Neospora caninum is a leading cause of abortion in cattle, and is thus an important veterinary health problem of high economic significance. Vaccination has been considered a viable strategy to prevent bovine neosporosis. Different approaches have been investigated, and to date the most promising results have been achieved with live-attenuated vaccines. Subunit vaccines have also been studied, and most of them represented components that are functionally involved in (i) the physical interaction between the parasite and its host cell during invasion or (ii) tachyzoite-to-bradyzoite stage conversion. Drugs have been considered as an option to limit the effects of vertical transmission of N. caninum. Promising results with a small panel of compounds in small laboratory animal models indicate the potential value of a chemotherapeutical approach for the prevention of neosporosis in ruminants. For both, vaccines and drugs, the key for success in preventing vertical transmission lies in the application of bioactive compounds that limit parasite proliferation and dissemination, without endangering the developing fetus not only during an exogenous acute infection but also during recrudescence of a chronic infection. In this review, the current status of vaccine and drug development is presented and novel strategies against neosporosis are discussed.

A Neospora sp. was isolated from the brains of two aborted bovine foetuses and grown continuously in vitro in bovine cell cultures. A comparison of the antigenic reactivity of in vitro cultivated tachyzoites with polyclonal antisera to Neospora caninum, Hammondia hammondi or Toxoplasma gondii revealed that the bovine protozoal isolates were similar to N. caninum and antigenically distinct from T. gondii. Tachyzoites of both bovine isolates had similar ultrastructural features, including an apical polar ring, conoid, electron-dense rhoptries and micronemes. The orientation of the micronemes, presence of micropores and a large number of electron-dense granules in the posterior portion of the bovine isolate tachyzoites differed from previous descriptions of N. caninum in vivo. Tachyzoites of the bovine isolates were ultrastructurally more similar to in vitro cultivated N. caninum tachyzoites than to tachyzoites of T. gondii or H. hammondi. The antigenic and ultrastructural similarities between N. caninum and the protozoal parasites isolated from aborted bovine foetuses in this study support the proposition that these parasites belong to the genus Neospora.

The protozoan parasites Eimeria spp. Toxoplasma gondii and Neospora caninum are significant causes of disease in livestock worldwide and T. gondii is also an important human pathogen. Drugs have been used with varying success to help control aspects of these diseases and commercial vaccines are available for all three groups of parasites. However, there are issues with increasing development of resistance to many of the anti-coccidial drugs used to help control avian eimeriosis and public concerns about the use of drugs in food animals. In addition there are no drugs available that can act against the tissue cyst stage of either T. gondii or N. caninum and thus cure animals or people of infection. All three groups of parasites multiply within the cells of their host species and therefore cell mediated immune mechanisms are thought to be an important component of host protective immunity. Successful vaccination strategies for both Eimeria and Toxoplasma have relied on using a live vaccination approach using attenuated parasites which allows correct processing and presentation of antigen to the host immune system to stimulate appropriate cell mediated immune responses. However, live vaccines can have problems with safety, short shelf-life and large-scale production; therefore there is continued interest in devising new vaccines using defined recombinant antigens. The major challenges in devising novel vaccines are to select relevant antigens and then present them to the immune system in an appropriate manner to enable the induction of protective immune responses. With all three groups of parasites, vaccine preparations comprising antigens from the different life cycle stages may also be advantageous. In the case of Eimeria parasites there are also problems with strain-specific immunity therefore a cocktail of antigens from different parasite strains may be required. Improving our knowledge of the different parasite transmission routes, host-parasite relationships, disease pathogenesis and determining the various roles of the host immune response being at times host-protective, parasite protective and in causing immunopathology will help to tailor a vaccination strategy against a particular disease target. This paper discusses current vaccination strategies to help combat infections with Eimeria, Toxoplasma and Neospora and recent research looking towards developing new vaccine targets and approaches.

Hammondia heydorni is regarded as a protozoan parasite that uses canids, e.g. dogs and foxes, as definitive hosts, but clinical signs of infection are rare. This study therefore took advantage of the opportunity to study an oocyst population from the faeces of a dog suffering from intermittent bouts of diarrhoea. Oocysts from the naturally infected dog were shown to be H. heydorni by using the polymerase chain reaction combined with DNA sequencing as a diagnostic tool.The nucleotide sequence data reported in this paper are available from GenBank under the following Accession numbers DQ183058, DQ183059 and DQ022687. A comparison of the first internal transcribed spacer (ITS1) sequence of ribosomal DNA obtained with those from other dog and fox oocysts, previously regarded as H. heydorni, showed these oocysts contained identical ITS1 sequences. However, the oocyst DNA from the fox and dog differed by the presence/absence of a 9 bp insertion/deletion within intron 1 of the alpha tubulin gene, and this difference was conserved across a number of different oocyst populations from the 2 species of host. A PCR assay was established that takes advantage of this insertion/deletion and is able to differentiate between the 2 oocyst populations. This study therefore provides evidence that H. heydorni oocysts from dogs and foxes represent 2 distinct genetic lineages that can be differentiated using a PCR, which targets the alpha tubulin locus.

Studies using antibodies to immunolocalize the Toxoplasma gondii dense granule protein GRA3, have shown that this protein associates strongly with the parasitophorous vacuole membrane (PVM). However, as there was no predicted membrane-spanning domain this highlighted an unanswered paradox. We demonstrate that the previously published sequence for GRA3 is actually an artificial chimera of 2 proteins. One protein, of molecular weight 65 kDa, shares the C-terminus with published GRA3 and possesses no significant sequence similarity with any protein thus far deposited in Genbank. The second, with a predicted molecular weight of 24 kDa shares the N-terminal region, is recognized by the monoclonal antibody 2H11 known to react with the dense granules of T. gondii and is therefore the authentic GRA3. The corrected GRA3 has an N-terminal secretory signal sequence and a transmembrane domain consistent with its insertion into the PVM. Antibodies to recombinant GRA3 recognize a protein of 24 kDa in T. gondii excretory–secretory antigen preparations. The signal peptide is necessary and sufficient to target GFP to the dense granules and parasitophorous vacuole. A homologue was identified in Neospora caninum. Finally, GRA3 possesses a dilysine ‘KKXX’ endoplasmic reticulum (ER) retrieval motif that rationalizes its association with PVM and possibly the host cell ER.