Horst Taraschewski

Chemotherapy of fish parasites

There are few agents on the market that control fish parasites. These are substances that are mainly used in other hosts; due to the different metabolism of fish, they often have only moderate effects on fish parasites. Therefore, the research and development of fish-specific antiparasitic compounds is needed to avoid the high losses suffered by commercial fish hatcheries. Drugs similar to toltrazuril would perhaps be promising, due to their broad spectrum of efficacy.

Treatment of fish parasites: 5. The effects of sym. triazinone (Toltrazuril) on fish parasitic ciliophora (Ichthyophthirius multifiliis FOUQUET, 1876, Apiosoma amoebea GRENFELL, 1884, Trichodina sp. EHRENBERG, 1831).

For chemotherapy of fish parasitized by different Ciliophora (Protozoa) toltrazuril was tested in vivo and in vitro against skin and gill parasitizing species (e.g. Ichthyophthirius multifiliis, Apiosoma amoebea and Trichodina sp.). For in vitro treatment naturally infected fish were incubated at 20 °C for 0.3,1,2,4.5 h in water containing 0,1,5,10,20 or 50 μg toltrazuril/ml. Lethal damages already occurred in about one third of the trophozoites of I. multifiliis after incubation in 5 μg/ml for 4.5 h, and in more than two thirds of the trophozoites after incubation in 10 μ/ml for 2 h. A few trophozoites, however, were still able to leave their hosts, to encyst and to produce theronts. However, when treatment was done by intermittent therapy (1×10 μg/ml for 2 h, first day; 1 × 20 μg/ml for 1 h, second day; 1 × 20 μg/ml for 1 h, third day) all the trophozoites were lethally damaged. The damages consisted in the destruction of the outer cell membranes, of the cilia, and of the mitochondria, as well as in the complete abolishment of the ribosomes and in the enlargement of the nuclear space. After in vitro treatment (10 μg/ml, 2 h) all the trophozoites were lethally damaged. In contrast to the trophozoites, the free-swimming theronts of I. multifiliis were not affected by the drug. In vivo treatment starting with 20 μg/ml for 1 h led to severe damages in A. amoebea, which were intensified after treatment with 50 μg/ml for 0.3 h. The specimens were heavily contracted, and the oral cilia were redrawn. Furthermore, the pellicular pores seen in the surface of controls were not detectable. Despite the affections caused by the treatment the parasites did not drop off their hosts. In vivo treatment against Trichodina sp. led to a reduced motility in these parasites starting with 10 μg/ml for 2 h. Incubation with 20 μg/ml for 1 h caused a complete stop of motion in most of the specimens. The highest dose (50 μg/ml; 0.3 h) only caused a dropping off in about one third of the specimens from their hosts. Treated specimens of Trichodina sp. had a more flattened appearance compared to untreated controls, and the epistomial disc was drastically enlarged. From these experiments it is suggested that treatment against the trophozoites of I. multifiliis should be done by intermittent therapy according to the following scheme: 1st day: 10 μg/ml, 2 h, 2nd day: 20 μg/ml, 1 h; 3rd day: 20 μg/ml, 1 h. In the cases of Apiosoma spp. and Trichodina spp. infected fish incubating with 50 μg/ml for only 20 minutes is recommended.

Identification and characterization of the proteolytic enzymes in the developmental stages of the eel-pathogenic nematode Anguillicola crassus

The proteolytic activities of homogenates prepared from the second larva (L2) and the third larva (L3) as well as the adult stage of the eel-pathogenic nematodeAnguillicola crassus were examined using hemoglobin, azocoll, elastin-orcein, and keratin azure as substrates. Whole bodies of L2 larvae, the anterior third of the bodies of L3 larvae, and the anterior fifth of the bodies of adults were studied. Extracts of L2 contained a trypsin-like proteinase exhibiting a molecular weight of 38000 Da on gelatin-substrate gel electrophoresis. The proteinase showed a pH optimum at 8 and activity against azocoll and keratin. An apparent molecular weight of 25000 Da was determined for the trypsin-like proteinase of the L3. This enzyme possessed collagenolytic, keratinolytic and slight elastinolytic activity at an optimal pH of 8. Samples of adults contained an aspartyl proteinase with a molecular weight of 90000 Da. When hemoglobin was used as the substrate, the enzyme displayed optimal activity at pH 5. It was concluded that the proteinases of the larval stages are penetration enzymes, whereas that of the adult stage is a digestive enzyme.

Treatment of fish-parasites: 7. Effects of sym. triazinone (toltrazuril) on developmental stages of Myxobolus sp. Bütschli, 1882 (myxosporea, myxozoa): A light and electron microscopic study.

In research for chemotherapy of fish parasitized by myxosporeans toltrazuril(1) was tested in vivo against Myxobolus sp., parasitizing the connective tissue in gills of the bream, Abramis brama. Naturally infected breams were incubated at 20°C in water containing 0, 5, 10 or 20 μg toltrazuril/ml or the pure solvent for 4 or 2 h, respectively. After replacing the fish into fresh water, they were sacrificed on day 2 after the treatment and the pseudocysts were studied by means of transmission electron microscopy. It was found that the drug caused severe damages in all developmental stages of Myxobolus sp. except for the mature spores. Starting with a dose of 5 ug/ml and 4 h exposure, a destruction of the mitochondria and a decrease in the number of ribosomes were observed in the uni- and multicellular stages. In sporoblasts while developing the polar capsules the perinuclear space was enlarged in most of the specimens. The karyoplasms were more electron lucent compared to those of untreated controls. A focal lysis of the karyoplasm was observed in early pansporoblasts (characterized by primordia of the polar tubes). Treatment with 10 μ/ml for 4 h had more pronounced effects. The shape of these unicellular and multicellular stages was, however, not changed after treatment with 5 or 10 μg toltrazuril for 4 h, whereas 20 μg toltrazuril/ml for 2 h altered the surface of both stages and led to considerable degenerations of the cytoplasm. In premature pansporoblasts an extrusion of the polar capsules (leaving the capsulogenic cells) was caused when incubated in 20 μm toltrazuril/ml. The pure solvent had no effects on developmental stages of Myxobolus sp.. From these experiments it is suggested that chemotherapy against Myxobolus sp. should be accomplished by bathing the fish in aerated containers with 10 μg toltrazuril/ml for 4 h. This treatment will decrease considerably the output of spores. However, since the mature spores are not affected, the treatment has to be repeated within several months. Fish with extended skin lesions, caused by net-catching or infections by fungi, should be treated under careful observation, because these factors decrease the tolerance to the drug.

In vitro study of the migratory and adherent responses of fish leucocytes to the eel-pathogenic acanthocephalan Paratenuisentis ambiguus (van Cleave, 1921) Bullock et Sammuel, 1975 (Eoacanthocephala: Tenuisentidae)

The migratory response of fish leucocytes to infective larvae of the eelpathogenic acanthocephalan Paratenuisentis ambiguus was tested in vitro . Leucocytes of the accidental hosts carp ( Cyprinus carpio ) and rainbow trout ( Oncorhynchus mykiss ) showed a significantly higher response than the leucocytes of the natural final host, eel ( Anguilla anguilla ). Carp leucocytes caused damage to the tegument of the parasite, manifest as electron-lucent cavities in the striped layer and localised detachment of the surface coat. It is postulated that in carp the cellular defense system limits the longevity of P. ambiguus and thus plays a role in the determination of the host-specificity of this acanthocephalan.

Autoradiographic and morphological studies on the uptake of the triglyceride [3H]-glyceroltrioleate by acanthocephalans

Two eoacanthocephalans, four palaeacanthocephalans, including the infective larvae of one species, and one archiacanthocephalan were exposed in vitro to a labelled triglyceride. The nutrient was almost exclusively incorporated by the tegument of the presoma. The uptake of [3H]-glyceroltriolete started at the anterior half of the presoma, followed by a consecutive labelling of the entire presoma tegument and the lemnisci. In the archiacanthocephalan, however, the general uptake was comparatively low. In the final host of the acanthocephalans, the absorbed nutrient must derive from the hosts intestinal wall, injured by the parasite. In the eoacanthocephalans and the palaeacanthocephalans, the labelled nutrient was most intensively taken up by the tegument of the hooks. Inside the hook tegument, the basal membrane and the outer membrane form a labyrinth of entangling crypts and protuberances. In the surrounding proboscis tegument as well as in the neck tegument, the lipid seemed to be transported along these two membranes. In addition to its absorption by the presoma tegument, the labelled nutrient was intensively incorporated by the apical organ and the paired lateral organ of the eoacanthocephalan presoma and by the terminal part of the uterine endothelium of all female eoacanthocephalans and palaeacanthocephalans. The use of the nutrient by the parasites is discussed.

Transmission experiments on the host specificity ofHeterophyes species in 16 potential definitive hosts

Sixteen different species of piscivorous mammals and birds were tried as experimental definitive hosts forHeterophyes heterophyes, H. aequalis andH. dispar. The hosts were classified in four categories, by fluke longevity, recovery and size, and the number of uterine eggs (embryonated/unembryonated): (1) Canidae and the cat were highly susceptible hosts for all three species ofHeterophyes; (2) several mammals and herons showed a reduced susceptibility to infection (H. aequalis, 6 species;H. dispar, 1 species;H. heterophyes, 0 species); (3) In a group of hosts specific to each trematode, flukes were recovered up to 14 days post infection, but their uterine eggs did not become embryonated; (4) In a fourth category of hosts, chiefly Mustelidae, flukes could not be recovered. Taking also the literature into account it is concluded that man is a highly susceptible host forH. heterophyes, and that probablyH. aequalis andH. dispar may reach reproductive maturity in humans also. The described wide host range ofH. aequalis appears to be more typical for Heterophyidae than the comparably narrow host range ofH. heterophyes.

Loperamid, an efficacious drug against fish-pathogenic acanthocephalans

A total of 20 drugs were tested for their efficacy in the treatment of infections involving the two major acanthocephalans infesting rainbow trout in European trout farms. In in vitro experiments, the antidiarrhoeic loperamid and the well-known anthelminthic drug niclosamide showed the best efficacy. Worms treated with loperamid contracted the posterior end of their body, in which severe necrosis of the tegument caused the death of the worms. In in vivo experiments, loperamid was the most efficacious drug: 50 mg/kg given to rainbow trout on 3 consecutive days led to a complete cure. According to preliminary tolerance tests in water baths, the toxicity of this drug is low as compared with that of niclosamide, which is very toxic. Thus, loperamid can be recommended as the drug of choice for therapy of acanthocephalan infections in fish.

Licht- und elektronenmikroskopische Untersuchungen zur Histopathogenität vonMacracanthorhynchus hirudinaceus (Archiacanthocephala) in experimentell infizierten Hausschweinen

One and 12 weeks after infection the lesions in the small intestine of two domestic pigs caused byMacracanthorhynchus hirudinaceus were studied by means of light and electron microscopy. Worms of 7 d.p.i. were found to have penetrated with their presoma into the tunica propria and partly into the tunica muscularis where they had caused local mechanic destructions and heavy eosinophilic-like and hemorrhagic reactions. Most eosinophilic-like granulocytes were attracted to the proboscis hooks, at the base of which an osmiophilic substance apparently was excreted. Worms of 84 d.p.i. had penetrated with their proboscis deeply into the tunica muscularis. The presoma had become encapsulated by a solid capsule of necrotic/inflammatory tissue in its inner part and by filamentous connective tissue in its outer part. The predominant leucocytes in the inflammatory tissue now were plasma cells indicating a progressive immune reaction. No worms were found to have perforated the entire intestinal wall as was described from human patients in China.ZusammenfassungDie vonMacracanthorhynchus hirudinaceus verursachten Läsionen im Dünndarm von experimentell infizierten Hausschweinen (7 Tage p.i. und 84 Tage p.i.) wurden licht- und elektronenmikroskopisch untersucht. Die Würmer der 84 Tage alten Infektion waren tiefer in die Darmwand eingedrungen als die 7 Tage alten Würmer. Ihr Präsoma wurde von Entzündungsbzw. Bindegewebe umhüllt, das zahlreiche Plasmazellen enthielt. Bei 7 Tage alten Infektionen wurde noch keine Fibrose nachgewiesen. Eosinophilen-ähnliche Granulozyten, die an den Proboscishaken akkumulierten, herrschten in der Läsion vor. Eine osmiophile Gleitflüssigkeit, die offenbar an der Basis der Proboscishaken ausgeschieden wurde, schien eine stark antigene Wirkung auf die Leukozyten des Schweins auszuüben. In keinem Fall wurden Würmer angetroffen, die die gesamte Darmwand perforiert hatten, wie es in China bei Menschen beschrieben wurde.

Autoradiographic and morphological investigations on the uptake and incorporation of tritiated lysin by acanthocephalans

Four palaeacanthocephalans and two eoacanthocephalans from fish were autoradiographically investigated for their in vitro uptake of tritiated lysin. All worms took up the labelled amino acid at their surface without showing a species-specific pattern. The tegument of the metasoma was more heavily labelled than the presomal tegument, suggesting that the worms predominantly absorb the nutrient from the food inside the hosts gut in vivo. Within the metasomal tegument the nuclei revealed the most intense labelling. In the muscles the outer myogenic belt absorbed more radioactivity than did the enclosed cytoplasmic core. In female worms the ovarian balls and eggs that showed incomplete eggshell formation were highly labelled. In male worms the cement glands showed the most intense labelling of all organs inside the body cavity. We conclude that the investigated acanthocephalans require lysin for protein synthesis and for the coding of protein synthesis in the tegumental nuclei.