New host record of Thelazia callipaeda (Nematoda: Spirurida) with a notably wide host range and shared zoonotic lineage in Japan.

Thelazia callipaeda (Nematoda: Spirurida: Thelaziidae) parasitizes the eyes of dogs, cats, humans, and various wild mammals, and is transmitted by drosophilid flies. In Japan, T. callipaeda is considered an emerging parasite that has expanded its endemic region northward. However, reports of its detection in mammals other than domestic animals and humans are scarce. This study reports the detection of T. callipaeda in Japanese red fox (Vulpes vulpes japonica), masked palm civet (Paguma larvata), Japanese badger (Meles anakuma), Japanese black bear (Ursus thibetanus japonicus), raccoon (Procyon lotor), Japanese raccoon dog (Nyctereutes viverrinus), domestic dog (Canis lupus familiaris), domestic cat (Felis silvestris catus), and human. Of these, the Japanese red fox, masked palm civet, Japanese badger, and Japanese black bear have been reported as novel host records. Sequence analysis of the cytochrome c oxidase subunit I gene of T. callipaeda revealed two unique lineages specific to Japan, with no regional or host species differences. These results suggest a wide host range for T. callipaeda, highlighting the significant role of wildlife as a reservoir for this parasite in Japan.

Aberrant sparganosis in cat caused by Spirometra mansoni (Cestoda: Diphyllobothriidae): a case report.

BACKGROUND: Sparganosis is a rare zoonotic disease caused by plerocercoid larvae of the genera Spirometra or Sparganum (Cestoda: Diphyllobothriidae). The larvae of Spirometra generally do not undergo asexual reproduction, whereas those of Sparganum can induce proliferative lesions in infected tissues. This paper presents an unusual case of proliferative sparganosis due to infection with Spirometra mansoni in a cat, normally considered a definitive host of the species. CASE PRESENTATION: A 9-year-old male domestic cat was presented with a mass on the right side of the face that underwent progressive enlargement for 1 month. The morphological and histopathological examinations revealed multiple asexual proliferative cestode larvae in the lesions, suggestive of proliferative sparganosis. Next-generation sequencing analysis of formalin-fixed and paraffin-embedded specimens of surgically excised tissue indicated that the worm was Spirometra mansoni. CONCLUSION: Although S. mansoni a common tapeworm species found in the small intestine of domestic cats and dogs in Japan, proliferative sparganosis is extremely rare. This is the first confirmed case of proliferative sparganosis due to infection with S. mansoni in cat.

A new species of Demodex (Acari: Demodecidae) from the skin of golden-handed tamarins, Saguinus midas (Primates: Cebidae).

Two captive-bred golden-handed tamarins, Saguinus midas L., 1758 (Primates: Cebidae), kept in households in Japan, presented with psoriasis-like plaques on their faces, along with scale, alopecia, and itching. Histopathological examination revealed numerous Demodex mites in the hair follicles, and the clinical symptoms in both cases improved after treatment with fluralaner. Based on the morphological and genetic characteristics of the mites collected from tamarins, we describe a new species of Demodex. This new species is the fifth valid Demodex species recorded from primates.

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.

Molecular characterization of oriental eyeworm (Thelazia callipaeda) detected from raccoon (Procyon lotor) and Japanese raccoon dog (Nyctereutes viverrinus) in Kanto region, Japan.

BACKGROUND: The oriental eyeworm Thelazia callipaeda (Spirurida: Thelaziidae) is an emerging parasitic ocular nematode of carnivores and humans. In domestic animals and humans, the infection causes varying degrees of inflammation and lacrimation, and wild carnivores represent an important reservoir. In this study we examined the infection status and molecular characterization of T. callipaeda in two urban carnivores, raccoons Procyon lotor and wild Japanese raccoon dogs Nyctereutes viverrinus, in the Kanto region of Japan. METHODS: From January 2020 to December 2021, 193 carcasses including 178 raccoons and 15 raccoon dogs were examined for the presence of worms in the eye. The worms from infected animals (one worm per host) were morphologically identified as T. callipaeda. Worms (1-5 worms per host) were subjected to genetic analysis using mitochondrial cytochrome c oxidase subunit I gene sequences. RESULTS: The prevalence of T. callipaeda in raccoons and Japanese raccoon dogs was 20.2% (36/178) and 13.3% (2/15), respectively. The cox1 sequences from 56 worms from 38 animals revealed three haplotypes (h9, h10, and h12). Analysis of multiple worms for five raccoons showed co-infection of two different haplotypes (h9 and h10) in a single host. Comparing our data with published sequences, three sequences obtained from raccoons and raccoon dogs shared the same haplotypes as those reported in humans, dogs, and cats in Japan. CONCLUSIONS: Our findings show a high prevalence of T. callipaeda in raccoons, suggesting that this invasive carnivore species serves as an important natural reservoir of T. callipaeda in the Kanto region of Japan, an area with the highest human population of the country.

Detection and characterization of Hyaloklossia kasumiensis (Apicomplexa: Sarcocystidae) from Nagoya Daruma pond frog, Pelophylax porosus brevipodus.

The Nagoya Daruma pond frog (Pelophylax porosus brevipodus) is an endangered anuran species endemic to the semi-aquatic environment of Japan. On June 11, 2021, four of the six Nagoya Daruma pond frogs captured from the wild and kept in an aquarium died in rapid succession. Pathological and/or parasitological examinations of two of the four carcasses revealed protozoan infection in their kidneys. The pathogen identified as Hyaloklossia kasumiensis (Apicomplexa: Sarcocystidae: Hyaloklossiinae) based on their morphological and molecular characterization. This is a new host record of H. kasumiensis. Mitochondrial cox1 sequence analysis suggest that the present species detected from Nagoya Daruma pond frog belongs to the same lineage as those found in the Tokyo Daruma pond frog (P. p. porosus) from Ibaraki Prefecture, Japan. This particular lineage may be prevalent among Pelophylax porosus ssp. distributed in Japan.

Three new species of Eimeria (Apicomplexa: Eimeriidae) from the Amami rabbit, Pentalagus furnessi (Mammalia: Leporidae).

The Amami rabbit, Pentalagus furnessi (Mammalia: Lagomorpha: Leporidae), is a relict and endangered species endemic to the Amami-Oshima and Tokunoshima Islands, located in southwestern Japan. Here, we described three new species of Eimeria (Apicomplexa: Eimeriidae) parasites detected from fecal samples of wild Amami rabbits. Eimeria furnessi n. sp., recorded in 21 (58.3%) samples, has ellipsoidal oocysts with two walls and micropyle, 26.0 × 16.6 µm, and elongate-ovoidal sporocysts, 13.1 × 6.3 µm, with Stieda body. Eimeria hilleri n. sp., recorded in 9 (25.0%) samples, has ellipsoidal oocysts with two walls and micropyle, 34.7 × 21.4 µm, and elongate-fusiform to elongate-ovoidal sporocysts, 15.7 × 8.3 µm, with Stieda and substieda bodies. Eimeria sagentae n. sp., recorded in 13 (36.1%) samples, has ellipsoidal oocysts with two walls and micropyle, 20.9 × 14.5 µm, and elongate-ovoidal sporocysts, 10.4 × 5.0 µm, with Stieda body. The three new species can be distinguished by the size and color of their oocysts. Further studies related to the pathogenicity of these parasites can improve the breeding and propagation procedures of the Amami rabbit.

Black-spotted pond frog Pelophylax nigromaculatus as a new host for the renal coccidian genus Hyaloklossia (Alveolata: Apicomplexa).

Hyaloklossia Labbé, 1896 (Apicomplexa: Sarcocystidae) is a renal coccidium that infects anuran species. The genus consists of two species: H. lieberkuehni, recorded from Pelophylax kl. esculentus, Pelophylax ridibundus, and Rana temporaria in Europe; and H. kasumiensis, recorded from Pelophylax porosus porosus in Japan. However, there have been no reports of Hyaloklossia in the other anurans in Japan. On June 2021, we examined a total of 58 adult frogs comprising 2 P. p. porosus, 23 Pelophylax nigromaculatus, 8 Rana japonica, 3 Glandirana rugosa (Ranidae), 13 Fejervarya kawamurai (Dicroglossidae), and 9 Buergeria buergeri (Rhacophoridae) for infection by Hyaloklossia. Microscopic examination of kidney tissues revealed a high infection incidence of 47.8% (11/23) in P. nigromaculatus, but the other frog species were negative for Hyaloklossia. Morphological and molecular analyses using nuclear ribosomal and mitochondrial genes confirmed the infective species as H. kasumiensis. This is a new host record for H. kasumiensis.

Subcutaneous cysticercosis due to Taenia crassiceps (Cestoda: Taeniidae) in an imported steppe lemming in Japan.

This study describes a subcutaneous proliferative cysticercosis in a pet steppe lemming, Lagurus lagurus (Rodentia: Cricetidae), bred and imported from Czech Republic into Japan. Numerous metacestodes were collected from the subcutaneous cystic lesion of the left medial thigh. Four surgical removals were coupled with anthelmintic treatment but ended with recurrence. Based on morphological features and mitochondrial DNA sequences, the metacestodes were identified as the larval stage of Taenia crassiceps (Zeder, 1800). This is the first case of infection with larval T. crassiceps in rodents of the genus Lagurus, and becomes the third case of the parasite detected from imported animals in Japan. Related public health concerns are discussed.

Genetic characterization of Trichomonas gallinae (Rivolta, 1878) in companion birds in Japan and the genotypical relationship in the Asia region.

BACKGROUND/PURPOSE: Avian trichomonosis is a parasitic infection that affects a wide range of avian species, including free-ranging and pet birds worldwide, and Trichomonas gallinae has been considered as the only causative agent for decades. The sequence of the 5.8S ribosomal RNA with internal transcribed spacer (ITS) regions was widely used for identifying genotypes and determining inter-specific and intra-specific diversity. Moreover, the sequence of Fe-hydrogenase (FeHyd) was proposed as the second genetic marker for providing improved resolution of strain subtyping discrimination. Though the correlation between genetic variability and strain virulence is controversial, FeHyd analyses seemed to be useful to investigate the host or geographic origin of isolates. This study aimed to investigate the genetic characteristics of avian Trichomonas spp. METHODS: Forty-seven oral swabs and crop lavage fluids were collected from 9 avian genera, which were diagnosed as Trichomonas-positive by microscopy in animal hospitals in Japan, were analyzed. RESULTS: Genetic analysis of clonal isolates revealed the prevalence of the single genotype, ITS-OBT-Tg-1, by ITS region analysis, while two different subtypes, A2 and novel A3, were suggested by FeHyd gene analysis among Japanese companion birds. Phylogenetic analyses of available ITS sequences obtained from the Asia region (China, Iran, Iraq, and Saudi Arabia) were also preformed, revealing endemic ITS-OBT-Tg-1, ITS-OBT-Tg-2, ITS-OBT-Ttl-1, genotype III, and Saudi Arabia's unique lineages. Furthermore, ITS-OBT-Tg-2 predominance in these countries indicates different strains origination from Japan. CONCLUSION: This is the first report of the genetic characterization of T. gallinae in Japan with discovery of novel subtype A3.

Haemoproteus columbae infection in a straggler racing pigeon sheltered in Japan.

A racing pigeon (Columba livia var. domestica), a straggler from Taiwan, was sheltered in Nara Prefecture, Japan in 2020. This pigeon showed hemolysis and elevated levels of hepatobiliary and muscle enzymes. Gametocytes of Haemoproteus columbae (Apicomplexa: Haemosporida) were observed within the host erythrocytes in thin blood smears. A partial sequence of the mitochondrial cytochrome b gene amplified from blood DNA was identical to the lineage HAECOL1 previously reported from pigeons worldwide. This is the first record of H. columbae infection in a sheltered bird in Japan.

Hyaloklossia Labbé, 1896 (Alveolata: Apicomplexa) in frogs: Description of a new species and proposing a new subfamily to accommodate these enigmatic parasites.

Hyaloklossia Labbé ,1896 (Alveolata: Apicomplexa) is a monotypic genus of renal coccidia found in anurans, particularly in the edible frog Pelophylax kl. esculentus (Amphibia: Anura: Ranidae), distributed in different parts of Europe. Here we propose a new Hyaloklossia species from the Tokyo daruma pond frog, Pelophylax porosus porosus. The coccidium detected in the renal tissue of P. p. porosus shared some morphological characteristics with the type species, Hyaloklossia lieberkuehni (Labbé, 1894), reported from P. kl. esculentus. However, in addition to size differences in several oocyst and sporocyst features between these parasites, phylogenetic analysis of gene fragments from two nuclear ribosomal loci and the mitochondrial cytochrome c oxidase subunit 1, exposed distinct genetic differences between H. lieberkuehni and our new species. Although our analysis validated the monophyly of Hyaloklossia with some members of the Toxoplasmatinae Biocca, 1957, Cystoisosporinae Frenkel et al., 1987, and Eumonosporinae Chou et al., 2021 (Sarcocystidae Poche, 1913), comparison of genetic differences between Hyaloklossia species from P. p. porosus and H. lieberkuehni revealed the presence of a greater number of polymorphisms than that observed when comparing inter-species (Heydornia spp., Besnoisita spp.) or inter-genus (Toxoplasma vs. Neospora, Neospora vs. Hammondia, and Neospora vs. Heydornia) variabilities among members of the Sarcocystidae. This indicates that Hyaloklossia, as re-erected and defined by Modrý et al. (2001, Int. J. Syst. Evol. Microbiol. 51, 767-772), with its homoxenous life cycle, requires placement in its own subfamily. Thus, we propose a new subfamily, Hyaloklossiinae n. subfam., to accommodate two species, H. lieberkuehni from Europe and Hyaloklossia kasumienesis n. sp. which we describe here from P. p. porosus in Japan.

Theileria infection with severe anemia and unhealed fracture in a sika deer Cervus nippon aplodontus (Cervidae: Cetartiodactyla).

An adult female sika deer (Cervus nippon aplodontus) inhabiting Nara Park, Nara, Japan, had broken bone injuries from a car accident. During its treatment, we found that the sika deer had severe anemia and the fracture remained unhealed throughout. Peripheral blood smear revealed piroplasms in the erythrocytes, which were identified as merozoites of undescribed Theileria species, widely found in sika deer in Japan. This is the report of a clinical case of Theileria infection, accompanied by severe anemia in a sika deer.

Detection of Eumonospora henryae (Apicomplexa: Sarcocystidae) from Falco columbarius (Falconiformes: Aves): Comparison of host-parasite phylogram and comments on the family Sarcocystidae Poche, 1913.

The genus Eumonospora Allen, 1933 (Apicomplexa: Sarcocystidae), an avian coccidia, is characterized by monosporocystic and octasporozoic oocysts without Stieda and substieda bodies. Some members of Eumonospora, which infect several raptor species, exhibit high levels of pathogenicity, making eumonosporiosis the leading cause of death in captive-bred raptors. The host specificity of these species appears to be mesostenoxenous, as evidenced by unsuccessful transmission between different orders of avian hosts. However, several studies have detected Eumonospora spp. in taxonomically distant avian hosts, indicating that some of these species may be euryxenous. In the current study, diarrheic fecal examination of a captive-bred juvenile merlin (Falconiformes: Aves) in Tokyo, Japan, was conducted, and a large number of oocysts were morphologically and molecularly identified as E. henryae (Yakimoff and Matschulsky, 1932), a coccidia species reported only in Strigiformes. This is a new recorded host for this coccidia. Phylogenetic analyses via Bayesian inference and maximum likelihood methods using concatenated genomic datasets consisting of nuclear 18S rDNA, nuclear 28S rDNA and mitochondrial cytochrome C oxidase subunit 1 gene, revealed a well-supported monophyletic clade of Eumonospora spp. belonging to the family Sarcocystidae Poche 1913, which largely corresponded to the avian host phylogram. Therefore, based on distinguishable oocyst morphology, a new subfamily, Eumonosporinae, within the family Sarcocystidae, is proposed, and a reconsideration of the definition of Sarcocystidae is suggested. Further molecular characterization of this emerging pathogen, as well as clarification of its complete life cycle, including cyst-forming ability, is required for more appropriate generic assessment.

Three new Prototapirella species, Opisthotrichum janus, and Troglocorys cava add to Entodiniomorphida (Ciliophora, Trichostomatia) diversity in mountain gorillas in Rwanda.

Recently, a high number of Entodiniomorphida species was discovered in Virunga mountain gorillas (Gorilla beringei beringei) in Rwanda compared to other primates. Thirteen species of five genera (Troglocorys, Gorilloflasca, Prototapirella, Troglodytella, and Opisthotrichum) were identified with 10 species in Gorilloflasca, Prototapirella, and Troglodytella being host-specific, to our current knowledge. The remaining three species have been described for other herbivorous mammals; Prototapirella gorillae for western lowland gorillas; Troglocorys cava for western lowland gorillas, chimpanzees, and guinea baboons; Opisthotrichum janus in the family Ophryoscolecidae is regarded as specific to African antelopes. Here, we show the first indication that wild herbivorous hindgut fermenting mammals might have rumen ophryoscolecids in the intestine sympatric with their native intestinal ciliates. We also describe three new Prototapirella species, P. sabyinyo, P. sulcata, and P. curiosa. The somatic ciliary zones of the three new species are atypical of Prototapirella while their buccal infraciliature is not different from that of other Prototapirella species.

First record of infestation of a pet sloth in Japan with the exotic tick Amblyomma geayi (Acari: Ixodidae).

On December 11, 2018, a single unengorged adult tick was found on the body surface of the trunk of an imported wild-caught Linnaeus's two-toed sloth (Choloepus didactylus) during a routine health check in an animal clinic in Tokyo, Japan. The tick was identified as Amblyomma geayi based on the morphological and molecular characteristics. This is the first case of the introduction of an Amblyomma species to Japan via an imported pet sloth. The present study highlights the current loopholes in Japan's regulatory system for animal imports.

Resurrection of the genus Eumonospora (Apicomplexa: Sarcocystidae) for Caryospora species without Stieda body.

The coccidian genus Eumonospora Allen, 1933 is re-established. Despite morphological features and host preference among species, coccidian with octasporozoic and monosporocystic oocysts are traditionally consider to belonging in the genus Caryospora Léger, 1904 (Apicomplexa: Eimeriidae). Recently, the genus Avispora Schuster et al., 2016 was proposed for above caryosporoids parasitizing birds based on combined morphological and phylogenetic analyses. However, diagnostic morphological characters of the genus Avispora, the absence of Stieda and substieda bodies, has already been mentioned in the description of the genus Eumonospora Allen, 1933 (Apicomplexa: Sarcocystidae), and thus Avispora is considered to be a junior synonym of Eumonospora. In this study, caryosporoid coccidians were detected from five owl species; Bubo scandiacus, Ptilopsis leucotis, Athene noctua, Strix nebulosa, and Pulsatrix perspicillata (Strigiformes: Strigidae) and identified as Avispora henryae (Yakimoff & Matikaschwaili, 1932) described from Bubo bubo (Strigiformes: Strigidae). Eumonospora henryae (Yakimoff & Matikaschwili, 1932) comb. nov. is redescribed for this species based not only on morphological features but also on phylogenetical analyses. The key of the genus Eumonospora and a list to the species known at present are also provided.

Detection and molecular characterization of Cryptosporidium species in wild-caught pet spiny-tailed lizards.

Uromastyx is a genus of the herbivorous agamid lizards, also known as spiny-tailed lizards or mastigures, which are found in parts of Africa and the Middle East. Currently, several species of this genus are available in the international pet trade in Japan. In this study, two imported wild-caught spiny-tailed lizards (Arabian blue mastigure, Uromastyx ornata philbyi, and Sudan mastigure, Uromastyx dispar flavifasciata) were diagnosed with a Cryptosporidium (Apicomplexa: Cryptosporidiidae) infection based on the presence of the oocysts in the rectal feces using sucrose flotation and light microscopy examination at a local animal hospital in Tokyo, Japan. One of the lizards had died, and histopathological examination revealed enteritis with the Cryptosporidium parasite. Sequence analyses using the small subunit ribosomal RNA, actin, and 70-kDa heat shock protein genes indicated that the lizards had contracted a novel variant of C. avium that commonly infects avian species.

Alien parasitic infections in the endangered Ryukyu long-furred rat (Diplothrix legata) on Amami-Oshima Island, Japan.

There is limited knowledge about parasites of the endangered Ryukyu long-furred rat, Diplothrix legata (Murinae, Rodentia) endemic to Okinawa, Tokunoshima, and Amami-Oshima Islands in Japan. In the present study, postmortem histopathological examination of an individual found on Amami-Oshima Island revealed a mixed helminth infection of Calodium hepaticum, Hydatigera taeniaeformis, and Angiostrongylus cantonensis. These helminths are considered non-native to Amami-Oshima Island and are maintained by invasive mammals, such as non-native rats and outdoor cats. This observation presents a new host record for C. hepaticum and H. taeniaeformis and the first record of A. cantonensis in Ryukyu long-furred rat on Amami-Oshima Island.

Neospora caninum antigens displaying virus-like particles as a bivalent vaccine candidate against neosporosis.

Neospora caninum is a causative and transmissible agent of dog and bovine neosporosis. The resulting reproductive failures in infected cattle lead to significant economic losses worldwide. However, there is no satisfactory treatment or vaccine currently available to combat this pathogen. Thus, the development of appropriate vaccines to manage its infection and transmission is urgently needed. In this study, we expressed Rous sarcoma virus-like particles (RSV-LP) that displayed dual N. caninum antigens in silkworms. The antigen candidates are modified by adding a transmembrane domain of GP64 protein from Bombyx mori nucleopolyhedrovirus (BmNPV) to the C-terminus of surface antigen 1 (NcSAG1) and SAG1-related sequence 2 (NcSRS2). The NcSRS2 alone or the NcSAG1/NcSRS2 bivalent form displaying RSV-LPs were purified using sucrose density gradient centrifugation. These purified VLPs were then used for immunizations in gerbils, Meriones unguiculatus, to evaluate the anti-N. caninum effects in vivo. The results demonstrated that antigens displaying RSV-LPs in immunized gerbils produced the antigen-specific antibody, leading to a relatively lower parasite load after infections of N. caninum. To the best of our knowledge, this is the first study to present an RSV-LP vaccine displaying bivalent antigens from neosporosis. Taken together, our strategy suggests that silkworm-expressed virus-like particles (VLPs) are promising bivalent vaccine candidates against N. caninum infections.