Strategies for anti-coccidial prophylaxis.

Coccidiosis, a serious disease resulting from infection with parasitic protozoa of the genus Eimeria, causes significant economic losses to the poultry industry, where intensive rearing facilitates transmission of infectious oocysts via the fecal/oral route. Current control relies primarily on prophylactic drugs in feed but, whilst cost effective, the rise of drug resistance and public demands for residue-free meat has encouraged development of alternative control strategies. Chickens that recover from infection with Eimeria develop solid immunity that is directed against the early asexual stages of the parasite life cycle. This has allowed development of a number of vaccines that utilize deliberate infection with controlled doses of virulent oocysts or reproductively attenuated lines of Eimeria. The latter are immunogenic but non-pathogenic. The realization that both prophylactic drugs and attenuated vaccines control but do not eradicate infection with Eimeria encouraged development of a vaccine based upon maternal immunity. Laying hens exposed to Eimeria are able to transfer protective antibodies to hatchlings via egg yolks and these antibodies have been used to identify parasite proteins that are conserved across the genus. When delivered maternally, these provide an economical means of preventing coccidiosis, offering immediate protection to newly hatched chicks.

Characterisation of the antigenic and immunogenic properties of bacterially expressed, sexual stage antigens of the coccidian parasite, Eimeria maxima.

Coccidiosis in poultry is caused by the intestinal parasite Eimeria; it causes significant financial losses to the commercial poultry industry worldwide. CoxAbic is the first commercially available subunit vaccine against coccidiosis. The vaccine consists of affinity purified sexual stage (gametocyte) antigens (APGA) isolated from Eimeria maxima. Production of this vaccine is time-consuming and laborious and, therefore, a recombinant subunit vaccine substitute for CoxAbic is desirable. The genes encoding the two immunodominant components of CoxAbic, gam56 and gam82, were cloned into the bacterial expression vector, pTRCHisB, and the proteins expressed and purified. Both recombinant proteins were recognised by protective chicken antibodies that were raised to APGA, by immunoblotting. In a competitive ELISA, a combination of the recombinant proteins inhibited the binding of anti-APGA antibodies to APGA by 76%, which was comparable to the inhibition of 98% observed when APGA was used as the competing protein in the assay. In two breeds of chicken (Australorp and Cobb500), the recombinant proteins alone, or in combination, elicited a dose-dependent, antibody response that recognised APGA by ELISA, and gametocytes by immunoblotting. Together, the results suggested that the development of a recombinant subunit vaccine that maintains the antigenic and immunogenic properties of the native protein vaccine, CoxAbic, is feasible.

Eimeria maxima TRAP family protein EmTFP250: subcellular localisation and induction of immune responses by immunisation with a recombinant C-terminal derivative.

EmTFP250 is a high molecular mass, asexual stage antigen from Eimeria maxima strongly associated with maternally derived immunity to this protozoan parasite in hatchling chickens. Cloning and sequence analysis has predicted the antigen to be a novel member of the thrombospondin-related anonymous protein (TRAP) family of apicomplexan parasites. Members of the TRAP family are microneme proteins and are associated with host cell invasion and apicomplexan gliding motility. In order to assess the immunogenicity of EmTFP250, a C-terminal derivative encoding a low complex, hydrophilic region and putative transmembrane domain/cytosolic tail was expressed in a bacterial host system. The recombinant protein was used to immunise mice and chickens and found to induce strong IgG responses in both animal models as determined by specific ELISAs. Using Western blotting, protective maternal IgG antibodies previously shown to recognise native EmTFP250 recognised the recombinant protein and, in addition, antibodies raised against the recombinant protein were shown to recognise native EmTFP250. Localisation studies employing immuno-light microscopy and immuno-electron microscopy showed that antibodies to the recombinant protein specifically labeled micronemes within merozoites of E. maxima. Furthermore, antibodies to the recombinant EmTFP250 derivative showed similar labeling of micronemes within merozoites of Eimeria tenella. This study is further suggestive of a functional importance for EmTFP250 and underscores its potential as a candidate for a recombinant vaccine targeting coccidiosis in chickens.

Molecular characterisation of EmTFP250: a novel member of the TRAP protein family in Eimeria maxima.

We have previously described a high molecular mass, asexual stage antigen from Eimeria maxima (EmTFP250), implicated as a target of maternal antibodies produced by breeding hens infected with this protozoan parasite. Following partial purification of the protein by ion exchange chromatography, N-terminal and internal peptide sequences were generated and used in the design of degenerate PCR primers. Using a rapid amplification of cDNA ends PCR-based strategy, the cDNA encoding EmTFP250 has been cloned and sequenced. Translation predicts a mature polypeptide with a molecular mass of 246kDa and an isoelectric point of 4.2. Analysis of the amino acid sequence has revealed a novel member of the TRAP (thrombospondin-related anonymous protein) family, containing 16 thrombospondin type-1 repeats and 31 epidermal growth factor-like calcium binding domains. EmTFP250 also contains two low complex, hydrophilic regions rich in glutamic acid and glycine residues, and a transmembrane domain/cytosolic tail associated with parasite gliding motility that is highly conserved within apicomplexan microneme proteins. The protein has 61% identity (71% similarity) with EtMIC4, a 218kDa microneme protein of Eimeria tenella also rich in epidermal growth factor-like and thrombospondin type-1 domains. Using Southern blotting, the gene encoding EmTFP250 has been determined to be present as a single copy within the genome, and reverse transcriptase-PCR has shown that expression is confined to the asexual stages of development. By employing a PCR-based method, a region of the E. maxima Houghton strain EmTFP250 gene was found conserved in Australian isolates of several (at least four) Eimeria species that parasitise chickens. The characterisation of EmTFP250 adds to the expanding apicomplexan TRAP family and suggests a functional significance for the protein.