The effect of naturally acquired rumen fluke infection on animal health and production in dairy and beef cattle in the UK.

The incidence of paramphistomosis, caused by the rumen fluke, Calicophoron daubneyi, has greatly increased within Europe in the last 15-20 years. However, the production impacts of this disease are poorly understood. This study firstly aimed to investigate the prevalence of rumen fluke in England and Northern Ireland (NI) by conducting an abattoir survey of dairy and beef cattle which also allowed the impact of rumen fluke on carcass weight, conformation and fat classification to be assessed. Secondly, an experiment aimed to assess the impact of C. daubneyi infection on diarrhea score, production loss and welfare in dairy heifers, while also evaluating the impacts of treating infected heifers with oxyclozanide. Rumen fluke prevalence was greater in NI than in England, with 53.8% (95% CI 51.9 - 55.9%) of the NI cattle carcases sampled being infected compared to 16.3% (95% CI 15.8 - 16.8%) and 17.9% (95% CI 17.4 - 18.4%) detected at the two abattoirs in England. However, there was no significant difference (P > 0.05) in the cold carcass weight between infected and non-infected cattle. Similarly, carcass conformation and fat classification were unaffected (P > 0.05) by the presence of rumen fluke. In the second experiment, daily live weight gain (DLWG), diarrhea score and welfare score were also unaffected (P > 0.05) by rumen fluke infection and by oxyclozanide treatment against rumen fluke. The farms in this experiment were managed to a high standard and animals had no intercurrent disease. Therefore, these findings suggest that on well-managed farms, production losses (growth rates) should not be compromised as a result of sub-clinical rumen fluke infection.

The Effect of Frequency of Fresh Pasture Allocation on the Feeding Behaviour of High Production Dairy Cows.

For ruminants, grazing and ruminating activities are essential in nutrient capture and ultimately animal performance however these activities can demand significant time and energy. This study evaluated the effect of three different pasture allocation frequencies (PAF's; 12, 24 and 36 h) on the feeding behaviour of grazing dairy cows. Eighty-seven spring calving dairy cows were divided into three treatments. Animals were rotationally grazed with fixed paddock sizes of 0.14 ha, 0.28 ha and 0.42 ha paddocks for the 12 h, 24 h and 36 h treatments, respectively. Animals (14 per treatment) were fitted with behaviour halters that monitored feeding activity. Diurnal feeding patterns were evident for all animals irrespective of PAF, concentrating the majority of grazing during daytime (90%) and ruminating activity during night (73%). Treatment significantly affected feeding behavior patterns. Peak grazing activity coincided with fresh pasture allocation in the 12 h and 24 h treatments. In the 36 h treatment, grazing was more evenly distributed over each 24 h period with peak grazing activity witnessed daily between 17:00 and 19:00 regardless of fresh pasture allocation, suggesting lack of anticipation of fresh feed delivery. In the 12 h treatment primiparous animals exhibited greater grazing and ruminating activity relative to multiparous animals in the 12 h treatment highlighting the impact of competition for resources within each feed on lower dominance animals.

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

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

The Effect of Frequency of Fresh Pasture Allocation on Pasture Utilisation and the Performance of High Yielding Dairy Cows.

Pasture allocation frequency (PAF) can influence pasture availability and grazing behaviour, which subsequently may impact on animal performance. Limited research to-date has investigated grazing management methods to improve the performance of high production dairy cows whilst also achieving high grass utilisation rates. This study evaluated the effect of three different PAF's (12, 24 and 36 h) on pasture utilisation, the performance of high yielding dairy cows and the interaction with parity. The experiment included two 60-day periods, 90 spring calving dairy cows (27 primiparous animals) in period one and 87 (24 primiparous animals) in period two. The average pre-grazing sward height (11.4 cm) was similar for all treatments in both periods. In period one, pasture utilisation rate was significantly higher (8%) in the 36 h compared to the 12 h treatment. In period two, milk energy output was significantly greater for primiparous animals in the 36 h treatment relative to the other treatments.

The cellular and molecular origins of extracellular vesicles released by the helminth pathogen, Fasciola hepatica.

Parasitic helminths secrete extracellular vesicles (EVs) which have potent immunomodulatory effects. Whilst the cargo of EVs has been characterised for many species, we know little about the mechanisms that govern their biogenesis and release. Using antibodies raised against a panel of Fasciola hepatica EV (FhEV) marker proteins, we have identified multiple sites of EV production in the parasite. Discrete immunofluorescence patterns were observed within the gastrodermal cells and tegumental syncytium for different marker proteins whilst the protonephridial (excretory) system and parenchymal-type 2 cells were identified as additional sites of production (or transit) of FhEVs. Ligation was used to mechanically block the oral sucker, excretory pore, or both, to determine the effect on FhEV release from live adult flukes in vitro. This revealed that FhEVs are predominately derived from the gut, whilst the tegument releases EVs to a lesser extent. The data also suggest that the protonephridial system contributes to the small (120 K) EV sub-population. Sphingomyelinase (SMase) activity is a key driver of EV biogenesis in mammalian cells and we have previously identified SMases in FhEVs by mass spectrometry. SMase activity associated with isolated FhEVs was susceptible to the chemical inhibitor GW4869 and treatment of adult flukes with GW4869 led to a significant reduction in 120 K EV release in vitro, suggesting that a ceramide-dependent mechanism could drive 120 K EV formation. In contrast, the release of the larger 15 K EVs was only moderately impacted, indicating that they form independently of SMase activity. Ultrastructural observation of GW4869-treated F. hepatica tissue showed severe disruption to the parenchyma and vacuolation of the tegument, gastrodermal cells and epithelial lining of the excretory ducts. This work establishes that targeted disruption of EV biogenesis and release in helminths is possible, and provides proof-of-concept for future studies investigating EV secretion as a target for parasite control.

Surface molecules of extracellular vesicles secreted by the helminth pathogen Fasciola hepatica direct their internalisation by host cells.

Helminth parasites secrete extracellular vesicles (EVs) that can be internalised by host immune cells resulting in modulation of host immunity. While the molecular cargo of EVs have been characterised in many parasites, little is known about the surface-exposed molecules that participate in ligand-receptor interactions with the host cell surface to initiate vesicle docking and subsequent internalisation. Using a membrane-impermeable biotin reagent to capture proteins displayed on the outer membrane surface of two EV sub-populations (termed 15k and 120k EVs) released by adult F. hepatica, we describe 380 surface proteins including an array of virulence factors, membrane transport proteins and molecules involved in EV biogenesis/trafficking. Proteomics and immunohistochemical analysis show that the 120k EVs have an endosomal origin and may be released from the parasite via the protonephridial (excretory) system whilst the larger 15k EVs are released from the gastrodermal epithelial cells that line the fluke gut. A parallel lectin microarray strategy was used to profile the topology of major surface oligosaccharides of intact fluorogenically-labelled EVs as they would be displayed to the host. Lectin profiles corresponding to glycoconjugates exposed on the surface of the 15 K and 120K EV sub-populations are practically identical but are distinct from those of the parasite surface tegument, although all are predominated by high mannose sugars. We found that while the F. hepatica EVs were resistant to exo- and endo-glycosidases, the glyco-amidase PNGase F drastically remodelled the surface oligosaccharides and blocked the uptake of EVs by host macrophages. In contrast, pre-treatment with antibodies obtained from infected hosts, or purified antibodies raised against the extracellular domains of specific EV surface proteins (DM9-containing protein, CD63 receptor and myoferlin), significantly enhanced their cellular internalisation. This work highlights the diversity of EV biogenesis and trafficking pathways used by F. hepatica and sheds light on the molecular interaction between parasite EVs and host cells.

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

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

Optimized conditions for the in vitro excystment of Calicophoron daubneyi metacercariae.

Paramphistomosis, caused by Calicophoron 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 study C. daubneyi in vitro to improve our understanding of rumen fluke biology. Towards this, we aimed to identify a suitable protocol for in vitro excystment of C. daubneyi metacercariae. Six methods that have been used to excyst metacercariae from a number of trematode species were tested with C. daubneyi metacercariae. 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 of C. daubneyi NEJs in vitro is 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.

Paramphistomosis of Ruminants: An Emerging Parasitic Disease in Europe.

Whilst historically regarded as being of minor importance in European livestock, recent evidence suggests that the prevalence of paramphistomosis is greater than that of fasciolosis in parts of the UK. In order to address this emerging threat to ruminant farming systems, and associated risks for food security posed by rumen fluke infection, it is imperative that we develop a better understanding of the basic biology of this parasite and how it interacts with its ruminant host. In this Opinion article we review recent progress in tracking the spread of rumen fluke infection in Europe, and propose some research questions that should be addressed if we are to develop tools to diagnose and treat paramphistomosis more effectively in the future.

Comparison of the microbial population in rabbits and guinea pigs by next generation sequencing.

This study aimed to determine the microbial composition of faeces from two groups of caecotrophagic animals; rabbits and guinea pigs. In addition the study aimed to determine the community present in the different organs in the rabbit. DNA was extracted from seven of the organs in wild rabbits (n = 5) and from faecal samples from domesticated rabbits (n = 6) and guinea pigs (n = 6). Partial regions of the small ribosomal sub-unit were amplified by PCR and then the sequences present in each sample were determined by next generation sequencing. Differences were detected between samples from rabbit and guinea pig faeces, suggesting that there is not a microbial community common to caecotrophagic animals. Differences were also detected in the different regions of the rabbits' digestive tracts. As with previous work, many of the organisms detected were Firmicutes or unclassified species and there was a lack of Fibrobacteres, but for the first time we observed a high number of Bacteroidetes in rabbit samples. This work re-iterates high levels of Firmicutes and unclassified species are present in the rabbit gut, together with low number of Fibrobacteres. This suggests that in the rabbit gut, organisms other than the Fibrobacteres must be responsible for fibre digestion. However observation of high numbers of Bacteroidetes suggests that this phylum may indeed have a role to play in digestion in the rabbit gut.