Transcriptome and secretome analysis of intra-mammalian life-stages of the emerging helminth pathogen, Calicophoron daubneyi reveals adaptation to a unique host environment.

Publication history: Accepted - 20 October 2020; Published online - 20 October 2020.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.This work was supported by an Industrial Partnership Award (to M.W.R) from the Biotechnology and Biological Sciences Research Council (BB/N017757/1) with additional financial support from Agrisearch and AHDB Beef & Lamb. N.A.M.O. was supported by a postgraduate studentship from the Department for the Economy (DfE) Northern Ireland

Dissection-independent production of Plasmodium sporozoites from whole mosquitoes.

Progress towards a protective vaccine against malaria remains slow. To date, only limited protection has been routinely achieved following immunisation with either whole-parasite (sporozoite) or subunit-based vaccines. One major roadblock to vaccine progress, and to pre-erythrocytic parasite biology in general, is the continued reliance on manual salivary gland dissection for sporozoite isolation from infected mosquitoes. Here, we report development of a multi-step method, based on batch processing of homogenised whole mosquitoes, slurry, and density-gradient filtration, which combined with free-flow electrophoresis rapidly produces a pure, infective sporozoite inoculum. Human-infective Plasmodium falciparum and rodent-infective Plasmodium berghei sporozoites produced in this way are two- to threefold more infective than salivary gland dissection sporozoites in in vitro hepatocyte infection assays. In an in vivo rodent malaria model, the same P. berghei sporozoites confer sterile protection from mosquito-bite challenge when immunisation is delivered intravenously or 60-70% protection when delivered intramuscularly. By improving purity, infectivity, and immunogenicity, this method represents a key advancement in capacity to produce research-grade sporozoites, which should impact delivery of a whole-parasite based malaria vaccine at scale in the future

Prospects for enhancing malaria vaccine efficacy by combining pre-erythrocytic antigens

Malaria causes almost half a million deaths each year. Existing interventions will almost certainly not be enough to tackle this enormous public health problem on their own. An effective vaccine is urgently needed. The leading malaria vaccine, RTS,S, confers suboptimal protective efficacy, and in addition targets only Plasmodium falciparum and not the other major species of human malaria, P. vivax. This thesis investigates the potential of combining pre-erythrocytic malaria vaccines as a means of enhancing protective efficacy. A novel mathematical model was developed which expresses probability of protection as a function of vaccine-induced humoural and cellular responses. The model predicts that combining partially effective vaccines should result in more than additive improvements in protective efficacy. This was supported by an experiment combining Rv21, a P. vivax circumsporozoite virus-like particle, with viral vectored P. vivax TRAP, the two leading pre-erythrocytic malaria vaccine antigens; this combination raised protective efficacy from 50% and 0%, respectively, to 100% sterile protection. It was also found that antigenic interference, a reduction in anti-CSP titres when Rv21 and PvTRAP are combined, occurred only in the presence of Matrix M adjuvant, and not when using alum, AddaVax or no adjuvant. With a view to creating a single-component multi-antigen vaccine, which would be more cost-effective than a multi-component vaccine, experiments were carried out to establish the virus-like particle Q&beta; as a platform capable of eliciting protective immunity via the display of short peptides derived from the CSP repeat region of both P. vivax and P. falciparum. For the first time, a tetramer peptide derived from the CSP repeat region of P. vivax VK210, AGDR, was shown capable of eliciting protective immunity alone. Finally, five novel linear B-cell epitopes were discovered, one from P. falciparum CSP, three from P. vivax TRAP and one from TRSP, each capable of conferring partial protection on mice. These epitopes were identified using novel screening methods, using sera from whole-protein vaccinated mice or by exploiting conservation within invasion protein sequences. Two of the protective epitopes, (NANP)6 and (ADGN long) were combined and found to enhance protective efficacy as predicted by the mathematical model. Thus this thesis lays the groundwork for the development of a single-component multi-epitope malaria vaccine with enhanced protective efficacy.</p

RNA interference in adult Ascaris suum - an opportunity for the development of a functional genomics platform that supports organism-, tissue- and cell-based biology in a nematode parasite

AbstractThe sustainable control of animal parasitic nematodes requires the development of efficient functional genomics platforms to facilitate target validation and enhance anthelmintic discovery. Unfortunately, the utility of RNA interference (RNAi) for the validation of novel drug targets in nematode parasites remains problematic. Ascaris suum is an important veterinary parasite and a zoonotic pathogen. Here we show that adult A. suum is RNAi competent, and highlight the induction, spread and consistency of RNAi across multiple tissue types. This platform provides a new opportunity to undertake whole organism-, tissue- and cell-level gene function studies to enhance target validation processes for nematode parasites of veterinary/medical significance

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

Microcrystalline Tyrosine (MCT®): A Depot Adjuvant in Licensed Allergy Immunotherapy Offers New Opportunities in Malaria.

Microcrystalline Tyrosine (MCT®) is a widely used proprietary depot excipient in specific immunotherapy for allergy. In the current study we assessed the potential of MCT to serve as an adjuvant in the development of a vaccine against malaria. To this end, we formulated the circumsporozoite protein (CSP) of P. vivax in MCT and compared the induced immune responses to CSP formulated in PBS or Alum. Both MCT and Alum strongly increased immunogenicity of CSP compared to PBS in both C57BL/6 and BALB/c mice. Challenge studies in mice using a chimeric P. bergei expressing CSP of P. vivax demonstrated clinically improved symptoms of malaria with CSP formulated in both MCT and Alum; protection was, however, more pronounced if CSP was formulated in MCT. Hence, MCT may be an attractive biodegradable adjuvant useful for the development of novel prophylactic vaccines