Far-East Asian Toxoplasma isolates share ancestry with North and South/Central American recombinant lineages.

Toxoplasma gondii is a global protozoan pathogen. Clonal lineages predominate in Europe, North America, Africa, and China, whereas highly recombinant parasites are endemic in South/Central America. Far East Asian T. gondii isolates are not included in current global population genetic structure analyses at WGS resolution. Here we report a genome-wide population study that compared eight Japanese and two Chinese isolates against representative worldwide T. gondii genomes using POPSICLE, a novel population structure analyzing software. Also included were 7 genomes resurrected from non-viable isolates by target enrichment sequencing. Visualization of the genome structure by POPSICLE shows a mixture of Chinese haplogroup (HG) 13 haploblocks introgressed within the genomes of Japanese HG2 and North American HG12. Furthermore, two ancestral lineages were identified in the Japanese strains; one lineage shares a common ancestor with HG11 found in both Japanese strains and North American HG12. The other ancestral lineage, found in T. gondii isolates from a small island in Japan, is admixed with genetically diversified South/Central American strains. Taken together, this study suggests multiple ancestral links between Far East Asian and American T. gondii strains and provides insight into the transmission history of this cosmopolitan organism.

Searching for new molecules involved in Anopheles mosquitoes' response to Plasmodium infection.

Plasmodium parasites within mosquitoes are exposed to various physiological processes, such as blood meal digestion activity, the gonotrophic cycle, and host responses preventing the entry of parasites into the midgut wall. However, when in vitro-cultured ookinetes are injected into the hemocoel of mosquitoes, Plasmodium parasites are not affected by the vertebrate host's blood contents and do not pass through the midgut epithelial cells. This infection method might aid in identifying mosquito-derived factors affecting Plasmodium development within mosquitoes. This study investigated novel mosquito-derived molecules related to parasite development in Anopheles mosquitoes. We injected in vitro-cultured Plasmodium berghei (ANKA strain) ookinetes into female and male Anopheles stephensi (STE2 strain) mosquitoes and found that the oocyst number was significantly higher in males than in females, suggesting that male mosquitoes better support the development of parasites. Next, RNA-seq analysis was performed on the injected female and male mosquitoes to identify genes exhibiting changes in expression. Five genes with different expression patterns between sexes and greatest expression changes were identified as being potentially associated with Plasmodium infection. Two of the five genes also showed expression changes with infection by blood-feeding, indicating that these genes could affect the development of Plasmodium parasites in mosquitoes.

Molecular phylogenetic analysis of Echinococcus multilocularis from horses raised in Canada or Japan, using mitochondrial cytochrome b gene-targeted PCR.

Alveolar echinococcosis is a zoonotic disease caused by a larval-stage Echinococcus multilocularis infection. Geographical haplotyping targeting the parasite's mitochondrial cytochrome b (cob) gene has been reported for isolates from definitive and intermediate hosts (wild canids and rodents); however, there are limited reports on strain typing for the dead-end host, the horse, which could act as a sentinel for E. multilocularis. Accordingly, we investigated the diversity of E. multilocularis in isolates obtained from slaughtered Japanese and Canadian horses originating from the Iburi and Hidaka regions in Hokkaido and from Alberta, respectively, with PCR and haplogroup analyses targeting cob gene sequences obtained. Seventy horses were diagnosed with alveolar echinococcosis based on histopathology and cob-gene PCR testing. The E. multilocularis detected in these horses was classified as either an Asian (for Hokkaido-raised horses) or a European (for Alberta-raised horses) haplogroup, based on the obtained cob-gene sequence analysis. In addition, haplotype network analysis revealed that E. multilocularis isolated from Hokkaido-raised horses is highly homologous to Kazakhstan isolates, and E. multilocularis isolated from Alberta-raised horses is highly homologous to Austrian isolates. The results of this study suggest that cob-gene-targeted PCR analysis could be useful for the geographical genetic characterization of E. multilocularis isolated from horses.

Detection and molecular analysis of Philopinna higai (Digenea: Didymozoidae) from domestically introduced Sarcocheilichthys fishes in the Tohoku region, Japan.

Philopinna higai is a species of Didymozoidae (Digenea: Hemiuroidea). The definitive hosts of this parasite only belong to the fish genus Sarcocheilichthys. Sarcocheilichthys fishes are endemic to Lake Biwa and southwestern Japan and were introduced into the northeastern (Tohoku) region. However, P. higai parasitism has not been investigated in the Tohoku region. In this study, we surveyed the distribution of P. higai in the Tohoku region and sequenced 28S rDNA (994 bp) and cytochrome oxidase subunit 1 (CO1) gene (721 bp) of P. higai. We also sequenced mitochondrial cytochrome b (581 bp) of Sarcocheilichthys fishes from the Tohoku region and Lake Biwa. Our findings confirmed the distribution of P. higai in all seven surveyed river systems in the four prefectures of the Tohoku region. The 28S rDNA sequence of P. higai did not differ among regions, whereas 10 haplotypes of CO1 were identified and clustered into two major clades. The haplotypes of Sarcocheilichthys fishes introduced in the Tohoku region were identical to the dominant haplotypes in Lake Biwa. Thus, P. higai from Lake Biwa and the Tohoku region were genetically the same species, although genetically differentiated populations formed in the Tohoku region.

Formation of free oocysts in Anopheles mosquitoes injected with Plasmodium ookinetes.

Malaria needs new strategies for its control. Plasmodium spp., the causative agent of malaria, is transmitted by mosquitoes. These parasites develop into oocysts and sporozoites in the body of the mosquitoes. A deeper understanding of oocysts that produce the infectious form of the parasite, sporozoites, can facilitate the development of novel countermeasures. However, the isolation of Plasmodium oocysts is challenging as these are formed between midgut epithelial cells and basal lamina after gametocytes enter the mosquito's body through blood feeding. Further research on oocysts has been impeded by issues related to oocyst isolation. Therefore, in this study, we injected Plasmodium into mosquitoes-an artificial and unique method-and aimed to clarify how oocysts were formed in mosquitoes after Plasmodium injection and whether free oocysts were formed from the mosquito tissue. Plasmodium berghei (ANKA strain) ookinetes cultured in vitro were injected into the thoracic body cavity (hemocoel) of female and male Anopheles stephensi mosquitoes. Oocysts were formed in the body of female and male mosquitoes at 14 days post injection. In addition, oocysts formed as a result of injection developed into sporozoites, which were infectious to mice. These findings suggest that P. berghei can complete its developmental stage in mosquitoes by injection. Some of the oocysts formed were free from mosquito tissue, and it was possible to collect oocysts with minimal contamination of mosquito tissue. These free oocysts can be used for investigating oocyst proteins and metabolism.

Molecular detection of Lotmaria passim in honeybees in Japan.

Crithidia mellificae (C. mellificae) and Lotmaria passim (L. passim) are trypanosomatids that infect Apis mellifera. We analyzed the prevalence of C. mellificae and L. passim in six regions of Japan from 2018 to 2019. The detection rate of C. mellificae was 0.0% in all regions, whereas L. passim was detected in 16.7%-66.7% of the honeybees. L. passim was detected at a significantly lower rate in the Cyugoku-Shikoku region than in other regions. Furthermore, phylogenetic analysis of the internal transcribed spacer 1 (ITS1) locus of related species was performed. All the samples in this study could be assigned to the L. passim clade. This study reveals that L. passim infection is predominantly prevalent in Japan. Further epidemiological surveys are needed to clarify the prevalence of C. mellificae infection in honeybees in Japan.

Dermocystid infection in Japanese fire-bellied newt, Cynops pyrrhogaster.

Here, we report details of a new infectious disease in wild-caught Japanese fire-bellied newts (Cynops pyrrhogaster), a Near Threatened species. Skin lesions consisting of numerous masses were found in the animals near Lake Biwa, Shiga Prefecture, Japan. The gross appearance of the skin lesions showed blister-, cyst-, and/or tumor-like morphology. Various sizes of skin lesions were observed on their entire body surface. Histologically, spherical basophilic cysts, including numerous spores, were observed in the dermis layer. Ultrastructural analysis indicated the presence of main bodies of flagellated zoospores within the spores. While 18s rRNA gene sequencing indicated that the skin lesions were due to dermocystid infection. To our knowledge, this is the first report of dermocystid infection in this amphibian in Japan. Further studies are needed to prevent epidemics and to establish diagnostic and treatment methods.

Plasmodium berghei Brca2 is required for normal development and differentiation in mice and mosquitoes.

BACKGROUND: Malaria is a major global parasitic disease caused by species of the genus Plasmodium. Zygotes of Plasmodium spp. undergo meiosis and develop into tetraploid ookinetes, which differentiate into oocysts that undergo sporogony. Homologous recombination (HR) occurs during meiosis and introduces genetic variation. However, the mechanisms of HR in Plasmodium are unclear. In humans, the recombinases DNA repair protein Rad51 homolog 1 (Rad51) and DNA meiotic recombinase 1 (Dmc1) are required for HR and are regulated by breast cancer susceptibility protein 2 (BRCA2). Most eukaryotes harbor BRCA2 homologs. Nevertheless, these have not been reported for Plasmodium. METHODS: A Brca2 candidate was salvaged from a database to identify Brca2 homologs in Plasmodium. To confirm that the candidate protein was Brca2, interaction activity between Plasmodium berghei (Pb) Brca2 (PbBrca2) and Rad51 (PbRad51) was investigated using a mammalian two-hybrid assay. To elucidate the functions of PbBrca2, PbBrca2 was knocked out and parasite proliferation and differentiation were assessed in mice and mosquitoes. Transmission electron microscopy was used to identify sporogony. RESULTS: The candidate protein was conserved among Plasmodium species, and it was indicated that it harbors critical BRCA2 domains including BRC repeats, tower, and oligonucleotide/oligosaccharide-binding-fold domains. The P. berghei BRC repeats interacted with PbRad51. Hence, the candidate was considered a Brca2 homolog. PbBrca2 knockout parasites were associated with reduced parasitemia with increased ring stage and decreased trophozoite stage counts, gametocytemia, female gametocyte ratio, oocyst number, and ookinete development in both mice and mosquitoes. Nevertheless, the morphology of the blood stages in mice and the ookinete stage was comparable to those of the wild type parasites. Transmission electron microscopy results showed that sporogony never progressed in Brca2-knockout parasites. CONCLUSIONS: Brca2 is implicated in nearly all Plasmodium life cycle stages, and especially in sporogony. PbBrca2 contributes to HR during meiosis.

Morphological and molecular phylogenetical identification of Tricodectes pinguis from Japanese black bears (Ursus thibetanus japonicus) in Aomori Prefecture, Japan.

Trichodectes pinguis, referred to commonly as the bear-biting louse, has been reported in several bear species. However, graphical (blurred or coarse) and genetic information on the louse is limited. In this study, we identified T. pinguis collected from Japanese black bears in the Aomori Prefecture, Japan. We confirmed 12S rDNA sequences derived from the collected T. pinguis and performed molecular phylogenetic analysis based on 12S rDNA. The analysis revealed the parasitic louse to be T. pinguis. Interestingly, the body size of T. pinguis found in this study was smaller than the previous recorded body size of them in Japan and Turkey. To better understand the biting louse infesting bears, morphometric and genetic information from other bear hosts needs to be accumulated.

Resolution of cryptic species complexes within the genus Metagonimus (Trematoda: Heterophyidae) in Japan, with descriptions of four new species.

A nationwide fish survey was conducted in Japan to detect metacercariae of the genus Metagonimus (Trematoda: Heterophyidae). The metacercariae were subjected to DNA barcoding for molecular species identification. A phylogeny inferred from the sequences of mitochondrial cytochrome c oxidase subunit 1 (cox1) prompted us to recognize three cryptic species complexes (i.e., the M. miyatai complex, the M. takahashii complex, and the M. katsuradai complex). Each complex included one or two undescribed species. For morphological description, adult flukes of each species were raised through the experimental infections of immunosuppressed mice. We propose M. saitoi n. sp., M. kogai n. sp., M. shimazui n. sp., and M. kinoi n. sp., based on their phylogeny, morphology, biogeography, and ecology (host-parasite relationships). The originally described species, M. miyatai, was split into M. miyatai sensu stricto and M. saitoi n. sp. The former is distributed mainly in eastern Japan and uses the sweetfish (Plecoglossus altivelis) and daces (Pseudaspius hakonensis and Ps. sachalinensis) as principal second intermediate hosts, while the latter is in western Japan and its principal fish hosts are the dark chub (Nipponocypris temminckii) and the pale chub (Opsariichthys platypus). The present survey resolves a long-standing controversy on the microtaxonomy of Metagonimus in Japan since the first discovery of Metagonimus yokogawai in 1912, and shows that 10 species of Metagonimus are still distributed in Japan, although human metagonimiasis is almost eradicated.

Identification and phylogenetic analysis of Babesia parasites in domestic dogs in Nigeria.

The present study examined the presence of Babesia parasites in 104 domestic dogs in Nigeria. Sequentially, Babesia parasites infecting domestic dogs underwent genetic and phylogenetic analyses. The results of nested PCR based on the Piroplasmida 18S rRNA gene illustrated that 13.5% (14/104) of the samples were positive. The obtained positive samples determined the nucleotide sequences of the 18S rRNA genes. In the genetic and phylogenetic analyses, four of five nucleotide sequences were similar to Babesia canis rossi, and one sample exhibited a close similarity to a Babesia sp. isolated from a raccoon in Hokkaido, Japan. The present study revealed the widespread presence of B. canis rossi among domestic dogs in Nigeria.

Role of Plasmodium berghei ookinete surface and oocyst capsule protein, a novel oocyst capsule-associated protein, in ookinete motility.

BACKGROUND: Plasmodium sp., which causes malaria, must first develop in mosquitoes before being transmitted. Upon ingesting infected blood, gametes form in the mosquito lumen, followed by fertilization and differentiation of the resulting zygotes into motile ookinetes. Within 24 h of blood ingestion, these ookinetes traverse mosquito epithelial cells and lodge below the midgut basal lamina, where they differentiate into sessile oocysts that are protected by a capsule. METHODS: We identified an ookinete surface and oocyst capsule protein (OSCP) that is involved in ookinete motility as well as oocyst capsule formation. RESULTS: We found that knockout of OSCP in parasite decreases ookinete gliding motility and gradually reduces the number of oocysts. On day 15 after blood ingestion, the oocyst wall was significantly thinner. Moreover, adding anti-OSCP antibodies decreased the gliding speed of wild-type ookinetes in vitro. Adding anti-OSCP antibodies to an infected blood meal also resulted in decreased oocyst formation. CONCLUSION: These findings may be useful for the development of a transmission-blocking tool for malaria.

Raccoon dogs (Nyctereutes procyonoides) are the new natural definitive hosts of Metagonimus hakubaensis.

The definitive hosts of Metagonimus hakubaensis are reported to be hamsters, rats, mice, dogs, cats, chickens, and quails in experimental infection and Japanese water shrews in natural infection. Here we report that raccoon dogs are new natural definitive hosts of M. hakubaensis, based on morphological and molecular analyses of Metagonimus flukes collected from the host species from Aomori Prefecture, Japan. Moreover, M. hakubaensis recovered from raccoon dogs showed higher fecundity than those recovered from Japanese water shrews. Therefore, raccoon dogs were considered as a more suitable natural definitive host of M. hakubaensis than Japanese water shrews.

Comb Atrophy after Bile Duct Ligation in Chickens.

Gross, histological, and immunohistochemical changes in the combs of chickens after bile duct ligation (BDL) are described. Gross reductions in comb size and volume and lower serum testosterone levels were evident in chickens after BDL. Histologically, atrophic combs were characterized by reduced blood capillary diameter, decreased acid mucopolysaccharides, thinning of the stratum germinativum of the epidermis and dermis, and reduced immunostaining intensity of androgen receptors. These results suggest that the affected cells in atrophic combs are androgen targets. BDL caused testicular atrophy in chickens, a primary complication of liver disease, and the resultant low serum testosterone levels subsequently caused atrophy of the comb. In other words, the atrophy of the comb observed in BDL chickens was a secondary complication of liver dysfunction that simulated the effects of liver disease.

Serological survey of Encephalitozoon cuniculi infection in cats in Japan.

Antibodies to Encephalitozoon cuniculi (E. cuniculi) were examined by enzyme-linked immunosorbent assay using E. cuniculi PTP2 recombinant protein from serum samples that had been collected from a total of 295 cats in Japan. Of these samples, 6.1% (18/295) had antibodies against E. cuniculi, which included 6.3% (6/96) of the male cats and 6.0% (12/199) of the female cats. The incidence was slightly higher in feral cats (8.3%, 11/132) compared to domesticated cats (4.3%, 7/163). This suggests the possibility that the cats of our country have become a reservoir of E. cuniculi. This study is the first to demonstrate the prevalence of E. cuniculi infection in cats in Japan.

Identification of immunodiagnostic antigens for cerebrospinal filariasis in horses by western blot analysis.

In the present study, the serum and cerebrospinal fluid of horses diagnosed with Setaria digitata cerebrospinal filariasis were analyzed by western blot. The results revealed S. digitata protein bands measuring 65, 34, 22, and 18 kDa in molecular weight. In particular, the 18 kDa band is a possible candidate for clinical immunodiagnosis on the basis of western blot findings.

Efficacy of levamisole alone and in combination with mebendazole against Gongylonema pulchrum infection in rabbits.

Gongylonema pulchrum is an important parasite of captive primates. Twelve rabbits were infected with 30 third-stage larvae of G. pulchrum. At 4-7 months post-infection, animals were administered levamisole at a single dose of 12 mg/kg, levamisole at 8 mg/kg three times at 2-day intervals, levamisole at a single dose of 8 mg/kg after administration of mebendazole at 70 mg/kg for 3 days or 8 ml of distilled water for 3 days (control). Necropsy at 14 days after treatment revealed that single and multiple dosages of levamisole reduced nematode burdens by 68.4% and 89.5%, respectively. The combined regimen of mebendazole and levamisole exhibited high efficacy for treating G. pulchrum located widely within the upper digestive tract, with a reduction of 98.2%. These results suggest that this combined chemotherapy treatment may be effective against G. pulchrum infection, including buccal and lingual gongylonemiasis in primates.

Reduced efficacy of ivermectin treatments in gastrointestinal nematode infections of grazing cattle in Japan.

Fecal egg count reduction tests (FECRT) and larval migration inhibition tests (LMIT) were conducted to assess the efficacy of ivermectin (IVM) against gastrointestinal nematodes on 2 cattle farms in northern Japan in 2009 and 2010. Twelve to 20 calves on each farm were treated topically with 0.5 mg IVM/kg 2 (Farm 2) or 4 times (Farm 1) during the grazing season (May-October). On Farm 1, fecal egg count (FEC) reduction at 14 days post-treatment ranged from 16 to 87% in 2009 and from 24 to 96% in 2010, with relatively low reductions in August and October (16-53%). Conversely, IVM treatment on Farm 2 reduced FEC by 97% in September 2009. Larvae obtained from fecal cultures and identified by PCR-RFLP analysis revealed that the dominant species on both farms prior to IVM administration was Cooperia oncophora. In 2009, the FEC reduction of C. oncophora on Farm 1 decreased from 85% in May to 56% in August. In 2010, the reduction in C. oncophora in August was 28%. In the LMIT using larvae collected from the fecal cultures on Farm 1 in May and August 2009, the EC50 value of IVM in C. oncophora in August (0.892 µg/ml) was 3 times higher than that in May (0.296 µg/ml). The results of the LMIT corroborated the FECRT data, indicating the presence of IVM-resistant C. oncophora on Farm 1, at least in August. This is the first report of IVM-resistant nematodes in Japanese cattle.

Repression of hepatic cytochrome P450 2D expression in mice during Babesia microti infection.

To examine the effect of Babesia infection on the level of the drug-metabolizing enzyme hepatic cytochrome P450 (CYP) 2D, we intraperitoneally inoculated Babesia microti into male ICR mice. CYP2D protein and CYP2D9 mRNA were significantly decreased at 12 days after infection with B. microti. The activity of bunitrolol 4-hydroxylase, which is catalyzed by CYP2D, was also significantly decreased. The mRNA levels of transcriptional regulators of CYP2D9, hepatocyte nuclear factor 4α and signal transducer and activator of transcription 5b, were markedly suppressed. These results suggest that Babesia infection represses CYP2D expression in the mouse liver. The decline in CYP2D-dependent drug metabolism might be involved in the incidence of adverse drug reactions in patients with babesiosis.