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.

Introducing the land snail Bradybaena pellucida increased infection risk of the avian parasite Postharmostomum commutatum in the Kanto region of Japan.

The trematode Postharmostomum commutatum is a parasite of the chicken Gallus gallus domesticus. Its heavy infection can cause inflammation and hemorrhage in the cecum of host birds. We found a severe infection of metacercariae of P. commutatum, which was identified based on DNA barcodes with morphology, in the introduced land snail Bradybaena pellucida and its related species in the Kanto region of Japan. Our field survey revealed that metacercariae were detected in 14 of 69 sampling locations in this region. B. pellucida was thought to be the major second intermediate host of metacercariae of the trematode because this snail was most frequently found in the study area and the prevalence and infection intensity were higher than those of the other snail species. The observed increase in metacercariae in introduced populations of B. pellucida can enhance the infection risk of chickens and wild host birds, probably owing to the spillback effect. Our seasonal field study showed that the prevalence and infection intensity of metacercaria seemed to be high in populations of B. pellucida during the summer and early autumn. Therefore, chickens should not be bred outdoors during these seasons to prevent severe infection. Our molecular analysis, based on cytochrome c oxidase subunit I sequences, showed a significantly negative value for Tajima's D in P. commutatum, suggesting an increase in its population size. Thus, P. commutatum distributed in the Kanto region may have increased its population size with the introduction of the host snail.

Morphological and molecular characterization of Eurytrema spp. Looss, 1907 detected in domestic water buffaloes and cattle in northern Vietnam.

Eurytrema spp. are pancreatic flukes belonging to the Dicrocoeliidae family. They are the cause of neglected diseases in Vietnam and are responsible for economic losses in ruminant production, particularly in water buffaloes and cattle. Eurytrema spp. have been widely reported in several Asian countries. Recently, morphological and molecular analyses to discriminate Eurytrema spp. have been conducted in Brazil, China, Bangladesh, Nepal, and Indonesia; however, similar analyses have not been performed in Vietnam. In the present study, we identified Eurytrema flukes collected from water buffaloes and cattle in northern Vietnam based on their morphology. Morphometric analyses were conducted on 15 samples each of Eurytrema cladorchis and Eurytrema coelomaticum. Representative samples from both species were selected for molecular analyses, and the nucleotide sequences of the 18S ribosomal RNA (18S rRNA) gene and internal transcribed spacer 2 (ITS2) were determined. Phylogenetic analyses based on 18S rRNA sequences revealed that E. cladorchis from Vietnam belongs to the same clade as that from Bangladesh. Similarly, E. coelomaticum isolates from Vietnam and China belonged to the same clade. Both clades were isolated from E. pancreaticum. This is the first study to describe the coexistence of E. cladorchis and E. coelomaticum in Vietnam and the first report of the ITS2 nucleotide sequence for E. coelomaticum, which can be used for molecular species discrimination.

Sex-inducing effects toward planarians widely present among parasitic flatworms.

Various parasitic flatworms infect vertebrates for sexual reproduction, often causing devastating diseases in their hosts. Consequently, flatworms are of great socioeconomic and biomedical importance. Although the cessation of parasitic flatworm sexual reproduction is a major target of anti-parasitic drug design, little is known regarding bioactive compounds controlling flatworm sexual maturation. Using the planarian Dugesia ryukyuensis, we observed that sex-inducing substances found in planarians are also widespread in parasitic flatworms, such as monogeneans and flukes (but not in tapeworms). Reverse-phase HPLC analysis revealed the sex-inducing substance(s) eluting around the tryptophan retention time in the fluke Calicophoron calicophorum, consistent with previous studies on the planarian Bipalium nobile, suggesting that the substance(s) is likely conserved among flatworms. Moreover, six of the 18 ovary-inducing substances identified via transcriptome and metabolome analyses are involved in purine metabolism. Our findings provide a basis for understanding and modifying the life cycles of various parasitic flatworms.

Molecular and phylogenetic analysis of E. vermicularis in appendectomy specimens from Iran.

Human infection with Enterobius vermicularis occurs worldwide, particularly in children. The role of E. vermicularis in appendicitis is neglected. This study was designed to investigate genotypes of E. vermicularis detected from appendectomy specimens in the human population from Iran and clarify the intra-species variation of the parasite. Seventy appendectomies for acute clinical appendicitis isolates from Azerbaijan and North Khorasan of Iran were used in the present study. The genetic information of Tehran and Hamedan regions was also obtained from GenBank for comparison and analysis. The nucleotide sequence of cytochrome c oxidase subunit 1 (cox1) gene was analyzed to perform genetic differentiation, haplotype network analysis, and population structure. Phylogenetic analysis of all the isolates were included in type B haplogroup. The number of haplotypes in all geographical locations of Iran is not much. Network analysis of sequences for regions such as Thailand, Iran, Denmark, and Poland show three classified subtypes B1, B2, and B3 in the B haplogroup. It seems that the haplotypes of E. vermicularis detected from appendectomy are B type, and divided into three subtypes. Further research using another genetic marker is required to elucidate the genetic variation of the parasites in detail.

Assessment of genetic markers for multilocus sequence typing (MLST) of Fasciola isolates from Iran.

BACKGROUND: Several markers have been described to characterise the population structure and genetic diversity of Fasciola species (Fasciola hepatica (F. hepatica) and Fasciola gigantica (F. gigantica). However, sequence analysis of a single genomic locus cannot provide sufficient resolution for the genetic diversity of the Fasciola parasite whose genomes are ∼1.3 GB in size. OBJECTIVES: To gain a better understanding of the gene diversity of Fasciola isolates from western Iran and to identify the most informative markers as candidates for epidemiological studies, five housekeeping genes were evaluated using a multilocus sequence typing (MLST) approach. METHODS: MLST analysis was developed based on five genes (ND1, Pepck, Pold, Cyt b and HSP70) after genomic DNA extraction, amplification and sequencing. Nucleotide diversity and phylogeny analysis were conducted on both concatenated MLST loci and each individual locus. A median joining haplotype network was created to examine the haplotypes relationship among Fasciola isolates. RESULTS: Thirty-three Fasciola isolates (19 F. hepatica and 14 F. gigantica) were included in the study. A total of 2971 bp was analysed for each isolate and 31 sequence types (STs) were identified among the 33 isolates (19 for F. hepatica and 14 for F. gigantica isolates). The STs produced 44 and 42 polymorphic sites and 17 and 14 haplotypes for F. hepatica and F. gigantica, respectively. Haplotype diversity was 0.982 ± 0.026 and 1.000 ± 0.027 and nucleotide diversity was 0.00200 and 0.00353 ± 0.00088 for F. hepatica and F. gigantica, respectively. There was a high degree of genetic diversity with a Simpson's index of diversity of 0.98 and 1 for F. hepatica and F. gigantica, respectively. While HSP70 and Pold haplotypes from Fasciola species were separated by one to three mutational steps, the haplotype networks of ND1 and Cyt b were more complex and numerous mutational steps were found, likely due to recombination. CONCLUSIONS: Although HSP70 and Pold genes from F. gigantica were invariant over the entire region of sequence coverage, MLST was useful for investigating the phylogenetic relationship of Fasciola species. The present study also provided insight into markers more suitable for phylogenetic studies and the genetic structure of Fasciola parasites.

Fumarate respiration of Fasciola flukes as a potential drug target.

Fascioliasis is a neglected tropical zoonotic disease caused by liver flukes belonging to the genus Fasciola. The emergence of resistance to triclabendazole, the only World Health Organization-recommended drug for this disease, highlights the need for the development of new drugs. Helminths possess an anaerobic mitochondrial respiratory chain (fumarate respiration) which is considered a potential drug target. This study aimed to evaluate the occurrence of fumarate respiration in Fasciola flukes. We analyzed the properties of the respiratory chain of Fasciola flukes in both adults and newly excysted juveniles (NEJs). Fasciola flukes travel and mature through the stomach, bowel, and abdominal cavity to the liver, where oxygen levels gradually decline. High fumarate reductase activity was observed in the mitochondrial fraction of adult Fasciola flukes. Furthermore, rhodoquinone-10 (RQ10 Em'= -63 mV), a low-potential electron mediator used in fumarate respiration was found to be predominant in adults. In contrast, the activity of oxygen respiration was low in adults. Rotenone, atpenin A5, and ascochlorin, typical inhibitors of mitochondrial enzymes in complexes I, II, and III, respectively, inhibit the activity of each enzyme in the adult mitochondrial fraction. These inhibitors were then used for in vitro viability tests of NEJs. Under aerobic conditions, NEJs were killed by rotenone or ascochlorin, which inhibit aerobic respiration (complex I-III), whereas atpenin A5, which inhibits complex II involved in fumarate respiration, did not affect NEJs. Moreover, ubiquinone-10 (UQ10 Em'= +110 mV), which is used in oxidative respiration, was detected in NEJs, in addition to RQ10. In contrast, under anaerobic conditions, rotenone and atpenin A5, which inhibit fumarate respiration (complex I-II), were crucial for NEJs. These findings demonstrate that NEJs have active hybrid respiration, in which they can properly use both oxygen and fumarate respiration, depending on oxygen availability. Thus, fumarate respiration is a promising drug target for Fasciola flukes, because it plays an essential role in both adults and NEJs.

The first genetic characterization of Setaria marshalli (Nematoda, Spirurida) with reliable DNA barcoding based on a mitochondrial genetic marker.

Setaria marshalli is a mosquito-borne filarial nematode that causes infection in calves younger than two years old. In the present study, nematodes were obtained from a calf in Japan and morphologically identified as S. marshalli. Additionally, the partial cytochrome oxidase subunit I (COI) region (596 bp) was analyzed for the first time to establish a reliable DNA barcode. Nucleotide sequences of COI were identical among the seven worms obtained. The COI region can be a useful marker for species discrimination in the case of S. marshalli since nucleotide variations observed between the closest congener, Setaria cervi (51/596 bp), were sufficient to allow species discrimination. However, the phylogenetic relationship of S. marshalli with its congeners was unclear in a maximum likelihood tree. We found that the partial COI sequence of S. marshalli analyzed in the present study matched a relevant section of the complete mitochondrial genome of S. labiatopapillosa that was deposited in the International Nucleotide Sequence Database. This finding suggests that S. marshalli was misdiagnosed as S. labiatopapillosa in a previous study. It is crucial to conduct accurate morphological analyses to obtain reliable molecular information regarding Setaria nematodes.

Title: Première caractérisation génétique de Setaria marshalli (Nematoda, Spirurida) avec un code-barres ADN fiable basé sur un marqueur génétique mitochondrial. Abstract: Setaria marshalli est une filaire transmise par les moustiques qui provoque une infection chez les veaux de moins de deux ans. Dans la présente étude, les nématodes ont été obtenus à partir d'un veau au Japon et identifiés morphologiquement comme S. marshalli. De plus, la région partielle de la sous-unité I (COI) de la cytochrome oxydase (596 pb) a été analysée pour la première fois afin d'établir un code-barres ADN fiable. Les séquences nucléotidiques de COI étaient identiques parmi les sept vers obtenus. La région COI peut être un marqueur utile pour la discrimination des espèces dans le cas de S. marshalli puisque les variations de nucléotides observées avec le congénère le plus proche, Setaria cervi (51/596 pb) étaient suffisantes pour permettre la discrimination des espèces. Cependant, la relation phylogénétique de S. marshalli avec ses congénères n'était pas claire dans un arbre à maximum de vraisemblance. Nous avons constaté que la séquence COI partielle de S. marshalli analysée dans la présente étude correspondait à une section pertinente du génome mitochondrial complet de S. labiatopapillosa qui a été déposée dans la base de données internationale de séquences de nucléotides. Cette découverte suggère que S. marshalli a été diagnostiqué à tort comme S. labiatopapillosa dans une étude précédente. Il est crucial de mener des analyses morphologiques précises pour obtenir des informations moléculaires fiables concernant les nématodes du genre Setaria.

Dispersal direction of Malaysian Fasciola gigantica from neighboring southeast Asian countries inferred using mitochondrial DNA analysis.

Fasciola gigantica and hybrid Fasciola flukes, responsible for the disease fasciolosis, are found in Southeast Asian countries. In the present study, we performed molecular species identification of Fasciola flukes distributed in Terengganu, Malaysia using multiplex PCR for phosphoenolpyruvate carboxykinase (pepck) and PCR-restriction fragment length polymorphism (RFLP) for DNA polymerase delta (pold). Simultaneously, phylogenetic analysis based on mitochondrial NADH dehydrogenase subunit 1 (nad1) was performed for the first time on Malaysian Fasciola flukes to infer the dispersal direction among neighboring countries. A total of 40 flukes used in this study were identified as F. gigantica. Eight nad1 haplotypes were identified in the F. gigantica population of Terengganu. Median-joining network analysis revealed that the Malaysian population was related to those obtained from bordering countries such as Thailand and Indonesia. However, genetic differentiation was detected using population genetics analyses. Nevertheless, the nucleotide diversity (π) value suggested that F. gigantica with the predominant haplotypes was introduced into Malaysia from Thailand and Indonesia. The dispersal direction suggested by population genetics in the present study may not be fully reliable since Fasciola flukes were collected from a single location in one state of Malaysia. Further studies analyzing more samples from many locations are required to validate the dispersal direction proposed herein.

Development of novel DNA marker for species discrimination of Fasciola flukes based on the fatty acid binding protein type I gene.

BACKGROUND: Multiplex polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) for nuclear phosphoenolpyruvate carboxykinase (pepck) and polymerase delta (pold), respectively, have been used to differentiate Fasciola hepatica, F. gigantica, and hybrid Fasciola flukes. However, discrimination errors have been reported in both methods. This study aimed to develop a multiplex PCR based on a novel nuclear marker, the fatty acid binding protein type I (FABP) type I gene. METHODS: Nucleotide sequence variations of FABP type I were analyzed using DNA samples of F. hepatica, F. gigantica, and hybrid Fasciola flukes obtained from 11 countries in Europe, Latin America, Africa, and Asia. A common forward primer for F. hepatica and F. gigantica and two specific reverse primers for F. hepatica and F. gigantica were designed for multiplex PCR. RESULTS: Specific fragments of F. hepatica (290 bp) and F. gigantica (190 bp) were successfully amplified using multiplex PCR. However, the hybrid flukes contained fragments of both species. The multiplex PCR for FABP type I could precisely discriminate the 1312 Fasciola samples used in this study. Notably, no discrimination errors were observed with this novel method. CONCLUSIONS: Multiplex PCR for FABP type I can be used as a species discrimination marker in place of pepck and pold. The robustness of the species-specific primer should be continuously examined using a larger number of Fasciola flukes worldwide in the future since nucleotide substitutions in the primer regions may cause amplification errors.

Population structure, molecular characterization, and phylogenetic analysis of Fasciola gigantica from two locations in Uganda.

Fasciola gigantica is a major pathogen that causes fasciolosis in Africa. A recent study in Uganda demonstrated that Fasciola flukes were present in 65.7% of slaughtered cattle. However, molecular identification of Fasciola species has not yet been performed in the country. In the present study, 292 Fasciola flukes were collected from Kampala and Gulu, Uganda. The samples were identified as F. gigantica using a multiplex polymerase chain reaction (PCR) assay for phosphoenolpyruvate carboxykinase (pepck) and a PCR-restriction fragment length polymorphism (RFLP) assay for DNA polymerase delta (pold). A significant genetic difference between F. gigantica obtained from cattle slaughtered at Kampala and Gulu was observed by analyzing the mitochondrial markers NADH dehydrogenase subunit 1 (nad1) and cytochrome C oxidase subunit 1 (cox1). Fasciola collected from Gulu had a more diversified population than that collected from Kampala, probably because of differences in livestock management systems. One of the possible reasons for this observation is that cattle slaughtered in Gulu were reared under an extensive communal grazing system, which is suitable for maintaining parasite diversity, whereas cattle slaughtered in Kampala mainly originated from fenced/closed farms, which limits parasite diversity. However, the cause of the difference between these two locations was not clearly defined by the results of this study. The F. gigantica population from Uganda was related to that obtained from Zambia. A star-like phylogeny was detected in a median-joining network analysis, which indicated rapid population expansion and suggested that the F. gigantica populations from both countries are maintained by domestic ruminants in eastern Africa. Interestingly, the F. gigantica population from Uganda was not related to those from Egypt and Nigeria. The results of the present study suggest that F. gigantica populations in African countries are indigenous to each country or region.

Morphological and molecular characterization of Calicophoron raja (Näsmark, 1937) collected from wild Bovidae in South Africa.

Paramphistomes, commonly known as rumen flukes, are digenean parasites that infect ruminants. Accurate morphological identification of paramphistome species is challenging and often neglected. For instance, it requires sagittal midline sections of adult flukes, which are difficult to prepare. Therefore, the majority of the genetic information on paramphistomes found in the International Nucleotide Sequence Database is not supported by morphological descriptions, and the DNA barcodes of paramphistome species remain unreliable. In the present study, both morphological and molecular characterizations were simultaneously performed to ensure the reliability of the DNA information for the paramphistome species Calichophoron raja (Näsmark, 1937). The morphological characteristics of the sagittal and horizontal sections of adult flukes from a black wildebeest (Connochaetes gnou) and a waterbuck (Kobus ellipsiprymnus) in South Africa were identical to those previously described for Ca. raja. Additionally, this study represents a new host record of the species from Co. gnou. All sequences of the internal transcribed spacer 2 region of ribosomal DNA were 100% identical among the 18 flukes analyzed in the present study. A single nucleotide mutation was observed between Ca. raja in this study and Ca. raja detected in domestic ruminants in Kenya.

Leptospiral meningoencephalitis in a raccoon dog.

Neuroleptospirosis is a rare disease caused by pathogenic Leptospira interrogans in humans; however, it has not been fully studied in animals. A young wild raccoon dog was found convulsing in the recumbent position and died the next day. Histologic examination revealed nonsuppurative meningoencephalitis in the cerebrum, cerebellum, midbrain, and medulla oblongata. The lesions consisted of mixed infiltrates of Iba1-positive macrophages and CD3-positive T cells, with a small number of CD79α-positive B cells and myeloperoxidase-positive neutrophils. In the frontal cortex, perivascular cuffs and adjacent microglial nodules were distributed diffusely, especially in the molecular layer. Glial nodules were comprised of Iba1- and myeloperoxidase-positive activated microglia. Immunohistochemistry revealed leptospires in mononuclear cell perivascular cuffs, but not in glial nodules. Neuroleptospirosis was accompanied by Leptospira-related nonsuppurative interstitial nephritis, pulmonary edema and hemorrhage, and coronary periarteritis, as well as Toxocara tanuki in the small intestine and nonspecific foreign-body granulomas in the lungs and stomach.

Identification of novel yolk ferritins unique to planarians: planarians supply aluminum rather than iron to vitellaria in egg capsules.

All animals, other than Platyhelminthes, produce eggs containing yolk, referred to as "entolecithal" eggs. However, only Neoophora, in the phylum Platyhelminthes, produce "ectolecithal" eggs (egg capsules), in which yolk is stored in the vitelline cells surrounding oocytes. Vitelline cells are derived from vitellaria (yolk glands). Vitellaria are important reproductive organs that may be studied to elucidate unique mechanisms that have been evolutionarily conserved within Platyhelminthes. Currently, only limited molecular level information is available on vitellaria. The current study identified major vitellaria-specific proteins in a freshwater planarian, Dugesia ryukyuensis, using peptide mass fingerprinting (PMF) and expression analyses. Amino acid sequence analysis and orthology analysis via OrthoFinder ver.2.3.8 indicated that the identified major vitellaria-specific novel yolk ferritins were conserved in planarians (Tricladida). Because ferritins play an important role in Fe (iron) storage, we examined the metal elements contained in vitellaria and ectolecithal eggs, using non-heme iron histochemistry, elemental analysis based on inductively coupled plasma mass spectrometry and transmission electron microscopy- energy-dispersive X-ray spectroscopy analysis. Interestingly, vitellaria and egg capsules contained large amounts of aluminum (Al), but not Fe. The knockdown of the yolk ferritin genes caused a decrease in the volume of egg capsules, abnormality in juveniles, and increase in Al content in vitellaria. Yolk ferritins of D. ryukyuensis may regulate Al concentration in vitellaria via their pooling function of Al and protect the egg capsule production and normal embryogenesis from Al toxicity.

Geographic origin of Explanatum explanatum (Creplin, 1847) Fukui, 1929 detected from domestic water buffaloes in Sri Lanka.

The adult stage of Explanatum explanatum has economic importance in the production of ruminants, especially water buffaloes. This species has been widely reported in the Indian sub-continent. Recently, molecular analyses to reveal the dispersal route of this species were performed in Bangladesh, Nepal, and India. In the present study, we focused on E. explanatum distributed in Sri Lanka. A total of 52 flukes were collected from water buffaloes in Sri Lanka and identified as E. explanatum based on the internal transcribed spacer 2 (ITS2) region of nuclear ribosomal DNA. Analysis of the mitochondrial NADH dehydrogenase subunit 1 (nad1) gene from DNA samples detected 18 haplotypes, and five of them were identical to those from the Indian E. explanatum. The pairwise fixation index value indicated that the Sri Lankan population had a comparatively closer relationship with the Indian population than with the Bangladeshi or Nepalese populations. The Sri Lankan population showed significantly lower genetic variability than the Indian population, suggesting that the Indian population was the ancestor of the Sri Lankan population. The movement of host ruminants, including water buffaloes, was probably involved in the introduction of the fluke into Sri Lanka. The results of our study provide useful information for elucidating the geographic origin of E. explanatum distributed in the Indian subcontinent.

Enzyme-linked immunosorbent assay (ELISA) using recombinant Fasciola cathepsin L1 for the diagnosis of human fasciolosis caused by Fasciola hepatica/gigantica hybrid type.

Recombinant Fasciola cathepsin L-1 (rCatL1) was evaluated in enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of human fasciolosis in Japan. Quality characteristics of the test were accessed by receiver operating characteristic (ROC) analysis, with sera from fasciolosis patients (n = 10), patients with no evidence of parasitic infections (n = 29), and patients with other helminth infections (n = 119). Both the sensitivity and specificity of the test achieved 100% with the control samples. To test the performance of the assay in an authentic situation, 311 serum samples, which had been sent to our laboratory for the diagnosis of parasitic infections from January 2018 to February 2019, were re-assessed using the rCatL1 ELISA. In this case, the sensitivity of the rCatL1 ELISA was 100%, giving positive results to all fasciolosis sera (n = 7), and the specificity was 99.0%, in which three of the 304 non-fasciolosis samples were judged positive. Careful re-examination of the laboratory data and medical imaging of these three patients revealed that one of the patients, who had been diagnosed as having larva migrans syndrome, was judged to be infected with Fasciola, in addition to ascarid nematodes. Thus the true specificity of the assay in the authentic reached 99.3% (302/304). As the rCatL1 ELISA exhibited a highly significant positive likelihood ratio (152.0) and negative likelihood ratio (0.0), calculated from the 311 sample data, this rCatL1 ELISA can be used for routine screening and definitive diagnosis test for fasciolosis in reference laboratories.

Molecular characterization and phylogenetic analyses of Fasciola gigantica of buffaloes and goats in Punjab, Pakistan.

Fasciola gigantica is considered to be a major pathogen causing fasciolosis in the Indian subcontinent, resulting in production losses of millions of dollars in the livestock industry. Understading the dispersal origin and the patterns of spread of F. gigantica is important. A total of 53 Fasciola flukes collected from buffaloes and goats in Punjab, Pakistan between 2017 and 2018 were identified as F. gigantica based on the multiplex PCR for the phosphoenolpyruvate carboxykinase (pepck) and the PCR-restriction fragment length polymorphism (RFLP) for DNA polymerase delta (pold). A significant genetic difference between F. gigantica from buffaloes and goats was indicated by the genetic analyses of mitochondrial markers, NADH dehydrogenase subunit 1 (nad1) and cytochrome C oxidase subunit 1 (cox1). Phylogenetic analysis of the seventeen nad1 haplotypes of F. gigantica from Pakistan with those in neighbouring countries of the Indian subcontinent revealed that all the haplotypes identified in Pakistan were clustered in haplogroup A. fasciola gigantica with the eight haplotypes might be expanded in Pakistan from Indian origin, along with the migration of the domestic animals, since they were related to Indian haplotypes. In contrast, the remaining nine haplotypes were not shared with any neighbouring countries, suggesting independent origin, probably from neighbouring Middle East countries. However, cautious interpretation is required due to the very limited samples size of this study. Our study provides a proof of concept for a method that could be used to investigate the epidemiology of F. gigantica.

Molecular detection of filarial nematode parasites in Japanese black bears (Ursus thibetanus japonicus) from Iwate Prefecture, Japan.

This study aimed to detect filarial parasites in blood samples of Japanese black bears (Ursus thibetanus japonicus) collected from Iwate Prefecture, Japan. Positive amplicons were obtained from 26 out of 30 samples by nested PCR targeting 18S ribosomal RNA gene and first internal transcribed spacer regions. DNA sequences of Mansonella sp. close to M. ozzardi and Dirofilaria sp. were detected for eight and 11 positive amplicons, respectively. Co-infection was detected for the remaining seven amplicons. Dirofilaria sp. was identified as D. ursi by further genetic analysis of 5S ribosomal RNA gene sequence. The results of this study will contribute to further investigations of Japanese black bears for monitoring their risk as a reservoir of possible zoonotic filarial parasites.

Seroprevalence of fasciolosis in Hokkaido sika deer (Cervus nippon yesoensis) from Hokkaido Prefecture, Japan revealed by ELISA using recombinant cathepsin L1.

Fasciolosis, a zoonotic disease caused by liver flukes of the genus Fasciola, has been reported in Hokkaido (Yezo) sika deer (Cervus nippon yesoensis) in Hokkaido Prefecture, Japan; however, the actual seroprevalence in the animal has not been adequately evaluated. The objective of the present study was to analyze the seroprevalence of the disease among Hokkaido sika deer. Recombinant cathepsin L1 (rCatL1) was used as an antigen for an indirect enzyme-linked immunosorbent assay (ELISA) to detect antibodies against Fasciola flukes. The sensitivity and specificity of the ELISA were 84.6% and 100%, respectively. The average seroprevalence in 1109 Hokkaido sika deer from 20 locations in Hokkaido Prefecture was 43.9%. Mature deer showed higher seroprevalence than younger individuals; however, even younger animals may act as a reservoir for the disease. Monitoring infection levels in the Hokkaido sika deer population is important not only for the livestock industry, but also for preventing human fasciolosis.

Molecular characterization and phylogenetic analysis of Fasciola hepatica from high- plateau and steppe areas in Algeria.

A previous study based on mitochondrial DNA markers reported the presence of Fasciola hepatica in Algeria. However, a precise species identification is still required. In this report, a total of 68 Fasciola isolates, collected from high-plateau (Bordj-Bou-Arreridj) and steppe (Djelfa) areas of Algeria, were identified at the species level by multiplex PCR and PCR-restriction fragment length polymorphism (RFLP) for nuclear phosphoenolpyruvate carboxykinase (pepck) and DNA polymerase delta (pold), respectively. The result of the multiplex PCR conflicted with that of the PCR-RFLP; however, subsequent nucleotide sequencing of pepck clearly showed that all isolates should be classified as F. hepatica. The two mitochondrial markers, NADH dehydrogenase subunit I (nad1) and cytochrome c oxidase subunit 1 (cox1), revealed a close relationship between the parasite populations from the plateau and those from the steppe. A dispersal direction from the high plateau to the steppe was indicated because the former population was more diversified than the latter. Moreover, these populations were more closely related to populations from Spain than those from Egypt or Afghanistan. Given the population characteristic of F. hepatica in Spain and the history of cattle trade, it seems likely that the parasite was introduced to Algeria from Europe through a route across the Mediterranean Sea.