Anticoccidial vaccine development: the importance of genetic diversity and delivery strategy

Lead Research Organisation: Royal Veterinary College
Department Name: Comparative Biomedical Sciences CBS

Abstract

Chickens are the most numerous livestock animals, with >50 billion reared annually. They are the livestock species most widely kept by the poorest people in the world, providing both dietary protein of animal origin and a currency for local marketing and trade. In many countries, including many parts of India, the role of women in backyard and traditional poultry production systems is crucial and diseases that compromise the health or survival of poultry impact on many of the poorest members of society. In India, coccidiosis is consistently ranked in the top three causes of bird mortality but vaccination is only rarely used in the organised sector and little control of any sort is applied in the unorganised sector. Coccidiosis is a devastating disease of poultry caused by protozoan parasites of the genus Eimeria, estimated to incur global costs in excess of £1.5 billion per annum and ranked in the top ten diseases of livestock based upon impact on the poor in South Asia. Cost-effective, multivalent recombinant vaccines are becoming a realistic prospect due to the identification of immunoprotective antigens and development of transfection technologies. However, genetic resistance to anticoccidial drugs can occur rapidly in the field and a similar fate could befall novel anticoccidial vaccines that rely on a small number of antigens if these are polymorphic and selectable. To predict the likely efficacy and longevity of such vaccines in the field it is important to know the prevalence of naturally-occurring genetic (antigenic) diversity, rate of multiple infections and frequency of genetic exchange between parasites. It is crucial also to have strong geographical networks in place, both for collecting parasites for laboratory studies and to ensure efficient dissemination, delivery and support of traditional and new diagnostics and therapeutics (drugs and vaccines) to the world's poorest rural economies. A small panel of immunoprotective parasite antigens has ben identified in two Eimeria species. It is proposed to identify genes coding for equivalent antigens in other economically important Eimeria species and to sequence these genes from a panel of field strains collected across India, and from strains collected from other parts of the world (available through an extensive global network of contacts at IAH). In total, ten genomic regions will be sequenced from each Eimeria field strain, seven of which are hypothesised to encode antigens under immune selection and three 'housekeeping' or 'neutral' loci. Using this information genetic diversity and Eimeria field population structure will be investigated, providing data for the construction of mathematical models to predict the occurrence and relevance of cross-fertilisation during natural infection. Quantitative PCR targeting genetically distinct sub-populations within each Eimeria field population will be used to test and develop the early models. The identification of effective vaccine antigens is just one step towards a successful vaccine. Using genetically distinct Eimeria strains, protective immunity induced by infection will be compared with immune responses stimulated through vaccination with one or more test antigens delivered as a recombinant protein, DNA vaccine, or using a transgenic Eimeria line as a vehicle. Homologous and heterologous challenge systems will be characterised. Data generated will help predict the likely responses of Eimeria field populations to the introduction of novel vaccines with a view towards ensuring sustainable efficacy against all Eimeria species. The development of new cheaper anticoccidial vaccines will have a huge impact on commercial poultry production and has the potential to be used as a tool by governments in developing countries to minimise the impact of infectious diseases on poultry. The provision of free vaccination to the poorest sectors of society can have a massive impact on the alleviation of poverty.

Technical Summary

Coccidiosis is a disease caused by the protozoan Eimeria species. Resistance has been detected among Eimeria field populations to every anticoccidial drug within one year of introduction. As novel vaccination strategies based upon small numbers of antigens become closer to reality it is clear that the Eimeria species could evolve under selection to evade vaccine-induced host immune responses. The scope for evasion will be determined by a series of variables including the (i) extent of naturally occurring genetic diversity, (ii) frequency of polyclonal infection in the field and (iii) rate of cross-fertilisation, none of which are known for the Eimeria species. In order to define these variables and inform the development of sustainable anticoccidial strategies with direct relevance to alleviating the cost of coccidiosis on the poor we will collect a panel of Eimeria field strains from nine states across India. These strains will be supplemented by Eimeria strains from around the world available through an extensive global network of contacts at IAH. Sequences orthologous to those that code for the most promising immunoprotective antigens discovered in lab studies at IAH will be identified from multiple Eimeria species. The prevalence of coding polymorphism will be determined from multiple Indian and international strains. Through expansion of the sequencing targets to a panel of ten genotyping loci per species the data generated will be used to characterise genetic diversity among Eimeria field populations and model the relevance of in vivo cross-fertilisation on population structure. Based on these data allele-specific qPCR will be developed to monitor field sample clonality and inform mathematical model construction. The in vivo immunogenicity of genotypically distinct strains will be determined and compared to single antigen vaccination strategies. Antigen delivery using transgenic Eimeria as a vehicle will be investigated.

Publications

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Blake DP (2015) Eimeria genomics: Where are we now and where are we going? in Veterinary parasitology

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Blake DP (2012) EmaxDB: Availability of a first draft genome sequence for the apicomplexan Eimeria maxima. in Molecular and biochemical parasitology

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Blake DP (2017) Recombinant anticoccidial vaccines - a cup half full? in Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases

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Blake DP (2015) Population, genetic, and antigenic diversity of the apicomplexan Eimeria tenella and their relevance to vaccine development. in Proceedings of the National Academy of Sciences of the United States of America

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Clark EL (2016) Cryptic Eimeria genotypes are common across the southern but not northern hemisphere. in International journal for parasitology

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Clark EL (2017) Are Eimeria Genetically Diverse, and Does It Matter? in Trends in parasitology

 
Description All key findings have been reported previously
Exploitation Route All have been previously reported
Sectors Agriculture, Food and Drink

 
Description Findings from the parasite prevalence survey have been used to increase awareness in vets and poultry producers in India about the relative risks of severe coccidiosis in different types of poultry production. Training has been disseminated to improve on-farm and in-vet diagnosis of disease and to inform vets and producers about optimisation of control. Findings have been published in a variety of journals - several local Indian journals as well as international journals, with a good number of local researchers having first-author papers. In 2016 we received £60K funding from grant BB/GCRF-IAA/19 to accelerate impact derived from this BBSRC CIDLID grant and a subsequent BBSRC/FADH grant BB/L00478X/1. Having developed strong links to poultry researchers and producers in India, we used the IAA funds to extend our network to Bangladesh, Pakistan and Sri Lanka. The goal was to articulate major barriers and limitations to efficient, secure and safe poultry production across the region. Specific objectives included 1) review of literature relating to poultry production systems, behaviours and practices, economic, social and public health impacts in each of the 4 countries 2) engage and discover the opinions of key stakeholders through focus groups, questionnaires and semi-structured interviews 3) organise a pilot workshop to explore key strengths/gaps in epidemiology, biosecurity, agrihealth economics, One Health, social science research etc. and begin to identify the major barriers to safe/secure poultry production 4) gauge appetite and applicability of distance learning training modules run in the UK and in India 5) engage students and early career researchers to participate and help organise activities. Collectively, the literature reviews, focus groups, questionnaires and workshop provided considerable evidence that there is an acute need for co-ordinated research and development activity to help meet the extremely rapid growth in demand for poultry products in the region in a safe/secure manner. Many limitations and serious concerns about the current state of poultry production were identified by participants (who included senior researchers from academia and government labs as well as senior managers of poultry production companies). A major outcome was the identification of short-medium- and long-term research and development objectives that could address specific barriers to safe/secure intensification of production. In 2017 we received a further £40K from grant BB/GCRF-IAA/17/19 which was used to follow up on one of the identified objectives: the need to develop stronger capacity and capability in poultry disease diagnostics and in biosecurity training. This was achieved through the running of bespoke training workshops, as was reported to BBSRC in detail at the end of the grant period. The diagnostics training was essentially a laboratory-based South-South exercise, with expert virologists at UVAS, Lahore and CVASU, Chittagong running in depth virology diagnostics training for 12 students (all veterinarians working for regional diagnostics centres in Sri Lanka, Bangladesh and Pakistan). The International training Course on 'Developing Biosecurity for the Poultry Sector' was prepared by RVC educators in collaboration with University of Peradenya, Sri Lanka. The pilot course ran in Feb 2018 with 22 students completing an online course, following by one week of residential field training in Sri Lanka. Both activities were hugely successful and we have been impressed by the willingness of colleagues across all countries to engage with this training initiative, even in the face of some difficult adminstrative issues. Much of the knowledge and networking that we gained through the IAA and IAA-extension awards was subsequently utilised in the RVC-led bid for a GCRF Interdisciplinary Hub on Poultry and One Health, a proposal that we are delighted has now been funded.
First Year Of Impact 2010
Sector Agriculture, Food and Drink
Impact Types Economic

 
Description Farmed Animal Diseases and Health (FADH)
Amount £632,704 (GBP)
Funding ID BB/L00478X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 06/2014 
End 11/2017
 
Description GCRF One Health Poultry Hub
Amount £18,152,563 (GBP)
Funding ID BB/S011269/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 02/2019 
End 05/2024
 
Description GCRF-IAA
Amount £60,000 (GBP)
Funding ID BB/GCRF-IAA/19 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2016 
End 03/2017
 
Description GCRF-IAA extension
Amount £40,000 (GBP)
Funding ID BB/GCRF-IAA/17/19 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2017 
End 03/2018
 
Description Response mode highlight 'Vaccine'
Amount £400,104 (GBP)
Funding ID BB/P003931/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 12/2016 
End 12/2019
 
Description iCASE PhD studentship
Amount £100,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2014 
End 09/2018
 
Title Disease prevalence and epidemiology 
Description Design and development of field sampling protocols and processes by which samples can be submitted by poultry owners/workers for identification of coccidial parasites in a flock. Samples are accompanied by a detailed questionnaire designed by the TANUVAS/RVC team; the paper-based questionnaire was transferred to the EpiCollect epidemiology web application (via RVC eMedia and Dr David Aanensen, Imperial College London). 
Type Of Material Biological samples 
Year Produced 2011 
Provided To Others? Yes  
Impact Questionnaires are shared between Indian labs in this CIDLID project, and have been used also by collaborators in sub-saharan Africa, Nigeria and Uganda. Tools developed in this project are now available for paralletl projects within TANUVAS and IVRI parasitology departments. 
 
Title Laboratory protocols for Eimeria 
Description Protocols specific for the passage, purification and manipulation of Eimeria parasites were introduced to TANUVAS, Chennai and IVRI, Bareilly. Development and validation of methods for this project at TANUVAS, IVRI and RVC yielded a comprehensive protocol describing sample collection, preparation and diagnostic analysis for use with EImeria (for the first time). Protocols specific to the development of attenuated vaccines using local parasite strains and to culture of E. tenella in embryonic chicks and cell culture were provided to TANUVAS in support of parallel projects. 
Type Of Material Biological samples 
Year Produced 2011 
Provided To Others? Yes  
Impact Support and expansion of parasitology research at TANUVAS and IVRI; provision of robust sampling methods for parasite surveillance activities; publication of several papers in local and international journals 
 
Title Mathematical models 
Description A model based on Hamiltonian paths to described the mechanism of chicken movement in different production systems 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Provided To Others? No  
Impact Likely to contribute to understanding the general flow of faecal-oral pathogens within hen houses and potentially also the efficacy of vaccination using live vaccines that depend on faecal-oral routes of transmission 
 
Title Quality Control 
Description Protocols to provide quality control of all samples collected under the project from point of collection to data analysis and publication. All protocols were shared between labs and a proportion of samples were cross-checked (molecular diagnostics) for quality assurance 
Type Of Material Improvements to research infrastructure 
Year Produced 2012 
Provided To Others? Yes  
Impact Application of GRP across all partner labs 
 
Title Transfer of diagnostics technologies to partners in India 
Description Protocols for single and multiplex PCRs (species-specific identification of Eimeria species), for qPCR using SYBR green and TaqMan probe-based chemistry (genus and species-specific identification) and for multi-locus sequence tagged characterisation (Eimeria genotyping) were all introduced and developed within partner labs in Inida. These are fully embedded in these labs and are being offered as a biotechnological service for poultry veterinarians and pharmaceutical companies across India 
Type Of Material Technology assay or reagent 
Year Produced 2012 
Provided To Others? Yes  
Impact Improved diagnostics tools for poultry producers in India 
URL http://www.coccialertnetwork.org
 
Description New India partnerships 
Organisation Institute of Bioinformatics and Applied Biotechnology (IBAB)
Country India 
Sector Academic/University 
PI Contribution One of the team (Dr Srinivas Rao) initiated a new collaboration on mathematical modelling of Eimeria with Prof Yathindra of IBAB Bangalore. A gorup of masters-qualified students received dedicated training in modelling through engagement with project work designed by Dr Rao.
Collaborator Contribution see above
Impact training of students
Start Year 2012
 
Description New India partnerships 2 
Organisation Anand Agricultural University
Department Department of Animal Biotechnology
Country India 
Sector Academic/University 
PI Contribution Through our work with the TANUVAS team as part of the CIDLID grant, we developed ideas for continuing our work on gut infections in poultry; specifically to investigate the influence of host and parasite genetic diversity on the holistic development of gut microbiomes. Prof Joshi is a leading Indian expert on microbiome sequencing (and a TANUVAS alumnus) and after discussion he agreed to become part of our growing UK-Indian network on poultry gut health.
Collaborator Contribution Prof Raman from TANUVAS helped to facilitate the new partnership with Prof Joshi and colleagues at Anand
Impact One paper (on a topic not directly related to the grant) has been published between RVC and Anand. A large body of data has been generated by Prof Joshi and his group at Anand and the disciplines covered include next generation sequencing, bioinformatics, avian genetics, bacteriology and parasitology.
Start Year 2014
 
Description Nigeria partnership 1 
Organisation Federal University of Agriculture, Abeokuta
Country Nigeria 
Sector Academic/University 
PI Contribution One MSc student trained in field sampling and parasite preservation for coccidiosis screening by microscopy
Collaborator Contribution none
Impact none so far
Start Year 2012
 
Description Nigeria partnership 2 
Organisation Ahmadu Bello University
Country Nigeria 
Sector Academic/University 
PI Contribution One PhD student visited RVC for one month and was trained in field sample, parasite preservation, Eimeria manipulation, molecular diagnostics and phylogenetics. SInce then there has been collaboration on the identification of novel 'cryptic' genotypes of parasites isolated from Nigerian poultry
Collaborator Contribution none
Impact Collaboration contributed to following publications: PMID: 27809980 PMID: 27368611 PMID: 27259544 PMID: 26354122
Start Year 2012
 
Description Tanzania partnership 
Organisation Sokoine University of Agriculture
Country Tanzania, United Republic of 
Sector Academic/University 
PI Contribution One technician trained in field sampling and parasite preservation for coccidiosis screening by microscopy One RVC summer student trained
Collaborator Contribution none
Impact One manuscript published which describes the burden of coccidiosis on small scale and backyard chicken flocks of up to 2000 birds. Data from the project were also submitted to the government veterinary surgeon, Kibaha, Tanzania for dissemination.
Start Year 2011
 
Description Zambia partnerships 
Organisation University of Zambia
Department School of Veterinary Medicine, University of Zambia
Country Zambia 
Sector Academic/University 
PI Contribution Two technicians trained in flied sampling for coccidiosis screening by microscopy One RVC masters student trained
Collaborator Contribution none
Impact One paper describing the burden imposed by coccidiosis on small scale and backyard chicken flocks of up to 2000 birds.
Start Year 2011
 
Description 16th International Scientific Conference (ISCon XVI), Chattogram Veterinary and Animal Sciences University (CVASU) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Invited presentation at an international conference focused One Health. Included a mixed audience of academics, students, government, industry and practioners.
Year(s) Of Engagement Activity 2019
 
Description Emerging Eimeria species and chicken genetics in Africa, University of Kwazulu-Natal 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact ~75 students and staff attended an invited plenary presentation at the University of Kwazulu-Natal, Durban, South Africa. Discussed project outcomes and follow up regarding student projects and possible project supervision. Dissemination of results to a wider community.
Year(s) Of Engagement Activity 2019
 
Description Husbandry pamphlets 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Six pamphlets focusing on veterinary and general husbandry, poultry production and pathogen control, diagnosis and control of coccidiosis, were produced during the course of this project. All pamphlets were translated into 11 Indian languages or dialects (including Tamil, English, Hindi, Malayalam, Kannada, Telugu and Marathi). These were distributed via TANUVAS and IVRI outreach workers to poultry producers and veterinarians across South and North India

A major target for this activity was the backyard sector of poultry production, in particular the predominantly female population of small chicken producers. Uptake particularly via the TANUVAS outreach department was high in this sector, where small changes in practice can lead to highly signficant improvements in terms of economics (improved weight gain of chickens).
Year(s) Of Engagement Activity 2012,2013,2014
 
Description Infomedia - requests for research information 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact A collection of activities throughout the programme aimed at increasing awareness in India of parasitic infections of poultry. Dr M Raman (TANUVAS) gave a radio talk on All India Radio about the emergence of parasitic infections due to climatic and meterological changes; Dr P. Banerjee (IVRI) gave two radio interviews specifically about the project; Dr D. Blake gave one TV interview on a closely related topic (impact of endemic parasite infection). In the media, there was a featured article in the Hindi magazine 'Kheti' on coccidiosis control in chickens.

not known
Year(s) Of Engagement Activity 2011,2012,2013,2014
 
Description Practioner workshop, University of Kwazulu-Natal 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact ~30 industry representatives and veterinary practioners attended a workshop at the University of Kwazulu-Natal, Durban, South Africa. Discussed project outcomes and follow application to the field. Dissemination of results to a wider community, capacity/capability building.
Year(s) Of Engagement Activity 2019
 
Description Student workshops and training, University of Kwazulu-Natal 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact ~20 students (undergraduate and postgraduate) attended a series of taught sessions and practical workshops focused on Eimeria, parasite, microbiome and chicken genetics at the University of Kwazulu-Natal, Durban, South Africa. Dissemination of results to a wider community, capacity/capability building.
Year(s) Of Engagement Activity 2019
 
Description World Veterinary Poultry Association CPD day 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact ~150 delegates attended a CPD training day organised by the World Veterinary Poultry Association, Malaysia branch. The day featured a series of talks from academics, industry representatives and veterinarians. Two talks decribing BBSRC funded studies were presented, focusing on (i) occurrence and genetic diversity of Eimeria, and (ii) the poultry enteric microbiome and links to chicken genetics.
Year(s) Of Engagement Activity 2020