Infectious organisms can cause significant losses in farm ruminant production as a result of abortion, embryonic damage or maternal infertility. One of the principal agents causing protozoal abortion or still-birth in cattle is Neospora caninum12. Infection affects approximately 12% of Swiss cattle 3 and may frequently be passed from mother to calf (vertical transmission) with no signs of disease. Problems occur when the parasite multiplies in the developing calf or the affected placenta and causes sufficient damage to trigger stillbirth or abortion, respectively. Preliminary research suggests that infection of the foetus early in gestation is more likely to be fatal to the conceptus than infection later in gestation 4. However it also appears that infection is more likely to be transmitted in late rather than early pregnancy. Thus the majority of infections are not symptomatic and inapparent infections are maintained and linearly expanded in a herd. Control of bovine neosporosis is difficult. Effective vaccines to protect cattle from abortion or vertical parasite transmission are currently not available 15. Pharmacologically active compounds are known to kill N. caninum6. Thus, toltrazuril and ponazuril proved high efficacy to prevent parasite dissemination and subsequent cerebral lesion formation in a murine experimental model 7. To assess treatment efficacy, mice were clinically investigated for symptoms; histologically by searching respective cerebral lesions and molecular biologically by detection of parasite DNA using the polymerase-chain-reaction (PCR). Efficacy of treatment was demonstrated to eliminate the parasite, and to reduce but not to abrogate parasite-specific humoral immunity in mice.

With regard to controlling infection, disease or diaplacental parasite transmission in cattle, a chemotherapeutical approach has not been studied yet. Therefore, we designed now an initial explorative approach to document a basic effectiveness at first. This experiment represents a simple first step, to be followed with a more extensive, but also more expensive future study to provide a statistically evidence of the effect in large numbers of animals. To monitor treatment efficacy in the experimentally infected calves of this study, we used the same parameters as outlined above in the mouse model.

Neosporosis is a disease affecting predominantly the fetal development in bovine hosts, it may also cause neuromuscular dysfunction in infected new born calves and pups 2. In cattle, the outcome of a N. caninum infection appears largely dependent upon the effectiveness of the immune system of the mother animal 15. Thus, in a certain number of cases, immunity appears insufficient to prevent vertical transmission to the bovine fetus 15.

Chemotherapeutical treatment of neosporosis may be an issue, provided that an appropriate drug is made available. In this respect, we have previously described the use of a mouse model to demonstrate the efficacy of toltrazuril and ponazuril for the prevention of parasite dissemination and subsequent cerebral lesion formation 7. We now addressed – in a first step – the question, if the same drug would exhibit a basic effect in the experimentally infected bovine host. This question was tackled in view to justify and plan future trials for the treatment of experimentally and naturally acquired bovine neosporosis.

With regard to our experimental design, the high costs of experimental calves did not allow to directly plan experiments with number of calves allowing statistical analyses. Thus, the project was basically designed as an explorative study, only allowing to document descriptively the effect of the treatment in experimentally infected calves. Conversely to our previous study in mice, where we had started chemotherapy 6 hours prior to infection, we now provided a 24 hour cycle to the parasite to establish and multiply within the animals, before medication was started. Chemotherapy itself was either performed as a short term, one-day medication or as a 6-consecutive-day medication. Assessment of serum levels of ponazuril showed that the two medication schedules considerably influenced the maximal serum concentrations in the animals. The relatively long half-life time of ponazuril led to an accumulation of the compound in 6-day-treated animals, so that the maximal peak was approximately 3 to 4-times higher than in 1-day-medicated calves. Also the duration of ponazuril detectability in serum was markedly longer in 6-day-treated animals. Nevertheless, no differences appeared between the two treatment schedules as in none of the treated animals N. caninum became detectable by PCR. With regard to the non-medicated control calves, which had received the same infection dose, we had to notify that only 3 out of 6 animals reached the infection stage where the parasite became detectable by PCR in the brain and muscles. Calves therefore seemed to exhibit, constitutively, a relatively low susceptibility to experimental infection. Similar observations had already been reported by others 16. Nevertheless, the absence of any DNA detectability in treated calves versus the 50% detectability in non-treated calves indicate efficacy of ponazuril to suppress parasite dissemination and establishment in peripheral organs such as brain and muscles. Histology and immunohistochemistry, probably because of the low infection intensity, did not result in the demonstration of parasites and respective lesions in the PCR positive tissues. Uggla et al. 16 had orally infected calves with N. caninum-tachyzoites. Similarly to our results, although their animals seroconverted upon infection, and some became positive by PCR, neither pathognomonic tissue damages nor parasites themselves could be demonstrated by histology and immunohistochemistry.

In our mouse model 7, we had shown that toltrazuril/ponazuril treatment, in parallel to abrogating parasite dissemination, also predominantly influenced the kinetics of the humoral immune response. Similarly now in calves, experimentally infected and non-treated animals developed a marked humoral immune response, seroconversion occurred between days 10 and 20 p.i., and maximal serum antibody concentrations were reached between days 30 and 40 p.i.. One-day medication of infected calves – although abrogating parasite detectability by PCR – did not change the time point of seroconversion and the antibody concentration levels when compared to non-treated animals. Conversely, the 6-day-treatment markedly reduced the concentration maximum of serum antibodies, and delayed the time point of seroconversion by approximately one week. These findings indicate that the one-day treatment may not have killed the parasite as efficiently or rapidly as the 6-day treatment. A higher antibody concentration may be linked either to a higher antigen concentration (indicating post-infectional parasite multiplication despite treatment) or to a longer lasting stimulation with identical antigen concentrations (indicating a longer survival of the inoculated parasite population). This question will require appropriate experiments to be addressed.

When tackling clinical parameters during the first phase of the experiments, it became apparent that non-treated as well as treated calves responded with fever to the infection. However, treated animals had milder fever as expressed by average lower peak levels and shorter fever periods. As the first fever occurred only 1–2 days after experimental infection in non-treated and 2–3 days p.i. in treated animals, we can exclude endotoxin activity by inoculated N. caninum tachyzoites. Principally, endotoxin induced fever is expected to occur a few hours after inoculation of e.g. lipopolysaccharides (LPS) 14. Inoculation of an appropriate number of Vero-cells in non-infected control animals did not result in fever, indicating that fever was indeed related to the parasite inoculum. In consequence, we can conclude that ponazuril therapy inhibited the parasite proliferation in a way to reduce fever. To elucidate the fever aspect more in details, we will have to perform experimental inoculation of the same dose but of killed parasites (such as done in the previous mouse experiments; 7). With these experiments we will be able to show if fever is directly associated to viable trophozoites or not.

Bovine N. caninum infection, although not demonstrating any acute-phase symptoms, may result in chronic phase consequences, as indicated by reduced gain of body weight in seropositive versus seronegative calves 18. In our experiments, however, no respective differences became apparent. While planning the experiments, we hypothesized that a putative intensive parasite dissemination in non-treated calves should result in tissue and organ damages, respectively, and thus also in the modification of blood parameters. Therefore, we analyzed serum glucose, GLDH and γ-GT to address liver functions, AST and CK for muscle damages, urea for kidney damages and triglycerides and NEFA to check the lipid status. All results were within the normal range of concentrations 192021, thus indicating the absence of any pathology that could have been demonstrated with the respective laboratory tools 22. These findings support again our hypothesis on the relatively low susceptibility of calves for experimental N. caninum infection. Therefore, even if the parasite became disseminated and subsequently established in the brain and muscles of at least 3 from 6 infected and non-treated calves, the clinical consequences of this infection became not detectable.

Based upon our findings in this initially explorative approach that indicate a basic effectiveness of ponazuril against experimental N. caninum infection in calves, we plan to follow our chemotherapeutical intervention strategy to control bovine neosporosis with a subsequent more extensive field study. This will now tackle naturally infected calves derived from N. caninum-positive mothers. This field study will be designed in view to include a large number of animals of appropriate statistical significance. Parameters to asses efficacy of treatment will include the same as used in this study. Conceptually, there is a general acceptance that healthy newborn calves from N. caninum-positive mothers are suspected to stay life-long chronically infected 23, with reactivations occurring during pregnancy. Elimination of the parasite after birth by chemotherapy may provide negative off-spring and thus N. caninum-free breeding lines. Furthermore, as the milk yield and gain of body weight appeared to be reduced in persistently infected cows 18, creation of Neospora-free breeding lines may also improve other economic aspects.

Author 1 (SK) carried out the experimental infection and treatment studies, and performed PCR, ELISA and histological/immunohistochemical analyses. Author 2 (HS) participated in the clinical investigations and the molecular and serological assays. Author 3 (JB) carried out the pathophysiological testings. Author 4 (RK) performed the determination of the ponazuril serum levels. Author 4 (GG) participated in the design of the study. Author 5 (BG) conceived the study and its design and supervised its coordination.