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Understanding the population structure of Fasciola flukes in domestic and wild animals is important for determining the extent of cross-infection between them. Although the parthenogenetic Fasciola flukes in Japan have been shown to comprise five genetic types based on the ribosomal internal transcribed spacer 1 (ITS1) and mitochondrial NADH dehydrogenase subunit 1 (nad1) regions, these genetic regions are not suitable for analyzing their population structure. In the present study, the genetic diversity and population structure of the parthenogenetic Fasciola flukes in Japan were studied using microsatellite DNA, ITS1, and nad1 regions. A total of 144 parthenogenetic Fasciola flukes, obtained from cattle and sika deer in 16 localities, were individually analyzed using PCR-RFLP for ITS1, PCR-direct sequence analysis for nad1, and post-labeling PCR and capillary electrophoresis for microsatellite DNA regions. The flukes showed higher genetic diversity in the microsatellite DNA regions than ITS1 and nad1. The population structures of parthenogenetic Fasciola flukes were unclear, however, it was suggested that the flukes are more diverse populations. We hypothesized that their distribution throughout Japan is closely related to livestock movement dependent on human activity. Moreover, it is considered that cross-infection of the flukes between cattle and sika deer possibly has occurred in the past.

We analyzed the nuclear ribosomal internal transcribed spacer (ITS) 1 and ITS2 sequences for Bangladesh isolates of Ascaridia galli, and we determined that the sequences were unreliable as molecular markers for distinguishing A. galli from other Ascaridia species, because the sequences showed high identity with that of A. columbae. However, the ITS1 sequences were available for designing PCR primers distinguishable between Ascaridia galli and Heterakis spp. Bangladesh isolates of A. galli constituted a monophyletic clade along with other geographical isolates in the cytochrome c oxidase subunit I (COI) phylogenetic tree, however, we could not clarify the phylogenetic relationships between A. galli and other Ascaridia spp., because their available sequences in GenBank were very few. The developed PCR method using DNA from A. galli and Heterakis spp. eggs would enable differential diagnosis of the individual infections in the future.

Trematodes of the genus Dicrocoelium are one of the most common parasites in ruminant animals; however, their life cycles in Japan are unclear. To find the sporocysts of D. chinensis in the natural field, we sampled 269 land snails (14 species) at a location with high level infection of sika deer in Gifu Prefecture, Honshu Island, Japan in autumn between 2017 and 2019. During the sampling period, we found mother sporocysts in the hepatopancreas of Aegista vulgivaga and Cyclophorus herklotsi. DNA barcoding based on the sequences of cytochrome c oxidase subunit 1 showed that the sporocysts from A. vulgivaga belonged to D. chinensis, indicating that this snail has potential as the first intermediate host of D. chinensis at this location.

The poultry infections caused by Dispharynx nasuta and Cheilospirura hamulosa nematodes are difficult to be diagnosed by fecal examination because of their egg similarity. In this study, we analyzed DNA sequences of nuclear ribosomal 18S-ITS1-5.8S-ITS2-28S region of D. nasuta and C. hamulosa and developed conventional multiplex PCR method using species-specific primers for discriminating between the two species. The method amplified 455-bp and 319-bp fragments specific to D. nasuta and C. hamulosa, respectively, and did not produce them against the other chicken nematode species, Ascaridia galli, Oxyspirura mansoni, Heterakis gallinarum, Heterakis beramporia, and Heterakis indica, suggesting that the multiplex PCR is sensitive and available for species diagnosis.

In this paper, lymnaeid snails collected from 107 localities in Hokkaido, Japan were examined for infection by Fasciola larvae. DNA sequences from the snails were analysed phylogenetically based on...

Ticks serve as important vectors of a variety of pathogens. Recently, the viral and prokaryotic microbiomes in ticks have been explored using next-generation sequencing to understand the physiology of ticks and their interactions with pathogens. However, analyses of eukaryotic communities in ticks are limited, owing to the lack of suitable methods. In this study, we developed new methods to selectively amplify microeukaryote genes in tick-derived DNA by blocking the amplification of the 18S rRNA gene of ticks using artificial nucleic acids: peptide nucleic acids (PNAs) and locked nucleic acids (LNAs). In addition, another PCR using non-metazoan primers, referred to as UNonMet-PCR, was performed for comparison. We performed each PCR using tick-derived DNA and sequenced the amplicons using the Illumina MiSeq platform. Almost all sequences obtained by conventional PCR were derived from ticks, whereas the proportion of microeukaryotic reads and alpha diversity increased upon using the newly developed method. Additionally, the PNA- or LNA-based methods were suitable for paneukaryotic analyses, whereas the UNonMet-PCR method was particularly sensitive to fungi. The newly described methods enable analyses of the eukaryotic microbiome in ticks. We expect the application of these methods to improve our understanding of the tick microbiome.

Precise discrimination of Echinostoma species within the ‘revolutum’ group is quite difficult because of their morphological similarities. The objective of this study was to precisely characterize the echinostomes of ducks from Bangladesh based on both morphological and molecular characteristics. Two Echinostoma species were identified: E. revolutum and E. robustum. In the phylogenetic trees (ITS2 and nad1), E. revolutum and E. robustum belonged to their respective Eurasian clade, which is distinct from the American clade. These results suggest that both species have two distinct and geographically separated lineages, Eurasian and American. Our molecular and morphological data combined with previously published data supports the synonymy of E. robustum, E. miyagawai, and E. friedi previously based on either molecular or morphological evidence. This study thus improves our understanding of species diversity of the ‘revolutum’ group, particularly in Asia.

Green-banded broodsacs of Leucochloridium sporocysts were obtained from land snails, Succinea lauta, collected in Esashi, Esashi District, Hokkaido, Japan. The broodsacs were similar to those of L. paradoxum, which have never been found on the Japanese archipelago. Here, we compare morphological and molecular genetic characteristics of the green-banded broodsacs to those of L. paradoxum. The broodsacs described in this study and those of L. paradoxum showed three equal parts; however, band characteristics of the second parts differed. Moreover, the sequences of nuclear 18S and 28S rRNA genes of the Leucochloridium flukes were not identical with those of L. paradoxum, and the sequences of the mitochondrial cytochrome c oxidase subunit 1 gene were paraphyletic to the species. These findings suggest that the broodsacs described in this study were distinct from those of L. paradoxum. In the future, genetic analyses on adult flukes from avian hosts should be performed, upon precise identification according to morphology, in order to clarify relationships between adult flukes and sporocysts of Leucochloridium species occurring in Japan.

Paragonimiasis is an important food-borne zoonosis caused by Paragonimus flukes and is endemic to western Japan. However, there have been few epidemiological studies in the Tohoku district of northeastern Japan. In this study, Paragonimus metacercariae (mc) was detected in Geothelphusa dehaani (Japanese freshwater crab or Sawagani) in Iwate Prefecture. Out of the 207 Sawagani collected from 35 localities, 12 individuals from six localities were infected with Paragonimus mc. The mc were identified as P. skrjabini miyazakii based on the sequences of nuclear ribosomal internal transcribed spacer 2 and mitochondrial cytochrome c oxidase I. This is the first report of P. s. miyazakii mc infection in Sawagani in Iwate Prefecture.

Ticks transmit many pathogens with public health and veterinary importance. Despite the wide distribution of tick-borne pathogens in Sudan, the information on the tick–pathogen relationship needs to be updated, particularly using modern molecular techniques. This cross-sectional study, conducted between September and November 2019, used morphology, PCR, and sequencing to confirm the identity of adult cattle ticks (male and female, n = 536) from Khartoum State (n = 417) and East Darfur State (n = 119). Moreover, the presence of Theileria annulata, Babesia bigemina, B. bovis, Anaplasma marginale, and Ehrlichia ruminantium was detected and confirmed in each tick using species-specific PCR or nested PCR and sequencing. The most economically important tick genera, Rhipicephalus, Hyalomma, and Amblyomma, were prevalent in the study area, and 13 different tick species were identified. The most prevalent tick species were Rhipicephalusevertsi evertsi (34.3%) and Hyalomma anatolicum (57.3%) in Khartoum State, and Rhipicephalus annulatus (27%), Rhipicephalus decoloratus (25%), and Hyalomma rufipes (29%) in East Darfur State. We detected all five pathogens in both states. To the best of our knowledge, this is the first study to report the presence of E. ruminantium, its vector Amblyomma variegatum, and B. bovis in Khartoum State. Further, this is the first report on most tick and pathogen species identified in East Darfur State. Our findings indicate the migration of some tick and pathogen species beyond their distribution areas in the country, and this consideration is necessary to develop future control strategies.

Trypanosoma theileri is considered a non- or low-pathogenic trypanosome that generally causes latent infection in apparently healthy cattle; however, T. theileri propagates in the bloodstream and may cause clinical disease in pregnant animals or co-infection with bovine leukemia virus or Theileria orientalis. In the current study, a monthly survey of T. theileri infection over one year was carried out in a research dairy farm in Hokkaido, Japan to determine the 1) seasonal variations in the prevalence, 2) genetic characterization of T. theileri, and 3) associations of milk and blood parameters in dairy cattle with T. theileri infection, including data of metabolic profile tests and dairy herd performance tests, using linear mixed models. We found that 1) the prevalence of T. theileri infection was significantly higher in summer and winter than in other seasons; 2) T. theileri possibly showed genetic diversity in Eastern Hokkaido; and 3) T. theileri infection was associated with significantly lower levels of blood urea nitrogen, milk protein, and solids-not-fat, which are caused by a low rumen fermentation level. This is the first study to report the negative impact of T. theileri infection in dairy cattle, and our study indicates that control of T. theileri infection can improve the productivity of dairy cattle.

The metacercarial infections of door snails (Gastropoda: Clausiliidae) with unknown species of the genus Brachylaima (Trematoda: Brachylaimidae) have recently been reported in eastern Honshu and Kyushu, Japan. A large scale snail survey was carried out to clarify their taxonomic status. From the period of 2015 to 2020, a total of 1239 land snails (768 door snails and 471 others) were collected from 32 localities in Honshu, Shikoku, and Kyushu. The resulting trematode isolates were identified as Brachylaima sp. by mitochondrial DNA barcoding. The sporocysts were found only a few from Megalophaedusa sublunellata (Clausiliidae), Tauphaedusa subaculus (Clausiliidae), and Aegista trochula (Camaenidae), while the metacercariae were frequently detected from 14 species of Clausiliidae and 2 species of other families. Although Brachylaima sp. showed a broad range of intermediate hosts, door snails seem to be very important to drive the life cycle. The gravid adults of Brachylaima sp. was experimentally raised from metacercariae using immunosuppressed mice. Morphological, phylogenetical, and ecological considerations prompted us to propose Brachylaima phaedusae n. sp. for this unknown species. The definitive hosts of the new species are completely unknown. The wide geographic distribution and high genetic diversity of the new species suggest a possibility that the definitive host is ground-foraging birds, which prefer door snails.

In Sudan, donkeys are important animals, providing transportation and income possibilities. However, the prevalence of parasites in donkeys in Sudan has not been thoroughly characterized. Accordingly, in this study, we aimed to detect selected hemoprotozoan parasites in donkeys in West Omdurman, Khartoum State, Sudan, wherein people depend mainly on donkeys for their daily life. In total, 198 blood samples collected from donkeys in a local market in West Omdurman, were screened using serological and molecular diagnostic techniques. Serologically, 52 (26.3%), 56 (28.3%), and 19 (9.6%) samples were positive for trypanosomosis using Card Agglutination Test for Trypanosoma evansi, Trypanosoma evansi crude antigen -based enzyme-linked immuno sorbent assay (ELISA) and recombinant Trypanosoma evansi GM6-4r-based ELISA, respectively. ELISA for equine piroplasmosis revealed 156 (78.8%) and 10 (5.1%)Theileria equi- and Babesia caballi-positive samples, respectively. PCR detected Trypanosoma congolense, subgenus Trypanozoon, Theileria equi, and Babesia caballi in 18 (9.1%), 77 (38.9%), 18 (9.1%), and 8 (4%) samples, respectively. Of the 77 Trypanozoon-positive samples, 35 (45.5%) were confirmed as Trypanosoma evansi type A. To our knowledge, this is the first report of detection of Trypanosoma congolense in donkeys outside of tsetse-infested areas in Sudan.

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.

We investigated here, the prevalence of Nosema microsporidia infections in the honeybees, Apis cerana japonica and Apis mellifera, in the Tohoku region of Japan. We detected Nosema ceranae DNA in 14 (2.8%) of 509 A. cerana japonica and in 34 (21.9%) of 155 A. mellifera honeybees from Aomori, Iwate, Akita, Yamagata, and Fukushima prefectures. Nosema apis DNA was undetectable in A. cerana japonica and A. mellifera. The unidentifiable Nosema species that genetically differed from N. apis, N. ceranae, and N. neumanni in terms of small subunit (SSU) rDNA, large subunit rDNA, and internal transcribed spacer sequences was identified in 105 (20.6%) of 509 A. cerana japonica and in 1 (0.6%) of 155 A. mellifera honeybees, and from Iwate prefecture. A phylogenetic tree based on SSU rDNA sequences showed that the Nosema sp. belonged to the same clade as N. thomsoni detected in moth and solitary bees in North America and N. pieriae found in cabbage butterfly in Turkey, which have not hitherto been detected in honeybees. The morphological characteristics of the spores should be analyzed to enable species identification of the Nosema sp.

Heterakis gallinarum, H. beramporia, and H. indica are common nematodes in gallinaceous poultry in Asian countries, and the infections occasionally lead to declining health of the hosts. These three Heterakis spp. can be identified by the morphological characteristics of the male worms; however, the female worms and eggs cannot be identified because they have no reliable morphological characteristics for discrimination. In addition, H. gallinarum is a well-known vector of fetal protozoan Histomonas meleagridis, making the discrimination between these three Heterakis species important in basic and clinical veterinary parasitology. We analyzed nuclear ribosomal 18S-ITS1−5.8S-ITS2−28S DNA sequences of these three Heterakis species. The 18S, 5.8S, and 28S DNA sequences had very high homology between the species; however, the ITS1 and ITS2 sequence similarity was 68.5 %–93.2 %. H. gallinarum, H. beramporia, and H. indica were divided into separate clades in the ITS1 and ITS2-concatenated phylogenetic tree. Therefore, to develop a multiplex PCR method for discriminating between the three Heterakis species, we designed species-specific reverse primers within the ITS2 region as follows: H. gallinarum-specific HgI2-R, H. beramporia-specific HbI2-R5, and H. indica-specific HiI2-R. The multiplex PCR amplified 396-bp, 272-bp, and 482-bp fragments specific to H. gallinarum, H. beramporia, and H. indica DNA, respectively, and did not amplify the fragments using other chicken nematode DNAs such as Ascaridia galli, Oxyspirura mansoni, Dispharynx nasuta, and Cheilospirura hamulosa. These results suggest that the multiplex PCR would serve as a useful tool for identifying and diagnosing infections of H. gallinarum, H. beramporia, and H. indica in poultry.

Fasciolosis is a public health problem originally transmitted from livestock. Although molecular analysis of the nuclear and mitochondrial genes allow discrimination between the two known species of Fasciola, F. hepatica and F. gigantica, these markers do not permit the detection of hybrid forms. On the other hand, molecular analysis of the pepck and pold genes from Fasciola do permit the detection of hybrid isolates and this study has not yet been performed on specimens from human and domesticated animals in Iran. Therefore, the aim of this study was to molecularly analyze Fasciola isolates using both conventional and the two new genetic markers mentioned above. Fifty-three adult Fasciola worms were isolated from the livers of 28 domesticated animals, including sheep, cattle and goat. Moreover, an adult worm was obtained from the bile duct of an infected woman during endoscopic retrograde cholangio-pancratography (ERCP). Targeted fragments from the ITS-1, NDI and COX1 genes were amplified using specific primers. PCR products were sequenced and genetically analyzed using MEGA v.7 and DnaSP software. Additionally, the pepck and pold regions were amplified and analyzed using multiplex PCR and RLFP-PCR, respectively. Multiple alignment of sequenced fragments showed highest similarity among the ITS-1 sequences isolated from all four hosts in comparison to the other genes. Furthermore, sequence diversity across the COX1 was higher than the NDI and ITS-1. Diversity among sequences isolated from cattle was higher than those from sheep and goat in all three genes. Tajima's D and Fu's Fs were negative and statistically significant for all the genes except ITS-1. Phylogenetic trees showed that the human F. hepatica isolate was closer to sheep isolates. The results of the pepck and pold analyses showed that all isolates were F. hepatica and there were no hybrid forms among samples. The molecular analyses corroborated this finding.

A variety of helminths have been found in domestic chickens in Bangladesh, but little is known about their gene sequences. Here, parasitic nematodes and trematodes were collected from the eyes of domestic chickens and analyzed for their morphological and morphometric characteristics, and characterized molecularly. The helminths were identified as Oxyspirura mansoni and Philophthalmus gralli. The ITS1 and ITS2 sequences of O. mansoni were 532 bp and 306 bp in length, respectively, and showed low identity (50.7–62.7%) with those of O. petrowi and O. conjunctivalis. Furthermore, the O. mansoni CO1 sequences (393 bp) showed five haplotypes (97.5–99.5% similarity) that formed a monophyletic clade. With respect to P. gralli, the ITS1 (452 bp) and ITS2 (736 bp) sequences showed 100% similarity with the reference sequences in GenBank. Both the ND1 and CO1 phylograms showed that P. gralli from Bangladesh, Costa Rica and Peru form a monophyletic clade, distinct from the clades of P. lucipetus and P. lacrymosus. Our data show that, Philophthalmus gralli isolates from Bangladesh, Costa Rica and Peru are genetically close to each other.

All 225 Fasciola flukes obtained from domestic animals (73 cattle, 7 sheep and 1 pig) of 18 distinct geographic areas in Ecuador-South America, were identified as Fasciola hepatica, based on molecular analyses of nuclear pepck and pold genes, and mitochondrial nad1gene as well as the morphological observation of sperm within the seminal vesicles. Fasciola gigantica and parthenogenic Fasciola forms endemic to Asian countries were not found in this study, although zebu cattle and water buffalos have introduced into South America from Asia; this could be due to the absence of suitable intermediate host snails. The results of pepck analysis using multiplex PCR developed previously showed that 32 of the flukes could not be confirmed as F. hepatica, suggesting that the method is unreliable for the accurate discrimination of F. hepatica, and that pepck gene of the species consists of multiple loci, not a single locus. The results of genetic diversity, phylogenetic, and network analyses based on mitochondrial nad1 sequences suggest that F. hepatica populations in South America, including Ecuador, formed from the ancestral F. hepatica individuals introduced into the continent along with anthropogenic movement of livestock infected with the species.

The number of wild sika deer (Cervus nippon yesoensis) continues to increase in Hokkaido Prefecture, Japan. The major concern for the livestock industry is the transmission of pathogens between sika deer and cattle. Fasciolosis is an important disease that can occur in both animals. The aim of this study was to examine the possible mutual transmission of this disease in Hokkaido Prefecture. A total of 105 Fasciola flukes were obtained from sika deer and 96 from domestic cattle. The Fasciola flukes in Japan are reported to possess no mature sperm. However, in this study, 14 flukes from sika deer and eight flukes from cattle contained mature sperm in their seminal vesicles. All the Fasciola flukes from the two host animals had Fh/Fg type in nuclear phosphoenolpyruvate carboxykinase (pepck) gene, with a mixed fragment pattern derived from F. hepatica and F. gigantica, which are considered to be hybrid Fasciola flukes. However, almost all the flukes had Fsp1 haplotype in NADH dehydrogenase subunit 1 (nad1) gene, indicating that their maternal lineage was F. hepatica. A new haplotype, Fsp3, was detected in one fluke obtained from cattle and differed in one nucleotide from Fsp1. Therefore, the Fasciola flukes detected in both host species had almost identical molecular characteristics. These findings suggest the mutual transmission of Fasciola flukes between sika deer and domestic cattle in Hokkaido.

In Hokkaido, Japan, wild sika deer are highly infected with Fasciola flukes, suggesting that the flukes complete their life cycle via intermediate host snails and definitive host animals occurring in the natural environment. However, infected snails have been found only in cattle farms contaminated with fasciolosis. This study reports the first Fasciola larva infection in Galba truncatula snails occurring in the Shoro and Atsuma rivers in the natural environment. Molecular analysis revealed that the nad1 haplotype of the larvae was consistent with that of Fasciola adults obtained from sika deer in Hokkaido. These results indicated that Fasciola flukes complete their life cycle via G. truncatula and sika deer occurring in the natural environment.

Honeybee acarapiosis and vorrosis were designated as Notifiable Infectious Diseases in the Act on Domestic Animal Infectious Diseases Control by the Minister of Agriculture, Forestry and Fisheries of Japan in 1997. However, the prevalences of A. woodi and V. destructor in Japan, especially in the Tohoku region, have not been sufficiently elucidated. This study was designed to clarify the prevalence of A. woodi and V. destructor mites in Apis cerana japonica and Apis mellifera in the Tohoku region and the characteristics of their mitochondrial cytochrome c oxidase I (COI) DNA. Acarapis woodi mites were detected from 13.5% of A. c. japonica and 0% of A. mellifera. Aomori prefecture, Japan is a new distribution locality for A. woodi. None of the honeybees examined showed infection by V. destructor mites. The COI sequences (1638 bp) of A. woodi were identical and phylogenetically closely related to those of A. woodi from Japan and the UK, suggesting that the mite would have been introduced into Japan with A. mellifera during the last 150 years and spread throughout the country.

Learedius learediPrice, 1934, is a blood fluke found in sea turtles, and the adult fluke parasitizes the cardiovascular system of the host. In this study we surveyed 46 green sea turtles, Chelonia mydas, on the Ogasawara Islands, Japan, and blood flukes were detected in the heart and blood vessels of 26 turtles. The flukes were identified as L. learedi based on a detailed morphological description. In addition, molecular identification and characterization of the parasite were performed. The nucleotide sequences of nuclear internal transcribed spacer 2 (ITS2) regions were almost identical to those of L. learedi reported previously, but not to those of Hapalotrema spp., which is the closest related genus. The nucleotide sequences of the 28S ribosomal DNA region formed a single clade with those of the reference L. learedi in the phylogenetic tree, but not with those of Hapalotrema spp. Therefore, the nucleotide sequences of ITS2 and 28S are robust markers for distinguishing L. learedi from other species. The nucleotide sequences of the mitochondrial cytochrome c oxidase subunit 1 (COI) region were analyzed to evaluate the genetic variations in L. learedi. The COI haplotypes revealed the extremely high genetic diversity of the species as well as the host turtles on the Ogasawara Islands. The haplotype frequency in the mitochondrial DNA of the green sea turtles on the Ogasawara Islands is known to be significantly different from those in other Pacific rookeries. Although the number of analyzed flukes is small in this study, no haplotype was close to that in other areas; on the basis of the data, we hypothesized that L. learedi differentiated along with the host turtles on the Ogasawara Islands.

Adult schistosomes were detected in the veins or capillaries of the large intestine, mesentery, liver, and adrenal glands in eight of 13 whooper swans (Cygnus cygnus) examined in Iwate Prefecture, Japan. However, neither eggs nor severe tissue injuries were observed in any of the swans. The schistosomes were definitively identified as Allobilharzia visceralis based on the nucleotide sequences of the ribosomal internal transcribed spacer (ITS) region. Allobilharzia visceralis infections have been reported in whooper swan in Iceland and tundra swan (Cygnus columbianus) in North America. These detections suggest that A. visceralis is distributed extensively along the swan flyways because the swans are migratory birds. To the best of our knowledge, this is the first report of A. visceralis infection in Asia.