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: kinetoplastida--current level of knowledge and outlook.

The possibilities for treating haemoflagellate infections (African trypanosomiasis) are very limited (Table 1; Mehlhorn and Schrevel 1995; Croft 1997; Hunter 1997; Wang 1997; Trouiller and Olliaro 1998). All the available drugs have severe side-effects in humans and animals. Vaccination is not really an option, in view of the wide antigen variability. At present, there are several drug combinations in clinical trials: suramin/eflornithine, suramin/metronidazole, suramin/pentamidine, melarsoprol/pentamidine, melarsoprol/nifurtimox and nifurtimox/eflornithine. Some of these combinations were successful in treating resistant Trypanosoma brucei rhodesiense and/or T. b. gambiense infections (Keiser et al. 2001). In leishmaniasis, the tendency is still to resort to the old antimony compounds, with their severe side effects. At present, miltefosine is in clinical phase and is the first oral drug against visceral leishmaniasis (Jha et al. 1999). Two drugs are currently used against Chagas' disease, although these do not cure chronic effects. There is no prospect of novel drugs in this indication either (Pecoul et al. 1999; Morel 2000).

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).

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.

Studies of resistance to anticoccidials in Eimeria field isolates and pure Eimeria strains.

Ten Eimeria field isolates from North Germany were studied in battery tests for sensitivity to selected anticoccidials. A high percentage of the Eimeria field isolates (9 out of 10) showed resistance to anticoccidials, mostly multiple resistance. Partial or complete resistance to maduramicin was found in 7 field isolates, to monensin in 6, to salinomycin in 5, to nicarbazin in 8, to halofuginone in 7, to robenidine and toltrazuril in 1, and to diclazuril in 2 field isolates. Multiple resistance had developed in 7 of the 10 isolates. Cross-resistance between maduramicin, monensin, and salinomycin occurred in 5 Eimeria isolates. One isolate showed cross-resistance between diclazuril and toltrazuril. From the resistant isolates 15 pure E. acerculina and 5 pure E. brunetti strains were obtained by single oocyst infections. Seven of the E. acerculina and 4 of the E. brunetti strains showed resistance or partial resistance that was also present in the original isolate. Ten of 11 resistant strains were multiply resistant.

Chemotherapy of human and animal coccidioses: state and perspectives.

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 later 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.

Intraspecific polymorphisms of Eimeria species due to resistance against anticoccidial drugs.

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.

Possible mode of action of toltrazuril: studies on two Eimeria species and mammalian and Ascaris suum enzymes.

The anticoccidial properties of toltrazuril in Eimeria falciformis-infected mice were potentiated by the simultaneous application of pyrimethamine, trimethoprim, or sulfadimidine. The same drugs potentiate the effect of toltrazuril by killing E. 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 nematode Ascaris 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.

[Omega-aminoacyl compounds against malaria]. / Untersuchungen an malariawirksamen omega-Aminoacyl-Verbindungen.

Investigations on diphenylthioether derivatives led to compounds with a high antimalarial (P. berghei) activity. 58 new compounds were synthetisized in order to study structure-efficacy relationships. General formula: Efficacy was optimal in compounds with R1 = NO2. Some of such compounds were at least half as effective as chloroquine and fully effective against drug resistant strains of P. berghei. But also other radicals proved to be suitable as long as S was not replaced by O. But two diphenylthioether derivatives (R1 = NO2 resp. CN) were found to be mutagenic in Salmonella/Microsome Ames test. Replacement of S by O caused a loss of mutagenicity but also a markedly drop of antimalarial activity.

Causes of death in lethal rat malaria.

The involvement of the brain, lungs and kidneys was studied in a lethal rat malaria. Lewis inbred rats were infected with Plasmodium berghei K173. The disease proved fatal within 10-14 days. Parasitaemia showed an increase of up to 43% parasitised red blood cells on Day 10 p.i. The haematocrit decreased from 50% to 12%. The systolic blood pressure dropped from 99 to 56 mmHg. The lactate dehydrogenase activity rose to 2,543 U/l. BUN and serum creatinine doubled during the course of the disease. The transaminases increased tenfold and the cholinesterase decreased from 943 U/l to 271 U/l. Morphologically the kidneys showed an immune complex glomerulo-nephritis with a normal tubulo-interstitial system. The brain, heart and lungs were normal by light microscopic examination. Marked anaemia and shock were the main causes of death in the above-mentioned specimen rat, showing that the course of the disease is significantly different from lethal infections in humans with Plasmodium falciparum who show severe pulmonary, renal and cerebral complications.

Observations on malaria in European perching birds (passeriformes).

893 passeriformes birds belonging to 44 species and 14 families were investigated for blood parasites during a study running over 5 years. More than 1700 blood samples were taken. An average of 14% of the birds proved to be infected, most frequently with Leucozytozoon and Haemoproteus ( Parahaemoproteus ). Plasmodium sp. as well as other blood parasites were rarely found. Positive findings were correlated to the number of birds investigated per species, to the frequency and mode of inspections, the species of birds, the age of birds, and to the season. The latter is especially important for Haemoproteidaes . The distribution of exoerythrocytic schizonts in various organs is dealth with. The malaria infections only very rarely were fatal to the birds.

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

The development of three chicken coccidia (Eimeria tenella, E. acervulina and E. maxima) was studied by means of light and electron microscopy. One group of chickens infected with 6000-20,000 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 endoplasmic 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.

Control of an outbreak of mouse coccidiosis in a closed colony.

In 1979 severe coccidiosis occurred within a closed colony of C57bl/6J Bom mice. The coccidiosis caused diarrhoea, weight loss and deaths. Pathogenicity and response to chemotherapy were studied in experimentally infected Bor: CFW1 (SPF) mice. Histological and other studies indicated that infection was caused by more than 1 Eimeria species which have not yet been determined. Treatment with different anticoccidial drugs was unsuccessful, but use of 2 triazinone-derivatives (Bay g 7183, Bay i 9142) added to the food (15 ppm) succeeded in completely eradicating the organisms from the breeding houses. Experimental findings on the sensitivity of the coccidia to other anticoccidial drugs are described.

Ultrastructure of developing gamonts of Eimeria contorta Haberkorn, 1971 (Protozoa, Sporozoa) with emphasis on the host-parasite interface.

Gamonts of Eimeria contorta were observed in intestinal epithelial cells of rats on days 6 and 7 postinfection. Early gamonts of both sexes were bound by a single limiting membrane, underlain locally by additional membranes. Mature microgamonts were limited by a single membrane as well, whereas macrogametes had a three-membrane pellicle. Amylopectin was found in gamonts of both sexes. Microgametes developed while connected to the microgamont's surface by a reinforced stalk. Flagellar basal bodies contained microtubules arranged in triplets. Macrogamonts contained labyrinthine wall-forming bodies (type 2) within cisternae of the rough endoplasmic reticulum before homogeneous wall-forming bodies (type 1) were found in the cytoplasm. In both sexes, the host cell membrane of the parasitophorous vacuole showed numerous blebs and intravacuolar folds; the latter appeared to branch occasionally. Intravacuolar tubules (IT) were connected to the surface of macrogametes and also interconnected with each other. The IT may have functions other than nutrient uptake. A third type of intravacuolar structure found within macrogametes resembled coils of longitudinally striated tubules. In cross section, these coils consisted of a series of microtubules interconnected by membranous bridges. In advanced stages, the coils were arranged parallel to the macrogamete's surface, thus forming an "exterior layer." They may have a role in oocyst wall formation.

Experimental chemotherapy of mammalian coccidiosis with Bay g 7183.

Bay g 7183, a substituted sym. triazintrione derivative has a known high efficacy against poultry coccidia. Experimental investigations using the mouse coccidia E. falciformis indicated an activity also against mammalian coccidia. Minimum effective daily doses range between 0.25 to 10 mg/kg. Eimeria species used: E. falciformis (mouse(, E. contorta (rat), E. chinchillae (Mastomys), E. irresidua, E. magna, E. media, E. perforans, E. stiedae (rabbit), E. ashata, E. arloingi, E. faurei, E. ninakohlyakimovi, E. parva (sheep). A very appropriate mode of treatment, e.g. for rabbits is the dermal one (pour on) beside the oral route by using a stomach tube or with medicated food. Bay g 7183 is active both on schizogonic stages and gamonts. This explains the high efficacy even of single doses. If given e.g. once weekly it prevents or stops development of even massive experimental infections and their clinical symptoms. The value of E. falciformis as a test model is discussed.

Electron microscopic studies on Blastocrithidia triatomae Cerisola et al. 1971 (Trypanosomatidae).

In the bug Triatoma infestans developmental stages of Blastocrithidia tritomae, which can be mistaken for T. cruzi during xenodiagnosis, were studied by means of electron microscopy. It turned out that three different forms appeared during the progressive divisions: "micro-", "pro-" and epimastigotes. The morphological study revealed significant criteria for a distinction from T. cruzi stages. Furthermore cyst-like bodies were observed often attached to the flagella of the different stages. As revealed by the presence of labyrinthine structures found in flagellated stages and in the cyst stages the latter were suggested as being specific cysts to be excreted with the feces and thus to establish new infections in other bugs. Apart from the cyst-like structures a comparison with the organelles known from other Trypanosomatidae showed only slight variations. A tubular cytopharynx-like structure, which was lined by microtubules, and the way of attachment of the flagellum to the surface of the host cell has not been described in Blastocrithidia before.