Mapping the Spatial Distribution of the Rumen Fluke Calicophoron daubneyi in a Mediterranean Area

Rumen flukes (Calicophoron daubneyi) represent a growing threat to the animal health, productivity and welfare of ruminants. The present study aimed to assess the spatial distribution of C. daubneyi infections in ruminants and to develop a predictive model of the environmental suitability for rumen flukes in a Mediterranean area. A cross-sectional coprological survey was conducted in 682 sheep, 73 goat and 307 cattle farms located in the Basilicata region (southern Italy). Faecal samples collected were analysed using the FLOTAC technique. Geographical Information Systems (GIS) and statistical models were developed to determine environmental risk factors and to delimitate the areas at highest risk of infections in small ruminants. The results showed 7.9% (95% CI 6.05–10.27) of sheep farms, 2.7% (95% CI 0.48–10.44) of goat farms and 55.0% (95% CI 49.62–60.99) of cattle farms were infected by C. daubneyi. The areas with high predicted risk were situated in the western part of the region. The soil texture, land use and the presence of streams and brooks were the variables statistically significant (p < 0.05) in explaining the C. daubneyi distribution in the study area. The study confirms the importance of geospatial technology in supporting parasite control strategies in livestock and demonstrates that a combined use of different geostatistical techniques can improve the prediction of the C. daubneyi infection risk in ruminants.

Modulation of Rumen Microbes Through Extracellular Vesicle Released by the Rumen Fluke Calicophoron daubneyi

Parasite derived extracellular vesicles (EVs) have been proposed to play key roles in the establishment and maintenance of infection. Calicophoron daubneyi is a newly emerging parasite of livestock with many aspects of its underpinning biology yet to be resolved. This research is the first in-depth investigation of EVs released by adult C. daubneyi. EVs were successfully isolated using both differential centrifugation and size exclusion chromatography (SEC), and morphologically characterized though transmission electron microscopy (TEM). EV protein components were characterized using a GeLC approach allowing the elucidation of comprehensive proteomic profiles for both their soluble protein cargo and surface membrane bound proteins yielding a total of 378 soluble proteins identified. Notably, EVs contained Sigma-class GST and cathepsin L and B proteases, which have previously been described in immune modulation and successful establishment of parasitic flatworm infections. SEC purified C. daubneyi EVs were observed to modulate rumen bacterial populations by likely increasing microbial species diversity via antimicrobial activity. This data indicates EVs released from adult C. daubneyi have a role in establishment within the rumen through the regulation of microbial populations offering new routes to control rumen fluke infection and to develop molecular strategies to improve rumen efficiency.

In vitro screening as an anthelmintic discovery pipeline for Calicophoron daubneyi: nutritive media and rumen environment-based approaches

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.

Transcriptome and secretome analysis of intra-mammalian life-stages of the emerging helminth pathogen, 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. Following 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 larvae then migrate upwards, 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 to avoid the host immune response. Here, transcriptome analysis of four intra-mammalian life-cycle stages, integrated with secretome analysis of the NEJ and adult parasites (responsible for acute and chronic disease 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 whilst a family of cathepsins B with varying S2 sub-site residues (indicating distinct substrate specificities) are 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 up-regulated in adult worms, although they are underrepresented in the secretome. The most abundant proteins in adult fluke secretions were helminth defence molecules (HDMs) that likely establish an immune environment permissive to fluke survival and/or neutralise pathogen-associated molecular patterns (PAMPs) 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 recognise antigens from other helminths commonly found as co-infections with rumen fluke.

Paramphistomidose – eine Übersicht zu Epidemiologie und klinischer Symptomatik

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.