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Calicophoron daubneyi is the primary rumen fluke (RF) found in Europe in ruminants and infection is more common in cattle than in sheep. The incidence of RF has appeared to increase greatly throughout Europe in the last 10-15 years, with outbreaks of clinical paramphistomosis confirmed in ruminants in many countries, including Great Britain and Ireland. Clinical disease, due to immature stages developing in the small intestine, appears infrequently but can occur, usually in the autumn or winter within weeks of beginning to graze wet pasture. Although disease due to adult RF has not been proven, subclinical production losses have been attributed to adult RF infection by some researchers. As the intermediate host for RF and the liver fluke (Fasciola hepatica) is the mud snail (Galba truncatula), similar habitats and environmental conditions favour both parasites. There may, however, be differences in parasite development and interactions within both the final and intermediate hosts. No anthelminthic product is licensed for treatment of ruminants for RF in the UK. However, oxyclozanide, licensed for the treatment of adult F. hepatica infection, has been shown to have activity, but it may be more effective against the adult than the immature stages. The future prevalence of RF due to climate change and limited treatment options is unpredictable. Infection and clinical disease could become more common and RF is worthy of further research.

This study was carried out to determine the paramphistome species parasitizing water buffaloes (WBs) grazing in the Kizilirmak Delta in Samsun Province, Turkey, and the intermediate hosts of the parasites. Between August 2016 and July 2018, abattoirs in Samsun Province were visited weekly and 139 slaughtered WBs were examined for paramphistome species. In the same period, 550 snails (300 Galba truncatula, 200 Physella (Physa) acuta and 50 Planorbis planorbis) were collected from pastures grazed by WBs during monthly sampling in the spring and autumn. Adult parasites were identified through the use of histological and molecular methods and larval stages were identified with a molecular method. Forty-five of the 139 WBs (32.4%) were infected with species of the family Paramphistomidae and a total of 4761 (mean 105.8) parasites were collected from them. The genera Paramphistomum and Calicophoron were distinguished from each other by examining the development status of the pars musculosa and the degree of lobulation of the testes in histological sections. Calicophoron daubneyi was present in all the infected animals and Paramphistomum cervi in only 3 of the same animals. There were larval forms in only 19 of the specimens identified as G. truncatula. Calicophoron daubneyi was molecularly diagnosed in 12 of the 19 infected G. truncatula and this result was confirmed by PCR and PCR–RFLP. Calicophoron daubneyi was also identified molecularly as being present in all 45 infected adult WBs and as developmental stages in 12 of 300 (4%) intermediate hosts, G. truncatula. The DNA sequences from the adult parasites in the definitive hosts and larval forms in intermediate hosts were allocated the codes MH939278 and MH939279, respectively, in GenBank. For the first time in Turkey, C. daubneyi was identified molecularly and its intermediate host was identified as G. truncatula. Calicophoron daubneyi was identified as the overwhelmingly dominant paramphistome species in WBs in the Kizilirmak Delta, with P. cervi found in mixed infections in only three animals.

Little is known about the genetic and morphological characters of Paramphistomum epiclitum. For the first time in Pakistan, adult flukes were morphologically characterized and the sequence variation in the nuclear ribosomal DNA (rDNA) region, including the first internal transcribed spacers (ITS1) and the 5.8S gene of the Paramphistomum epiclitum were studied. Adult amphistomes were examined by light microscopy and sequences of ITS1 and 5.88S rDNA genes were obtained. Twenty adult flukes were measured, 13.17 ± 1.19 mm in length and 5.28 ± 1.34 mm in width. Seventeen adult flukes were sequenced and high sequence variability was observed in 5′ end of ITS1 region. The 5.8S and 3′ end of ITS1 sequences had 100% identity among the samples. A comparative analysis revealed that different types and numbers of repeats were found within each ITS1 region. The 3′ end of ITS1 region from P. epiclitum showed 98% homology with P. cervi from China and formed a subclade with genetic distance of 0.1663. The 5.8S gene showed 100% identity within Paramphistomidae family and formed a sub-clade with P. epiclitum, P. leydeni, P. cervi and Cotylophoron cotylophorum species isolated from China, India and Uruguay. This work provides new information on morphological identity and genetics of P. epiclitum from Pakistan.

Fasciolosis due to Fasciola hepatica is one of the dominant pathologies in Algeria. On the other hand, gastroduodenal paramphistomosis are little studied and little known. Our work consisted of an epidemiological survey in the Souk-Ahras region to investigate these two parasites in cattle. Regarding the epidemiological investigation, it took place in the Souk-Ahras abattoirs, where 530 cattle were inspected for the presence of both parasites. The prevalence of fasciolosis was 12.3%, while the prevalence of paramphistomosis was 7.9% in cattle. Elderly animals were more infected than those under two years of age and females were more infected than males and for both parasites. Therefore, it is important to take into consideration both parasitic diseases and appropriate control measures are strongly recommended to improve cattle productivity.

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.

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.

ZusammenfassungDie Paramphistomidose ist eine weltweit auftretende parasitäre Erkrankung bei diversen Wiederkäuern, ausgelöst durch verschiedene Pansenegelarten (u. a. Paramphistomum cervi, Calicophoron daubneyi und Paramphistomum leydeni). In Europa sind lokale Vorkommen von Pansenegelinfektionen bei Haus- und Wildwiederkäuern seit Jahrzehnten beschrieben. Mittlerweile gibt es Hinweise, dass die Paramphistomidose an Bedeutung gewinnt, denn in Großbritannien, Irland, Frankreich, Spanien, Belgien und den Niederlanden wurden teilweise hohe Prävalenzen festgestellt. Aktuelle Prävalenzdaten aus Deutschland liegen nicht vor. In neueren Untersuchungen in Norddeutschland, Hessen und Bayern konnte Calicophoron daubneyi, die auch in Europa aktuell am häufigsten vorkommende Pansenegelart, nachgewiesen werden. Die Entwicklung des Pansenegels ist an Zwischenwirte aus der Familie der Wasserlungenschnecken gebunden. Calicophoron daubneyi und der Große Leberegel Fasciola hepatica teilen im Laufe ihrer Entwicklung den gleichen Zwischenwirt, die Zwergschlammschnecke Galba truncatula. Der Endwirt nimmt die infektiösen Metazerkarien auf. Im Dünndarm schlüpfen die jungen Pansenegel und heften sich im Duodenum an. Danach wandern sie in den Pansen ein, wo sie als Adulte anfangen, Eier auszuscheiden. Die Infektion kann während der intestinalen Phase zu schwerem Durchfall und bei hoher Befallsintensität zum Tod führen. Die ruminale Paramphistomidose verläuft in den meisten Fällen subklinisch. Aktuell steht zur Diagnostik der koproskopische Einachweis mittels Sedimentationsverfahren zur Verfügung. Aufgrund der Ähnlichkeit besteht die Gefahr der Verwechslung mit Eiern des Großen Leberegels. Die Paramphistomidose kann mit Oxyclozanid behandelt werden. Über die Wirksamkeit anderer Wirkstoffe liegen widersprüchliche Ergebnisse vor. Umso wichtiger erscheint die Prophylaxe dieser Parasitose. Bekämpfungsvorschläge richten sich aufgrund der ähnlichen Epidemiologie nach den Empfehlungen, die zur Vorbeugung der Fasziolose abgegeben werden. Ob es sich bei der Paramphistomidose auch in Deutschland um eine sich ausbreitende Infektionskrankheit handelt, kann aktuell noch nicht beurteilt werden.

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.

In order to ensure global food security a rationale approach is required to control all those factors which directly or indirectly affect the food productivity. The neglected helminthic diseases alone are responsible for huge economic losses to the agrarian stakeholders. The problem is further compounded by the emerging drug resistance in flukes against the commonly used anthelmintics like triclabendazole. Therefore, the search for alternatives including the nano-based approaches has become a necessity to develop future control strategies. In the present study the effect of biologically synthesized silver nanoparticles (AgNPs) was investigated on an economically important amphistome parasite, Gigantocotyle explanatum, obtained from the infected liver of the Indian water buffaloes, Bubalus bubalis. In vitro treatment of the adult worms with different doses of AgNPs severely affected the worm motility and caused ROS mediated damages in the treated flukes. The antioxidant system and the detoxification ability of the worms appeared to be disrupted along with pronounced DNA damage in the treated worms as compared to the controls. Following the treatment of worms with different concentrations of AgNPs there was a significant (p

Infections with the liver fluke Fasciola hepatica may result in considerable economic losses in the dairy livestock industry worldwide. Infections have been associated with detrimental impacts on milk production and milk quality as well as reduced fertility. However, most related data rely on examinations on herd level and the rather few studies on individual cow level are based solely on antibodies as measure for F. hepatica infections. This entails the risk of including false-positives as anti-F. hepatica antibodies persist for months even if the infection is cleared. Therefore, the presented study aimed to overcome this issue by assessing the association between F. hepatica infections measured via faecal egg counts (FEC) and milk production as well as fertility parameters in individual dairy cows. In total, 2006 faecal samples from 1166 Black and White dairy cows from 17 small and medium-sized German grassland farms were examined in July and September 2015. The relationship between patent F. hepatica infections and the milk production parameters milk yield, milk protein content, milk fat content and somatic cell score (SCS) was assessed in a linear mixed model using test-day records of individual cows. Patent F. hepatica infections were found on 35.3% (7/17) of farms with an individual cow prevalence of 10.1% (97/963) in July and 9.1% (95/1036) in September. Patent rumen fluke infections were detected on 17.6% (3/17) farms with an individual cow prevalence of 0.4% (4/963) in July and 0.7% (9/1036) in September. No significant association was found between F. hepatica infection status and either SCS as an indicator of udder health or milk production parameters, despite 0.06 and 0.10% lower values for milk protein and fat content in patently infected cows. Linear mixed models and generalized linear mixed models were established to estimate the impact of fasciolosis on the fertility parameters calving to first service (CTFS), calving interval (CI), success in first insemination (SFI) and 56-day nonreturn rate (NRR56). A significantly higher average CTFS of 4.69 days was detected in F. hepatica infected cows (P = 0.025), but no significant relationship was found for the other fertility parameters.

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.

Paramphistomosis can lead to morbidity and mortality of ruminant livestock within tropical and sub-tropical climates. In recent decades, rumen fluke has become an emerging infection in temperate climates across Western Europe, with Calicophoron daubneyi, the primary species present. Clinical outbreaks with C. daubneyi larvae are reported and adults might be responsible for production losses. There is not currently a widely licensed anthelmintic product available to control C. daubneyi. In this study, three existing flukicide anthelmintics were tested for efficacy against mature C. daubneyi, comparing a standard in vitro culturing assay and a new more relevant rumen fluid based in vitro compound screening protocol. The new rumen based screen confirmed that oxyclozanide was active against adult C. daubneyi and identified activity with praziquantel. The study highlighted the downstream value of incorporating relevant in vitro screening for anthelmintic discovery pipelines.

Paramphistomosis is a parasitic disease endemic in ruminants nearly worldwide. In the present study, anin vitroscreening of the main anthelmintics used in Mexico was carried out to determine the mean lethal dose for rumen fluke eggs from cattle in a humid, warm region. Rumen flukes were obtained from cattle slaughtered in the states of Tabasco and Chiapas in Mexico. Eggs were collected using a 37-μm sieve and quantified. Then, anin vitroincubation study was performed: 100 eggs were placed into the wells of polystyrene microtiter plates. Anthelmintic products were tested on the eggs at concentrations ranging from 0.0015 to 3.0 mg/ml for rafoxanide, 0.0025 to 10.20 mg/ml for nitroxinil and 0.0015 to 3 mg/ml for closantel to determine the median lethal dose (LD50) and maximum lethal dose (LD99). A control group (water) was included in each plate. Three different species of rumen flukes (Calicophoron brothriophoron,Calicophoron clavulaandParamphistomum cervi) belonging to five isolates were identified. Nitroxinil had the highest efficacy against rumen fluke eggs, with an LD50of 0.11 to 65 μg/ml, whereas rafoxanide showed the lowest efficacy with an LD50ranging from 500 to 1713 μg/ml. Closantel showed high variability in the LD50among the different analysed isolates (17 to 122 μg/ml). The evaluated flukicidal drugs presented differential efficacy against the development of rumen fluke eggs. The efficacy of the drugs will vary depending on the geographical area of origin of the animals.

European beaver (Castor fiber L. 1758) is the biggest rodent living in Europe. It is a semi-aquatic animal known for building dams and burrows. European beaver is a potential host for a wide range of parasites and other infectious diseases. In Slovakia, there is an increasing number of beavers but the data about their parasitic fauna are missing. Our work is the first documentation about the beaver's parasitofauna in Slovakia. In a 1-year study, we collected and examined 19 beaver fecal samples from the vicinity of beaver burrows inhabiting three particular localities at the Danube, Topľa, and Laborec rivers in Slovakia. In these fecal samples, 4 different species of intestinal endoparasites were detected as follows: oocysts of Cryptosporidium, cysts of Giardia, eggs of Stichorchis subtriquetrus, and eggs and larvae of Travassosius rufus. Parasites were confirmed only in samples collected at river Topľa. Based on our results, we can conclude that European beaver can be an important source of parasitic contamination of surface waters especially in the localities shared by people.

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.

In the present study, the 75% alcoholic fractionation of excretory-secretory (ES) antigen isolated from 200 to 300 live P. epiclitum was assessed for its diagnostic potential for the detection of caprine amphistomosis by using antibody detection enzyme immunoassay. Prior to enzyme immunoassay, 75% alcoholic fractionation of excretory-secretory (ES) antigen was subjected to SDS- PAGE and western blot analysis for the presence of immunoreactive polypeptides. SDS-PAGE analysis of ES antigen resolved a total 7 polypeptides bands of size 56, 27, 25, 22.5, 12, 11 and 10 kDa. Western blot analysis revealed only two immunoreactive polypeptides (11 kDa and 12 kDa) when polypeptides resolved in SDS-PAGE were probed with known positive pooled serum. None of the polypeptides showed reactions with pooled known negative serum. The working dilutions of antigen, sera and conjugates were determined by checkerboard titration for employing ELISA and cut-off O.D. was calculated 0.616 by using the mean absorbance value of 11 negative kid sera. The sensitivity and specificity of ELISA was found to be 100% and 86.76%, respectively. As per kappa value estimation, the strength of agreement was found to be good. Antibodies to 75% alcoholic fractionation of ES antigen was detected in 20% goats (n = 160) of either sex, although faecal examination detected 10.6% goats to be infected with amphistomosis. The study confirmed that 75% alcoholic fractionation of ES antigen of P. epiclitum based ELISA had good value for serodiagnosis of caprine amphistomosis.

Paramphistomosis, caused byCalicophoron daubneyi, is an emerging infection of ruminants throughout Western Europe. Despite its prevalence, many questions remain regarding the basic biology of this parasite and how it interacts with its host. Consequently, there is a need to develop methods to studyC. daubneyi in vitroto improve our understanding of rumen fluke biology. Towards this, we aimed to identify a suitable protocol forin vitroexcystment ofC. daubneyimetacercariae. Six methods that have been used to excyst metacercariae from a number of trematode species were tested withC. daubneyimetacercariae. Three of these achieved an average of >50% excystment whilst one method, which included an acid-pepsin treatment, incubation in reducing conditions and an alkaline/bile salt solution to activate the larvae, consistently gave >80% excystment. The latter protocol also showed no detrimental effect on the motility of newly excysted juvenile (NEJ) parasites when observed for up to 24 h in RPMI 1640 medium post-excystment. The successful production ofC. daubneyiNEJsin vitrois a significant step forward, and will enable the discovery of infective stage-specific parasite antigens and facilitate drug screening trials, to aid the development of much needed diagnostic and therapeutic options for paramphistomosis.

SUMMARYIn this study, we have produced a monoclonal antibody (MoAb) against 16 kDa antigen ofParamphistomum gracile(16 kDaAgPg), and developed an accurate sandwich enzyme-linked immunosorbent assay (sandwich ELISA) for the detection of circulating 16 kDaAg in the serum and fecal samples from cattle naturally infected withP. gracile. MoAb 1D10 was immobilized on a microtitre plate, and the antigen in the samples was captured and detected with biotinylated rabbit anti-16 kDaAgPg antibody. The lower detection limit of sandwich ELISA was 3·5 pg mL−1, and no cross-reaction with other parasite antigens was evaluated. The reliability of the assay was examined using the serum and fecal samples from cattle naturally infected withP. gracile, Fasciola gigantica, Moniezia benedeni, Trichurissp.,Strongyloidessp., strongylids and non-infected animals. The sandwich ELISA showed the sensitivity, specificity and accuracy at 98·33, 100 and 99·55% (serum samples), and 96·67, 100 and 99·09% (fecal samples). Therefore, this detection method is a rapid and excellent potential assay for the accurate diagnosis of paramphistomosis.

The two ruminant parasites, Paramphistomum cervi and Carmyerius gregarius, were collected from fresh-slaughtered native cattle at local abattoirs in Sadat district, Menoufia province and identified morphologically, then molecularly by sequencing the nucleotides of 18S ribosomal RNA gene (18S rRNA). The nucleotide sequences of the two isolates were 456 (P. cervi) and 401 bases for (C. gregarius). The data were used along with those of several other helminth species from the GenBank to identify these two species genetically. The nucleotide sequences were aligned using multiple sequence alignments of nucleotides by Clustal W 12.1 V and construct their relationship. Neighbor-joining analytical method was used showing sister relationship between C. gregarius from Sadat district and Gastrodiscoides hominis (EF027096) with relative identity of (98%) due to the presence of single nucleotides polymorphisms (SNPs) in the form of indels as nine nucleotides positions. But when clustering of P. cervi Sadat isolate with Paramphistomoidea sp. S4 isolate P5 (GU735643), this relationship shows complete identity (99%) between them. The homology and diversity was done using Bayesian analyses in MrBayes v3.1. This work will give a useful guide for other researchers for the molecular taxonomic position of Paramphistomatidae spp. in Sadat district among the different species around the world.

The anthelmintic activity of column fractioned Dicranopteris linearis (Burm.f.) Underw. extracts was used for in vitro incubation study against adult Gastrothylax crumenifer. The qualitative analysis showed ethanol extract performed well to express 12 secondary metabolites studied except anthocyanin. Quantitative phytochemical analysis of terpenoids, phenols, flavonoids and tannins showed predominant amount of terpenoids (97.0±1.15)mg/g and tannins (30.8±0.44)mg tannic acid equivalents/gram. GC-MS analysis of fraction separated under column (Fraction V-79.5%) showed presence of QUERCETIN 7,3',4' TRIMETHOXY, a flavonoids (polyphenol derivative), PHYTOL, a diterpenoids, feren-8-ene, a triterpenoids, hexdecanoic acid, a fatty acid derivative shown to have potent anthelmintic property. 25ml of Hedon-Fleig (H-F) salt solution containing various concentrations (1, 2, 3, 4 and 5mg/ml) respectively as test extracts, Standard control Oxyclozanide @1(*)% (0.25g/25ml) and negative control H-F salt solution, distributed to 7-Petri plates in an incubator with 5% CO2 at 37°C. Twenty five amphistomes were incubated and the motility of control and drug treated flukes was observed under dissection microscope at a regular time interval of 0, 10, 15, 30min and 1hr respectively. The motility response of the parasites was categorized with specific score 3, 2, 1, 0 respectively. Relative Motility (RM) value based on mean mortality and drug concentration for analysis of Lethal Concentration (LC50) and were determined by Probit regression analysis. In vitro incubation study revealed death of all trematodes, lethal at 10min incubation time at 5mg/ml concentration, indicated RM value is 0, equivalent effective to Standard control Oxyclozanide, where RM value is 0 at 10min. Confirmative study on gross morphology, histopathology, and ultra-structural changes showed considerable effect on suckers, teguments and internal organs and were dose dependent. It is indicative that presence of terpenoids, flavonoids, tannins and phenolics which are contributing factors for anthelmintic activity, producing various degree of damage to suckers, teguments and internal organs. Hence D. linearis (Burm.f.) Underw. can be a lead for synthesis of a new trematodicidal drug as alternative for other drugs and overcome anthelmintic resistance and cost effective.

Paramphistomosis, caused by paramphistomid flukes, is a gastrointestinal parasitic disease of domestic and wild ruminants. Originally thought to be limited to the tropics and subtropics, the disease has recently been reported in temperate regions. Here we describe the concurrent infection of a red deer doe (Cervus elaphus) with Paramphistomum leydeni and Paramphistomum cervi. This is the first report of P. leydeni in Croatia. Flukes were identified on the basis of morphological keys (tegumental papillae) and sequencing of the internal transcribed spacer region 2 in ribosomal DNA. Our results confirm that the absence of tegumental papillae allows P. cervi to be differentiated morphologically from other paramphistomid species in Europe based on incident light stereomicroscopy. Nevertheless the limitations of morphological identification and taxonomic issues suggest that previous findings on paramphistomid infection should be interpreted carefully. The possible worldwide distribution of these pathogens means that paramphistomosis may be more common and its economic impact greater than previously thought.

A number of monoclonal antibodies (MoAbs) against the 16 kDa antigen of Paramphistomum gracile (16 kDaAgPg) were produced in vitro by hybridoma technique. Reactivity and specificity of these MoAbs were evaluated by ELISA and immunoblotting assays. Seven MoAb clones were selected from the stable hybridoma clones, namely 1D10, 2D7, 3B10, 3D9, 4F1, 4G4, and 5G12. It was found to be IgM and kappa light chain isotypes. By immunoblotting and ELISA, all MoAbs reacted with purified 16 kDaAgPg at molecular weight (MW) of 16 kDa and with the native 16 kDa antigen at MW of 16 kDa in the whole body (WB) and excretory-secretory (ES) fractions, but not with tegumental antigens (TA) of adult fluke. All of these MoAbs showed no cross-reactions with antigens of other parasites commonly found in ruminants, including Eurytrema pancreaticum, Gigantocotyle explanatum, Schistosoma spindale, Moniezia benedeni, Avitellina centripunctata, Haemonchus placei, Trichuris sp., and Setaria labiato-papillosa. Localization and distribution of the native 16 kDaAg in adult P. gracile by immunohistochemistry, using MoAbs as probes, showed that the native 16 kDaAg was present in high concentration in the cytoplasm of vitelline cells, eggshell globules, and the shells of eggs, but not in the tegument, muscle, parenchymal cells, and cecum of adult fluke. This finding indicated that the 16 kDaAg is a copiously expressed parasite protein that is released into the ES; thus, 16 kDaAg and its MoAb could be a good candidate for immunodiagnosis of paramphistomosis in ruminants.

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

In this article, the main amphistome species infecting domestic and wild ruminants in East and Southern Africa, their snail intermediate hosts and epidemiological features are reviewed and discussed. Twenty-six amphistome species belonging to nine genera from three families occur in domestic and wild ruminants in the region under review and over 70% of them belong to the genera Calicophoron, Carmyerius and Cotylophoron . Of the amphistome species, 76.9% are shared between domestic and wild ruminant hosts – an important observation when considering the different options for control. Seven freshwater snail species belonging to four genera from two families act as intermediate hosts of the identified amphistome species, with the genus Bulinus contributing 57% of the snail species. Some of the snails are intermediate hosts of amphistome species belonging to the same genus or to different genera; a phenomenon not yet fully elucidated as some snails are reported to be naturally infected with amphistome cercariae of unidentified species. Only nine (34.6%, 9/26) of the amphistome species have known snail intermediate hosts, while most (65.4%, 17/26) have unknown hosts. Species of intermediate hosts and the potential of the flukes to infect these hosts, the biological potential of the snail hosts, the definitive hosts management systems and their grazing habits are considered to be the main factors influencing the epidemiology of amphistomosis. Based on the epidemiological features of amphistome infections, various practical control options are discussed. Further research is necessary to determine amphistome–snail associations, develop diagnostic tests that can detect prepatent infections in the definitive host, determine the burden and economic importance of amphistomosis in domestic and wild ruminants and the efficacy of different anthelmintics in the treatment of patent infections.

Paramphistomes are important parasites in veterinary medicine. There are few anthelmintic drugs available against them. The development of new drugs is urgently needed and this process can be accelerated through the development of rodent models for in vivo testing. Among the few paramphistomes that develop in rodents is the caecal fluke Zygocotyle lunata, a species with which several biological studies have been performed over several decades. Nevertheless, its use as a model for evaluation of anthelmintic drugs had not yet been evaluated. In the present study, we evaluated the efficacy of praziquantel (PZQ 300 mg/kg 5x), albendazole (ABZ 200 mg/kg 5x) and closantel (CLO 50 mg/kg single dose, 50 mg/kg 3x and 25 mg/kg 3x) for treatment of mice experimentally infected with Z. lunata. The animals were infected with 20 metacercariae of the parasite and were treated 30 days post-infection. Untreated groups were maintained as controls. Seven days after the treatments, the animals were euthanized for recovery and counting of parasites. We found that PZQ and ABZ, at the dosages and therapeutic schedule employed here, did not cause significant alterations in worm burden [worm counts 16.0 ± 2.8 (13–19), 17.6 ± 2.1 (14–19) and 16.2 ± 1.9 (13–18) (p = 0.51) in PZQ, ALB and control, respectively]. CLO 50 mg/kg in a single dose caused significant reduction in the number of parasites [treated: 1.8 ± 0.9 (1–3); control: 15.6 ± 2.5 (12–19)], although it did not result in complete elimination of the parasites in any animal. Despite the fact that three doses of CLO 50 mg/kg or CLO 25 mg/kg caused complete elimination of the parasites in most surviving animals, there was significant host mortality. In general, results here obtained are concordant with those of studies performed on ruminant paramphistomes. Given that Z. lunata can be maintained in laboratory rodents, it is a suitable model for screening anthelmintic drugs against paramphistomes.

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., Graphical abstract, Highlights • The first transcriptome data for intramammalian stages of Calicophoron daubneyi. • Comparative secretome profiles of infective stage and adult flukes. • New mechanistic insights into infectivity and pathogenicity. • Development of the first coproantigen-based ELISA for paramphistomosis., In brief Paramphistomosis, caused by Calicophoron daubneyi, is a parasitic infection of ruminant livestock currently spreading throughout Western Europe. Here, transcriptome analysis of four intramammalian life-cycle stages, integrated with secretome profiling of the infective stage and adult parasites (responsible for acute and chronic diseases, respectively), revealed how selected families of unique virulence factors and immunomodulators are regulated in accordance with fluke development and migration. This analysis allowed the development of the first coproantigen-based ELISA for paramphistomosis.

An antigenic component of adult Paramphistomum gracile was characterized by means of indirect enzyme-linked immunosorbent assay (indirect ELISA), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using sera from cattle naturally infected with P. gracile, Eurytrema pancreaticum, Fasciola gigantica, Moniezia benedeni, strongylids, Trichuris sp. and Strongyloides sp. The whole body (WB) extracts of P. gracile were fractionated by gel filtration chromatography in a Sephadex G-200 column. It was found that the WB extract fractions, F1-F3 were highly antigenic, F5 was moderately antigenic and F4 was poorly antigenic. For SDS-PAGE and immunoblotting, the antigenic molecules of WB extract and all five fractions were mostly at molecular weights (MW) ranging from 12 to 150 kDa. One antigenic protein of 16 kDa detected in WB extract and F1-F3 was found to give a consistent reaction with sera from infected cattle. The antigenicity of the purified 16 kDa protein was confirmed by immunoblotting and indirect ELISA using a pool of sera and individual serum samples from infected cattle (at 1 : 78 125 dilution) and hyperimmunized rabbit (at 1 : 390 625 dilution). This finding suggests that the 16 kDa protein may be a potential antigen for the immunodiagnosis of cattle paramphistomosis caused by P. gracile.

There is concern about the probable recent introduction, increased prevalence and potential economic impact of rumen fluke infection of United Kingdom cattle. A study of 339 cattle slaughtered in a Scottish red meat abattoir was undertaken with the aims of describing the prevalence and geographical distribution of rumen fluke infection, estimating its effect on production, and evaluating faecal egg counts (FECs) as a tool to diagnose infection in live animals and study the epidemiology of the disease. The overall proportion of cattle consigned to the abattoir from northern United Kingdom with rumen fluke infection in the forestomachs was 0.29. Rumen flukes were distributed predominantly in the cranial sac of the rumen and adjacent to the reticular groove. Overall, a mean of 213 and median of 44 rumen flukes was identified in the forestomachs of rumen fluke-positive cattle. The mean and median FECs of animals were 26.01 and 5.20 eggs per gram (epg), respectively. There was a significant difference between the mean FECs per rumen fluke of 0.08 and 0.13 epg during summer/autumn and winter sampling periods, respectively. The overall correlation between rumen fluke FECs and the number of flukes in the forestomach was high, albeit lower in the summer/autumn than in the winter period. The sensitivities of rumen fluke FECs for the identification of flukes in the forestomach during the summer/autumn and winter sampling periods were 0.65 and 0.85, respectively. These results will aid in the interpretation of rumen fluke FECs when monitoring cattle health and production and studying the parasite's epidemiology in a temperate environment, thereby informing rational, precise and sustainable disease control.

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.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.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.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.

BackgroundFasciolosis, caused by the liver fluke Fasciola gigantica, and paramphistomosis are widespread in cattle in Tanzania, and the use of trematocides is encouraged by the Government livestock extension officers. However, reduced efficacy of oxyclozanide against Fasciola gigantica and amphistomes (rumen flukes), and albendazole against F. gigantica, has been reported in some regions. This study was conducted to assess the efficacy of different trematocides against F. gigantica and amphistome infections in cattle at Iringa Rural and Arumeru Districts.MethodsCattle found with concurrent infection of F. gigantica and amphistomes were randomly grouped into six experimental groups. One control group was left untreated while five treatment groups were treated with one of five trematocides that include: albendazole, nitroxynil, oxyclozanide, closantel and triclabendazole. Post-treatment faecal sample collection was done on the day of treatment and again at 7, 14 and 28 days, from each cattle. The samples were processed by Flukefinder® method to recover and identify eggs. Assessment of the efficacy of the trematocides against F. gigantica and amphistomes was conducted using faecal egg count reduction (FECR) tests.ResultsThe findings of the present study in both districts indicate that nitroxynil, oxyclozanide, closantel and triclabendazole are effective against patent F. gigantica infection, as the calculated FECR% for each trematocide was 100% by day 14 post-treatment. However, albendazole found to have reduced efficacy of against F. gigantica, as FECR% was 49% in Arumeru District and 89% in Iringa Rural District by day 14 post-treatment. Oxyclozanide was the only trematocide found to be effective against amphistomes with FECR of 99%.ConclusionsAlbendazole had reduced efficacy against F. gigantica in cattle in Arumeru and Iringa Rural Districts, Tanzania. The reduced efficacy was prominent in Arumeru, where cattle are commonly treated with anthelmintics, than in Iringa Rural, where cattle are seldom treated.

SUMMARYCross-species infection among humans, chimpanzees (Pan troglodytes) and baboons (Papio spp.) is potentially a significant public health issue in Africa, and of concern in the conservation of P. troglodytes. However, to date, no statistical comparisons have been made between the prevalence, richness and composition of parasite communities in sympatric populations of baboons and P. troglodytes. We compared parasite communities in sympatric P. troglodytes and Papio papio living in a wilderness site, in the Republic of Senegal, West Africa. We asked whether, in the absence of humans, there are significant differences between these hosts in their interactions with gastrointestinal parasites. We tested whether host, location, or time of collection accounted for variation in prevalence, richness and community composition, and compared prevalence across six studies. We concluded that, despite being closely related, there are significant differences between these two hosts with respect to their parasite communities. At our study site, prevalence of Balantidium, Trichuris and Watsonius was higher in P. papio. Papio papio harboured more parasites per host, and we found evidence of a positive association between Trichuris and Balantidium in P. troglodytes but not P. papio.

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.

As evidence is growing that in many temperate areas paramphistome infections are becoming more common and widespread, this study was undertaken to determine the role of deer as reservoirs for rumen fluke infections in livestock. A total of 144 deer faecal samples (88 from fallow deer, 32 from red deer and 24 samples from sika, sika/red deer hybrids) were screened for the presence of fluke eggs. Based on the ITS-2 rDNA locus plus flanking 5.8S and 28S sequences (ITS-2+), fluke eggs were identified to species level. Our results indicate that, of the 3 deer species, fallow deer had the highest fluke infection rates. Two rumen fluke species, Calicophoron daubneyi and Paramphistomum leydeni, with morphologically distinct eggs, were identified. Concurrent infections of the two paramphistome species and liver fluke, Fasciola hepatica, were common. Considering the comparatively low egg burdens observed in this study, it is unlikely that deer represent a significant source of infection for Irish livestock.

Paramphistome infections have been associated with significant morbidity, caused chiefly by the activity of juvenile flukes in the intestine of the ruminant final host. Most cases have been reported in tropical and sub-tropical areas. However, recent reports of an apparent increase in the incidence of rumen fluke and its geographical range in Europe have renewed interest in a parasite previously thought to be of little significance in temperate regions. Moreover, the identity of rumen flukes present in the British Isles is currently being revised. As a result, work is underway throughout Europe to review and re-assess the clinical and economic significance of rumen flukes. During the present study, historical diagnostic laboratory records were interrogated for recent changes in the incidence of rumen fluke in Ireland. Three cattle herds were monitored for the presence of paramphistome eggs using coprological analysis over a period of 2 months (in the case of a group of housed steers) and 14 months (in the case of two extensively operated farms), respectively. Adult rumen fluke collected following slaughter were weighed and typed in two loci. We found that Calicophoron daubneyi is the most common if not only paramphistome species present in Ireland and that infections in cattle are now much more prevalent than was the case five or six years ago. The pylogenetic relationship of our isolates to the only published sequence and to C. daubneyi isolates from Northern Ireland was analysed. Genetic heterogeneity was similar all over the island and comparable to that of Fasciola hepatica, a fact that may have implications for the parasite's ability to develop resistance to the very limited number of drugs currently available for treatment. The same haplotypes predominated throughout the island. Although the clinical significance of C. daubneyi is still uncertain, considering the apparent pervasiveness of the parasite, rumen fluke should be considered a differential diagnosis when treating scour or ill-thrift in young calves, and goats and sheep of any age.

An evaluation of Gastrothylax crumenifer crude antigen preparation viz., Somatic Antigen (SAg), Excretory Secretory Antigen (ESAg) and Egg Antigen (EAg) in serodiagnosis of disease was undertaken. Test sera samples were obtained from 30 Paramphistomiasis Positive and 30 Gastrothylax free sheep slaughtered at Hazratbal Kashmir. The referral antigenic preparation were evaluated against Paramphistomiasis positive sera, via., control negative sera, using double immunodiffusion test (DID), (IEP) Immunoelectrophoretic assay and ELISA. The performance of referral antigens, as assessed from percent sensitivity and specificity, revealed an increasing trend from DID (Double immunodiffusion-An immunological technique used in the detection, identification and quantification of antibodies and antigens) to IEP (immunoelectrophoresis-A general name for a number of biochemical methods for separation and characterization of proteins based on electrophoresis and reaction with antibodies), followed by ELISA, detecting higher number of sheep positive for paramphistomiasis. In ELISA the ESAg and SAg were evaluated as most reactive antigens with no significant difference and EAg was the least antigenic. In IEP, EAg had the higher sensitivity (60%) and analogous specificity of SAg and ESAg. The formation of the preceptin lines in the proximity to EAg containing wells (cathode end) in IEP was suggestive of higher molecular weight of G. crumenifer specific protein molecules with slower rate of migration. Purification and characterization of G. crumenifer and identification of specific antigenic molecules, particularly in EAg has been suggested for qualitative improvement of diagnostic value of the antigens in the tests used here in.

Limited information is available on diagnostic stages of parasites in Florida manatees (Trichechus manatus latirostris). We examined 67 fecal samples from captive and wild manatees to define the diagnostic stages of the parasite fauna known to occur in Florida manatees. Parasite eggs were freshly extracted ex utero from identified mature helminths and subsequently characterized, illustrated, and matched to those isolated from fecal samples. In addition, coccidian oocysts in the fecal samples were identified. These diagnostic stages included eggs from 5 species of trematodes (Chiorchis fabaceus, Chiorchis groschafti, Pulmonicola cochleotrema, Moniligerum blairi, and Nudacotyle undicola), 1 nematode (Heterocheilus tunicatus), and oocysts of 2 coccidians (Eimeria manatus and Eimeria nodulosa).

Helminth infections are the cause of morbidity in Cambodian cattle but other factors such as nutritional deficiencies and concurrent diseases may enhance the effects of parasites. The present study aimed to investigate the impact of anthelmintic treatment, feed supplementation, or both on gastrointestinal strongyle (GIS) and trematode infections as well as on morbidity parameters in Cambodian village cattle. At the beginning of the dry season, cattle populations in six villages were randomly assigned to a group: (A) receiving anthelmintic treatment (ivermectin+clorsulon) at week 0; (P) feed pellet supplementation during week 0-13 or both (AP). On five visits (week 0-29), faecal and blood samples were obtained for parasitological examination and haematocrit determination, respectively. Body condition (BCS), hind quarter fouling (HQFS), diarrhoea (DS), and conjunctiva colour (FAMACHA©) were scored and heart girth circumference was determined. To investigate the impact of treatment over time (week 0-29), a mixed model was used with treatment, time, and their interaction as fixed effects, and animal and village as random factors. At baseline, the proportion of GIS positive animals was high (67.9%), whereas trematode infections were low (Paramphistomum: 8.8%; Fasciola: 2.6%). Very thin to emaciated cattle (BCS 1-2) were more prevalent (11.4%) and FAMACHA© scores of ≤3 or below (65.8%) less prevalent than in an earlier study in the region. A Time ⨯ Treatment interaction was present for faecal egg counts (FEC) of GIS, GIS prevalence (both p

Objective: Clerodendrum viscosum, Eryngium foetidum, Lippia javanica, and Murraya koenigii are one among the common wild edible plants in Northeast India which are also used as antidiabetic, stomach-ache relieving drugs, etc., The present study was aimed to reveal the phytochemical, antioxidant, and anthelmintic activity of the plants. Materials and Methods: The antioxidant capacity of methanolic extract of plants was studied by 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferric reducing antioxidant power, TBARS, and total antioxidant activity (TAA). Total phenolics, flavonoids, Vitamin C, carbohydrate, and protein are also estimated following standard protocols. Anthelmintic activity of the extracts has also been studied in vitro against trematode parasites. Results: The result showed that the methanolic extracts of plants possess a substantial quantity of alkaloids, phenolics, flavonoids, proteins, carbohydrates, and Vitamin C. Phenolics, flavonoids, and Vitamin C contents were found higher in C. viscosum followed by M. koenigii, L. javanica, and E. foetidum. The in vitro antioxidant assays revealed substantial free radical scavenging property in all the plants. TAA increased in the order C. viscosum > M. koenigii > L. javanica > E. foetidum. Similarly, C. viscosum displayed a better antioxidant capacity with IC50 values 29.74 ± 3.63 μg and 148.77 ± 18.38 μg for DPPH and thiobarbituric acid reactive species, respectively. In addition, the plant extracts also showed good anthelmintic activity against Paramphistomum sp. Time taken for paralysis and death were 0:56 ± 0:09 h and 1:35 ± 0:07 h for L. javanica at 50 mg/mL concentration. Conclusion: The study therefore suggests the importance of tested plants as a natural source of free radical scavenger and plausible veterinary uses.

SUMMARY Paramphistomoids are ubiquitous and widespread digeneans that infect a diverse range of definitive hosts, being particularly speciose in ruminants. We collected adult worms from cattle, goats and sheep from slaughterhouses, and cercariae from freshwater snails from ten localities in Central and West Kenya. We sequenced cox1 (690 bp) and internal transcribed region 2 (ITS2) (385 bp) genes from a small piece of 79 different adult worms and stained and mounted the remaining worm bodies for comparisons with available descriptions. We also sequenced cox1 and ITS2 from 41 cercariae/rediae samples collected from four different genera of planorbid snails. Combining morphological observations, host use information, genetic distance values and phylogenetic methods, we delineated 16 distinct clades of paramphistomoids. For four of the 16 clades, sequences from adult worms and cercariae/rediae matched, providing an independent assessment for their life cycles. Much work is yet to be done to resolve fully the relationships among paramphistomoids, but some correspondence between sequence- and anatomically based classifications were noted. Paramphistomoids of domestic ruminants provide one of the most abundant sources of parasitic flatworm biomass, and because of the predilection of several species use Bulinus and Biomphalaria snail hosts, have interesting linkages with the biology of animal and human schistosomes to in Africa.

More than 70 species of the family Paramphistomatidae, have been identified in ruminants in different parts of the world. Most are pathogenic, causing amphistomosis. Adult flukes of this Family have a predilection for the rumen, liver or bile duct of ruminants where they may cause damage to the epithelium. Identification of adult paramphistomes to the species level based on morphology alone requires specialized knowledge, whereas, molecular genetic marker analysis is more precise and transferable. In the present study, we performed both morphological and molecular characterisation of fifteen adult flukes collected from the liver of domesticated buffalo in the Punjab province of Pakistan. The morphology of five of these flukes was examined in detail and on this basis these were identified as either Explanatum explanatum or Explanatum bathycotyle. PCR and sequencing of the ITS-2 rDNA region from these 5 flukes, plus 10 others, revealed a single haplotype in all cases. This differed by just a single nucleotide polymorphism from a previously described E. explanatum ITS-2 rDNA sequence. Phylogenetic comparison of these E. explanatum ITS2-rDNA sequences with those from other Paramphistomatidae, Fasciola and Dicrocoelium species was performed to assesss within and between species variation and validate the use of ITS-2 rDNA as a robust species-specific marker for E. explanatum. This work provides a validated species-specific marker of E. explanatum and the first report of this parasite species from Pakistan.

The liver fluke, Fasciola hepatica, is common in many parts of Great Britain. To detect liver fluke infection and to assess whether fasciolicide treatment has been successful, the faecal egg count (FEC) and faecal egg count reduction test (FECRT) are widely used. Rumen fluke is also increasingly reported from Great Britain, but its species identity is yet to be determined. Liver fluke and rumen fluke eggs are morphologically similar, which may lead to erroneous diagnoses of liver fluke infection or treatment failure. As an alternative to FEC, a coproantigen ELISA (cELISA) can be used. The potential for this test to cross-react with rumen fluke species from Great Britain has not been evaluated. Rumen fluke specimens from cattle and sheep in Scotland were identified to species level using DNA sequencing of the ITS-2 region. Subsequently, rumen and liver fluke obtained from naturally co-infected sheep were subjected to immunohistochemistry using antibodies from a commercially available cELISA kit for F. hepatica. Finally, faecal samples from naturally co-infected sheep flocks were examined by FEC and cELISA. Rumen fluke from imported and home-bred cattle and sheep in Scotland belonged to the species Calicophoron daubneyi, rather than Paramphistomum cervi, the species presumed to be most common in Great Britain. Intense staining of the gastrodermis was observed in F. hepatica but cross-reactivity with C. daubneyi was not seen. Faecal samples that contained rumen fluke eggs but not liver fluke eggs were all negative by cELISA. We conclude that C. daubneyi is the most common rumen fluke of domestic ruminants in Scotland and that cELISA reduction testing may be a valuable alternative to FECRT in herds or flocks that are co-infected with liver and rumen fluke.

8 páginas, 8 figuras, 1 tabla., Paramphistomosis and Fasciolosis caused by Calicophoron daubneyi and Fasciola hepatica, respectively, are frequent and important trematodoses in ruminant livestock worldwide. Both parasites use the same snail, Galba truncatula, as intermediate host. The aim of this study was to develop and validate an analytical method based on a mitochondrial DNA (mtDNA) multiplex PCR technique which would allow the early and specific identification, in one step, of C. daubneyi and F. hepatica infection in G. truncatula. First of all, a 1035 bp fragment of mtDNA from adult C. daubneyi worms was obtained. Then two pairs of specific mtDNA primers, which amplified a DNA fragment of 885 pb in the case of C. daubneyi, and of 425 pb in that of F. hepatica, were designed. By means of the multiplex PCR technique developed, there was always a specific amplification in samples from adult F. hepatica and C. daubneyi, but not from Calicophoron calicophorum, Cotylophoron cotylophorum, Cotylophoron batycotyle or Dicrocoelium dendriticum. Likewise, specific amplifications of the expected DNA fragments happened in all samples from snails harbouring larval stages of C daubneyi or F. hepatica, previously detected by microscopy. However, amplifications were not seen when DNA from snails harbouring other Digenea (Plagiorchiidae, Notocotylidae and furcocercous cercariae) was analysed. Moreover, DNA from G. truncatula molluscs free from infection was not amplified. The multiplex PCR assay permitted infection in the snails experimentally infected with 4 miracidia to be detected as early as day 1 p.i. in the case of F. hepatica and with only 2 miracidia from day 2 p.i. in both, C daubneyi and F. hepatica. Nevertheless it was necessary to wait until days 29 and 33 p.i. to see C. daubneyi and F. hepatica immature redia, respectively, using microscope techniques. The detection limit of the PCR technique was very low: 0.1 ng of DNA from C daubneyi and 0.001 ng of DNA from F. hepatica. This allowed infection by either F. hepatica or C daubneyi to be detected even when pools made up with only 1 mu l (60 ng of DNA) from infected snail plus 99 mu l from non-infected ones were analyzed. Moreover, simultaneous detection of both parasites was experimentally possible in pools made up with uninfected (98 mu l), C. daubneyi infected (1 mu l) and F. hepatica infected (1 mu l) snails. The most precise and early diagnosis of the infections using the multiplex PCR technique designed will allow more realistic epidemiological models of both infections to be established and consequently a better strategic control., This study was financially supported by the Spanish INIA, Project FAU2006-00021-C03-00 and by the European Social Funds, as well as by the Castile and León Autonomy (Spain) Project LE023A10-2.A.M. Martínez-Ibeas is supported by the ‘Castilla y León’ Autonomy (Spain) and the European Social Funds (Contract for young researchers). M. Martínez- Valladares is supported by the Spanish National Research Council (CSIC) and the European Social Funds (JAE Doc Contract). B. Mi˜nambres was supported by ‘Ramón y Cajal’ contract.

The immunogenic components of adult Paramphistomum cervi excretion–secretion (ES) fraction were revealed by SDS–PAGE and immunoblotting technique using sera from cattle naturally infected with P. cervi, Fasciola gigantica, strongylids, Trichuris sp., and Strongyloides sp. By SDS–PAGE, it was found that the ES fraction comprised 13 distinct protein bands. Immunoblotting analysis of these proteins exhibited nine prominent antigenic bands which were recognized by paramphistomosis antisera. These antigenic proteins had molecular weights ranging from 10–170 kDa. One antigenic protein band of 40 kDa was found to give a consistent reaction with sera from all infected cattle. Its diagnostic sensitivity, specificity and accuracy using this test were 100%, 98.9% and 99.3%, respectively. The positive and negative predictive values were 98% and 100%, respectively. The 40 kDa antigen was partially purified by gel filtration and ion-exchange chromatography. The antigenicity of 40 kDa protein for diagnosis of P. cervi infection was confirmed by immunoblotting and indirect ELISA (at 1:78,125 dilution) using a pool of sera and individual serum samples from infected cattle. The present findings suggest that the 40 kDa protein may be used as a diagnostic antigen for paramphistomosis.

The analysis of infection by Paramphistomidae trematodes was conducted in two agricultural regions with different knowledge on this parasitosis. Faecal and blood samples were collected from 374 cattle in Salto (NW Uruguay) where there is a lack of information about paramphistomosis. A total of 429 cattle from Galicia (NW Spain), an area with previous records of infection by gastric flukes, were sampled. Diagnostics of trematodosis was developed by using a copromicroscopic probe and an ELISA with excretory/secretory antigens collected from adult Calicophoron daubneyi (Paramphistomidae) specimens. Results were evaluated according intrinsic and extrinsic factors. In the Uruguay, the percentage of cattle passing Paramphistomidae-eggs by faeces was 7% (95% Confidence Interval 5, 10). A significantly higher prevalence of paramphistomosis in the Hereford × Angus cattle (OR = 3.5) was recorded, as observed for the oldest ruminants (>3.5. years). An overall seroprevalence of 29% (25, 34) was obtained by ELISA, with the highest values in the Friesians (OR = 3), the youngest bovines (

12 páginas, 5 figuras, 4 tablas., The present study explored various basic aspects of the epidemiology of paramphistomosis in Galicia, the main cattle producing region in Spain. In total, 589 cows from different farms located across the region were selected at random in the slaughterhouse for examination of the rumens and reticula for the presence of Paramphistomidae flukes. Paramphistomes were found in 111 of 589 necropsied cows (18.8%; 95% CI: 15.7-21.9%), with higher prevalences of infection in beef cows than in dairy cows (29.2% vs 13.9%). Although the number of flukes per animal was generally low (median = 266 flukes), some cows harboured large parasite burdens (up to 11,895 flukes), which may have harmful effects on their health or productivity. Cows with higher parasite burdens also excreted greater numbers of fluke eggs in their faeces, which suggests that heavily parasitized mature cows play an important role in the transmission of paramphistomosis. This role may be particularly important in Galicia, where the roe deer, which is the only wild ruminant in the study area, was found not to be a reservoir for the infection. The use of morpho-anatomical and molecular techniques applied to a large number of fluke specimens provided reliable confirmation that Calicophoron daubneyi is the only species of the family Paramphistomidae that parasitizes cattle in Galicia. The environmental data from the farms of origin of the necropsied cows were used in Bayesian geostatistical models to predict the probability of infection by C. daubneyi throughout the region. The results revealed the role of environmental risk factors in explaining the geographical heterogeneity in the probability of infection in beef and dairy cattle. These explanatory factors were used to construct predictive maps showing the areas with the highest predicted risk of infection as well as the uncertainty associated with the predictions., Ministerio de Economia y Competitividad FAU2006-00021-C03-00. Junta de Castilla y Leon (Spain). European Social Funds

peer-reviewed Background Rumen flukes are trematode parasites found globally; in tropical and sub-tropical climates, infection can result in paramphistomosis, which can have a deleterious impact on livestock. In Europe, rumen fluke is not regarded as a clinically significant parasite, recently however, the prevalence of rumen fluke has sharply increased and several outbreaks of clinical paramphistomosis have been reported. Gaining a better understanding of rumen fluke transmission and identification of risk factors is crucial to improve the control of this parasitic disease. In this regard, a national prevalence study of rumen fluke infection and an investigation of associated risk factors were conducted in Irish sheep flocks between November 2014 and January 2015. In addition, a molecular identification of the rumen fluke species present in Ireland was carried out using an isolation method of individual eggs from faecal material coupled with a PCR. After the DNA extraction of 54 individual eggs, the nuclear fragment ITS-2 was amplified and sequenced using the same primers. Results An apparent herd prevalence of 77.3 % was determined. Several risk factors were identified including type of pasture grazed, regional variation, and sharing of the paddocks with other livestock species. A novel relationship between the Suffolk breed and higher FEC was reported for the first time. The predominant rumen fluke species found was C. daubneyi. Nevertheless, P. leydeni was unexpectedly identified infecting sheep in Ireland for the first time. Conclusions An exceptionally high prevalence of rumen fluke among Irish sheep flocks has been highlighted in this study and a more thorough investigation is necessary to analyse its economic impact. The isolation of individual eggs coupled with the PCR technique used here has proven a reliable tool for discrimination of Paramphistomum spp. This technique may facilitate forthcoming studies of the effects of paramphistomosis on livestock production. The most noteworthy finding was the identification of P. leydeni affecting sheep in Ireland, however further studies are required to clarify its implications. Also, a significant relationship between Suffolk breed and a heavier infection was found, which can be used as a starting point for future research on control strategies of rumen fluke infection. This study was funded by Irish Department of Agriculture, Food and the Marine research stimulus funding; project reference 13/ S/405.

In the present study, we sequenced and analyzed the mitochondrial (mt) genome of Gastrothylax crumenifer and compared it with other selected trematodes. The full mt genome of G. crumenifer was amplified, sequenced, assembled, analyzed and then subjected to phylogenetic analysis. The complete mt genome of G. crumenifer is 14,801 bp in length and contains two rRNA genes, two non-coding regions (LNR and SNR), 12 protein-coding genes, and 22 transfer RNA genes. The gene organization of the G. crumenifer mt genome is the same as that of other trematodes, except for Schistosoma haematobium and Schistosoma spindale. All the genes are transcribed in the same direction and rich in “A + T”, which is in accordance with other trematodes, such as Fasciola hepatica, Paramphistomum cervi, and Fischoederius elongatus. Phylogenetic analysis using concatenated amino acid sequences of the 12 protein-coding genes showed that G. crumenifer is closely related to F. elongatus. The availability of mt genome sequence of G. crumenifer can provide useful DNA markers for studying the molecular epidemiology and population genetics of this parasite and other paramphistomes.

Adults Fischoederius cobboldi are conical-shaped, concave ventrally and convex dorsally, measures about 8–10 mm in length and 4–6 mm in width across the mid section. Scanning electron microscopy (SEM) of entire body showed that the tegumental surface exhibits highly corrugation and transverse folds alternating with grooves and without spines. At higher magnification, the surface of each fold is further increased with a meshwork of ridges separated by irregular-sized pits. The ventral surface has more complex corrugations and invaginations than those of the dorsal surface of the body. Both anterior and posterior suckers have thick edges covered with transverse folds and appear spineless. The genital pore is located at the anterior one-third of the body. There are two types of sensory papillae on the surface: type 1 is bulbous in shape and nipple-like tips, measuring 10–15 μm in diameter at the base, and also type 2 is a similar shape and has short cilia on tips. These sensory papillae occur in large clusters, each having between 7 and 25 units depending on the region of the body. Clusters of papillae on the ventral surface and around the anterior suckers tend to be more abundant and larger in size. The dorsal side of the body exhibit similar surface features, but papillae appear less numerous and are smaller. Corrugations and invaginations of the dorsal aspect are also less extensive than those on the ventral surface of the body.

Aim of present study was to screen medicinal plants for flukicidal activity in vitro to develop alternative sources of treatment for trematodes infection. For this purpose, crude methanolic extracts (CME) of Cymbopogn jwarancusa and Conyza canadensis were prepared and live adult flukes viz; Fasciola gigantica, and Paramphistomum cervi isolated from liver and bile ducts of slaughtered buffalo were subjected to different drug concentrations as well as positive and negative control. Motility inhibition and paralysis leading to the death of parasites was considered as flukicidal activity of plants extracts. The results revealed that CME of C. jwarancusa and C. canadensis showed significant (P0.05) flukicidal activity compared to positive control. Also there was a significant effect of different concentrations (P0.05) and exposure of time on the flukes (P0.05). Furthermore, ED50 for C. jwarancusa and C. canadensis against F. gigantica were 13.1 and 41.4 mg/ml, respectively. In the case of P. cervi, it was 10.8 and 29.0 mg/ml. It can be concluded that both tested plants showed greater flukicidal activity as compared to positive control with Albendazole till the 8(th) hr. These potent plants needs further studies invivo to elucidate their mode of action.

To develop a duplex PCR method for identifying Metagonimus yokogawai and Haplorchis taichui.ITS1 sequences of M. yokogawai and H. taichui, as well as those of their homologous species were obtained from GenBank, and two sets of specific primer pairs for M. yokogawai and H. taichui were designed accordingly using Primer Premier 5.0 software. PCR reaction system and conditions were optimized. The established duplex PCR method was applied in a pool of M. yokogawai, H. taichui, and 17 related species to examine its specificity. Sensitivity was evaluated through serial dilutions of plasmids containing their specific sequences. Finally, the duplex PCR was applied to identify M. yokogawai and H. taichui among trematodes collected from the viscera of 47 cats and 40 dogs to test its practicality.The duplex PCR method amplified target sequences of M. yokogawai and H. taichui, generating 648 bp and 279 bp products, respectively. No cross reaction was found with the following 17 related species: Haplorchis pumilio, Clonorchis sinensis, Pharyngostomum cordatum, the metacercaria of Metorchis sp. and Exorchis sp., Echinochasmus liliputanus, Echinochasmus perfoliatus, Echinostoma friedi, Hypoderaeum conoideum, Holostephanus sp., Diplodiscus sp., Anisakis sp., Metorchis orientais, Paragonimus westermani, Watsonius watsoni, Notocotylus sp. and Hysterothylacium sp, indicating a high specificity of this method. The detection limits for DNAs of M. yokogawai and H. taichui were 1.49 x 10(-1) pg and 1.14 x 10(-1) pg, suggesting a good sensitivity for this method. Further, the duplex PCR successfully identified M. yokogawai and H. taichui from cat and dog viscera, with no cross amplification of other trematodes.The duplex PCR is effective in identifying Metagonimus yokogawai and Haplorchis taichui.