Andrew Hemphill

Redescription of Neospora caninum and its differentiation from related coccidia

Neospora caninum is a protozoan parasite of animals, which before 1984 was misidentified as Toxoplasma gondii. Infection by this parasite is a major cause of abortion in cattle and causes paralysis in dogs. Since the original description of N. caninum in 1988, considerable progress has been made in the understanding of its life cycle, biology, genetics and diagnosis. In this article, the authors redescribe the parasite, distinguish it from related coccidia, and provide accession numbers to its type specimens deposited in museums.

A European perspective on Neospora caninum

Since the identification of Neospora caninum in 1984 as a parasite separate from Toxoplasma gondii by Bjerkas et al., and its subsequent characterization and classification in 1988 by Dubey and co-workers, this parasite has attracted increasing attention, primarily as an important causative agent of abortion in cattle and neuromuscular disease in dogs, but also as a complementary model system to T. gondii for investigating the basic biology of intracellular parasitism. During November 11-14, 1999, the COST 820 Annual meeting (Vaccines against coccidioses) took place in Interlaken, Switzerland. Almost half of the papers presented at that meeting were on N. caninum and neosporosis, reflecting the increasing awareness of the importance of this parasite on part of the scientific community in Europe. On the occasion of the meeting, participants in this COST Action involved in Neospora research in Europe were asked to participate in this invited review in order to document the growing interest in N. caninum and the disease it causes. Thus, this paper is a unique collection of contributions provided by several European experts in the field. It is comprised of 10 reviews or original papers on different aspects of Neospora research including epidemiology, immunology, application and development of serological tools, and molecular characterisation of the parasite currently carried out throughout Europe. In addition, two distinguished invited speakers from overseas (Milton McAllister and John Ellis) provided valuable contributions. This invited review demonstrates that the COST 820 Action has brought together scientists from all over Europe and other parts of the world, and has laid the basis for many fruitful collaborations. The studies described here will contribute in assessing the relevance of neosporosis as a potential risk factor not only for animals, but also for human health.

Adhesion and invasion of bovine endothelial cells by Neospora caninum

Neospora caninum is a recently identified coccidian parasite which was, until 1988, misdiagnosed as Toxoplasma gondii. It causes paralysis and death in dogs and neonatal mortality and abortion in cattle, sheep, goats and horses. The life-cycle of Neospora has not yet been elucidated. The only two stages identified so far are tissue cysts and intracellularly dividing tachyzoites. Very little is known about the biology of this species. We have set up a fluorescence-based adhesion/invasion assay in order to investigate the interaction of N. caninum tachyzoites with bovine aorta endothelial (BAE) cells in vitro. Treatment of both host cells and parasites with metabolic inhibitors determined the metabolic requirements for adhesion and invasion. Chemical and enzymatic modifications of parasite and endothelial cell surfaces were used in order to obtain information on the nature of cell surface components responsible for the interaction between parasite and host. Electron microscopical investigations defined the ultrastructural characteristics of the adhesion and invasion process, and provided information on the intracellular development of the parasites.

Nitazoxanide, a broad-spectrum thiazolide anti-infective agent for the treatment of gastrointestinal infections.

Colonisation of the gastrointestinal tract by anaerobic bacteria, protozoa, trematodes, cestodes and/or nematodes and other infectious pathogens, including viruses, represents a major cause of morbidity and mortality in Africa, South America and southeast Asia, as well as other parts of the world. Nitazoxanide is a member of the thiazolide class of drugs with a documented broad spectrum of activity against parasites and anaerobic bacteria. Moreover, the drug has recently been reported to have a profound activity against hepatitis C virus infection. In addition, nitazoxanide exhibits anti-inflammatory properties, which have prompted clinical investigations for its use in Crohn’s disease. Studies with nitazoxanide derivatives have determined that there must be significantly different mechanisms of action acting on intracellular versus extracellular pathogens. An impressive number of clinical studies have shown that the drug has an excellent bioavailability in the gastrointestinal tract, is fast acting and highly effective against gastrointestinal bacteria, protozoa and helminthes. A recent Phase II study has demonstrated viral response (hepatitis C) to monotherapy, with a low toxicity and an excellent safety profile over 24 weeks of treatment. Pre-clinical studies have indicated that there is a potential for application of this drug against other diseases, not primarily affecting the liver or the gastrointestinal tract.

The Trypanosoma brucei cAMP phosphodiesterases TbrPDEB1 and TbrPDEB2: flagellar enzymes that are essential for parasite virulence

Cyclic nucleotide specific phosphodiesterases (PDEs) are pivotal regulators of cellular signaling. They are also important drug targets. Besides catalytic activity and substrate specificity, their subcel‐lular localization and interaction with other cell components are also functionally important. In contrast to the mammalian PDEs, the significance of PDEs in protozoal pathogens remains mostly unknown. The genome of Trypanosoma brucei, the causative agent of human sleeping sickness, codes for five different PDEs. Two of these, TbrPDEBl and TbrPDEB2, are closely similar, cAMP‐specific PDEs containing two GAF‐do‐mains in their N‐terminal regions. Despite their similarity, these two PDEs exhibit different subcellular localizations. TbrPDEB1 is located in the flagellum, whereas TbrPDEB2 is distributed between flagellum and cytoplasm. RNAi against the two mRNAs revealed that the two enzymes can complement each other but that a simultaneous ablation of both leads to cell death in bloodstream form trypanosomes. RNAi against Tbr‐PDEB1 and TbrPDEB2 also functions in vivo where it completely prevents infection and eliminates ongoing infections. Our data demonstrate that TbrPDEB1 and TbrPDEB2 are essential for virulence, making them valuable potential targets for new PDE‐inhibitor based trypanocidal drugs. Furthermore, they are compatible with the notion that the flagellum of T. brucei is an important site of cAMP signaling.—Oberholzer, M., Marti, G., Baresic, M., Kunz, S., Hemphill, A., Seebeck, T. The Trypanosoma brucei cAMP phosphodiesterases TbrPDEB1 and TbrPDEB2: flagellar enzymes that are essential for parasite virulence. FASEB J. 21, 720–731 (2007)

Vaccination of mice against experimental Neospora caninum infection using NcSAG1- and NcSRS2-based recombinant antigens and DNA vaccines.

NcSAG1 and NcSRS2, the two major immunodominant tachyzoite surface antigens of the apicomplexan parasite Neospora caninum, were investigated for their potential as vaccine candidates in mice. Recombinant recNcSRS2 and recNcSAG1 were expressed in Escherichia coli as poly-histidine-tagged fusion proteins. Separate groups of mice were immunized with purified recNcSAG1, recNcSRS2, or a combination of both, and were then challenged with N. caninum tachyzoites. Subsequent experiments included intramuscular vaccination of mice with the eukaryotic expression plasmid pcDNA3 containing either NcSRS2 or NcSAG1 cDNA inserts, followed by a single booster with the corresponding recombinant antigens. Immunization with a crude somatic antigen (NC1-extract) was included in the experiments. Following challenge, the presence of the parasite in the different organs was assessed by a N. caninum-specific PCR, while the parasite burden in infected brain tissue was assessed by quantitative real-time PCR. Immunization of mice employing individual recombinant antigens, or combined recNcSAG1/recNcSRS2, resulted in a lower degree of protection against cerebral infection, when compared to combined DNA/recombinant antigen vaccination. Serological analysis showed that this protective effect was associated with the occurrence of antibodies directed against native parasite antigens in those animals receiving combined DNA/recombinant antigen vaccination. Conversely, mice immunized with recombinant antigens alone generated antibodies recognizing only the recombinant antigens. Mice experiencing clinical signs such as walking disorders, rounded back, apathy and paralysis were observed only in the untreated positive control groups, but never in the vaccinated groups. Our results suggest that a combined DNA/recombinant antigen-vaccine, based on NcSAG1 and NcSRS2, respectively, exhibited a highly significant protective effect against experimentally induced cerebral neosporosis in mice.

Susceptibility of B-cell deficient C57BL/6 (microMT) mice to Neospora caninum infection.

Neospora caninum is a coccidian parasite of veterinary importance by causing abortion or stillbirth in cattle among other problems in diverse animal species. We assessed an experimental murine model for its suitability to study the immune response to N. caninum infection. Thus, wild‐type (wt) C57BL/6 mice and B‐cell (and consequently antibody)‐deficient μMT mice were infected with N. caninum tachyzoites and sacrificed at days 10, 24 and 29–44 post infection (dpi). Various organs were collected for parasitological and pathological analysis, spleen and serum for immunological investigations. Splenocytes were in vitro‐stimulated with N. caninum (NC)‐ and T. gondii‐antigens for assessing T cell proliferation and cytokine production. While wt mice were resistant to disease, μMT mice died starting from 29 dpi onwards. Histological examination of brain tissue from μMT mice exhibited a high infection intensity with multifocal necrotic cerebral lesions, which were absent in the brains of wt mice. NC antigen‐stimulated spleen cells of both wt and μMT mice infected with N. caninum showed a marked proliferative depression at 10 dpi. At 24 dpi, this immunosuppression was still maintained in μMT mice whereas it was restored in wt mice. Stimulated splenocytes of infected μMT mice secreted significantly less IFN‐γ and less IL‐10 than corresponding wt splenocytes. For IL‐10, this difference increased with time. The susceptibility of μMT mice appeared associated to B‐cell deficiency, allowing the persistant spread of the parasite causing immunosuppression and finally resulting in a lethal outcome of infection.

The Host-Parasite Relationship in Neosporosis

Neospora caninum is an apicomplexan parasite which invades many different cell types and tissues. It causes neosporosis, namely stillbirth and abortion in cattle and neuromuscular disease in dogs, and has been found in several other animal species. N. caninum is closely related to Toxoplasma gondii, and controversial opinions exist with respect to its phylogenetical status. Initially, two stages of N. caninum had been identified, namely asexually proliferating tachyzoites and bradyzoites. The sexually produced stage of this parasite, oocysts containing sporozoites, has been found only recently. In order to answer the many open questions regarding its basic biology and its relationship with the host, a number of diagnostic tools have been developed. These techniques are based on the detection of antibodies against parasites in body fluids, the direct visualization of the parasite within tissue samples by immunohistochemistry, or the specific amplification of parasite DNA by PCR. Other studies have been aiming at the identification of specific antigenic components of N. caninum, and the molecular and functional characterization of these antigens with respect to the cell biology of the parasite. Clearly, molecular approaches will also be used increasingly to elucidate the immunological and pathogenetic events during infection, but also to prepare potential new immunotherapeutic tools for future vaccination against N. caninum infection.

Immunology and morphology studies on the proliferation of in vitro cultivatedEchinococcus multilocularis metacestodes

The larval stage ofEchinococcus multilocularis causes alveolar echinococcosis (AE) in various mammals, including humans. Traditionally metacestodes are maintained in the laboratory by serial transplantation passages into susceptible animals such as mice or gerbils. However, in animal models it has always been difficult to draw definite conclusions about the factors modulating metacestode differentiation, and investigations on gene expression and respective regulation have been hampered by the complexicity of the host-parasite interplay. This paper describes the maintenance and proliferation ofE. multilocularis metacestodes as well as the formation of protoscolices in a chemically defined medium devoid of host influence. The interactive role of a heterologous human cell line (CACO2) in the in vitro development of metacestodes was also assessed. The morphology and ultrastructure of in vitro-generated metacestodes was studied using scanning (SEM) and transmission electron microscopy (TEM). Different cultivation procedures were analyzed in terms of expression of B-and T-cell epitopes and of the relevant laminated layer-antigen Em2; the exact localization of this antigen was further demonstrated by immunogold electron microscopy.

Cellular and immunological basis of the host-parasite relationship during infection with Neospora caninum.

Neospora caninum is an apicomplexan parasite that is closely related to Toxoplasma gondii, the causative agent of toxoplasmosis in humans and domestic animals. However, in contrast to T. gondii, N. caninum represents a major cause of abortion in cattle, pointing towards distinct differences in the biology of these two species. There are 3 distinct key features that represent potential targets for prevention of infection or intervention against disease caused by N. caninum. Firstly, tachyzoites are capable of infecting a large variety of host cells in vitro and in vivo. Secondly, the parasite exploits its ability to respond to alterations in living conditions by converting into another stage (tachyzoite-to-bradyzoite or vice versa). Thirdly, by analogy with T. gondii, this parasite has evolved mechanisms that modulate its host cells according to its own requirements, and these must, especially in the case of the bradyzoite stage, involve mechanisms that ensure long-term survival of not only the parasite but also of the host cell. In order to elucidate the molecular and cellular bases of these important features of N. caninum, cell culture-based approaches and laboratory animal models are being exploited. In this review, we will summarize the current achievements related to host cell and parasite cell biology, and will discuss potential applications for prevention of infection and/or disease by reviewing corresponding work performed in murine laboratory infection models and in cattle.