Axel Haberkorn

Chemotherapy of human and animal coccidioses: state and perspectives.

Abstract The state and perspectives for chemotherapy of cyst-forming and non-cyst-forming coccidia in humans and animals are summarized. In toxoplasmosis the therapeutic care of transplacental infections, which have gone out of control because of immunodeficiency, is in the forefront of attempts at improvement. Predominant drugs in use are pyrimethamine combined with a sulfonamide or with clindamycin, or trimethoprim plus sulfamethoxazole. For reasons of tolerability in human immunodeficiency virus (HIV)-infected patients, after 3 months of therapy a maintenance treatment on 2 days a week has recently given very positive results. In cats, monensin and toltrazuril are effective against the intestinal developmental stages of Toxoplasma gondii, the latter drug affecting to a reasonable extent the extraintestinal stages as well. Attempts to treat neosporosis and sarcocystosis remain in the initial stages. The same is true for cryptosporidiosis in humans and animals. A number of highly effective drugs are available for prophylaxis of poultry coccidiosis. Increasing problems with resistance have led to new treatment schemes such as shuttle and rotation programs. In addition to a new polyether, semduramycin, a benzeneacetonitrile derivative (diclazuril) has been developed in recent years. After three decades a new drug (toltrazuril), a symmetrical triazinone derivative, has brought improvements for therapy and/or metaphylaxis in coccidiosis of poultry and mammals. The increasing possibilities for vaccination may result in new aspects for the use of chemotherapeutics, i.e., new combinations and/or shuttle or rotation programs.

Possible mode of action of toltrazuril: studies on twoEimeria species and mammalian andAscaris suum enzymes

The anticoccidial properties of toltrazuril inEimeria falciformis-infected mice were potentiated by the simultaneous application of pyrimethamine, trimethoprim, or sulfadimidine. The same drugs potentiate the effect of toltrazuril by killingE. tenella schizonts in chicken kidney-cell cultures. Activities of some enzymes of the respiratory chain, such as succinate-cytochrome C reductase and NADH oxidase and succinate oxidase from mouse liver, were reduced in the presence of toltrazuril. The same effects could be observed when the activities of NADH oxidase and fumarate reductase from the nematodeAscaris suum were determined in the presence of the drug. Vertebrate enzymes involved in pyrimidine synthesis, e.g., dihydrofolate reductase from chicken liver, were also affected by toltrazuril; however, this effect was 500 times weaker than that shown by pyrimethamine. Toltrazuril also showed an inhibitory effect on the dihydroorotate-cytochrome C reductase from mouse liver. Our results suggest that toltrazuril primarily affects the respiratory chain and secondarily, two enzymes involved in pyrimidine synthesis.

The effects of sym. Triazinones on developmental stages ofEimeria tenella, E. maxima andE. acervulina: A light and electron microscopical study

The development of three chicken coccidia (Eimeria tenella, E. acervulina andE. maxima) was studied by means of light and electron microscopy. One group of chickens infected with 6000–20000 oocysts received a single dose of 5 mg/kg body weight (comparable to approx. 25 ppm in the feed) Bay Vg 7183 or Bay Vi 9142 orally (on day 3 or 4 p.i.), whereas others received two doses (on days 3 and 4 or on days 4 and 5 p.i.). The animals were killed on days 3, 4, 5, 6 and 7 p.i. and parts of the mucosa were dissected from the caecum (E. tenella), the ileum (E. maxima) and the duodenum (E. acervulina). Significant damage was observed in comparison to the controls, affecting nearly all of the parasites in those animals that had been treated twice, whereas some of the parasites remained microscopically unchanged after only one treatment. In general, the perinuclear space, mitochondria and the endoplasmatic reticulum were found to be considerably enlarged. Nuclear divisions were disturbed in schizonts and microgamonts, thus resulting in a greatly reduced production of parasites. The most important damage occurring in the macrogamonts concerned the wall-forming bodies II. As they burst, the formation of intact oocyst-walls was hindered, even if fertilization had taken place.

Toltrazuril effective against a broad spectrum of protozoan parasites

Toltrazuril, a sym. triazinone (Bay Vi 9142), has been reported to be highly effective against coccidia (Haberkorn and Stoltefuss 1984, 1987). In an electron microscopic study, Mehlhorn et al. (1984) showed that especially the intracellular stages (schizonts, gamonts) were destroyed, thus proving that the drug could pass through the host cell membrane and the cytoplasm and thereby ensuring its use as a therapeutic (coccidiocidal) tool (Raether 1988). The fact that toltrazuril enters the host cell and is available in a water-soluble form led to the idea of testing it in vivo against a variety of protozoan parasites in fish and insects (Figs. 1-6).

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).

Sym. triazinone (toltrazuril) effective against fish-parasitizing Monogenea

Sym. triazinone (toltrazuril, Code No Bay Vi 9142), which has been shown to be an anticoccidial drug (Haberkorn 1986; Haberkorn and Stoltefuss 1987; Haberkorn etal. 1983; Mehlhorn etal. 1984), is also effective against Monogenea parasitizing the gills or external surface of fish. Naturally infected carps, breams, roaches, eels, and threespined sticklebacks parasitized by Dactylogyrus vastator, D. extensus, D. cornu, Diplozoon homoion, Pseudodactylogyrus bini, and Gyrodactylus arcuatus were incubated in water containing 0.5, 10, 20, 30, or 50 gg toltrazuril/ml for 0.3, 1, 2, 3, and 4 h. The pure solvent (4 ml/1000 ml water) had no effect on either the fish or parasites. Toltrazuril caused irreversible damage to the tegument of Dactylogyrus species and P. bini, beginning at a dose of 5 gg/ml and 4 h exposure. In vitro treatment with toltrazuril causes death in D. paradoxurn (host=chub) and D. homoion after 4-80 min (10 ~tg/ml), depending on the age of the parasites. Specimens of G. arcuatus were severely affected after treatment with 20 gg/ml for 1 h. In all species, the teguments of the prohaptor and peduncle showed marked vacuolization and disruptions. We recommend that infections in Gyrodactylus and Dactylogyrus species and P. bini be treated using a water bath with 10 gg toltrazuril/ml for 4 h at 20~ (breams and roaches: 15 ~ C) under good aeration. Fish with extensive skin lesions (due to other infections) should carefully be observed during treatment, because this factor decreases their drug tolerance. By means of light, scanning (SEM) and transmission electron microscopy (TEM), it was clearly demonstrated that toltrazuril caused damage to

Die Entwicklung vonEimeria falciformis (Eimer 1870) in der weißen Maus (Mus musculus)

SummaryExperiments were carried out with a strain ofEimeria falciformis on more than 3,000 mice to clarify the infectious behaviour.The course of development takes place mainly in the caecum and in the upper half of the colon. In the jejunum single schizonts can be found and in the ileum several are found more or less regularly, but gametocytes can be demonstrated very seldom in the small intestine. The greater the inoculum, the larger is the part of the intestine that becomes infected. Stadia of the first schizogony generation are often found in the epithelial cells of the villi and the later stadia of development above all in the crypts. A schizogony generation is completed in 1.5 to 2 days, the gametocytes need 2 days to mature. Gametocytes can already develop from the merozoites of the 1st generation. Normally however, they develop out of the 2nd or 3rd and rarely from the 4th schizogony generation.The duration of the oocyst production depends on the number of inoculated oocysts. It lasts, at a maximum, from the 4th to 16th day. After reinfection, the excretion of oocysts begins anew. A full immunity is reached only after the 4th inoculation. The sporulation of oocysts requires 36 hrs to 5 days at 22° C. The oocysts that are excreted during the first days take longer to sporulate than those found in the feces from the 8th day on. The material for infection does not loose its infectivity for 3–4 months when potassium bichromate is added and then kept at +6° C. After 16 months storage at least 10% of the oocysts are still infective. Sporulated oocysts generally survive deep-freeze conservation at −79° C only 11 days. Single specimen however could be kept considerably longer.Oral, subcutaneous, intramuscular, intraveneous and intraperitoneal inoculation of oocysts cause identical infections.The course of infection is neither influenced by splenectomy, nor by keeping the mice at a lower temperature (+15° C instead of 20–24° C) the dose of infection influences the clinical symptoms. The reproductive potential is inversely proportional to the number of inoculated oocysts.The merozoites are, in regard to their further development to gametocytes or schizonts, not determined. The sexual determination must be phenotypical, since infections with only one merozoite also lead to a complete development ofE. falciformis.ZusammenfassungDie Entwicklung vonE. falciformis in der Maus wird beschrieben. Die Infektionsdosis beeinflußt direkt proportional die Schwere der klinischen Symptome, die Dauer der Oocystenausscheidung und die Ausdehnung der Infektion im Darm. Sie hat dagegen keinen Einfluß auf die Anzahl insgesamt ausgeschiedener Oocysten. Die Anzahl der Schizogoniezyklen ist nicht endogen festgelegt. Die Merozoiten sind hinsichtlich ihrer weiteren Entwicklung zu Schizonten oder Gamonten nicht determiniert, wie Übertragungsexperimente zeigten. Die Geschlechtsbestimmung der Merozoiten erfolgt phänotypisch. Auch intravenöse, intraperitoneale, intramuskuläre und subkutane Inokulation von Oocysten führt bei der Maus zu einem normalen Infektionsverlauf.

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.