Development of a vaccine to control poultry red mite

Infestation of hen houses with the poultry red mite (PRM) costs the poultry industry an estimated 130 million euros per annum in the EU (approximately 0.43 euros per hen). The mites live, off-host, in inaccessible areas of the cages during daylight and emerge during darkness to feed on the hens, biting through the skin to feed on blood. Infestation of production facilities with this parasite therefore has important animal welfare implications including anaemia, increased irritation and restlessness, feather-pecking and an increased incidence of cannibalism. These behaviours have a considerable negative impact on productivity. Poultry red mites have also been implicated as carriers for a number of important avian diseases and have recently been recommended to be listed as an occupational hazard for poultry workers on the basis of their allergenicity.
Until recently, PRM infestation has been controlled with the use of pesticide sprays in hen houses during the period that the houses are not populated (i.e. before the introduction of new birds at the start of a laying cycle). Controlling mite populations is now a major problem, with most pesticides affording only limited or short-lived reduction in the population of mites. Also, because of concerns over safety and environmental contamination, many of the currently effective pesticides have been withdrawn from use and the emergence of resistance to the remaining compounds has exacerbated these problems with mite control.

Vaccination offers a safe, effective alternative to chemical treatments. It is now recognised that vaccines to blood-feeding parasites can result in effective and sustainable control and our previous work has clearly established that vaccination against PRM is feasible and deliverable. A commercially viable vaccine against PRM should contain one highly effective synthetic version of a mite protein ("antigen") or, less ideally, a cocktail incorporating a small number of such proteins. Previous studies at Moredun have shown that vaccination of laying hens with extracts of PRMs can induce mortality in mites feeding on the blood of vaccinated hens and control mite numbers. These studies have shown that: i) Antibodies to mite antigens in the blood can cause significant mite mortality, and this effect (mortality) occurs within 24 hours, and ii) a similar effect can be achieved with recombinant proteins which are easier to produce consistently and hence lend themselves to large scale production. The overall objectives of the proposed work are therefore: 1) To identify protective immunogenic sub-fractions of the PRM extract; 2) To identify the immunogenic antigen(s) in these sub-fractions by combined immunoblotting and proteomic analyses; 3) To produce a sub-set of these immunogenic proteins as recombinant proteins and raise antibodies against these recombinants in hens; 4) To test these antibodies for mite mortality and/or developmental effects in our lab-based feeding system; 5) To select the most potent recombinant antigens from this screen to test in combination, in a field vaccine trial.

If successful, this project will deliver the first recombinant vaccine to be developed against an ectoparasite with a worldwide distribution and, therefore, market. We will use innovative technologies, coupling Moredun's unique PRM genomic data and specialist protein analysis facilities to identify the protective antigens, produce them in recombinant systems and deliver them to the birds in the context of the most effective vaccinology that our industry partner can supply.

Our previous work has clearly established that vaccination against the poultry red mite, Dermanyssus gallinae, using both native and recombinant antigens, is feasible. Thus far, candidate antigens have been identified through either a "pragmatic" approach of fractionating extracts of the mites and using these as vaccines or a "rational" approach of selecting suitable antigens based on assumed orthology with protective antigens from other species of parasite. We will now interconnect the pragmatic and rational approaches using a suite of tools uniquely available to us.

In this project we propose to take a complex D. gallinae extract, which we have previously used to induce a protective antibody response in hens, and subfractionate that extract using anion exchange and size exclusion chromatography. These subfractions will be used to generate antibodies in hens which will then be fed in vitro, in a bloodmeal, to poultry red mites. Mite mortality following these feeds will be indicative of protective antigens in the subfraction used in immunisation. Two dimensional gel electrophoresis of the subfractions, accompanied by Western blotting and immunoprecipitation with antibodies from the immunised birds, will allow us to locate the immunoreactive proteins in the extracts and permit their identification by MALDI-ToF. Recently we performed considerable transcriptomic characterisation of D. gallinae using "next generation" sequencing, digital transcriptomics and subtractive libraries to identify genes upregulated by critical parasitic activities and we will interrogate this database for protein identification. Recombinant proteins will be produced for candidate antigens identified in this way and will be used to immunise hens. Antibodies generated by these hens will be tested for efficacy in the in vitro feeding systems singly and in combinations. Finally, candidate targets showing most promise will be taken forward to an in vivo field scale vaccine trial.

By contributing to development of a vaccine against an economically-important parasite we will aid control of a mite that has developed widespread acaricide resistance. Therefore, our work will have a significant impact on food security and on the rural and national economy by improving hen productivity and welfare. In the UK, the 6th largest egg producer in the EU, there were 36 million laying hens in 2010 with retail egg sales of £868M. In the UK, 87.5 % of poultry units have reported infestation with poultry red mite (PRM). Recent data suggests that PRM costs 37p per hen in control and production losses, equating to annual losses of > £13M in the UK. In addition, PRM is a major pest of egg production worldwide. Global egg production has tripled since 1970. The worldwide opportunities for a vaccine to control PRM are therefore substantial and growing. A successful vaccine against PRM may also open opportunities for vaccination against the Northern fowl mite, a major economic concern in North America.
Reducing acaricide use through vaccination will impact on operator safety and the presence of residues in foodstuffs. These factors combined will have the effect of improving competitiveness of the UK food industry in the global marketplace, contributing to greater food security and will inform future government policy on disease control. The knowledge gained would be applicable to other parasitic mites which have also developed acaricide resistance, by informing future design of vaccines to control these parasites. Finally, UK and international industry will benefit by the exploitation of IP arising from the project (i.e. vaccines, novel therapeutics). In terms of training skills, the PDRA will receive intensive and appropriate training. All applicants and the PDRA will be involved in impact activities (see below).

Short-term impact: Where appropriate, results will be presented in high-impact academic journals and at international scientific meetings. Moredun (MRI), has well established systems for knowledge exchange (KE), with a Communications team which facilitates and coordinates KE activities. Moredun's successful system of KE is exemplified by our relationship with farmer members of the Moredun Foundation, enabling the results of projects to be rapidly communicated to end users in an appropriate format. Moredun also has close working relationships with egg industry bodies such as the BEMB and the BEIC. These mechanisms will allow us to communicate the results of this project rapidly to veterinarians and the poultry industry in an appropriate format.
MRI, by the nature of its funding, has close links with Scottish Government: e.g. specific Policy Days are held annually and these are attended by politicians, civil servants and stakeholders. MRI provides a broad range of training for staff in scientific and generic skills which the member of staff will attend.

Medium/long-term impact: The major exploitable output from this project is a prototype recombinant subunit vaccine to control PRM which is likely to lead to a marketable vaccine product (and therefore economic impact), through the technical and administrative capabilites of the industrial partner, Pfizer, within 5-10 years of initial testing. Pfizer are expert in leading the route to market for such products. Once results have been published and/or IP secured, the next phase will be to perform validatory protection trials using the most appropriate method of vaccine delivery (formulation with existing vaccines in the avian production systems for example).

Exploitation: Pfizer Animal Health focuses on medicines and vaccines for livestock and companion animals and its premier position in the UK market involves discovery, development and marketing of a wide range of medicines including vaccines. Globally, Pfizer supports every major domesticated terrestrial animal species in >140 countries and is therefore in prime position to exploit the outcomes of the work