Evaluation of protective immune responses induced by DNA vaccines encoding Echinococcus granulosus EgM123 protein in Beagle dogs.

Introduction: Echinococcus granulosus, known as cystic echinococcosis, is a prominent zoonotic parasitic disease of significant global concern. The definitive hosts serves as the primary reservoir for the transmission of echinococcosis, as well as a main factor in the prevention and control of the disease. Unfortunately, there is currently no commercially available vaccine for these hosts. Nevertheless, DNA vaccines show potential as a feasible strategy for the control and management of parasitic diseases. Methods: In this study, the EgM123 antigen was selected for its well-documented immunogenic properties to develop a DNA vaccine aimed at combating E. granulosus infection in canines. Results: The results showed a marked increase in IgG levels in the group vaccinated with pVAX1-EgM123 DNA compared to the PBS group. Additionally, the cytokines IL-1, IFN-γ, IL-4, and IL-6 were significantly upregulated in the pVAX1-EgM123 DNA vaccine group. Furthermore, in comparison to the PBS control group, the EgM123 DNA vaccine group exhibited a notable 87.85% reduction in worm burden and a 65.00% inhibition in segment development. Discussion: These findings indicate that the pVAX1-EgM123 DNA vaccine shows promising immunogenicity, successfully eliciting a targeted immune response in canines. Moreover, it significantly diminishes the worm burden and hinders the progression of tapeworms in the pVAX1-EgM123 DNA vaccine group. These findings suggest that the pVAX1-EgM123 DNA vaccine holds promise as a potential candidate vaccine for combating E. granulosus infection in dogs.

Global distribution and definitive host range of Echinococcus species and genotypes: A systematic review.

Echinococcus species (spp.) are regarded as neglected cestodes causing several potential zoonoses of global public health. This systematic review critically appraises the worldwide distribution of Echinococcus spp. and genotypes (Echinococcus spp.: recognized species in the genus; genotypes: variants identified within E. granulosus sensu lato.) in definitive hosts. We analyzed 82 studies from major databases, comprising 24 individual host species, including canids, felids, and a hyenid species. Canids, particularly dogs, were the most studied group among the host species, with E. granulosus sensu stricto (G1-G3) being the most frequently reported. E. granulosus s.s. was distributed across five major continents, while other Echinococcus spp. and genotypes exhibited an uneven continental distribution. The highest overlap of species existed among Asia, Europe, and Africa. Among the reported host species, 4.2 % were endangered (e.g. Lycaon pictus), 12.5 % species were vulnerable (e.g. Panthera leo, Panthera pardus, and Acinonyx jubatus), and 4.2 % were near threatened (e.g. Speothos venaticus). Overall, our review highlights the significance of canids, particularly dogs, as the core focus of scientific investigations, with E. granulosus s.s. being the most widely distributed species across five major continents, emphasizing the urgent need for continued research and public health efforts.

First report of Paracapillaria philippinensis infection in flowerhorn cichlid in India.

The flowerhorn cichlid is a popular ornamental fish in many Asian countries. The present study reports the occurrence of Paracapillaria philippinensis (Chitwood et al., 1968), a parasitic nematode in flowerhorn cichlid (Cichlasoma sp.) from south India. The infected fish demonstrated clinical symptoms viz. dark coloration, poor appetite, lethargy, erratic swimming, and white stringy faeces. Microscopic observation of the intestinal content and faeces revealed the presence of adult worms, larvae, and unembryonated eggs. PCR amplification of eukaryotic 18S rRNA, P. philippinensis-specific SSU rRNA gene, and the subsequent sequence analysis confirmed the species identity as P. philippinensis. The generated sequences were submitted in the GenBank, NCBI, under the accession numbers, MK895507.1, MK895446.1, MW144993.1, and OR685675.1. This is the first scientific report of P. philippinensis in fish from India, and it confirms that the flowerhorn cichlid can act as a definitive host for P. philippinensis. This report alerts fish handlers and enthusiasts to undertake suitable precautionary measures while handling live flowerhorn cichlids to prevent possible transmission of P. Philippinensis, which has the potential to infect humans causing intestinal capillariasis.

Echinococcus species in wildlife.

Transmission of Echinococcus spp. in life cycles that involve mainly wildlife is well recognized for those species with small mammals as intermediate hosts (e. g. E. multilocularis), as well as for E. felidis and the 'northern' genotypes of E. canadensis (G8 and G10). In contrast, the remaining taxa of E. granulosus sensu lato are best known for their domestic life cycles, and the numerous wild mammal species (mainly ungulates) that have been recorded with cystic echinococcosis in the past were mainly considered a result of spill-over from the dog-livestock transmission system. This view was challenged with the advent of molecular characterization, allowing discrimination of the metacestodes, although the contribution of wild mammals to various Echinococcus life cycles has remained uncertain for scarcity of wildlife studies. Numerous records of cysts in wild ungulates date back to the 20th century, but cannot with certainty be allocated to the Echinococcus species and genotypes that are recognized today. This means that our current knowledge is largely restricted to studies of the past two decades that kept adding gradually to our concepts of transmission in various geographic regions. In particular, new insights were gathered in the past years on E. granulosus s.l. in wildlife of sub-Saharan Africa, but also on transmission patterns of E. multilocularis in previously neglected regions, e. g. North America. Here, an update is provided on the current state of knowledge on wild mammals as hosts for all Echinococcus species, listing >150 species of wild hosts with references, as well as estimates on their epidemiological impact and our current gaps of knowledge.

Proteomics analysis of Toxoplasma gondii merozoites reveals regulatory proteins involved in sexual reproduction.

Sexual reproduction plays a crucial role in the transmission and life cycle of toxoplasmosis. The merozoites are the only developmental stage capable of differentiation into male and female gametes, thereby initiating sexual reproduction to form oocysts that are excreted into the environment. Hence, our study aimed to perform proteomic analyses of T. gondii Pru strain merozoites, a pre-sexual developmental stage in cat IECs, and tachyzoites, an asexual developmental stage, using the tandem mass tag (TMT) method in order to identify the differentially expressed proteins (DEPs) of merozoites. Proteins functions were subjected to cluster analysis, and DEPs were validated through the qPCR method. The results showed that a total of 106 proteins were identified, out of which 85 proteins had quantitative data. Among these, 15 proteins were differentially expressed within merozoites, with four exhibiting up-regulation and being closely associated with the material and energy metabolism as well as the cell division of T. gondii. Two novel DEPs, namely S8GHL5 and A0A125YP41, were identified, and their homologous family members have been demonstrated to play regulatory roles in oocyte maturation and spermatogenesis in other species. Therefore, they may potentially exhibit regulatory functions during the differentiation of micro- and macro-gametophytes at the initiation stage of sexual reproduction in T. gondii. In conclusion, our results showed that the metabolic and divisional activities in the merozoites surpass those in the tachyzoites, thereby providing structural, material, and energetic support for gametophytes development. The discovery of two novel DEPs associated with sexual reproduction represents a significant advancement in understanding Toxoplasma sexual reproduction initiation and oocyst formation.

Case report: Infection with Dicrocoelium dendriticum in a Japanese Chin dog.

Dicrocoelium dendriticum is a trematode colonising the bile ducts of herbivores. Coproscopic findings in dogs are usually considered gastrointestinal passages of eggs after ingestion of unheated liver tissue or infected ruminant faeces. Here, a Japanese Chin presented with diarrhoea and weight loss. Eggs comparable to D. dendriticum were detected in faeces and infection was confirmed via PCR and by ruling out differential diagnoses. Egg excretion continued for a period of 10 months. Praziquantel (50 mg/kg body weight [BW]) was administered orally for four consecutive days. Egg excretion 10 days after treatment entailed further treatments with 100 mg/kg BW, again for four days. Faecal samples were negative ten days and four weeks afterwards, diarrhoea resolved, and the dog gained weight. In cases of repeated coproscopic positivity for D. dendriticum, an infection with dogs acting as definitive hosts should be considered. Treatment with praziquantel at a higher dosage may be required.

Metapopulation Structure of Two Species of Pikeworm (Triaenophorus, Cestoda) Parasitizing the Postglacial Fish Community in an Oligotrophic Lake.

In the present study, we estimated the levels of infestation of the main fish species that are hosts for two Triaenophorus species: T. crassus and T. nodulosus. The prevalence of T. crassus and T. nodulosus infestations in the intestine of their definitive host-pike Esox lucius was similar (71.0% and 77.4%, respectively). At the same time, the prevalence of T. crassus infestation in muscle tissue was significantly different between the second intermediate hosts, Coregonus lavaretus pidschian (31.4%) and Cor. l. pravdinellus (91.2%), due to considerable differences in their diets. For T. nodulosus, we found significant variations in the levels of prevalence among the second intermediate hosts-100% for Lota lota, 81.8% for Cottus sibiricus 31.9% for Thymallus arcticus, and 24.5% for Perca fluviatilis-that we also explained using different diets. Moreover, analysis of the symmetry of parasite infestations did not reveal any asymmetry between the number of cysts in the left and right body surfaces of the "planktivorous" form/species of whitefish, whereas in the ''benthivorous", an asymmetry of parasite infestations was found.

Eight cases of canine thelaziosis found in two localities in Korea.

Thelazia callipaeda, a parasitic nematode that causes thelaziosis in various mammals, including humans, is known to be endemic in Korea. However, life cycle-related information on the parasite, primarily from human infection and a few dog cases, is limited. This study reports additional cases of T. callipaeda infections in dogs from both rural and urban areas in Korea, indicating the potential for transmission to humans and other animals. We collected 61 worms from 8 infected dogs from Paju and Cheongju Cities and observed their morphological characteristics under a light microscope. The findings indicate that T. callipaeda infections in animals in Korea may be underestimated and are distributed close to human environments. Our results contribute to the growing knowledge of the reservoir hosts of T. callipaeda in Korea and highlight the importance of continued surveillance and research to prevent and control this emerging zoonotic disease.

Dynamic RNA profiles in the small intestinal epithelia of cats after Toxoplasma gondii infection.

BACKGROUND: Felids are the only definitive hosts of Toxoplasma gondii. However, the biological features of the feline small intestine following T. gondii infection are poorly understood. We investigated the changes in the expression of RNAs (including mRNAs, long non-coding RNAs and circular RNAs) in the small intestinal epithelia of cats following T. gondii infection to improve our understanding of the life cycle of T. gondii and cat responses to T. gondii infection. METHODS: Fifteen cats were randomly assigned to five groups, and the infection groups were inoculated with 600 tissue cysts of the T. gondii Pru strain by gavage. The small intestinal epithelia of cats were collected at 6, 10, 14, and 30 days post infection (DPI). Using high-throughput RNA sequencing (RNA-seq), we investigated the changes in RNA expression. The expression levels of differentially expressed (DE) genes and non-coding RNAs (ncRNAs) identified by RNA-seq were validated by quantitative reverse transcription PCR (qRT-PCR). Differential expression was determined using the DESeq R package. RESULTS: In total, 207 annotated lncRNAs, 20,552 novel lncRNAs, 3342 novel circRNAs and 19,409 mRNAs were identified. Among these, 70 to 344 DE mRNAs, lncRNAs and circRNAs were detected, and the post-cleavage binding sites between 725 ncRNAs and 2082 miRNAs were predicted. Using the co-location method, we predicted that a total of 235 lncRNAs target 1044 protein-coding genes, while the results of co-expression analysis revealed that 174 lncRNAs target 2097 mRNAs. Pathway enrichment analyses of the genes targeted by ncRNAs suggested that most ncRNAs were significantly enriched in immune or diseases-related pathways. NcRNA regulatory networks revealed that a single ncRNA could be directly or indirectly regulated by multiple genes or ncRNAs that could influence the immune response of cats. Co-expression analysis showed that 242 circRNAs, mainly involved in immune responses, were significantly associated with T. gondii infection. In contrast, 1352 protein coding RNAs, mainly involved in nucleic acid process/repair pathways or oocyte development pathways, were negatively associated with T. gondii infection. CONCLUSIONS: This study is the first to reveal the expression profiles of circRNAs, lncRNAs and mRNAs in the cat small intestine following T. gondii infection and will facilitate the elucidation of the role of ncRNAs in the pathogenesis of T. gondii infection in its definitive host, thereby facilitating the development of novel intervention strategies against T. gondii infection in humans and animals.

Molecular Identification of Sarcocystis rileyi and Sarcocystis sp. (Closely Related to Sarcocystis wenzeli) in Intestines of Mustelids from Lithuania.

The genus Sarcocystis is a group of numerous protozoan parasites having a two-host life cycle. Based on laboratory experiments and/or phylogenetic analysis results it was shown that seven Sarcocystis spp. producing sarcocsyts in bird tissues are transmitted via predatory placental mammals. To date the role of small mammals of the family Mustelidae in the distribution of avian Sarcocystis spp. have not been studied. During the current investigation, intestinal mucosa scrapings of 115 mustelids belonging to five species were tested for S. albifronsi, S. anasi, S. rileyi, and S. wenzeli infecting anseriforms and chickens. Microscopically, free sporocysts, sporulating oocysts, and loose oocysts were found in 61 samples (53.0%). Using nested PCR targeting the ITS1 region and sequencing, S. rileyi was confirmed in eight American minks, two European polecats and single European badger. Sarcocystis sp. was identified in one American mink and one European pine marten. Based on the partial ITS1 region this parasite showed that 100% identity to pathogenic Sarcocystis sp. caused a fatal infection in backyard chickens from Brazil. Phylogenetically, the Sarcocystis sp. identified in our study was most closely related to S. wenzeli parasitising domestic fowl (Gallus domesticus).

Seroprevalence and risk factors of Toxoplasma gondii in urban cats from China.

BACKGROUND: Toxoplasma gondii infects almost all warm-blooded animals, and cats play a crucial role in the epidemiology of T. gondii as the definitive host. Despite sporadic reports on the seroprevalence of T. gondii in domestic cats, systematic surveys are lacking and some regions remain in China uninvestigated. METHODS: A total of 1,521 serum samples were collected from 10 regions of China and analyzed by antibodies against T. gondii by ELISA with the purpose of identifying risk factors of T. gondii infection in cats across China and obtaining seroprevalence data from some previously uninvestigated areas. RESULTS: Antibodies to T. gondii were detected in 62 of 1,478 (4.2%) urban pet cats and in 9 of 43 (20.9%) stray cats. Among the regions examined, the prevalence was 13% in Sichuan, 12.8% in Chongqing, 6.4% in Hunan, 2.5% in Hubei and 0.9% in Guangdong. Additionally, this is the first report on the seroprevalence of T. gondii in urban pet cats from Qinghai (6.2%), Anhui (3.1%), Jiangxi (2.5%), Shaanxi (2.4%) and Ningxia (1.6%). The age and lifestyle (stray or pet) of cats were identified as the risk factors for seropositivity by multivariate analysis of the data. CONCLUSIONS: Our findings improve our understanding of seroprevalence and risk factors of T. gondii infection in cats across China, and provide useful information for the formulating of preventive and control measures against this widespread zoonotic parasite.

Metabolomics study of cat small intestine during the early stage of Toxoplasma gondii oocyst formation identifies potential biomarkers.

Toxoplasma gondii (T. gondii) is a zoonotic intracellular protozoan parasite that can invade, replicate and survive in almost all cells of warm-blooded animals. T. gondii infection threatens the life of the fetus or can cause morbidity in the infant. As the only definitive host of T. gondii, felids spread the pathogen mainly by forming oocysts in the small intestines and discharging the oocysts into the ambient environment, consequently polluting water, vegetables, and meat products. In this study, we used untargeted metabolomics technology to study the changes in metabolites that occurred during the early stage of oocyst formation in the cat small intestine following T. gondii infection and attempted to identify metabolic biomarkers that could potentially be used as diagnostic molecular markers in the future. Domestic cats (Felis catus) were infected with T. gondii Pru tissue cysts, and samples of their small intestinal epithelium were collected at 2 and 4 days post-infection (DPI) for metabolic analysis. LC-MS/MS and multivariate statistical analysis were employed to detect metabolomic signatures that discriminated between the infected and control groups. A total of 1673 ions and 1201 ions were obtained in the positive and negative modes, respectively. Of these ions, 175 were up-regulated and 127 were down-regulated in the positive ion mode; whereas, 123 were up-regulated and 81 were down-regulated in the negative ion mode. Three commonly altered ions (0.74_313.0414 m/z, 8.82_615.2621 m/z and 8.16_325.2362 m/z) were determined to have potential research value. Seventy common metabolic pathways were enriched at two time points, with arginine biosynthesis, pyrimidine metabolism, pantothenate and CoA biosynthesis being the three most significant pathways related to T. gondii. The area under the curve (AUC) of differential metabolites combined with relevant literature analysis showed that N-Methylpelletierine and 3,3-Difluoro-17-methyl-5alpha-androstan-17beta-ol have higher predictability and better potential application value than other metabolites. Our analysis of metabolic markers during the early stage of T. gondii oocyst formation in the small intestine of the definitive host (cat) provided novel insight for understanding oocyst development and a theoretical basis for the application of potential biomarkers.

[Progress of researches on infection with two species of Echinococcus causing human diseases in animal hosts and influencing factors].

Echinococcosis is a serious zoonotic parasitic disease caused by infections with larval Echinococcus. The life cycle of Echinococcus involves a variety of animal hosts, including hoofed animals and rodents as intermediate hosts and carnivores as definitive hosts. The transmission of human echinococcosis is closely associated with the life cycle of E. granulosus and E. multilocularis among animal hosts in nature. This review summarizes the recent advances in the prevalence and influencing factors of E. granulosus and E. multilocularis infections in animal hosts, so as to provide insights into precision control of echinococcosis.

Sarcocystis calchasi and other Sarcocystidae detected in predatory birds in California, USA.

Outbreaks of neurological disease associated with Sarcocystis calchasi have been observed in captive and free-ranging rock pigeons (Columba livia) in Europe and the United States as well as in wild Brandt's cormorants (Phalacrocorax penicillatus) and captive psittacines in California, USA. Experimental and field studies have identified northern goshawks (Accipiter gentilis) and European sparrowhawks (A. nisus) as definitive hosts in Europe while the definitive hosts elsewhere remain unknown. In this study, we aimed to identify the potential definitive host(s) of S. calchasi through molecular analysis of intestinal samples from seven predatory (n = 85) and one omnivorous (n = 11) bird species in California. In total, apicomplexan-generic 28S rRNA PCR products were obtained and sequenced for 42 raptors. Three of 16 (18.8%) Cooper's hawks (A. cooperii) and two of 26 (5.6%) red-tailed hawks (Buteo jamaicensis) also tested positive for the S. calchasi-specific ITS1 PCR and sequencing of the 28S rRNA PCR product was 100% homologous to S. calchasi. In addition to S. calchasi (5.9%; 5/85), other Sarcocystis spp. detected in raptors included: S. jamaicensis (21.2%; 18/85), S. columbae (8.2%; 7/85), S. turdusi (7.1%; 6/85), and S. halieti (4.7; 4/85%). Infections with closely related S. jamaicensis and S. (Frenkelia) microti (9.4%; 8/85) could not be distinguished for eight raptors. Eumonospora henryae (1.2%; 1/85) was detected in one raptor. Our results indicate for the first time that S. calchasi may have a definitive host range in North America that includes at least two raptors, Cooper's hawks and red-tailed hawks, within the family Accipitridae.

The Role of Birds of the Family Corvidae in Transmitting Sarcocystis Protozoan Parasites.

Members of the family Corvidae are ecologically flexible omnivorous birds, particularly adaptive to urban habitats, and living in proximity to humans; these birds may serve as definitive hosts (DH) for Sarcocystis spp., but research about this is lacking. In the present study, intestinal samples from 91 corvids collected in Lithuania were molecularly tested by species-specific PCR targeting the ITS1 and cox1 genes and subsequently sequenced for the presence of Sarcocystis spp. Under a light microscope, oocysts of Sarcocystis spp. were observed in 43 samples (47.3%), while molecular methods, detected Sarcocystis spp. in 77 birds (84.6%). Eleven Sarcocystis spp. (S. columbae, S. cornixi, potentially pathogenic S. halieti, S. kutkienae, S. lari, S. turdusi, S. wobeseri, S. arctica, S. lutrae, S. ovalis, and S. oviformis) were identified in the intestinal samples from six corvid species from Lithuania. Infections with multiple Sarcocystis spp. were detected in 79.2% of the infected corvid birds. Three of the identified Sarcocystis spp. use corvids as intermediate hosts (IH); therefore, corvids may serve as IH and DH of the same Sarcocystis species. Based on molecular results and on corvid diet, omnivorous corvids may play an important role in transmitting Sarcocystis spp.

NEW DEFINITIVE HOST RECORD FOR CHORDODES MORGANI (NEMATOMORPHA) IN NEBRASKA WITH NOTES ON ECOLOGY.

The life cycle and ecology of the horsehair worm Chordodes morgani (Nematomorpha) in Nebraska remain unknown. To identify its definitive host, we installed a series of pitfall traps along 3 first-order streams at 4 sites: Elk Creek, Upper Elk Creek, Maple Creek, and West Oak Creek, all located northwest of Lincoln, Nebraska. In addition, we opportunistically hand-collected insects at these sites, including wood cockroaches (Parcoblatta virginica), and maintained them in the lab until they passed adult worms. Two of these field-collected wood cockroaches each yielded 1 adult worm, which was identified as C. morgani by microscopy, showing that P. virginica serves as a definitive host. Experimental infections of captive-reared Parcoblatta americana supported this result. The wood cockroach was found at all 3 creeks, but C. morgani was not found at West Oak Creek, suggesting that the definitive host does not limit the distribution of C. morgani. Physical properties of the streams were measured to examine how these properties influenced the distribution of the worm. Flow rate and pH differed between the 3 sites where C. morgani was found and the West Oak Creek site, suggesting an important role for these abiotic factors in the distribution of this horsehair worm species.

MOLECULAR IDENTIFICATION OF JUVENILE NEOECHINORHYNCHUS SPP. (PHYLUM: ACANTHOCEPHALA) INFECTING OSTRACOD AND SNAIL HOSTS PROVIDES INSIGHT INTO ACANTHOCEPHALAN HOST USE.

The role of invertebrates in some acanthocephalan life cycles is unclear because juvenile acanthocephalans are difficult to identify to species using morphology. Most reports suggest acanthocephalans from turtle definitive hosts use ostracods as intermediate hosts and snails as paratenic hosts. However, laboratory studies of the life cycle suggest that ostracods and snails are both required hosts in the life cycle. To elucidate the role of ostracods and snails in acanthocephalan life cycles better, we collected 558 freshwater snails of 2 species, including Planorbella cf. Planorbella trivolvis and Physa acuta, from 23 wetlands in Oklahoma, U.S.A., and examined them for acanthocephalan infections. Additionally, we examined 37,208 ostracods of 4 species, Physocypria sp. (morphotype 1), Cypridopsis sp., Stenocypris sp., and Physocypria sp. (morphotype 2) for juvenile acanthocephalans from 2 wetlands in Oklahoma. Juvenile acanthocephalans were morphologically characterized, and the complete internal transcribed spacer (ITS) region of nuclear rDNA was sequenced from acanthocephalans infecting 11 ostracod and 13 snail hosts. We also sampled 10 red-eared slider turtles, Trachemys scripta elegans, and 1 common map turtle, Graptemys geographica, collected from Oklahoma, Arkansas, and Texas and recovered 1,854 adult acanthocephalans of 4 species. The ITS of 17 adult acanthocephalans of 4 species from turtle hosts were sequenced and compared to juvenile acanthocephalan sequences from ostracod and snail hosts from this study and GenBank to determine conspecificity. Of the 23 locations sampled for snails, 7 (30%) were positive for juvenile acanthocephalans in the genus Neoechinorhynchus. The overall prevalence and mean intensity of acanthocephalans in Planorbella cf. P. trivolvis and P. acuta were 20% and 2 (1-6) and 2% and 1 (1), respectively. In contrast, only 1 of 4 species of ostracods, Physocypria sp. (morphotype 1), was infected with larval/juvenile Neoechinorhynchus spp. with an overall prevalence of 0.1% and a mean intensity of 1 (1-2). Although 4 species of acanthocephalans infected turtle definitive hosts, including Neoechinorhynchus chrysemydis, Neoechinorhynchus emydis, Neoechinorhynchus emyditoides, and Neoechinorhynchus pseudemydis, all the ITS sequences from cystacanths infecting snail hosts were conspecific with N. emydis. In contrast, the ITS sequences from larval/juvenile acanthocephalans from ostracods were conspecific with 2 species of acanthocephalans from turtles (N. emydis and N. pseudemydis) and 1 species of acanthocephalan from fish (Neoechinorhynchus cylindratus). These results indicate that N. emydis infects freshwater snails, whereas other species of Neoechinorhynchus appear not to infect snail hosts. We document new ostracod and snail hosts for Neoechinorhynchus species, including the first report of an ostracod host for N. pseudemydis, and we provide novel molecular barcodes that can be used to determine larva, juvenile, and adult conspecificity of Neoechinorhynchus species.

Invasive American mink (Neovison vison) as potential definitive host of Sarcocystis elongata, S. entzerothi, S. japonica, S. truncata and S. silva using different cervid species as intermediate hosts.

Canids and scavenger birds were shown to act as definitive hosts of numerous Sarcocystis species using members of the Cervidae family as an intermediate host, whereas definitive hosts spreading closely related S. elongata, S. entzerothi, S. japonica, S. matsuoae, S. rangiferi, S. truncata, S. silva and S. tarandi remain unknown. In the current study, the intestine samples of 40 American minks (Neovison vison) were molecularly tested for the presence of the above-mentioned Sarcocystis spp. Species-specific PCR of cytochrome c oxidase subunit I (cox1) fragments and subsequent sequencing revealed the presence of sporocysts/oocysts of five species, S. elongata (n=2), S. entzerothi (n=10), S. japonica (n=4), S. silva (n=13) and S. truncata (n=21) in the analysed samples. Sarcocystis infection was confirmed in 32/40 (80%) examined samples. In addition, half of the infected animals (50%) were infected with multiple Sarcocystis species suggesting that American minks had access to meat of different deer species, such as roe deer, red deer and sika deer. This causes concern about compliance of hunters and game processing companies with game waste management rules. Further research on the involvement of mustelids in the transmission of various Sarcocystis spp. from different geographical locations is needed.

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

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

Cryptocotyle lata (Trematoda: Heterophyidae) Adult from a Korean Raccoon Dog, Nyctereutes procyonoides koreensis.

Total 513 heterophyid flukes were collected from a carcass of wild Korean raccoon dog, Nyctereutes procyonoides koreensis, in Korea. With morphological and molecular characteristics, the flukes were identified to Cryptocotyle lata. The adult C. lata were minute, transparent, pentagonal, 522 µm long by 425 µm wide. Ceca extended into post-testicular region. Ventrogenital sac elliptical, 79 µm by 87 µm with genital pore and ventral sucker. Two testes semielliptical and slightly lobed, located in the posterior region, right testis 173 µm by 155 µm, left testis 130 µm by 134 µm. In a phylogenetic tree, the fluke specimen of this study was grouped with C. lata divergent from Cryptocotyle lingua. We report here N. procyonoides koreensis first as a natural definitive host of C. lata.