Abstract
Spirometra infections in companion animals and wildlife in Japan have been diagnosed based on the morphology of the adult worms and eggs, and the etiological agent has been mainly ascribed to Spirometra erinaceieuropaei. However, recent studies have revealed that two other species, Spirometra mansoni and Spirometra asiana, coexist in Japan. Spirometra asiana is a new species recently discovered in Japan. Although morphological discrimination between these two species is difficult, molecular identification is useful. Therefore, to understand which species commonly parasitizes companion animals and wildlife in Japan, a preliminary study was performed based on mitochondrial DNA analysis. Eleven adult worms examined were identified as S. mansoni, suggesting that S. mansoni infects companion animals and wildlife commonly than S. asiana in Japan.
Keywords: companion animals, Japan, mitochondrial DNA analysis, Spirometra asiana, Spirometra mansoni, wildlife
Tapeworms of the genus Spirometra (Cestoda: Diphyllobothriidae), known as cat and dog tapeworms, are distributed worldwide and require two intermediate hosts to complete their life cycle [8]. Eggs excreted with feces of felids and canids enter water and develop into a coracidium within an egg, that is ingested when released into freshwater by zooplanktonic copepods, the first intermediate host, the coracidium develops into the procercoid. When copepods harboring the procercoid are ingested by the second intermediate hosts (e.g., tadpoles and frogs), the procercoid develops into a plerocercoid. The plerocercoid larva can survive for many years in various paratenic hosts, such as amphibians, reptiles, chickens, pigs, wild boars, and humans. The most common definitive hosts are felids and canids, which become infected by ingesting the second intermediate and/or paratenic hosts that plerocercoids infest. Upon infection, plerocercoid develops into adult worm in the small intestine of the definitive hosts and the adult worm can cause spirometrosis. The infestation due to adult worms is asymptomatic in most cases; however, anorexia, diarrhea, and malnutrition are occasionally observed in companion animals, and intestinal obstruction may be caused in more severe infections [12]. Extensive studies of Spirometra infections have been performed in companion animals and wildlife in Japan and the etiological agents have been identified as Spirometra erinacei [1, 16] or Spirometra erinaceieuropaei [3,4,5, 7, 11, 13,14,15, 17, 18,19,20,21] based on the morphology of adult worms and eggs found.
Molecular studies have recently revealed that two other species, Spirometra mansoni and Spirometra asiana, but not S. erinaceieuropaei, coexist in Japan [22, 23]. Spirometra asiana is a species that was first discovered in 2017 in Shimane Prefecture, southwestern Japan, and was described as a new species in 2024 [23]. Although these two species are morphologically similar, they are genetically distinguishable [22, 23]. Therefore, we preliminarily examined Spirometra infections in companion animals and mammalian wildlife by sequence analysis of mitochondrial cytochrome c oxidase subunit I gene (cox1).
Adult Spirometra worms were obtained from domestic cats (Felis silvestris catus) at animal clinics and welfare center. The parasite specimens were provided to us on the condition that the demographic information of the cats from animal hospitals would not be disclosed. No information was available on cats from animal welfare center. A specimen from a domestic dog (Canis familiaris) was given to us directly by the dog owner and the specimens from red foxes (Vulpes vulpes schrencki) were obtained from their carcasses. All parasite samples were provided as 70% ethanol-fixed specimens. We obtained 8 specimens from 8 cats, one specimen from a dog, and 2 specimens from 2 red foxes in different prefectures (Fig. 1, Table 1). Preparation of genomic DNA, PCR amplification of the complete cox1, and DNA sequencing were performed according to previously reported method [22]. Molecular phylogenetic analysis was performed using the maximum likelihood algorithm with Hasegawa-Kishino-Yano + G + I as the best substitution model implemented and bootstrap values (>90% in 1,000 replications) were calculated using MEGA X [10]. The evolutionary divergence between cox1 sequences was estimated using the maximum composite likelihood with a substitution model (d) implemented in MEGA X [10]. Dibothriocephalus nihonkaiensis was used as an outgroup.
Fig. 1.
Map showing localities (cities) where adult worms of Spirometra species were collected. Silhouettes indicate animal hosts from which Spirometra specimens were collected.
Table 1. Adult Spirometra worms examined in the present study.
| Sample no. | Animal hosts (age/sex/lifestyle) | Localities | Accession numbers for cox1 |
|---|---|---|---|
| 1 | Domestic cat (not available) | Fukui City, Fukui Pref. | LC738766 |
| 2 | Domestic cat (not available) | Tarumizu City, Kagoshima Pref. | LC738767 |
| 3 | Domestic cat (not available) | Katsuyama City, Fukui Pref. | LC738768 |
| 4 | Domestic cat (not available) | Katsuyama City, Fukui Pref. | LC738769 |
| 5 | Domestic cat (not available) | Shikokuchuo City, Ehime Pref. | LC738770 |
| 6 | Domestic cat (not available) | Fukui City, Fukui Pref. | LC738771 |
| 7 | Domestic cat (not available) | Fukui City, Fukui Pref. | LC738772 |
| 8 | Domestic cat (not available) | Kitami City, Hokkaido | LC738773 |
| 9 | Domestic dog (female, 8-year-old, asymptomatic) | Hamada City, Shimane Pref. | LC738774 |
| 10 | Red fox (unknown) | Sapporo City, Hokkaido | LC738896 |
| 11 | Red fox (unknown) | Sapporo City, Hokkaido | LC738897 |
The Spirometra specimens examined in this study showed remarkable intraspecific variations (evolutionary divergence, d=0.0006‒0.0343), but formed a monophyletic clade, and thus, all were identified as S. mansoni (Fig. 2). All cox1 sequence data reported in this study have been deposited in DDBJ under accession numbers (LC738766‒LC738897).
Fig. 2.
Maximum likelihood tree inferred from the complete cox1 sequences of Spirometra mansoni, Spirometra asiana, and the related species with accession numbers, prefecture/province and country names. Silhouettes indicate animal hosts from which Spirometra specimens were collected. Scale bar indicates the number of nucleotide substitutions/site.
This result suggests that S. mansoni commonly infect companion animals and mammalian wildlife, although the number examined is too small for the data to be concluded. It is known that S. mansoni is widely distributed in Asia [8, 22, 23] and S. mansoni infections in cats and dogs have also been reported in China [2] and South Korea [6]. The etiological agents in Chinese and Korean reports were identified due to morphology and molecular diagnosis, respectively. On the other hand, infection cases due to S. asiana have been confirmed in dogs and wild boars in Shimane Prefecture [22, 23] and human in Kochi Prefecture [9], respectively, in Japan. So far, S. asiana has been found in South Korea and Tanzania outside of Japan [23].
Spirometra mansoni and/or S. asiana cause Spirometra infection/spirometrosis in companion animals and wildlife in Japan [22, 23]. Despite these parasites being important pathogens in veterinary public health, there are only a few cases of molecular analysis of Spirometra infections in companion animals and wildlife in Japan [13, the present study]. The reason may be that S. erinaceieuropaei has been long claimed as the only species causing Spirometra infections; thus, there was no need for molecular identification. The cox1 sequence of Spirometra worm collected from a dog in Sapporo was reportedly from S. erinaceieuropaei, which was reasonable at the time [13]. However, the sequence exactly corresponds to that of S. mansoni according to the latest taxonomy.
Although it is morphologically difficult to differentiate between S. mansoni and S. asiana, it has become clear that molecular diagnosis is useful. However, molecular diagnosis is not performed in animal clinics. Therefore, it is recommended to discriminate between these species at parasitology-related research institutions to accumulate information on the distribution, clinical symptoms, infection risks, and epizoology for each species, including performing retrospective analysis, if Spirometra specimens are available.
In conclusion, veterinary professionals, pet owners, gibier-related businesses, and hunters should consider that Spirometra infections in companion animals and wildlife are caused by S. mansoni and/or S. asiana in Japan.
CONFLICTS OF INTEREST STATEMENT
The authors declare that no conflict of interest exists.
Acknowledgments
The authors thank veterinarians, Kyoko Ideishi (Tarumizu City, Kagoshima Prefecture, Japan), Takao Irie (Faculty of Veterinary Medicine, Miyazaki University, Miyazaki Prefecture, Japan), Hajime Kawaguchi (Katsuyama City, Fukui Prefecture, Japan), Hiroyuki Tabuchi (Kitami City, Hokkaido, Japan), Takehiro Uno (Shikokuchou City, Ehime Prefecture, Japan), Fukui Animal Protection Supervision Center (Fukui City, Fukui Prefecture, Japan), and local collaborators, Takashi Imada and Kayoko Inazawa (Hamada City, Shimane Prefecture, Japan) for providing the parasite specimens. This work was supported in parts by grants from the Ministry of Health, Labour and Welfare, Japan (grant no. 21KA1003 to HS) and the Japan Agency for Medical Research and Development (grant no. JP23fk0108639 to YM).
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