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.

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.

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.

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.

Toxoplasma gondii infection in Amami spiny rat on Amami-Oshima Island, Japan.

The Amami spiny rat (Tokudaia osimensis) is an endangered rodent species that is endemic to the forests of Amami-Oshima Island, Kagoshima, Japan. In July 2018, a deceased adult male Amami spiny rat was found on the Yuwandake Mountain Trail on the south-central coast of Amami-Oshima Island. Histopathological observations revealed protozoan infections in the liver, lungs, and heart. Nested or semi-nested PCRs targeting the B1, SAG3, GRA6, and ROP18 genes successfully detected the genomic DNA of Toxoplasma gondii in the formalin-fixed and paraffin-embedded specimen. Sequence analyses of the SAG3, GRA6, and ROP18 genes suggested that the strain detected in the study specimen was related to the type II strain of T. gondii. This is the first confirmed case of T. gondii infection in an Amami spiny rat.

Molecular and histopathological features of Cryptosporidium ubiquitum infection in imported chinchillas Chinchilla lanigera in Japan.

Long-tailed chinchillas Chinchilla lanigera are popular rodent species kept both in households, where they are hand-raised as pets, and in zoological facilities. From January 2016 to February 2017, 13 juvenile chinchillas from five facilities in Japan were diagnosed with cryptosporidiosis at the animal hospital. Eight of the cases were fatal. All of the animals were imported from the Czech Republic by the same vendor. Histopathological and multilocus sequence analyses using 18S ribosomal RNA, actin, 70-kDa heat shock protein, and 60-kDa glycoprotein genes confirmed Cryptosporidium ubiquitum of subtype XIId as the etiological agent. Multilocus analysis demonstrated the presence of two new sequence types closely related to the C. ubiquitum Xlld strain isolated from a human in the USA. This study indicated that potentially zoonotic Cryptosporidium is widespread and may have caused a high number of deaths among imported juvenile chinchillas.

Morphological and molecular characterization of Cystoisospora sp. from Asian small-clawed otters Aonyx cinereus.

In this study, we provide the first description of Cystoisospora infection in Asian small-clawed otters (Aonyx cinereus). In July 2017, three juvenile otters recently imported from the Republic of Indonesia showed severe diarrhea and were diagnosed with coccidial infection; two of them eventually died. Fecal examination revealed the presence of numerous oocysts. Sporulated oocysts showed typical Cystoisospora features, measuring 24.6 ±â€¯1.6 (22.0-27.0) × 21.8 ±â€¯1.4 (19.0-25.0) µm, with an oocyst length/width ratio of 1.1 ±â€¯0.1 (1.0-1.3). Each sporocyst contained four sporozoites in a head-to-tail arrangement. The Stieda body was absent, and the sporocyst residuum was present. These morphological characteristics differentiated this species from the other valid Cystoisospora species described from mustelids. Molecular analysis was conducted at two loci: the nuclear 18S ribosomal RNA and mitochondrial cytochrome c oxidase subunit I genes. The 18S sequence showed high similarity with canine Cystoisispora ohioensis (1-bp difference, 1422/1423 [99.9%]). At the cytochrome c oxidase subunit I gene locus, the sequence from otters was identical to that of feline Cystoisospora rivolta (847/847 [100%]). Phylogenetic analyses using concatenated data demonstrated that Cystoisospora sp. from otters and C. rivolta grouped together in the same Cystoisospora clade. Based on these data, we concluded that Cystoisospora sp. detected from otters appeared to be highly similar to C. rivolta.

Successful treatment of refractory demodicosis and transient papules with a single dose of fluralaner in a dog with uncontrolled severe endocrine disease.

A 12-year-old female Shih-Tzu with hyperadrenocorticism and hypothyroidism developed concurrent refractory generalized demodicosis that did not respond to doramectin treatment. Although amitraz treatment was effective, the dog developed severe diabetes, which resulted in the cessation of amitraz and trilostane. Attempts to control the diabetes were unsuccessful, and its hyperadrenocorticism was left untreated, leading to the recurrence of demodicosis. However, demodicosis went into complete remission with a single dose of fluralaner. Transient erythematous papules appeared on the trunk three days after the administration of fluralaner, but no other adverse reactions were noted. We demonstrated that fluralaner is a potent treatment for demodicosis, and skin eruptions are possible after the first dose of the drug.

A New Stubby Species of Demodectic Mite (Acari: Demodicidae) From the Domestic Dog (Canidae).

A new species of Demodex was detected in the earwax of a dog with otitis externa in Saitama Prefecture, Japan, in July 2010. The opisthosoma length of the mite was slightly shorter than 1/2 of its body length, which was different from the other species in domestic dogs, D. canis and D. injai, but was similar to the form of mites termed "short-bodied species", including D. cornei. However, the stubby external form was morphologically different from those of "short-bodied species", excluding a case without a species description reported from Greece. Among known species, the mite was similar to D. equi and D. acutipes.

First report of fatal disseminated microsporidiosis in two inland bearded dragons Pogona vitticeps in Japan.

Introduction.Encephalitozoon pogonae is a newly described pathogen belonging to the phylum Microsporidia. In Austria and the USA, this species has been isolated from fatal and disseminated cases of captive-bred inland bearded dragons. Here, we report the case of fatal disseminated microsporidiosis caused by E. pogonae in two bearded dragons in Japan. Case Presentation. The two lizards from different private households in Tokyo, Japan, had been brought to an animal hospital for examination. In both cases, the animal presented with a history of weight loss for several weeks. There were no improvements in clinical symptoms and the lizards deteriorated and finally died. Histopathological examination demonstrated necrotizing granulomatous inflammation attributed to disseminated microsporidian infection. Nucleotide sequencing of the nuclear ribosomal internal transcribed spacer region identified the microsporidian as E. pogonae with sequence identity of 100 %. Conclusion. We report the first case, to our knowledge, of disseminated microsporidiosis caused by E. pogonae in inland bearded dragons in Japan. Although it is difficult to diagnose prenatally since the signs are nonspecific, the disease should be considered in the differential diagnosis of chronic infections that do not respond to antibiotics.

Hepatozoon apri n. sp. (Adeleorina: Hepatozoidae) from the Japanese wild boar Sus scrofa leucomystax (Mammalia: Cetartiodactyla).

Hepatozoon apri n. sp. is described from Japanese wild boars Sus scrofa leucomystax in Japan. The gamonts in the peripheral blood leukocytes were 11.6 ± 1.4 × 6.7 ± 1.3 µm in size. The meronts in the muscle tissues were 35.0-47.5 µm in length and 26.5-30 µm in width. A high rate (53.0%) of infection was found by nested PCR using muscle specimens from 181 wild boars captured in Tokushima, Japan. A phylogenetic analysis based on 18S rRNA gene sequences revealed that H. apri n. sp. detected in wild boars is closely related to Hepatozoon spp. isolated from carnivores. This is the first description of a species belonging to the genus Hepatozoon detected in ungulates.

Isospora lunaris n. sp. (Apicomplexa: Eimeriidae) from the domestic Java sparrow in Japan.

Five individuals of the domestic Java sparrows, Lonchura oryzivora (Aves: Estrildidae), were examined for coccidian parasites. Sporulated oocysts had two sporocysts containing four sporozoites each. Sporulated oocysts (n=30) were spherical, with a two splinter-like polar granules. Oocyst size was 22.1×20.7 (20.0-25.0×20.0-22.5)µm. They had a thick wall that consisted of a pale yellow outer layer and a dark yellow inner layer, and lacked micropyle and residuum. Sporocysts (n=60) were elongated ovoid 14.1×9.8 (12.5-15.0×7.5-10.0)µm, smooth walled, and colorless, with crescent-shaped Stieda and indistinct substieda bodies. Sporocyst residuum was interspersed between sporozoites. Sporozoites were oriented transverse to the sporocyst longitudinal axis. On the basis of morphological data, the species isolated in the present study is a new species of Isospora and propose the name Isospora lunaris n. sp.

Evaluation of recombinant Neospora caninum antigens purified from silkworm larvae for the protection of N. caninum infection in mice.

Three antigens (NcSAG1, NcSRS2 and NcMIC3) from Neospora caninum were expressed using the BmNPV bacmid system in silkworm larvae and purified from the hemolymph. From 20 silkworm larvae, 1.5, 1.2 and 1.4 mg of purified recombinant NcSAG1, NcSRS2 and NcMIC3 were obtained, respectively. When each purified recombinant antigen was immunized with Freund's incomplete adjuvant (FIA) to mice, recombinant NcSAG1 induced a Th2 immune response in immunized mice and produced a SAG1-specific antibody. In the experiment where NcSAG1-immunized mice were challenged with N. caninum, the cerebral N. caninum burden was significantly reduced compared with that of either the FIA- or PBS-immunized mice. Recombinant NcSRS2 or NcMIC3 induced both Th1 and Th2 immune responses, but NcMIC3-immunization did not induce significant production of NcMIC3-specific antibodies. These results suggest that the silkworm can produce recombinant antigens of N. caninum, which can be used as a recombinant vaccine against N. caninum.

Bombyx mori nucleopolyhedrovirus displaying neospora caninum antigens as a vaccine candidate against N. caninum infection in mice.

Baculovirus display systems have been utilized for cell-specific gene transfer, regenerative medicine, and as vaccine vectors. In particular, baculovirus particles displaying surface antigens have been used as vaccines against some parasites and viruses. In this study, Bombyx mori nucleopolyhedrovirus (BmNPV) particles displaying Neospora caninum antigens (NcSAG1, NcSRS2, and NcMIC3) purified from the hemolymph or fat body of silkworm larvae were prepared to vaccinate mice against N. caninum. Each antigen was expressed on the surface of BmNPV particles through glycoprotein 64 transmembrane and cytoplasmic domains. Antigen-specific antibody production was induced in mice by immunization with each recombinant BmNPV particle. NcMIC3-displaying BmNPV particles purified from the fat body induced a lower antibody titer than particles purified from the hemolymph. Antigen-specific IgG2a was predominantly produced in mice by immunization with NcSAG1-displaying BmNPV particles compared to IgG1, and induction of IFN-γ was dominant, indicating that antigen-displaying BmNPV particles can elicit a Th1 immune response in mice. Semi-quantitative PCR analysis revealed that immunization with each antigen-displaying BmNPV particle partially protected mice from cerebral N. caninum infection. These results suggest that antigen-displaying BmNPV particles can provide an alternative method of controlling neosporosis in cattle and represent a new generation of N. caninum vaccines.

Booster responses by oral vaccination with transgenic plants against chicken leucocytozoonosis.

We developed a transgenic potato (TrP/R7) expressing the recombinant R7 (rR7) antigen for use as an oral vaccine to protect against a chicken protozoan disease, chicken leucocytozoonosis. The TrP/R7 potato was produced by Agrobacterium tumefaciens-mediated transformation and regeneration, and the R7 gene insertion into potato chromosomes was confirmed by genomic polymerase chain reaction and Southern hybridization. rR7 antigen expression in TrP/R7 potato was also confirmed by sandwich enzyme-linked immunosorbent assay and western blotting using an antibody against the second-generation schizont of Leucocytozoon caulleryi. A transgenic potato clone with the highest rR7 antigen expression (3 µg rR7 antigen per gram of fresh-weight potato leaves) was selected, cultivated, and used in oral administration experiments to examine its ability to boost immunity. Chickens were immunized with chicken leucocytozoonosis vaccine "Hokken" by injection, and chickens that developed moderate levels of antibody titres were fed with TrP/R7 leaves. Chickens fed with TrP/R7 leaves showed increased antibody responses. In contrast, chickens fed with non-transgenic potato leaves showed a continuous decrease in antibody titres. Furthermore, chickens fed with TrP/R7 potato leaves showed strong resistance against experimental challenge with L. caulleryi infection. This study demonstrates the use of a plant-based oral vaccine to boost immunity against a protozoan disease.

Development of two murine antibodies against Neospora caninum using phage display technology and application on the detection of N. caninum.

Neosporosis, caused by an intracellular parasite, Neospora caninum, is an infectious disease primarily of cattle and dogs. It occurs worldwide and causes huge damages to dairy farms. In this study, we immunized mice with recombinant surface-associated protein 1 of N. caninum (rNcSAG1) and developed two novel monoclonal antibodies, A10 and H3, against NcSAG1 using phage-display technology. Both clones bound to purified rNcSAG1 and the half maximal inhibitory concentrations of A10 and H3 are 50 and 72 nM of rNcSAG1, respectively. In immunofluorescence assays, both A10 and H3 Fabs bound to N. caninum parasites. Direct detection of N. caninum parasites was developed firstly using an enzyme-linked immunosorbent assay (ELISA) with A10 and H3. Binding of A10 and H3 antibodies to rNcSAG1 was also inhibited by some certain anti-N. caninum antibodies in the neosporosis-positive cattle sera, suggesting they might bind to the same epitopes of NcSAG1 with those anti-N. caninum antibodies of bovine. These antibodies were demonstrated to have a potential for monitoring the N. caninum parasites in a dairy farm, which may lead to protect livestock from parasite-infection.

Comparative study of protective activities of Neospora caninum bradyzoite antigens, NcBAG1, NcBSR4, NcMAG1, and NcSAG4, in a mouse model of acute parasitic infection.

Neospora caninum is an obligate intracellular protozoan parasite that causes severe neuromuscular diseases, repeated abortion, stillbirth, and congenital infection in livestock and companion animals. The development of an effective vaccine against neosporosis in cattle is an important issue due to the significant worldwide economic impact of this disease. We evaluated the immunogenicity of four bradyzoite antigens, NcBAG1 (first described in this study), NcBSR4, NcMAG1, and NcSAG4, using an acute infection mouse model to determine synergistic effects with the tachyzoite antigen as a candidate for vaccine production. Mice were inoculated with the recombinant vaccines (r-)NcBAG1, rNcBSR4, rNcMAG1, rNcSAG4, or phosphate-buffered saline (PBS) (adjuvant control group) in an oil-in-water emulsion with bitter gourd extract, a Th1 immune stimulator, or PBS alone as the infection control group. Mice inoculated with each vaccine developed antigen-specific IgG1 and IgG2a antibodies and isolated splenocytes from mice produced high levels of interferon-γ when infected with the N. caninum tachyzoite. The mice inoculated with rNcBAG1, rNcMAG1, or rNcSAG4 developed slight to moderate clinical symptoms but did not succumb to infection. In contrast, rNcBSR4 and both control groups developed severe disease and some mice required euthanasia. The parasitic burden in the brain tissues of vaccinated mice was assessed by N. caninum-specific real-time PCR at 5 weeks after infection. The parasite load in rNcBAG1-, rNcMAG1-, and rNcSAG4-inoculated mice was significantly lower than that in adjuvant and infection control mice. Therefore, these antigens may be useful for the production of a N. caninum-specific vaccination protocol.

Induction of a T-Helper 1 (Th1) immune response in mice by an extract from the Pleurotus eryngii (Eringi) mushroom.

To assess the effect of edible mushroom extracts on the induction of T-helper 1 (Th1) immunity, we examined differences in interferon-gamma (IFN-γ) and interleukin (IL)-4 production in mice induced by hot-water extracts of 15 species of edible mushroom. Extracts from Agaricus bisporus, Flammulina velutipes, Hypsizigus marmoreus, Lentinula edodes, and Lyophyllum decastes induced both IFN-γ and IL-4 production in mice, whereas extracts from Pleurotus ostreatus only induced IL-4. In contrast, extracts from Agaricus blazei, Grifola frondosa, Morchella esculenta, Pholiota nameko, Pleurotus citrinopileatus, and Pleurotus eryngii induced only IFN-γ production. In particular, the extract from P. eryngii induced high levels of IFN-γ and reduced levels of IL-4. We further investigated the use of a trial immunogen using the P. eryngii extract as a Th1 immunostimulator. An oil-in-water emulsion of the hot-water extract from P. eryngii (immunostimulator) and ovalbumin (OVA; antigen) was used as a trial immunogen. This immunogen induced strong OVA-specific IgG2a antibody production in mice compared with the negative controls. In addition, OVA-specific IgG1 antibody levels were lower than those for the negative controls. Marked increases in serum IFN-γ levels and high-level production of IFN-γ in the culture supernatant from the CD4(+) spleen cells in the trial immunogen group mice were observed. Our results suggested that the hot-water extract from P. eryngii induced Th1 immunity by acting as an immunostimulator.