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

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 been 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.

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.

Economic impact Eimeria species cause coccidiosis in all livestock, most notably poultry, where estimated costs in excess of £1.5 billion are incurred worldwide every year. Current methods of control rely on chemotherapy or vaccination with live parasites but increasing drug resistance, restrictive legislation or prohibitive costs limit the potential of both. New control strategies are urgently required. Immunoprotective antigens with potential to be used in novel vaccination strategies have been identified at IAH. The successful development of these antigens into effective vaccines will progress through the work described here. Our ability to maintain the efficacy of these vaccines in the long term will also be informed. Access by poultry producers to information describing coccidia-associated risks in relation to the environment and choice of control strategy will enhance efficiency, minimise losses and help to alleviate the impact of coccidia on poverty. Impact on the poor Chickens are the most numerous livestock animals (>50 billion reared annually). They provide staple ingredients in the diets of many of the world's people and are also increasingly recognised as the most important species kept by the landless poor and many smallholder famers. Among the poor, poultry are the most important source of dietary protein of animal origin as well as a currency for local marketing and trade. The role of women in backyard poultry production is crucial. The development of cheap and effective anticoccidial vaccines 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-at-point-of-use vaccination to the poorest sectors of society would have a massive impact on the alleviation of poverty. Food security The global human population is predicted to increase by a third in the next 25 years. Countries of the developing world will host 97% of this expansion. The provision of food is becoming ever more topical and poultry are the most efficient way of producing dietary animal protein. Control of pathogens such as the Eimeria species that undermine effective poultry production has never been so important. Animal welfare As well as their economic impact, Eimeria parasites induce serious disease that can compromise the welfare of infected chickens. The effective reduction of disease as a result of sustainable vaccination supports the Five Freedoms implicit to animal welfare as set out by the Farm Animal Welfare Council. General public and the environment Concerns about farm animal welfare, the use of chemicals in food production and the environment continue to gain importance with a large proportion of the general public. Studies promoting development of sustainable alternatives will improve the image of the UK poultry industry and poultry derived products. Reducing the cost of coccidiosis and its prevention will reduce the cost of poultry products to the consumer and lessen the impact of Eimeria on the poor who consume poultry products in the developed and developing worlds. Strategic relevance This project addresses the BBSRC research priority 'Animal health' making use of the relevant target species in the specialist facilities available at the IAH. The tools and data developed will underpin future biological studies with the Eimeria species and support a 'systems approach to biological research' focused on Eimeria. Studies supporting efficient production and disease control in poultry are directly relevant to BBSRC policy priorities 'economic and social impact' and 'welfare of managed animals' and DFID's commitment to sustainable agriculture. In line with DFID policy these studies will play a key role in assessing the relevance of genetic polymorphism and cross-fertilisation to future anticoccidial control, informing creation of a solution to coccidiosis and assisting effective delivery.