In-herd prevalence of Fasciola hepatica and Calicophoron / Paramphistomum spp. infections in German dairy cows with comparison of two coproscopical methods and establishment of real-time pyrosequencing for rumen fluke species differentiation.

Infections with liver and rumen flukes are among the most frequent parasitic diseases in cattle worldwide. In Europe, the predominant liver fluke species is Fasciola hepatica, and the recently rapidly spreading rumen flukes are mostly Calicophoron daubneyi and occasionally Paramphistomum leydeni. In this study, 1638 faecal samples from individual dairy cows from 24 northern and 18 southern German farms as well as one central German farm, all preselected for potential F. hepatica infection, were examined to determine in-herd prevalences of liver and rumen fluke infections. Furthermore, individual faecal egg counts (FECs) were determined in the northern and central German cows. On farms with patent F. hepatica infections, the mean in-herd prevalence was 15.8% in northern Germany, 41.6% in southern Germany and 14.0% in the central German farm. Rumen fluke infections resulted in high in-herd prevalences in all regions with a mean prevalence of 46.0% in northern, 48.4% in southern and 40.0% in central Germany. Individual FECs varied between 0.1 and 4.1 (mean 0.4) eggs per gram faeces (EPG) for F. hepatica and between 0.1 and 292.4 (mean 16.9) EPG for rumen flukes. Mean in-herd prevalence and mean FECs did not differ significantly between mono- and coinfected farms for either fluke species. Comparison of the classical sedimentation technique and the Flukefinder® method on a subset of 500 faecal samples revealed a similar number of positive samples, however, Flukefinder® mean FECs were three to four times higher for liver and rumen fluke eggs, respectively, with an increasing gap between EPG levels with rising egg counts. Fluke egg size measurement confirmed P. leydeni eggs on average to be larger in length and width (161.0 µm x 87.1 µm) than those of C. daubneyi (141.8 µm x 72.9 µm). However, due to overlap of measurements, morphological species identification based on egg size proved unreliable. For accurate identification, a real-time pyrosequencing approach was established, offering the advantage over classical Sanger sequencing of unambiguously identifying rumen fluke mixed species infections. Real-time pyrosequencing confirmed C. daubneyi (78.1% [50/64]) as the predominant rumen fluke species in Germany, while P. leydeni was detected in 12.5% (8/64) of sampled cows. A total of 9.4% (6/64) cows were infected with both C. daubneyi and P. leydeni, representing the first finding of a mixed infection in domestic ruminants in Europe to date.

Species identity and phylogeny of Paramphistomoidea Fischoeder, 1901 occurring in cattle and sheep in North Cameroon.

Paramphistomidae and Gastrothylacidae are parasitic flatworms occurring in wild and domestic ruminants in different parts of the world especially in Asia and Africa. In Central Africa, few studies have been done using molecular techniques to resolve taxonomical groupings and understand the epizootiology of these parasites. In this study, we molecularly characterized two hundred adult flukes collected from the fore stomachs of cattle and sheep in the Adamawa region of the northern Cameroon. PCR and sequencing of the nuclear ITS-2 of the ribosomal DNA gene and a portion of the mitochondrial cox-1 locus revealed the presence of at least nine species belonging to the genera of Cotylophoron, Calicophorn, Orthocoelium and Carmyerius. In Zebu cattle, we identified Ca. microbothrium, Ca. clavula, Ca. phillerouxi, Co. cotylophorum, Co. fuelleborni, O. scoliocoelium, Car. gregarius, Car. graberi and Car. mancupatus and one yet unknown Paramphistomoidea sp, whereas in sheep, only Ca. microbothrium was found. The present study also strongly suggests cross-hybridization between the two Cotylophoron species coexisting in cattle. These results have implications for the diagnosis and control of rumen flukes in the region and point to the need for accurate species identification to understand parasite distribution and population genetics.

Molecular detection of Fasciola, Schistosoma and Paramphistomum species from freshwater snails occurring in Gauteng and Free State provinces, South Africa.

Trematodiases are diseases caused by snail-borne trematode parasites that infect both animals and humans. Fascioliasis, schistosomiasis and paramphistomosis are some of these diseases and they affect millions of livestock, leading to significant economic losses. The aim of the study was to document freshwater snails occurring in selected study sites in the Free State and Gauteng provinces as well as identify and detect larval trematodes that they harbour. Samples were collected from a total of five study sites within two provinces of South Africa. Morphological features were used to identify snail species and were further confirmed genetically by polymerase chain reaction (PCR), sequencing and phylogenetic analysis. The larval trematodes were also detected by PCR, PCR-Restriction Length Fragment Polymorphism (PCR-RLFP), sequencing and phylogenetic analysis. A total of 887 freshwater snails were collected from Free State (n = 343) and Gauteng (n = 544). Five different genera of snails as well as species in the Succineidae family were documented. The snails in descending order of abundance were identified as: Physa (P.) spp. (51%), Succineidae spp. (20%), Galba (G.) truncatula (12%), Pseudosuccinea (Ps.) columella (10%), Planorbella (Pl.) duryi (6%) and Bulinus (B.) truncatus (1%). Approximately 272 DNA pools were created for genetic identification of snails and detection of trematode parasites. Schistosoma species were not detected from any of the snail species. A total prevalence of 46% was obtained for Fasciola hepatica in the identified snail species across all study sites. Overall, the highest prevalence of F. hepatica was obtained in Physa species (24%), whilst the lowest was observed in B. truncatus snails (1%). Forty three percent (43%) of the snail samples were PCR positive for Paramphistomum DNA. This is the first report of P. mexicana in South Africa. Fasciola hepatica was confirmed from all obtained snail species per study site. This is the first reported detection of F. hepatica in Pl. duryi and P. mexicana snails as well as the first confirmation of natural infection from P. acuta in South Africa.

Identification of Cotylophoron cotylophorum (Fischoeder, 1901) in cattle on St. Kitts, West Indies and its relationship with African and Asian populations.

There is limited information about the species of rumen fluke (Family Paramphistomidae) in the Caribbean. However, knowledge of species distribution is needed to better understand disease risk and epidemiology. Morphological identification is challenging with more recent DNA sequencing enabling a better understanding of rumen fluke distribution. In this study, rumen fluke specimens, collected between 2015 and 2016 from cattle on the island of St. Kitts, West Indies, were analysed. The ribosomal internal transcribed spacer 2 (ITS-2) region of rDNA was amplified using generic trematode primers. Results from Sanger sequencing were compared to reference sequences in GenBank and indicated the species was Cotylophoron cotylophorum with 100% sequence identity and 91% query cover. The ITS2 sequences were then compared to previously published ITS2 sequences for the Cotylophoron genus. When all the St. Kitts C. cotylophorum ITS2 sequences were compared with all other Cotylophoron sequences from India, Kenya, and Zimbabwe, three variable nucleotide sites, resulting in five unique haplotypes, were identified. Nine ITS2 sequences shared haplotype 1, which included all those from St. Kitts and single representatives from India and Kenya, potentially indicating global movement of this species.

The determination and relationship of four coexisting paramphistomes in perspective of integrative taxonomic investigation.

Co-infections with Orthocoelium species and other paramphistomes were found in different ruminant hosts from two provinces of Thailand. Whilst O. parvipapillatum coexisted with Paramphistomum epiclitum in the same cattle (Bos taurus) from Pathum Thani Province, Thailand, O. dicranocoelium and Fischoederius elongatus were found in water buffalo (Bubalus bubalis) from Chiang Mai Province. Morphological, histological, and tegumental surface features of both Orthocoelium species were intensively investigated for species differentiation. Statistical analysis of eight morphometric ratios presented morphological differences for three paramphistomes in the Paramphistomidae family and some relationships among paramphistomes in different definitive hosts. The genetic relationships of the co-infecting paramphistomes were investigated using p-distance and phylogenetic tree analyses. Genetic variations in the Orthocoelium co-infecting paramphistomes, P. epiclitum and F. elongatus, were calculated and compared to DNA sequence alignments based on internal transcribed spacer 2 (ITS2) and cytochrome c oxidase subunit I (COI) DNA markers. In addition, the phylogenetic tree constructions from both DNA markers and their concatenated sequence (ITS2 + COI) were used for species confirmation and the presentation of genetic relationships between co-infecting paramphistomes and other paramphistomes. This study improves the basic taxonomical description and understanding of parasite-parasite and host-parasite interactions from the perspectives of morpho-histological, morphometric, and genetic variation in co-infecting paramphistomes and Orthocoelium species in different hosts.

Differentiating paramphistome species in cattle using DNA barcoding coupled with high-resolution melting analysis (Bar-HRM).

Paramphistomosis is caused by paramphistome or amphistome parasites, including Fischoederius elongatus, Gastrothylax crumenifer, Orthocoelium parvipapillatum, and Paramphistomum epiclitum. The control and prevention of these parasite outbreaks are difficult because of the wide occurrence of these species. Besides, the clinical manifestations and their egg characteristics are similar to those of other intestinal flukes in the paramphistome group, leading to misdiagnosis. Here, we employed DNA barcoding using NADH dehydrogenase (ubiquinone, alpha 1) (ND1) and cytochrome c oxidase subunit I (COI), coupled with high-resolution melting analysis (Bar-HRM), for species differentiation. As a result, ParND1_3 and ParCOI4 resulted in positive amplification in the paramphistomes and Fasciola gigantica, with significantly different melting curves for each species. The melting temperatures of each species obtained clearly differed. Regarding sensitivity, the limit of detection (LoD) for all species of paramphistomes was 1 pg/µl. Our findings suggest that Bar-HRM using ParND1_3 is highly suitable for the differentiation of paramphistome species. This approach can be used in parasite detection and epidemiological studies in cattle.

Calicophoron daubneyi (Dinnik, 1962) (Digenea) in beef and dairy cattle in the Czech Republic: prevalence and drug efficacy.

A total of 1,724 beef and 2,941 dairy cattle older than one year from 66 beef and 67 dairy farms in the Czech Republic were examined for the presence of rumen and liver fluke eggs in 2019-2022. Out of 227 positive animals, all were positive for paramphistome and five for fasciolid eggs. Molecular analysis of the ITS2 rDNA revealed the presence of Calicophoron daubneyi (Dinnik, 1962) and Fasciola hepatica Linnaeus, 1758. Faecal egg count (FEC) showed low infection intensity (12 EPG) in animals infected with F. hepatica and high variability in C. daubneyi infections (2-589 EPG). Efficacy of oxyclozanide, albendazole, ivermectin, and closantel against C. daubneyi infection was evaluated at eight beef cattle herds. Faecal samples were collected from all positive animals at 0 and 21days post-treatment. Based on FEC, albendazole, ivermectin and closantel reduced the number of C. daubneyi eggs shed by 0-9.9%, with no effect on the number of infected animals. The use of oxyclozanide on two beef farms showed 100% efficacy against C. daubneyi and F. hepatica. Follow-up examination 5-6 months after drug application showed reinfection of most animals with C. daubneyi, but the FEC was significantly lower. The finding of four dairy cows infected with C. daubneyi housed in a stable without pasture suggests the possibility of the infection being introduced through roughage.

Complex morphological characterization and morphometric-molecular discrimination of two paramphistome species co-infecting cattle, Orthocoelium sp. and Paramphistomum epiclitum.

Co-infection by two paramphistome species, Orthocoelium sp. and Paramphistomum epiclitum, is found in cattle in Thailand. The morphological features of these and other paramphistomes under a light microscope are similar, resulting in misidentification and misdiagnosis. We classified these paramphistomes into three morphological variation types, namely Orthocoelium sp., P. epiclitum MV1 (immature), and P. epiclitum MV2 (matured). Ten morphological characteristics were investigated, and the values were transformed into 25 ratio criteria for statistical investigation. Morphometric analysis can classify the variation of these specimens using differences in the bifurcal level, the vitellaria starting level, the starting level of the anterior testis, and the center level of the posterior testis positions by body length ratios. These ratios can separate the samples into three morphologically different groups, whereas molecular analysis based on the nuclear internal transcribed spacer 2 (ITS2) region and the mitochondrial cytochrome c oxidase subunit I (COI) gene could only distinguish two specific groups. In addition, the Orthocoelium specimen, related to O. dicranocoelium and O. parvipapillatum according to morphological and histological analysis, was monophyletic grouped via ITS2 analysis. Our study provides a scientific basis for the taxonomic classification and clustering of morphologically varying species, improving the identification, detection, and diagnosis of co-infecting paramphistomes.

DNA barcoding of rumen flukes (Paramphistomidae) from bovines in Germany and Austria.

Rumen flukes have received growing veterinary attention in western and central Europe during the past two decades because of an increase in prevalence of infection in cattle and sheep, including cases of severe clinical disease. Historically, rumen fluke infections in Europe were assumed to be caused mainly by Paramphistomum cervi (or species, which were later considered to be synonymous with P. cervi), but more recently molecular studies demonstrated Calicophoron daubneyi to be the predominating species. For the present investigation, adult rumen flukes isolated from 23 cattle originating from ten farms in Germany (Saxony [1], Baden-Württemberg [4], Bavaria [5]) and one farm in Austria (Tyrol) were analyzed to establish partial sequences of the mitochondrial cytochrome c oxidase subunit I (COI) and the complete sequence of the nuclear internal transcribed spacer 2 (ITS2). Flukes of five animals (dairy cows from three farms in Bavaria) were determined as P. leydeni, and flukes of 18 animals (dairy cows or cattle from cow-calf operations from eight farms in Saxony [1], Baden-Württemberg [4], Bavaria [2], and Tyrol [1]) were identified as C. daubneyi. Based on the molecular analysis of adult rumen flukes collected from cattle, the results of this investigation confirm the common occurrence of C. daubneyi in Germany and reveal the first definitive findings of P. leydeni in Germany and C. daubneyi in Austria.

Transcriptome and Secretome Analysis of Intra-Mammalian Life-Stages of Calicophoron daubneyi Reveals Adaptation to a Unique Host Environment.

Paramphistomosis, caused by the rumen fluke, Calicophoron daubneyi, is a parasitic infection of ruminant livestock, which has seen a rapid rise in prevalence throughout Western Europe in recent years. After ingestion of metacercariae (parasite cysts) by the mammalian host, newly excysted juveniles (NEJs) emerge and invade the duodenal submucosa, which causes significant pathology in heavy infections. The immature flukes then migrate upward, along the gastrointestinal tract, and enter the rumen where they mature and begin to produce eggs. Despite their emergence, and sporadic outbreaks of acute disease, we know little about the molecular mechanisms used by C. daubneyi to establish infection, acquire nutrients, and avoid the host immune response. Here, transcriptome analysis of four intramammalian life-cycle stages, integrated with secretome analysis of the NEJ and adult parasites (responsible for acute and chronic diseases, respectively), revealed how the expression and secretion of selected families of virulence factors and immunomodulators are regulated in accordance with fluke development and migration. Our data show that while a family of cathepsins B with varying S2 subsite residues (indicating distinct substrate specificities) is differentially secreted by NEJs and adult flukes, cathepsins L and F are secreted in low abundance by NEJs only. We found that C. daubneyi has an expanded family of aspartic peptidases, which is upregulated in adult worms, although they are under-represented in the secretome. The most abundant proteins in adult fluke secretions were helminth defense molecules that likely establish an immune environment permissive to fluke survival and/or neutralize pathogen-associated molecular patterns such as bacterial lipopolysaccharide in the microbiome-rich rumen. The distinct collection of molecules secreted by C. daubneyi allowed the development of the first coproantigen-based ELISA for paramphistomosis which, importantly, did not recognize antigens from other helminths commonly found as coinfections with rumen fluke.

Cotylophoron marajoensis n. sp. (Digenea: Paramphistomidae) a parasite of Bubalus bubalis on Marajó Island, Pará, Brazilian Amazon.

The genus Cotylophoron belongs to the Paramphistomidae family and its definitive hosts are ruminants in general. This work describes the presence of a new species of the gender, a parasite in the rumen and reticulum of Bubalus bubalis, on Marajó Island in the Eastern Brazilian Amazon, using of light microscopy, scanning electronic microscopy and molecular biology techniques. One hundred and ten animals were analyzed, of which 4.54% were parasitized by flukes in their adult forms. The helminths were found fixed to the ruminal mucosa and present Liorchis-type pharynx, Cotylophoron-type genital sucker, oblique testicles larger than the ovary, uterus in rings full of eggs and Cotylophoron-type acetabulum. These morphologic characters do not fit into any previously described species. Thus, it is proposed that this is a new species in the genus Cotylophoron. The present work expands the record of parasitism by helminths in Bubalus bubalis, this being the first record of trematoda from the genus Cotylophoron for this host in the Brazilian Amazon.

A phylogenetic study of the cecal amphistome Zygocotyle lunata (Trematoda: Zygocotylidae), with notes on the molecular systematics of Paramphistomoidea.

Zygocotyle lunata inhabits the caecum of birds and mammals from the American continent. This amphistome parasite is easily maintained in the laboratory and serves as a model organism in life-cycle studies, but it has seldom been studied using molecular data. Neither the position of Z. lunata in the superfamily Paramphistomoidea nor the monophyly of the Zygocotylidae has been evaluated with molecular phylogenetic methods. In the present study, adult specimens of Z. lunata obtained experimentally in mice from Brazil were submitted to molecular studies. Partial sequences of nuclear (1261 bp of 28S and 418 bp of 5.8S-ITS-2) and mitochondrial (1410 bp of cytochrome c oxidase 1, cox1) markers were compared with published data. In the most well-resolved phylogeny, based on 28S sequences, Z. lunata clustered in a well-supported clade with Wardius zibethicus, the only other species currently included in the Zygocotylidae, thus confirming the validity of this family. Divergence of 28S sequences between these species was 2.2%, which falls in the range of intergeneric variation (0.9-5.6%) observed in the other two monophyletic groups in the 28S tree, i.e., representatives of Gastrodicidae and Neotropical cladorchiids (Cladorchiidae). Analysis of ITS-2 and two parts of the cox1 gene placed Z. lunata within poorly resolved clades or large polytomies composed of several paramphistomoid families, without clarifying higher-level phylogenetic relationships. The cox1 of a Brazilian isolate of Z. lunata is 99.6% similar to a Canadian isolate, confirming the pan-American distribution of the species. Finally, our phylogenetic reconstructions of Paramphistomoidea revealed a complex scenario in the taxonomic composition of some amphistome families, which highlights a need for further integrative studies that will likely result in rearrangements of traditional morphology-based classifications.

Cotylophoron marajoensis n. sp. (Digenea: Paramphistomidae) a parasite of Bubalus bubalis on Marajó Island, Pará, Brazilian Amazon / Cotylophoron marajoensis n. sp. (Digenea: Paramphistomidae) parasito de Bubalus bubalis na Ilha de Marajó, Pará, Amazônia Brasileira

Abstract The genus Cotylophoron belongs to the Paramphistomidae family and its definitive hosts are ruminants in general. This work describes the presence of a new species of the gender, a parasite in the rumen and reticulum of Bubalus bubalis, on Marajó Island in the Eastern Brazilian Amazon, using of light microscopy, scanning electronic microscopy and molecular biology techniques. One hundred and ten animals were analyzed, of which 4.54% were parasitized by flukes in their adult forms. The helminths were found fixed to the ruminal mucosa and present Liorchis-type pharynx, Cotylophoron-type genital sucker, oblique testicles larger than the ovary, uterus in rings full of eggs and Cotylophoron-type acetabulum. These morphologic characters do not fit into any previously described species. Thus, it is proposed that this is a new species in the genus Cotylophoron. The present work expands the record of parasitism by helminths in Bubalus bubalis, this being the first record of trematoda from the genus Cotylophoron for this host in the Brazilian Amazon.

Resumo O gênero Cotylophoron pertence à família Paramphistomidae e possui como hospedeiros definitivos ruminantes em geral. Este trabalho descreve a presença de uma espécie nova do gênero, parasito do rúmen e retículo de Bubalus bubalis, na Ilha de Marajó, Amazônia oriental brasileira, a partir das técnicas de microscopia de luz, microscopia eletrônica de varredura e biologia molecular. Foram analisados 110 animais, dos quais 4,54% estavam parasitados por trematódeos na sua forma adulta. Os helmintos foram encontrados fixados à mucosa ruminal, apresentando faringe do tipo Liorchis, ventosa genital do tipo Cotylophoron, testículos oblíquos maiores que o ovário, útero em alças repleto de ovos, e acetábulo do tipo Cotylophoron. Estes caracteres morfológicos não se enquadram em nenhuma espécie previamente descrita. Assim, propõe-se uma nova espécie ao gênero Cotylophoron. O presente trabalho amplia o registro do parasitismo por helmintos em Bubalus bubalis, sendo este o primeiro registro de trematódeos do gênero Cotylophoron nesse hospedeiro para a Amazônia brasileira.

Morphological and molecular identification with frequency analysis of Calicophoron microbothrioides in central Chile / Identificação morfológica e molecular com análise de frequência de Calicophoron microbothrioides na região central do Chile

Abstract This research aimed to determine the presence of paramphistomids in cattle slaughtered in a slaughterhouse of the Ñuble Region of Chile, to identify flukes and to analyze the frequency of these parasites in the Maule, Ñuble, and Biobío administrative regions of Chile. Between October of 2016 and April of 2017, rumens of 494 cattle were examined for flukes in the forestomachs. Worms were identified morphologically and, in addition, molecular analysis of the internal transcriber spacer region 2 of the fluke's DNA was done and phylogenetic analyses were performed with Bayesian inference in 14 worms. The frequency was analyzed by locality (low- or highlands) and age. The overall frequency was 11.24%. The district with the highest frequency of presentation was Chillán Viejo (30.8%). Districts in the lowlands had similar frequencies to those in the mountain lands (p=0.1). The frequency of flukes was significantly higher in adult animals than in young ones (p<0.01). We obtained a 460 bp-length fragment of DNA that was identical to the sequences previously identified as Paramphistomum cervi and Calicophoron microbothrioides, and performed morphological analyses confirmed that our samples belonged to C. microbothrioides. This is the first published study of C. microbothrioides in Chile.

Resumo Este trabalho teve como objetivo determinar a presença de paramphistomídeos em bovinos abatidos em um matadouro da Região do Ñuble do Chile, para identificar parasitas e analisar a frequência desses parasitos nas regiões administrativas de Maule, Ñuble e Biobío, no Chile. Entre outubro de 2016 e abril de 2017, rúmens de 494 bovinos foram examinados à procura de vermes no pré-estômago. Os vermes foram identificados morfologicamente e, além disso, a análise molecular da região interna do espaçador do transcritor 2 do DNA e análises filogenéticas foram realizadas com inferência bayesiana em 14 vermes. A frequência foi analisada pela altitude da localidade (baixa ou alta) e idade. A frequência geral foi de 11,24%. O distrito com as maiores frequências de parasitismo foi Chillán Viejo (30,8%). Os distritos das terras baixas tinham frequências semelhantes às encontradas nas terras das montanhas (p=0,17). A frequência foi significativamente maior em animais adultos do que em jovens (p<0.01). Obtivemos um fragmento de DNA de 460 pb que era idêntico às sequências anteriores identificadas como Paramphistomum cervi e Calicophoron microbothrioides, e realizamos análises morfológicas que permitiram confirmar que nossas amostras pertenciam a C. microbothrioides. Este é o primeiro estudo publicado sobre C. microbothrioides no Chile.

Morphological and molecular identification with frequency analysis of Calicophoron microbothrioides in central Chile.

This research aimed to determine the presence of paramphistomids in cattle slaughtered in a slaughterhouse of the Ñuble Region of Chile, to identify flukes and to analyze the frequency of these parasites in the Maule, Ñuble, and Biobío administrative regions of Chile. Between October of 2016 and April of 2017, rumens of 494 cattle were examined for flukes in the forestomachs. Worms were identified morphologically and, in addition, molecular analysis of the internal transcriber spacer region 2 of the fluke's DNA was done and phylogenetic analyses were performed with Bayesian inference in 14 worms. The frequency was analyzed by locality (low- or highlands) and age. The overall frequency was 11.24%. The district with the highest frequency of presentation was Chillán Viejo (30.8%). Districts in the lowlands had similar frequencies to those in the mountain lands (p=0.1). The frequency of flukes was significantly higher in adult animals than in young ones (p<0.01). We obtained a 460 bp-length fragment of DNA that was identical to the sequences previously identified as Paramphistomum cervi and Calicophoron microbothrioides, and performed morphological analyses confirmed that our samples belonged to C. microbothrioides. This is the first published study of C. microbothrioides in Chile.

A high throughput deep amplicon sequencing method to show the emergence and spread of Calicophoron daubneyi rumen fluke infection in United Kingdom cattle herds.

The prevalence of C. daubneyi infection in the United Kingdom has increased, but despite the potential for rumen flukes to cause production loss in ruminant livestock, understanding of their emergence and spread is poor. Here we describe the development of a method to explore the multiplicity of C. daubneyi infection and patterns of the parasite's emergence and spread, based on Illumina MiSeq deep sequencing of meta barcoded amplicons of a fragment of the cytochrome c oxidase subunit I (mt-COX-1) locus. Our results show high levels of genetic diversity in 32 C. daubneyi populations derived from finished prime cattle consigned to slaughter from northern United Kingdom. The results are consistent with a single introduction of C. daubneyi infection to some of the farms where the cattle had been grazed during their lifetime and multiple introductions to most. The results illustrate the impact of high levels of animal movements in the United Kingdom, whereby multiple common mt-COX-1 haplotypes were identified in 26 populations in the absence of geographical clustering of clades. This has implications for the adaptability of environmental and intermediate host stages of the parasite to changing climatic and animal management conditions, or of parasitic stages to exposure to anthelmintic drugs; potentially allowing for greater pathogenicity, or the development of anthelmintic resistance, respectively.

Polyomic tools for an emerging livestock parasite, the rumen fluke Calicophoron daubneyi; identifying shifts in rumen functionality.

BACKGROUND: Diseases caused by parasitic flatworms of rumen tissues (paramphistomosis) are a significant threat to global food security as a cause of morbidity and mortality in ruminant livestock in subtropical and tropical climates. Calicophoron daubneyi is currently the only paramphistome species commonly infecting ruminant livestock in temperate European climates. However, recorded incidences of C. daubneyi infection in European livestock have been increasing over the last decade. Whilst clinical paramphistomosis caused by adult worms has not been confirmed in Europe, fatalities have been attributed to severe haemorrhagic enteritis of the small intestine resulting from the migration of immature paramphistomes. Large numbers of mature adults can reside in the rumen, yet to date, the impact on rumen fermentation, and consequently on productivity and economic management of infected livestock, have not been resolved. Limited publicly available nucleotide and protein sequences for C. daubneyi underpin this lack of biological and economic understanding. Here we present for the first time a de novo assembled transcriptome, with functional annotations, for adult C. daubneyi, which provides a reference database for protein and nucleotide sequence identification to facilitate fundamental biology, anthelmintic, vaccine and diagnostics discoveries. RESULTS: This dataset identifies a number of genes potentially unique to C. daubneyi and, by comparison to an existing transcriptome for the related Paramphistomum cervi, identifies novel genes which may be unique to the paramphistome group of platyhelminthes. Additionally, we present the first coverage of the excretory/secretory and soluble somatic proteome profiles for adult C. daubneyi and identify the release of extracellular vesicles from adult C. daubneyi parasites during in vitro, ex-host culture. Finally, we have performed the first analysis of rumen fluke impacting upon rumen fermentation parameters using an in vitro gas production study resulting in a significant increase in propionate production. CONCLUSIONS: The resulting data provide a discovery platform (transcriptome, proteomes, EV isolation pipeline and in vitro fermentation system) to further study C. daubneyi-host interaction. In addition, the acetate: propionate ratio has been demonstrated to decrease with rumen fluke infection suggesting that acidotic conditions in the rumen may occur.

Morphological and molecular characterization of Paramphistomum epiclitum of small ruminants.

Morphological and molecular identification can pave the way to design the most effective control measures against the Paramphistomum epiclitum in small ruminants. Morphology of the flukes had described the features of Paramphistomum genus. Body was conical with concave ventral and convex dorsal surface, tegumental spines all around the body in the immature stage, terminal funnel shape oral sucker, sub-terminal acetabulum, blind caeca with a serpentine course touching the anterior level of the acetabulum. Vitelline glands were at the lateral margins of the body extended from the pharynx to the posterior sucker. Testes were lobed and tandem, wavy post-testicular uterus and genital pore behind intestinal bifurcation. Sequence analyses of internal transcribed spacer (ITS)-2+ (PCR products of approximately 500 bp) of 10 flukes yielded 2 genotypes, Navsari isolate 1 and 2. In BLAST analysis, ITS-2+ genotypes were 97.3-99% similar with published sequences (KF564870, JF834888, KF642983 and JX678254) of P. epiclitum of Paramphistomatidae. Two genotypes depicted 4 single nucleotide polymorphisms (NPs) in the form of transitions (C-T at 10 and 18; G-A at 255; A-G at 367 locus), 1 triple NPs (CGT-GAA between 21-23 loci) and missing A base at codon 40 in the genotype 1. Average AT and GC content was 49.61% and 50.38%, respectively. Trees topology inferred by Neighbor Joining and Maximum Likelihood methods of ITS2+ of trematodes were similar, with small difference of bootstrap values. Navsari genotypes formed a tight cluster with the P. epiclitum, originated from different location with high bootstrap value and 0.004-0.011 estimated evolutionary divergence.

Detection of Galba truncatula, Fasciola hepatica and Calicophoron daubneyi environmental DNA within water sources on pasture land, a future tool for fluke control?

BACKGROUND: Increasing trematode prevalence and disease occurrence in livestock is a major concern. With the global spread of anthelmintic resistant trematodes, future control strategies must incorporate approaches focusing on avoidance of infection. The reliance of trematodes on intermediate snail hosts to successfully complete their life-cycle means livestock infections are linked to the availability of respective snail populations. By identifying intermediate snail host habitats, infection risk models may be strengthened whilst farmers may confidently apply pasture management strategies to disrupt the trematode life-cycle. However, accurately identifying and mapping these risk areas is challenging. METHODS: In this study, environmental DNA (eDNA) assays were designed to reveal Galba truncatula, Fasciola hepatica and Calicophoron daubneyi presence within water sources on pasture land. eDNA was captured using a filter-based protocol, with DNA extracted using the DNeasy® PowerSoil® kit and amplified via PCR. In total, 19 potential G. truncatula habitats were analysed on four farms grazed by livestock infected with both F. hepatica and C. daubneyi. RESULTS: Galba truncatula eDNA was identified in 10/10 habitats where the snail was detected by eye. Galba truncatula eDNA was also identified in four further habitats where the snail was not physically detected. Fasciola hepatica and C. daubneyi eDNA was also identified in 5/19 and 8/19 habitats, respectively. CONCLUSIONS: This study demonstrated that eDNA assays have the capabilities of detecting G. truncatula, F. hepatica and C. daubneyi DNA in the environment. Further assay development will be required for a field test capable of identifying and quantifying F. hepatica and C. daubneyi infection risk areas, to support future control strategies. An eDNA test would also be a powerful new tool for epidemiological investigations of parasite infections on farms.

First genetic evidence for the presence of the rumen fluke Paramphistomum epiclitum in Pakistan.

More than 70 species of the Superfamily Paramphistomoidea, have been identified in ruminants in different parts of the world. Most are pathogenic, causing amphistomosis. Adult flukes within this family have a predilection for the forestomach (rumen) or bile duct of the liver, where they may cause epithelial damage. Identification of adult Paramphistomum, Calicophoron, Gastrothylax and Fischoederius at the species level based on morphology requires specialised expertise, whereas molecular genetic marker analysis is more precise and transferable. In the present study, we performed molecular characterisation of twenty seven adult flukes collected from the forestomachs of buffalo, cattle and goats in the Punjab province of Pakistan. PCR and sequencing of the ITS-2 rDNA region revealed a single haplotype in all cases. Phylogenetic comparison of P. epiclitum ITS2-rDNA sequences with those from other Paramphistomum, Calicophoron, Gastrothylax and Fischoederius species was performed to assess within and between species variation and validate the use of ITS-2 rDNA as a robust species-specific marker for P. epiclitum identification. This work provides a validated species-specific marker of P. epiclitum and the first report of this parasite species from Pakistan. The results of this study also have implications for the diagnosis and control of rumen flukes in the region and the need for accurate species identification to understand parasite distribution and population genetics.