First Report of Mermithidae (Enoplea: Mermithida) Parasitizing Adult Stable Flies in Japan.

Mermithidae is a family of nematodes that parasitize a wide range of invertebrates worldwide. Herein, we report nematodes that were unexpectedly found in three of 486 adult stable flies (Stomoxys calcitrans) captured from three farms (F1, F2, and F3) in different regions of Gifu Prefecture, Japan. We aimed to characterize these nematodes both at the morphological and molecular level. Morphological studies revealed that the nematodes were juveniles of Mermithidae. Phylogenetic analysis based on 18S and 28S rDNA indicated that the mermithids from farms F1 and F2 could be categorized into the same cluster as Ovomermis sinensis and Hexamermis sp., whereas the mermithid from farm F3 clustered with Amphimermis sp. Additionally, these mermithids could be categorized within the same clusters as related mermithids detected in Japan that parasitize various arthropod orders. Our findings suggest that these stable flies may have been parasitized by mermithids already present in the region and that genetically distinct species of mermithids occur across Japan. To the best of our knowledge, this is the first report of mermithids parasitizing adult stable flies in Japan.

MORPHOLOGIC OBSERVATION AND FIRST MOLECULAR CHARACTERIZATION OF ENCYCLOMETRA JAPONICA (DIGENEA: ENCYCLOMETRIDAE) IN THE DIGESTIVE TRACT OF WILD TIGER KEELBACKS (RHABDOPHIS TIGRINUS) IN JAPAN.

Encyclometra japonica Yoshida and Ozaki, 1929 is a fluke that parasitizes the digestive tract of snakes. When the species was first reported, it was originally characterized on the basis of morphologic features, namely, the characteristics of the 2 testes located obliquely in the anterior-posterior direction, and the position of the ventral sucker one-fourth to one-third from the anterior extremity. Thereafter, more specimens with other morphologic variations were reported, and a new morphologic feature for species discrimination was proposed. However, the proposal is uncertain, and the criteria for morphologic discrimination between E. japonica and other Encyclometra species remain debatable. Thus, morphologically discriminable characteristics on the basis of a sufficient number of specimens are required, and molecular methods for species identification that allow for a more objective assessment need to be established. Therefore, in the present study, nucleotide sequences of 18S, 28S ribosomal deoxyribonucleic acid and cytochrome c oxidase subunit I (COI) of E. japonica were determined along with detailed morphologic observations for the first time. Key characteristics were observed in all specimens, whereas some morphologic variations were observed in the symmetry of the cecum. The COI sequences, which are known to be variable genetic regions, were identical among all specimens; therefore, all of them were considered the same species. This result clarifies the high morphologic variation in E. japonica. The 18S and 28S sequences were 99.78% and 99.11-99.19% similar to those of Encylometra colubrimurorum. The high degree of homology between these genes indicates that E. japonica and E. colubrimurorum are closely related but independent species. The present data will be used for the identification of E. japonica and to evaluate the relationships within the genus Encyclometra.

A serological survey of porcine reproductive and respiratory syndrome virus in wild boar in Gifu Prefecture, Japan.

Porcine reproductive and respiratory syndrome (PRRS) is an infectious swine disease caused by the PRRS virus (PRRSV) that results in economic loss to the pig-rearing industry. To study PRRSV infection in wild boars and pigs, we conducted a serological survey in Gifu Prefecture, Japan, from 2020 to 2021. Three out of 453 (0.7%) wild boar sera were positive for PRRSV antibodies in a commercial ELISA. However, given that PRRSV RNA was not detected in these three wild boars and the specificity and sensitivity of the test kit, these are considered as false positives. Although seropositive pigs were found in multiple pig farms in the study area, the role of wild boars as a source of PRRS to pig farms appeared to be minimal.

Contact between Mesocestoides vogae tetrathyridia induces their division.

Mesocestoides vogae is a cestode of the order Cyclophyllidea, and its second intermediate hosts are mammals, birds, amphibians, and reptiles. The parasite forms a tetrathyridium in the second intermediate host and multiplies asexually, sometimes to the point of filling the host's abdominal cavity. Proliferated tetrathyridium may cause lethal conditions in the host. During the asexual multiplication period, the scolex first replicates into two and then divides into two worms. In this study, to investigate the factors that promote the replication, tetrathyridia were cultured under various in vitro conditions. When several worms which already had two scolexes were cultured together, the division into two worm bodies was complete, but when single worm was cultured, the division hardly proceeded. The result indicates that the division progression of tetrathyridia with two scolexes requires the presence of other worms. In contrast, tetrathyridia with only one scolex did not initiate the division process, whether cultured together or alone. Then, the necessity of direct contact between the bodies of the worms to promote the division of tetrathyridia with two scolexes was assessed. For this, the well of the culture plate was partitioned into upper and lower parts using a mesh, and 20 worms in the upper part and single worm in the lower part were cultured. In all examined wells, worms in the upper part showed complete division, whereas the worms in the lower part rarely completed the division. Thus, direct contact between tetrathyridium promotes the division of tetrathyridia bearing two scolexes.

Prevalence of Toxoplasma gondii infection among small mammals in Tatarstan, Russian Federation.

Toxoplasma gondii is a zoonotic parasite with a wide host range that includes humans, domestic animals and wild animals. Small mammals serve as intermediate hosts for T. gondii and may contribute to the persistence of this parasite in the environment. Mass mortality in wild animals and deaths in rare endemic species make the study of this parasite of growing importance. In this study, T. gondii infection prevalence was evaluated in brain tissues from 474 small mammals captured at 26 trapping points in urban and rural areas of Tatarstan, Russian Federation. Nested PCR was used to detect the T. gondii B1 gene in the samples. Overall, 40/474 samples (8.44%) showed B1 gene positivity. T. gondii infection among the wild small mammals trapped in the rural area was significantly higher as a whole than that of the urban area as a whole. Multivariate logistical regression analysis also showed that the trapping area (rural or urban) significantly contributed to T. gondii positivity. Vegetation in the trapping points, small mammal species, sex, age or distance from the trapping points to the nearest human settlements did not significantly affect T. gondii positivity in the sampled small mammals.

Spatial distribution of anti-Toxoplasma gondii antibody-positive wild boars in Gifu Prefecture, Japan.

Toxoplasma gondii is a globally wide-spread parasite that infects almost all species of mammals and birds, including humans. We studied the spatial distribution of individual T. gondii-seropositive wild boar in Gifu Prefecture (10,621 km2), Japan. Altogether, 744 wild boars were captured at 663 points around human settlements in Gifu Prefecture. Serum samples were collected after recording the exact capture locations, along with each wild boar's body length and sex. We then used a commercial enzyme-linked immunosorbent assay kit for swine to measure anti-T. gondii antibodies in these animals. Among the 744 wild boars, 169 tested positive for T. gondii (22.7%). No significant difference in T. gondii seroprevalence was observed between the mountainous northern region with high winter snow cover and the mild-wintered geographical plain of the southern part of the prefecture. In contrast, 8 of the 11 wild boars that were captured in a public park surrounded by residential areas showed T. gondii seropositivity (72.7%), a value significantly higher than those of the wild boar populations in the other prefecture areas. This in-depth analysis, which spans the big city suburbs and rural areas of a whole prefecture, explains the seroprevalence of zoonotic T. gondii in wild boar and has public health implications.

Releasing latent Toxoplasma gondii cysts from host cells to the extracellular environment induces excystation.

Toxoplasma gondii, an obligate intracellular protozoan parasite, infects a wide variety of mammals and birds. Although T. gondii infects the brain and muscles in its latent cyst form containing bradyzoite stage parasites during chronic infection, when a chronically infected host becomes immunodeficient or is preyed upon by a predator, the latent cyst undergoes excystation. However, it is not yet known how T. gondii recognises the triggers of excystation in the microenvironment surrounding the cyst. In this study, we incubated T. gondii cysts from host cells in several solutions containing a variety of ionic compositions. Excystation occurred in a solution with an ionic composition which mimicked that of the extracellular environment. However, excystation did not occur in a solution that mimicked the intracellular environment. We also found that the specific Na+/K+ ratio and the presence of Ca2+, mimicking the extracellular environment, are required to trigger excystation. To examine whether the stage conversion of bradyzoite to tachyzoite occurs prior to egress, we constructed a gene-modified T. gondii strain expressing a green fluorescent protein specifically in the tachyzoite stage. During the process of cyst reactivation of this strain, green fluorescence was detected prior to excystation. This suggests that stage conversion from bradyzoite to tachyzoite occurs prior to cyst disruption. These results indicate that T. gondii bradyzoites monitor the ionic composition of their surroundings to recognise their expulsion from host cells, to effectively time their excystation and stage conversion.

Detection and molecular characteristics of Pyelosomum cochlear (Digenea: Pronocephalidae) in the urinary bladder of the green sea turtle (Chelonia mydas) in the Northwest Pacific Ocean.

The genus Pyelosomum consists of parasitic flukes occurring primarily in marine turtles; Pyelosomum cochlear Looss 1899 is the only species of this genus that parasitizes the urinary bladder. In this study, we detected flukes in the urinary bladders of 20 of 88 green sea turtles (Chelonia mydas) harvested in the Ogasawara Islands, in the Northwest Pacific Ocean. We identified the flukes as P. cochlear based on detailed morphological observations and comparisons of morphometric measurements of the species reported previously. Nucleotide sequences of nuclear ribosomal 18S and 28S regions and the mitochondrial cytochrome c oxidase subunit 1 (COI) region were determined for the flukes. The 18S and 28S phylogenetic trees revealed that the species of the superfamily Pronocephaloidea, including P. cochlear, constituted a single clade, but the species of the family Pronocephalidae did not constitute a single taxon. These findings suggest that Pronocephalidae is a paraphyletic group. The COI sequences of P. cochlear exhibited high genetic diversity, suggesting that they would be useful markers to understand the genetic structure of the parasite and its evolutionary relationship with the host turtle populations. This is the first study to provide the nucleotide sequences of Pyelosomum species; these data will be available for further molecular studies of this genus and its related taxa.

Morphological observation and first molecular characterization of Grassenema procaviae Petter, 1959 (Cosmocercoidea: Atractidae) in the stomach of Cape hyrax (Procavia capensis) raised in a zoo in Japan.

The superfamily Cosmocercoidea comprises three families: Cosmocericidae, Kathlaniidae and Atractidae. Information on the nucleotide sequences of the Cosmocercoidea is quite limited, and the molecular classification of the whole superfamily has been slow to progress. The genus Grassenema of the family Atractidae is a parasitic nematode group that occurs in the digestive tract of hyraxes and includes three species: Grassenema procaviae, G. dendrohyraci, and G. hyracis. The type species of the genus, G. procaviae, was isolated from the digestive tract of Cape hyraxes (Procavia capensis) and has the potential to cause gastric ulcers. Although G. procaviae is a common parasite of Cape hyraxes, no genetic information for the parasite is currently available. In this study, we obtained the first genomic sequences of G. procaviae and performed detailed morphological observations. Furthermore, molecular phylogenetic analysis was performed, and the taxonomic position of the parasite was evaluated using 18S and 28S rDNA sequences. Those data will be useful for molecular identification of G. procaviae and future phylogenetic analysis within the Atractidae.

Suppression of inflammatory genes expression in the injured host intestinal wall during Mesocestoides vogae tetrathyridium larvae migration.

Mesocestoides vogae is a cestode parasite of the family Mesocestoididae (order Cyclophyllidea). Its larvae, tetrathyridium, are approximately 1 mm long and 300 µm wide and infect a wide range of host species including humans. Tetrathyridium migrate through the intestinal wall to invade the peritoneal cavity. Despite intestinal penetration by such a large-sized parasite, symptomatic intestinal disorders are not common during the migration period. In this study, the dynamics of tetrathyridia migration and their pathogenicity towards intestinal tissues were examined in mice infected orally with these parasites. Most tetrathyridia were found to migrate through the intestinal wall, moving into the peritoneal cavity or liver 24 to 48 hours after the oral infections. Next, the pathogenicity of tetrathyridium in the intestinal wall was histopathologically evaluated, and tissue injury from tetrathyridium migration was confirmed. Inflammatory foci were observed as tetrathyridium migration tracks from 48 hours after oral infection; however, the number of inflammatory foci had decreased by half more than 48 hours later. Therefore, we examined the gene expression levels of the macrophage driving cytokine, IL-1ß, and the eosinophil recruiting chemokine, CCL11, by quantitative reverse-transcriptase PCR. The expression levels of these genes in the infected group were significantly lower than those of the non-infected group at 48 hours post-infection. Although the immunomodulating ability of the excretory-secretory products released from tetrathyridium has been previously shown by in vitro assays, the significance of this ability in their lifecycle has remained unclear. In this study, we discovered that tetrathyridium causes temporal inflammation in the intestinal wall during penetration and large-scale migration in this organ, but tetrathyridium simultaneously suppresses the host's inflammatory gene expression, might to be a strategy that reduces inflammatory responses and increases survival of the parasite.

A Toxoplasma gondii strain isolated in Okinawa, Japan shows high virulence in Microminipigs.

Toxoplasma gondii strains have been isolated all over the world and their virulence has been examined mainly using laboratory mice. However, T. gondii differs in virulence depending on the host animal species. Therefore, to evaluate the virulence of each strain in domestic animals, it is necessary to examine using not only mice but also the concerned animals. We have shown that TgCatJpOk4, a T. gondii strain recently isolated in Okinawa, Japan, has a high virulence against laboratory mice, comparable to highest virulent RH strain in mice; however, the virulence to domestic animals remains unknown. In this study, we examined the virulence using the Microminipig. After infection, four out of five infected pigs showed severe clinical symptoms: inappetence, hypoactivity and tachypnea. Eventually, three out of the five infected pigs succumbed before the end of the observation. Among the three dead pigs, histological analysis revealed that interstitial pneumonia and spotty necrosis in the liver indicating that the TgCatJpOk4 strain has a high virulence not only in laboratory mice, but in pigs as well.

Native SAG1 in Toxoplasma gondii lysates is superior to recombinant SAG1 for serodiagnosis of T. gondii infections in chickens.

Toxoplasma gondii can infect almost all mammals and birds, including chickens. The aim of this study was to identify an appropriate immunogenic antigen for serodiagnosis of T. gondii infections in chickens. We examined serum samples from chickens that were intravenously or intraperitoneally infected with 106-108 tachyzoites of T. gondii strains PLK, RH, CTG, ME49 or TgCatJpGi1/TaJ using enzyme-linked immunosorbent assays (ELISAs), latex agglutination tests (LATs) and western blotting. Regardless of parasite strain or infection dose and route, the commercial LAT was positive for almost all sera collected 1 week post-infection. However, at 2 weeks post-infection, LATs were negative in the same birds. ELISAs using the Escherichia coli-produced recombinant T. gondii antigens SAG1 and GRA7 showed strong signals at 1-2 weeks post infection, but thereafter diminished for the majority of serum samples. In contrast, western blotting against crude tachyzoite antigens showed a persistent band up to 4 weeks post-infection. Sera from these chickens reacted much more strongly with SAG1 from crude tachyzoite antigens than with recombinant SAG1. Even in experimentally-infected birds whose parasite burdens in tissue were undetectable, sera still reacted with native SAG1. We tested sera from free-range chickens on a small farm in Ghana, Africa, using western blotting and found that the serum of one bird reacted with a single band of approximately 27 kDa, the putative molecular weight of SAG1. Thus we conclude that native SAG1, but not E. coli-produced recombinant SAG1, is suitable for serodiagnosis of T. gondii infections in chickens.

Toxoplasma gondii seroprevalence in goats, cats and humans in Russia.

Toxoplasmosis, a most common zoonosis, is caused by the protozoan parasite Toxoplasma gondii. However, there is little epidemiological information on T. gondii infections in humans and livestock animals in Russia. Therefore, in this study, the seroprevalence of T. gondii in goats in Russia was investigated. A total of 216 goats from 32 farms were investigated and 95 of them were seropositive for T. gondii. The difference in seroprevalence between the examined regions was not statistically significant. We next collected serum samples from 99 cats and 181 humans in Kazan city, the state capital of the Republic of Tatarstan, Russia, and examined their T. gondii seroprevalences. Thirty-nine of the 99 cat samples and 56 of the 181 human samples showed seropositivity. Logistical regression analysis revealed that the cat breeding history of the human subjects, but not their sex or age is a significant risk factor for T. gondii seropositivity. These findings suggest that the natural environment in Russia may be widely polluted with T. gondii oocysts shed by cats, and ingestion of these oocysts provides a major route for human infection with this parasite.