcribed spacer region of nuclear ribosomal DNA and the cytochrome c oxidase subunit 1 gene [8]. To determine the chromosomal ploidy (diploid or triploid) of P. westermani, the 16S mitochondrial rRNA gene was also analyzed [1].

Lung cysts were found in one raccoon dog (2.6%; 1/39) and one Japanese badger (1.9%; 1/54). Only one fluke was isolated from a single cyst in the raccoon dog, and the fluke was used for molecular identification. In the Japanese badger, 58 flukes were isolated from many cysts, the number of which could not be counted because of adhesion, and two flukes were arbitrarily selected as representatives for species identification. The species of the lung fluke in the raccoon dog was molecularly identified as triploid P. westermani and that of the Japanese badger was molecularly identified as P. s. miyazakii (Table 1).

Table 1.

Prevalence of Paragonimus spp. in wild animals in Miyazaki Prefecture

		Ashizawa et al.† [7]	Ashizawa et al. [5]	Ashizawa et al.† [6]	Ashizawa et al.‡ [3]	Ashizawa et al. [2]	This study
		Sampling year:	Sampling year:	Sampling year:	Sampling year:	Sampling year:	Sampling year:
		1974–1975	1974–1976	1975–1976	1976–1978	1977–1978	2019–2021
Raccoon dogs	Paragonimus westermani positive	NA	NA	NA	NA	NA	1
	P. skrjabini miyazakii positive	NA	NA	NA	NA	NA	0
	Paragonimus spp. negative	NA	NA	NA	NA	NA	38
	Prevalence of Paragonimus spp. (%)	NA	NA	NA	NA	NA	2.6
Japanese badgers	P. westermani positive	NA	0	NA	NA	0	0
	P. skrjabini miyazakii positive	NA	3	NA	NA	2	1
	Paragonimus spp. negative	NA	7	NA	NA	4	53
	Prevalence of Paragonimus spp. (%)	NA	30.0	NA	NA	33.3	1.9
Weasels and martens	Paragonimus spp. positive	4	NA	10	10*	NA	NA
	Paragonimus spp. negative	22	NA	28	26	NA	NA
	Prevalence of Paragonimus spp. (%)	15.4	NA	26.3	27.8	NA	NA

* Cysts or lesions positive for Paragonimus spp., † Martens (Martes melampus melampus), ‡ Weasels (Mustela itatsi/M. sibirica coreana), NA stands for “data is not available”.

Freshwater crabs, Eriocheir japonica, were captured in a river near the location where the infected raccoon dog was found (approximately 270 m along the street). A total of 15 crabs were minced and artificially digested, and metacercariae were examined under a stereomicroscope. In total, 63 metacercariae were collected from the crabs and were molecularly identified as triploid P. westermani. With regard to the infection source of the positive Japanese badger, a survey targeting freshwater crab (Geothelphusa dehaani) was not performed because no part of the river was considered a suspected source of infection around the point where the positive carcass was found.

This study provides a description of the first case of a raccoon dog infected with triploid P. westermani in Miyazaki Prefecture. The prevalence was 2.6% in the surveyed raccoon dogs. This was lower than the value reported in previous studies although the surveying conditions were different; the triploid type was detected in 11% (1/9) of raccoon dogs from Yakushima Island in the 2000s [18], and the diploid type was detected in 8.6% (12/140) of raccoon dogs from Hyogo Prefecture in the 1980s [21].

Another zoonotic Paragonimus species, P. s. miyazakii, was found in 1.9% of Japanese badgers; in comparison, a 30–33% prevalence was recorded in the 1970s in Miyazaki Prefecture [2, 5]. Moria nipponica (Mori, 1937) (critically endangered, rare) is a first intermediate host for P. s. miyazakii and Geothelphusa dehaani (near threatened) is a second intermediate host for P. s. miyazakii and P. westermani; both of these species are now in the Red Data list (2015 edition) in Miyazaki Prefecture [15]. The current low endemicity of those two Paragonimus in wild animals may have been affected by decreasing population sizes and distribution of freshwater snails/crabs that serve as the first/second intermediate hosts in recent decades.

In addition to detecting triploid P. westermani in a raccoon dog, metacercariae of the fluke were also detected in freshwater crabs inhabiting a location close to where the infected raccoon dog was collected. Raccoon dogs generally do not have a clearly defined territory, but they have a home range of 8–111 ha depending on surroundings, season, sex, and age class [13, 17]. Therefore, we suppose that the raccoon dog may have used the location where freshwater crabs were investigated as watering places or feeding grounds. Moreover, the raccoon dog is known to sometimes consume crustaceans [9, 12]; therefore, the P. westermani lifecycle is suspected to be sporadically maintained via raccoon dogs and freshwater crabs in such riverside environments. Because the other raccoon dogs and Japanese badgers that were collected within a 1-km radius of the points where the positive carcasses were found were negative for Paragonimus spp. (data not shown), fluke-positivity could be influenced by individual preference for crustaceans or availability of other preferable food. In addition to the consumption of crustaceans, potential transmission via paratenic hosts of triploid P. westermani, e.g., wild boar and deer [21, 24], should be considered because approximately 50% of the carcasses of these animals are known to be visited and scavenged by raccoon dogs [19, 22].

Other carnivorous/omnivorous animals have been reported as definitive hosts of Paragonimus spp. in wildlife in Japan. In the early 1950s, P. westermani infection in feral dogs was reported at a prevalence of 7.2–21.1% [10, 14]. A Japanese red fox infected with diploid P. westermani was also reported [4]. Weasels and martens were also found to be infected with P. s. miyazakii with a prevalence of 15–28% in the 1970s [3, 6, 7]. In this study, we could not conclude the main definitive hosts for these Paragonimus species in nature. Further investigations into these wild animals could help clarify the current endemicity of these Paragonimus species.

In conclusion, our findings clearly show that zoonotic Paragonimus is distributed, albeit in low abundance, in raccoon dogs and Japanese badgers from Miyazaki Prefecture, and its lifecycle is sporadically maintained along the water where host animals inhabit and visit. To elucidate potential reservoirs for zoonotic Paragonimus in wildlife, further investigations should be conducted that target various animals in different areas.

POTENTIAL CONFLICTS OF INTEREST

The authors have no relationships or support that might be perceived as constituting a conflict of interest.