Fasciola hepatica products can alter the response of bovine immune cells to Mycobacterium avium subsp. paratuberculosis.

BACKGROUND: Fasciola hepatica causes economically important disease in livestock worldwide. The relevance of this parasitic infection extends beyond its direct consequences due to its immunoregulatory properties. OBJECTIVES: Given the importance of the T helper 1 (Th1) immune response in controlling infections with Mycobacterium avium subspecies paratuberculosis (MAP) in cattle, we aimed to establish the immunological consequences that co-infection with F. hepatica might have on the course of Johne's disease (JD). METHODS: This study compared the in vitro response of bovine immune cells to infection with MAP or exposure to MAP antigens following F. hepatica infection or stimulation with F. hepatica products. RESULTS: We found a decreased proliferation of peripheral blood mononuclear cells (PBMCs) after infection with F. hepatica. This reduction was inversely correlated with fluke burden. Pre-stimulation with F. hepatica molecules produced a significant reduction of ileocaecal lymph node leucocyte proliferation in response to MAP antigens. Additionally,F. hepatica products reduced expression of the CD14 receptor by macrophages and increased levels of apoptosis and bacterial (MAP) uptake. CONCLUSIONS: Overall, F. hepatica infection had little impact on the in vitro response of immune cells to MAP, whereas in vitro co-stimulation with F. hepatica molecules had a measurable effect. Whether this is likely to affect JD progression during in vivo chronic conditions remains unclear.

Epidemiological investigation of a severe rumen fluke outbreak on an Irish dairy farm.

Although the rumen fluke, Calicophoron daubneyi is now very common and widespread throughout Western Europe, reports of clinical cases are still rare. This study explores the epidemiological background to a severe rumen fluke outbreak in 6-month-old heifers on a dairy farm in Ireland. Sequence analysis of the cytochrome oxidase subunit 1 (Cox1) gene of the rumen fluke metacercariae on pasture failed to identify predominant, possibly pathogenic subtypes. However, estimates of metacercarial load indicated that the animals were exposed to a daily dose of about 5334 C. daubneyi metacercariae for a period of 3 weeks resulting in the build-up of very large numbers of immature worms in the small intestine. It is hypothesized that specific environmental conditions may favour this parasite over its competitor, the liver fluke, Fasciola hepatica, possibly by allowing it to emerge earlier. The possibility that C. daubneyi may be better adapted to the Irish climate than F. hepatica together with the fact that selective treatment against F. hepatica effectively frees the niche for C. daubneyi, may result in the gradual replacement of F. hepatica by C. daubneyi.

Migration of Fasciola hepatica newly excysted juveniles is inhibited by high-mannose and oligomannose-type N-glycan-binding lectins.

Fasciola hepatica has both zoonotic importance and high economic impact in livestock worldwide. After ingestion by the definitive host, the Newly Excysted Juveniles (NEJ) penetrate the intestine before reaching the peritoneal cavity. The role of some NEJ-derived proteins in invasion has been documented, but the role of NEJ glycans or lectin-binding receptors during initial infection in the gut is still unknown. To address these questions, the migration of NEJ through rat intestine was recorded at 30 min intervals up to 150 min by two ex vivo methods. Firstly, jejunal sheets were challenged with NEJ incubated with biotinylated lectins. Secondly, untreated NEJ were incubated with distal jejunum pre-treated with lectins. Both Concanavalin A (ConA) and Galanthus nivalis (GNL), which recognize mannose-type N-glycans, significantly inhibited NEJ migration across the jejunum. Most of the lectins bound to the tegument and oral sucker of the NEJ, but only ConA and GNL maintained this interaction over 150 min. None of the lectins examined significantly reduced NEJ migration when pre-incubated with jejunal sheets, suggesting that host glycans might not be essential for initial binding/recognition of the gut by NEJ. Agents capable of blocking mannose-type N-glycans on the NEJ tegument may have potential for disrupting infection.

Development of a versatile in vitro method for understanding the migration of Fasciola hepatica newly excysted juveniles.

Fasciola hepatica is a parasitic trematode that causes serious losses to livestock producers, and also zoonotic disease. The limitations of chemotherapy for the control of fasciolosis have led to significant interest in the development of vaccines to protect cattle and sheep from infection. However, relatively few studies have concentrated on the mechanisms of invasion of the gut by newly excysted juvenile liver flukes (NEJ) and the host response triggered by this event. The aim of this work was to develop an in vitro model to study invasion by NEJ, while also reducing the requirement for challenge infections of experimental animals. Fasciola hepatica metacercariae were excysted in vitro and placed into compartments containing rat distal jejunal sheets. Variations in incubation medium, chamber size and incubation temperature were used to identify optimal conditions for NEJ migration across the gut. Histological examination showed increased migration until 120 min post-incubation. The use of RPMI, without gassing at 39 °C, as the incubation medium was found to be optimal, with 40·5% of NEJ migrating after 150 min. This study describes a readily-reproducible method for studying the migration of F. hepatica NEJ within the definitive host. It will be useful for identifying potential drug and vaccine targets.

Fasciola hepatica Infection in Cattle: Analyzing Responses of Peripheral Blood Mononuclear Cells (PBMC) Using a Transcriptomics Approach.

The parasitic helminth Fasciola hepatica (liver fluke) causes economic loss to the livestock industry globally and also causes zoonotic disease. New control strategies such as vaccines are urgently needed, due to the rise of drug resistance in parasite populations. Vaccine development requires a comprehensive understanding of the immunological events during infection. Previous in vivo studies by our group have investigated global differentially expressed genes (DEGs) in ovine peripheral blood mononuclear cells (PBMC) in response to both acute and chronic F. hepatica infection. This work demonstrated that pathways involved in the pathogenesis of ovine fasciolosis included fibrosis, inhibition of macrophage nitric oxide production, and antibody isotype switching, among others. Transcriptomic changes in PBMC populations following F. hepatica infection in cattle, in which the disease phenotype is quite different, have not yet been examined. Using RNA sequencing we investigated gene expression changes in PBMC isolated from 9 non-infected and 11 F. hepatica-experimentally-infected calves immediately before infection, at 1 and at 14 weeks post-infection. Longitudinal time-course comparisons between groups revealed 21 and 1,624 DEGs driven exclusively by F. hepatica infection in cattle at acute and chronic stages, respectively. These results show that fewer DEGs at the acute stage of infection can be identified in cattle, as compared with sheep. In addition, the log2 fold-changes of these DEGs were relatively low (-1 to 3) reflecting the different clinical presentation of F. hepatica infection in cattle. Gene pathways for hepatic fibrosis and hepatic cholestasis along with apoptosis of antigen-presenting cells were enriched at chronic stages. Our results reflect the major differences in the disease phenotype between cattle and sheep and may indicate pathways to target in vaccine development.

In silico analyses of protein glycosylating genes in the helminth Fasciola hepatica (liver fluke) predict protein-linked glycan simplicity and reveal temporally-dynamic expression profiles.

Glycoproteins secreted by helminth parasites are immunogenic and represent appealing components of vaccine preparations. Our poor knowledge of the pathways that mediate protein glycosylation in parasitic flatworms hinders our understanding of how proteins are synthesised and modified, and our ability to target these pathways for parasite control. Here we provide the first detailed description of genes associated with protein glycosylation in a parasitic flatworm, focusing on the genome of the liver fluke (Fasciola hepatica), which is a globally important trematode parasite of humans and their livestock. Using 190 human sequences as search queries against currently available F. hepatica genomes, we identified 149 orthologues with putative roles in sugar uptake or nucleotide sugar synthesis, and an array of glycosyltransferase and glycosidase activities required for protein N- and O-glycosylation. We found appreciable duplication within these orthologues, describing just 87 non-redundant genes when paralogues were excluded. F. hepatica lacks many of the enzymes required to produce complex N- and O-linked glycans, which explains the genomic basis for the structurally simple glycans described by F. hepatica glycomic datasets, and predicts pervasive structural simplicity in the wider glycome. These data provide a foundation for functional genomic interrogation of these pathways with the view towards novel parasite intervention strategies.

Tegument Glycoproteins and Cathepsins of Newly Excysted Juvenile Fasciola hepatica Carry Mannosidic and Paucimannosidic N-glycans.

Recently, the prevalence of Fasciola hepatica in some areas has increased considerably and the availability of a vaccine to protect livestock from infection would represent a major advance in tools available for controlling this disease. To date, most vaccine-target discovery research on this parasite has concentrated on proteomic and transcriptomic approaches whereas little work has been carried out on glycosylation. As the F. hepatica tegument (Teg) may contain glycans potentially relevant to vaccine development and the Newly Excysted Juvenile (NEJ) is the first lifecycle stage in contact with the definitive host, our work has focused on assessing the glycosylation of the NEJTeg and identifying the NEJTeg glycoprotein repertoire. After in vitro excystation, NEJ were fixed and NEJTeg was extracted. Matrix-assisted laser desorption ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) analysis of released N-glycans revealed that oligomannose and core-fucosylated truncated N-glycans were the most dominant glycan types. By lectin binding studies these glycans were identified mainly on the NEJ surface, together with the oral and ventral suckers. NEJTeg glycoproteins were affinity purified after targeted biotinylation of the glycans and identified using liquid chromatography and tandem mass spectrometry (LC-MS/MS). From the total set of proteins previously identified in NEJTeg, eighteen were also detected in the glycosylated fraction, including the F. hepatica Cathepsin B3 (FhCB3) and two of the Cathepsin L3 (FhCL3) proteins, among others. To confirm glycosylation of cathepsins, analysis at the glycopeptide level by LC-ESI-ion-trap-MS/MS with collision-induced dissociation (CID) and electron-transfer dissociation (ETD) was carried out. We established that cathepsin B1 (FhCB1) on position N80, and FhCL3 (BN1106_s10139B000014, scaffold10139) on position N153, carry unusual paucimannosidic Man2GlcNAc2 glycans. To our knowledge, this is the first description of F. hepatica NEJ glycosylation and the first report of N-glycosylation of F. hepatica cathepsins. The significance of these findings for immunological studies and vaccine development is discussed.

Bovine paramphistomes in Ireland.

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.