Gisela Greif

Efficacy of toltrazuril and ponazuril against experimental Neospora caninum infection in mice.

Abstract Neosporosis is a disease affecting predominantly fetal development in cattle and dog hosts; and it may cause neuromuscular disfunction in infected new-born calves and pups. Predispositions – including, e.g. transient immunosuppression during pregnancy – may result in an increased dissemination of the parasite within the host or its offspring. Chemotherapeutic treatment of neosporosis may be an issue, provided that an appropriate drug is made available. In this respect, we describe the use of a mouse model for the evaluation of toltrazuril and ponazuril medication as a means of preventing parasite dissemination and subsequent formation of cerebral lesions. Toltrazuril- and ponazuril-treated mice were experimentally infected intraperitoneally (i.p.) with 2 × 106Neospora caninum tachyzoites. The infection was monitored at three levels: clinically, by assessing symptoms, histologically, by assessing the occurrence of cerebral lesions and parasites by immunohistochemistry, and on the molecular level, by detection of parasite DNA using PCR. Chemotherapy using either toltrazuril or ponazuril, both applied in a drinking-water formulation (20 mg toltrazuril or ponazuril kg−1 body weight day−1) completely prevented the formation of cerebral lesions in all treated animals, as assessed by immunohistochemistry. PCR analyses of these treated animals showed that DNA-detectability was reduced by 91% and 90% upon toltrazuril and ponazuril medication, respectively.

An explorative study to assess the efficacy of Toltrazuril-sulfone (Ponazuril) in calves experimentally infected with Neospora caninum

BackgroundNeospora caninum is an important cause of infectious abortion and stillbirth in cattle world-wide. Infection is common and may frequently be passed from mother to calf (vertical transmission) with no signs of disease. Based on our previous observation that N. caninum-infection can be efficiently controlled with Toltrazuril-sulfone (Ponazuril) in experimentally infected mice, we addressed the question if efficacy could also be obtained in experimentally infected calves.Material and MethodsThe study included 19 calves and represents an initial explorative approach to document a basic effectiveness at first. Fifteen animals received each 2 x 108N. caninum trophozoites, half of the dose being injected intravenously and the other half subcutaneously. Efficacy of treatment was assessed using molecular detection of parasite DNA with PCR and pathological alterations by immunohistochemistry in different organs of the animals. Assessment included also clinical, serological and pathophysiological parameters.ResultsIn those calves medicated with ponazuril (one, or six consecutive days, respectively, starting one day after infection), a complete abrogation of the parasite detectability was obtained in the brain and other organs, while 50% of non-treated calves became PCR-positive in brain and muscles. Clinically, ponazuril chemotherapy of infected calves – in comparison to non-treated infected animals – reduced symptoms (fever), but no differences were observed between treated and non-treated animals with regard to serum enzymes and metabolites. Efficacy of a six-day treament was also reflected by significantly lower anti-Neospora antibody concentrations developed after infection, when compared to non-treated animals.ConclusionBased on 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 with naturally infected calves.

Chemotherapeutic approaches to protozoa: Coccidiae – current level of knowledge and outlook

Progress in the treatment and prophylaxis of cyst-forming coccidial infections (Neospora, Sarcocystis Toxoplasma) and Cryptosporidium infections has been limited (Table 1: Haberkorn 1996: Croft 1997: Wang 1997). However, new possibilities have been opened up in the treatment of Eimeria-induced coccidiosis in poultry and mammals. due to improvements in treatment and, or metaphylaxis. A new polyether antibiotic. semduramycin, has recently been added to the range of effective prophylactic preparations. The development of resistance to anticoccidial agents is now posing similar problems to those encountered with malaria, coccidiosis in poultry being particularly affected. Because no new active ingredient from a new family of chemical substances has been developed for more than 10 years, the following approaches are being adopted to get round this problem: the use of older preparations which have not been used for a long time, the introduction of combinations such as narasin nicarbazin or methyl benzoquate clopidol and the alternating use of anticoccidial agents in rotation and shuttle programmes. The goal of a real alternative, i.e. vaccination, has been achieved to a certain extent in the form of live vaccines for laying hens and broiler breeders and is being practiced in some countries.

Chemotherapeutic approaches to protozoa: haemosporina--current level of knowledge and outlook.

Chloroquine and mefloquine are available for prophylactic treatment in malaria, against a background of the burgeoning problem of resistance developing to chloroquine and related drugs (Mehlhorn and Schrevel 1995). For this reason, highly specific national recommendations are given out regarding prophylaxis. The option of a viable vaccine is currently not available. More new compounds are therefore urgently required, since 2-5 million of the 200 300 million infected people die each year. At the moment, atovaquone and artemisinin derivatives are of great interest, as are drug combinations such as atovaquone/proguanil (since 1997), artemether/ benflumetol (since 1998?; Ciba-Geigy, patent WO9202217) and chlorproguanil/dapsone (since 2000?), as these compounds are also effective against multi-resistant strains of Plasmodium falciparum (Tables 1, 2; Croft 1997; Wang 1997). Pyronaridin (since 2000?) has been discovered in a Chinese academy and is in clinical trials (Trouiller and Olliaro 1998; Pecoul et al. 1999).

Immunity to coccidiosis after treatment with toltrazuril.

Abstract Some characteristics of immune sera after toltrazuril treatment which may be involved in the enhancement of immunity are as follows. Firstly, toltrazuril acts against all intracellular parasite stages from 1 to 168 h post-infection: intracellular schizonts of generations I, II and III and also intracellular gamonts. These damaged stages remain in the host cell for a prolonged time, during which they act as antigens which can be recognised by the immune system. Secondly, immune sera during therapeutic treatment with toltrazuril show a high antibody titre of 1:100 and 1:250 in IFAT after challenge. In the immunoblot, these sera show a strong reaction with low molecular protein fragments between 14 and 20 kDa. Toltrazuril does not interfere with the process of parasite protein recognition by the immune system during infection. Thirdly, there is a tendency for a higher IgG response after treatment with toltrazuril. Fourthly, a higher antibody titre in IFAT, immunoblot and ELISA correlates with a higher reduction in oocyst excretion, lesion scoring and increased weight gains in toltrazuril-treated animals.

Intraspecific polymorphisms of Eimeria species due to resistance against anticoccidial drugs

Abstract Resistance analyses were done on 15 Eimeria acervulina strains and 5 E. brunetti strains. In all, 55% of these strains proved to have complex profiles of resistance to anticoccidial drugs as judged by resistance-index (RI) evaluation. Genomic fingerprints generated by random amplified DNA (RAPD) with 16 primers via the polymerase chain reaction (PCR) revealed a high degree of similarity (SI) between nonresistant strains (SI up to 95%). Polymorphisms including band shifts, differences in banding intensity, and missing bands led to significantly low SI values (57%, 69%, 82%) in drug-resistant Eimeria strains. After experimental induction of diclazuril resistance (1, 2, and 4 ppm) in a laboratory isolate VT-1 primer 5′-CCC TGA GAT GGG AAC CTC-3′ amplified a polymorphic band of around 600 bp. Polymorphisms detected by RAPD-PCR will facilitate the selection of molecular markers and might lead to the design of diagnostic tests for drug-resistant genotypes.

Chemotherapeutic approaches to protozoa: Giardia, Trichomonas and Entamoeba-current level of knowledge and outlook.

The situation regarding the treatment of human Giardia and Trichomonas infections and the intestinal and tissue stages of Entamoeba histolytica with metronidazole and other 5-nitroimidazoles is currently satisfactory (Table 1; Mehlhorn 2000). Following correct and rapid diagnosis, the parasites are eliminated reliably and completely. The situation in cases of infection with Acanthamoeba (often involving the eyes) or with Naegleria (often involving the brain) is serious, however. In both cases, there is no drug of choice available. Treatment consists of relieving the symptoms and/or preventing local degeneration.

Excystation of Eimeria tenella Sporozoites Impaired by Antibody Recognizing Gametocyte/Oocyst Antigens GAM22 and GAM56

ABSTRACT Eimeria tenella is the causative agent of coccidiosis in poultry. Infection of the chicken intestine begins with ingestion of sporulated oocysts releasing sporocysts, which in turn release invasive sporozoites. The monoclonal antibody E2E5 recognizes wall-forming body type II (WFBII) in gametocytes and the WFBII-derived inner wall of oocysts. Here we describe that this antibody also binds to the stieda body of sporocysts and significantly impairs in vitro excystation of sporozoites. Using affinity chromatography and protein sequence analysis, E2E5 is shown to recognize EtGAM56, the E. tenella ortholog of the Eimeria maxima gametocyte-specific GAM56 protein. In addition, this antibody was used to screen a genomic phage display library presenting E. tenella antigens as fusion proteins with the gene VIII product on the surfaces of phagemid particles and identified the novel 22-kDa histidine- and proline-rich protein EtGAM22. The Etgam22 mRNA is expressed predominantly at the gametocyte stage, as detected by Northern blotting. Southern blot analysis in combination with data from the E. tenella genome project revealed that Etgam22 is an intronless multicopy gene, with approximately 12 to 22 copies in head-to-tail arrangement. Conspicuously, Etgam56 is also intronless and is localized adjacent to another gam56-like gene, Etgam59. Our data suggest that amplification is common for genes encoding oocyst wall proteins.

In silico analysis of the cyclophilin repertoire of apicomplexan parasites.

BackgroundCyclophilins (Cyps) are peptidyl cis/trans isomerases implicated in diverse processes such as protein folding, signal transduction, and RNA processing. They are also candidate drug targets, in particular for the immunosuppressant cyclosporine A. In addition, cyclosporine is known to exhibit anti-parasitic effects on a wide range of organisms including several apicomplexa. In order to obtain new non-immunosuppressive drugs targeting apicomplexan cyclophilins, a profound knowledge of the cyclophilin repertoire of this phylum would be necessary.ResultsBLAST and maximum likelihood analyses identified 16 different cyclophilin subfamilies within the genomes of Cryptosporidium hominis, Toxoplasma gondii, Plasmodium falciparum, Theileria annulata, Theileria parva, and Babesia bovis. In addition to good statistical support from the phylogenetic analysis, these subfamilies are also confirmed by comparison of cyclophilin domain architecture. Within an individual genome, the number of different Cyp genes that could be deduced varies between 7–9 for Cryptosporidia and 14 for T. gondii. Many of the putative apicomplexan cyclophilins are predicted to be nuclear proteins, most of them presumably involved in RNA processing.ConclusionThe genomes of apicomplexa harbor a cyclophilin repertoire that is at least as complex as that of most fungi. The identification of Cyp subfamilies that are specific for lower eukaryotes, apicomplexa, or even the genus Plasmodium is of particular interest since these subfamilies are not present in host cells and might therefore represent attractive drug targets.

NcGRA2-RT-PCR to Detect Live Versus Dead Parasites in Neospora caninum-Infected Mice

In the present work, we optimized a recently established NcGRA2-RT-PCR based on RNA to detect live Neo- spora caninum parasites in tissue, and compared the results with the conventional inoculation of diagnostic specimen onto cell culture. C57BL/6 mice were experimentally infected with Nc-1 tachyzoites and subsequently euthanized 6 or 12 days post infection (dpi). Selected organs were used to search for parasites by (i) PCR using genomic DNA (gDNA), (ii) PCR using cDNA and (iii) in vitro inoculation of cell culture. At 6 dpi, Neospora-gDNA was detected in 34 out of 36 organs. Viable parasites were detected in 11 (NcGRA2-RT-PCR) and 15 (in vitro cultivation) out of 36 organs. Comparison of NcGRA2-RT-PCR and in vitro detection gave a fair agreement (kappa 0.29), whereas comparison of PCR using gDNA and RT-PCR or in vitro detection resulted in a slight agreement (kappa 0.05 and 0.08, respectively) only. At 12 dpi, para- site gDNA was found in 10 out of 36 organs. In 7 of these organs viability of parasites was confirmed with NcGRA2-RT- PCR and growth of parasites in cell culture. Comparison of NcGRA2-RT-PCR and in vitro detection gave a substantial agreement (kappa 0.8), whereas comparison of PCR using gDNA and RT-PCR or in vitro detection resulted in a moderate agreement (kappa 0.59 and 0.77, respectively). As NcGRA2-RT-PCR is almost as sensitive as in vitro cultivation in de- tecting live parasites, it represents a fast, easy and safe method of viable parasite detection, and thus an attractive alterna- tive to the in vitro cultivation approach.