Damer P. Blake

Securing poultry production from the ever-present Eimeria challenge

The intestinal disease coccidiosis, caused by protozoan parasites of the genus Eimeria, is one of the most important livestock diseases in the world. It has a high impact in the poultry industry where parasite transmission is favoured by high-density housing of large numbers of susceptible birds. Coccidiosis control in poultry is achieved by careful husbandry combined with in-feed anticoccidial drugs or vaccination with live parasites. However, outbreaks of coccidiosis still occur and subclinical infections, which significantly impact on productivity and food security, are common due to widespread drug resistance, high parasite prevalence, and environmental persistence. Herein, we review some recent approaches for the production of cheaper third generation vaccines, based on robust methods for identification of immunoprotective antigens and the use of transgenic Eimeria.

Transfer of antibiotic resistance between commensal and pathogenic members of the Enterobacteriaceae under ileal conditions

Aim: To determine the rate of antibiotic resistance transmission between commensal and pathogenic representatives of the Enterobacteriaceae.

Influence of tetracycline exposure on tetracycline resistance and the carriage of tetracycline resistance genes within commensal Escherichia coli populations

Aims: To assess the influence of incremental tetracycline exposure on the genetic basis of tetracycline resistance within faecal Escherichia coli.

A Selective Review of Advances in Coccidiosis Research

Coccidiosis is a widespread and economically significant disease of livestock caused by protozoan parasites of the genus Eimeria. This disease is worldwide in occurrence and costs the animal agricultural industry many millions of dollars to control. In recent years, the modern tools of molecular biology, biochemistry, cell biology and immunology have been used to expand greatly our knowledge of these parasites and the disease they cause. Such studies are essential if we are to develop new means for the control of coccidiosis. In this chapter, selective aspects of the biology of these organisms, with emphasis on recent research in poultry, are reviewed. Topics considered include taxonomy, systematics, genetics, genomics, transcriptomics, proteomics, transfection, oocyst biogenesis, host cell invasion, immunobiology, diagnostics and control.

Genomic analysis of the causative agents of coccidiosis in domestic chickens

Global production of chickens has trebled in the past two decades and they are now the most important source of dietary animal protein worldwide. Chickens are subject to many infectious diseases that reduce their performance and productivity. Coccidiosis, caused by apicomplexan protozoa of the genus Eimeria, is one of the most important poultry diseases. Understanding the biology of Eimeria parasites underpins development of new drugs and vaccines needed to improve global food security. We have produced annotated genome sequences of all seven species of Eimeria that infect domestic chickens, which reveal the full extent of previously described repeat-rich and repeat-poor regions and show that these parasites possess the most repeat-rich proteomes ever described. Furthermore, while no other apicomplexan has been found to possess retrotransposons, Eimeria is home to a family of chromoviruses. Analysis of Eimeria genes involved in basic biology and host-parasite interaction highlights adaptations to a relatively simple developmental life cycle and a complex array of co-expressed surface proteins involved in host cell binding.

White-Nose Syndrome fungus introduced from Europe to North America

The investigation of factors underlying the emergence of fungal diseases in wildlife has gained significance as a consequence of drastic declines in amphibians, where the fungus Batrachochytrium dendrobatidis has caused the greatest disease-driven loss of biodiversity ever documented [1]. Identification of the causative agent and its origin (native versus introduced) is a crucial step in understanding and controlling a disease [2]. Whereas genetic studies on the origin of B. dendrobatidis have illuminated the mechanisms behind the global emergence of amphibian chytridiomycosis [3], the origin of another recently-emerged fungal disease, White-Nose Syndrome (WNS) and its causative agent, Pseudogymnoascus destructans, remains unresolved [2,4]. WNS is decimating multiple North American bat species with an estimated death toll reaching 5-6 million. Here, we present the first informative molecular comparison between isolates from North America and Europe and provide strong evidence for the long-term presence of the fungus in Europe and a recent introduction into North America. Our results further demonstrate great genetic similarity between the North American and some European fungal populations, indicating the likely source population for this introduction from Europe.

Genetic Mapping Identifies Novel Highly Protective Antigens for an Apicomplexan Parasite

Apicomplexan parasites are responsible for a myriad of diseases in humans and livestock; yet despite intensive effort, development of effective sub-unit vaccines remains a long-term goal. Antigenic complexity and our inability to identify protective antigens from the pool that induce response are serious challenges in the development of new vaccines. Using a combination of parasite genetics and selective barriers with population-based genetic fingerprinting, we have identified that immunity against the most important apicomplexan parasite of livestock (Eimeria spp.) was targeted against a few discrete regions of the genome. Herein we report the identification of six genomic regions and, within two of those loci, the identification of true protective antigens that confer immunity as sub-unit vaccines. The first of these is an Eimeria maxima homologue of apical membrane antigen-1 (AMA-1) and the second is a previously uncharacterised gene that we have termed ‘immune mapped protein-1’ (IMP-1). Significantly, homologues of the AMA-1 antigen are protective with a range of apicomplexan parasites including Plasmodium spp., which suggest that there may be some characteristic(s) of protective antigens shared across this diverse group of parasites. Interestingly, homologues of the IMP-1 antigen, which is protective against E. maxima infection, can be identified in Toxoplasma gondii and Neospora caninum. Overall, this study documents the discovery of novel protective antigens using a population-based genetic mapping approach allied with a protection-based screen of candidate genes. The identification of AMA-1 and IMP-1 represents a substantial step towards development of an effective anti-eimerian sub-unit vaccine and raises the possibility of identification of novel antigens for other apicomplexan parasites. Moreover, validation of the parasite genetics approach to identify effective antigens supports its adoption in other parasite systems where legitimate protective antigen identification is difficult.

Eimeria species parasites as novel vaccine delivery vectors: Anti-Campylobacter jejuni protective immunity induced by Eimeria tenella-delivered CjaA

Vaccination of poultry against coccidiosis caused by the Eimeria species is almost entirely based upon varied formulations of live parasites. The recent development of a series of protocols that support genetic complementation by transfection in Eimeria now provides an opportunity to utilise live anticoccidial vaccines to deliver additional vaccinal antigens. The capacity of Eimeria tenella to express an exogenous antigen and induce an immune response during in vivo infection which is protective against subsequent bacterial challenge has been tested here using the anti-Campylobacter jejuni vaccine candidate CjaA. Using restriction enzyme mediated integration (REMI) a transgenic E. tenella population expressing CjaA and the fluorescent reporter mCitrine has been developed. Vaccination of specific pathogen free chickens by single or multiple oral inoculation of E. tenella-CjaA oocysts induced 91% and 86% immune protection against C. jejuni challenge compared with unvaccinated and wild-type E. tenella vaccinated controls (p<0.001). Increasing vaccination number had no significant influence on the magnitude of protection. These results support the hypothesis that eimerian parasites can be developed as multivalent vaccine vectors and encourage the extension of these studies.

Population, genetic, and antigenic diversity of the apicomplexan Eimeria tenella and their relevance to vaccine development

Significance Sixty billion chickens are produced worldwide each year, and all are at risk from Eimeria, parasites that cause coccidiosis. Control relies widely on chemoprophylaxis, but pressure to reduce drug use in farming urges development of cost-effective vaccines. Antigens such as apical membrane antigen 1 (AMA1) offer promise as anticoccidial vaccine candidates, but experience with related apicomplexans such as Plasmodium, in which pre-existing antigenic diversity and incompatible population structure have undermined vaccine development, tempers confidence. Parasite genotyping identified enormous region-specific variation in haplotype diversity for Eimeria tenella but a contrastingly low level of polymorphism for EtAMA1. Although high levels of polyclonal Eimeria infection and hybridization indicate an ability to disseminate vaccine resistance rapidly, the low level of EtAMA1 diversity promotes vaccine development. The phylum Apicomplexa includes serious pathogens of humans and animals. Understanding the distribution and population structure of these protozoan parasites is of fundamental importance to explain disease epidemiology and develop sustainable controls. Predicting the likely efficacy and longevity of subunit vaccines in field populations relies on knowledge of relevant preexisting antigenic diversity, population structure, the likelihood of coinfection by genetically distinct strains, and the efficiency of cross-fertilization. All four of these factors have been investigated for Plasmodium species parasites, revealing both clonal and panmictic population structures with exceptional polymorphism associated with immunoprotective antigens such as apical membrane antigen 1 (AMA1). For the coccidian Toxoplasma gondii only genomic diversity and population structure have been defined in depth so far; for the closely related Eimeria species, all four variables are currently unknown. Using Eimeria tenella, a major cause of the enteric disease coccidiosis, which exerts a profound effect on chicken productivity and welfare, we determined population structure, genotype distribution, and likelihood of cross-fertilization during coinfection and also investigated the extent of naturally occurring antigenic diversity for the E. tenella AMA1 homolog. Using genome-wide Sequenom SNP-based haplotyping, targeted sequencing, and single-cell genotyping, we show that in this coccidian the functionality of EtAMA1 appears to outweigh immune evasion. This result is in direct contrast to the situation in Plasmodium and most likely is underpinned by the biology of the direct and acute coccidian life cycle in the definitive host.

The role of sialyl glycan recognition in host tissue tropism of the avian parasite Eimeria tenella.

Eimeria spp. are a highly successful group of intracellular protozoan parasites that develop within intestinal epithelial cells of poultry, causing coccidiosis. As a result of resistance against anticoccidial drugs and the expense of manufacturing live vaccines, it is necessary to understand the relationship between Eimeria and its host more deeply, with a view to developing recombinant vaccines. Eimeria possesses a family of microneme lectins (MICs) that contain microneme adhesive repeat regions (MARR). We show that the major MARR protein from Eimeria tenella, EtMIC3, is deployed at the parasite-host interface during the early stages of invasion. EtMIC3 consists of seven tandem MAR1-type domains, which possess a high specificity for sialylated glycans as shown by cell-based assays and carbohydrate microarray analyses. The restricted tissue staining pattern observed for EtMIC3 in the chicken caecal epithelium indicates that EtMIC3 contributes to guiding the parasite to the site of invasion in the chicken gut. The microarray analyses also reveal a lack of recognition of glycan sequences terminating in the N-glycolyl form of sialic acid by EtMIC3. Thus the parasite is well adapted to the avian host which lacks N-glycolyl neuraminic acid. We provide new structural insight into the MAR1 family of domains and reveal the atomic resolution basis for the sialic acid-based carbohydrate recognition. Finally, a preliminary chicken immunization trial provides evidence that recombinant EtMIC3 protein and EtMIC3 DNA are effective vaccine candidates.