The "bioeffect assessment index" (BAI). A concept for the quantification of effects of marine pollution by an integrated biomarker approach.

The "bioeffect assessment index" (BAI) is based on the integration of several pathological endpoints measured in the liver of European flounder (Platichthys flesus (L.)) during a long term study of biological effects of pollution in the German Bight. The BAI represents a modification of the "health assessment index" since it includes solely validated biomarkers reflecting toxically induced alterations at different levels of biological organisation in order to quantify the effects of environmental pollution. The concept of the BAI is based on the observation of progressive deleterious effects from early responses to late effects. Specific "key events" were detected, representing progressive stages of functional deterioration. The biomarkers selected from a whole battery of cellular markers for the BAI calculation reflect deleterious effects of various classes of contaminants such as heavy metals, organochlorines, pesticides, PAHs, and therefore reflect general toxicity in an integrative manner. Selected biomarkers were: lysosomal perturbations (reduced membrane stability), storage disorders (lipid accumulation) as early markers for toxic effects of liver cells, and the size of macrophage aggregates and their acid phosphatase activity. The latter two markers are indicative for the modulation of non-specific immune response which represents longer time scale responses after chronic exposure.

Purification of cytosolic glutathione transferases from Schistocephalus solidus (plerocercoid): interaction with anthelmintics and products of lipid peroxidation.

Glutathione (GSH) transferase isoenzymes have been partially resolved from the cytosol of Schistocephalus solidus (plerocercoid) by GSH affinity chromatography and chromatofocusing at pH 7-5. The presence of isomeric forms was also suggested by analytical isoelectric focusing and high-performance liquid chromatography (HPLC). Gel filtration and sodium dodecyl sulphate-polyacrylamide gel electrophoresis indicated that GSH transferase forms were dimers with a subunit size of approximately 24 kDa. The major GSH transferase form in S. solidus (plerocercoid) showed greater biochemical relationship to the Mu family of mammalian GSH transferase compared to the mammalian Alpha or Pi families. The major subunit purified by GSH affinity chromatography and reversed-phase HPLC also showed high N-terminal homology with the Mu family. A minor GSH transferase form appeared more biochemically related to the Alpha family with respect to substrate specificity and inhibitor sensitivity. The major GSH transferase was inhibited by haematin-related compounds, bile acids and a number of anthelmintics including members of the benzimidazole and phenol-based class of compounds. The major GSH transferase had conjugating activity with members of the trans, trans-2,4-alkadienal and trans-2-alkenal series, secondary products of lipid peroxidation.

Heterosporis schuberti n.sp., a new microsporidian parasite of aquarium fish.

Heterosporis schuberti n.sp. is described from the myocytes of an ornamental fish, Pseudocrenilabrus multicolor (Cichlidae). An apparently identical species was also found in Ancistrus cirrhosus (Loricariidae). Early meronts - uninucleate or plurinucleate - are perhaps responsible for dissemination of the infection throughout the muscle tissue. Later development of the microsporidian takes place in a structure encased with a thick envelope, for which the name sporophorocyst is proposed. At first, it contains merogony stages. Later, sporogony stages appear, too, which eventually prevail until a voluminous sporophorocyst is packed full with sporophorous vesicles with macrospores and rather rare microspores. Pleistophora anguillarumHoshina, 1951 reveals features similar enough to permit its reassignment to the genus HeterosporisSchubert, 1969.

Goussia carpelli (Protozoa: Coccidia) infection in stressed and immunosuppressed common carp Cyprinus carpio.

Goussia carpelli causes enteritic coccidiosis in juvenile carp. In nature it affects carp fry (1 to 2 mo old) and fish (3 to 4 mo old) which are subjected to environmental stress. Carp treated with corticosteroids or subjected to temperature stress in the laboratory produced higher numbers of oocysts during the primary infection. Resistance to reinfection via the fecal contamination route, however, was not reduced by the application of hydrocortisone, dexamethasone, or X-ray irradiation given both prior to and concurrently with reinfection. The administration of hydrocortisole or irradiation did not induce a relapse of a previous infection. Carp which had been immunosuppressed by hydrocortisole injection during a primary infection were also refractory to a secondary infection via fecal contamination. The results of these experiments suggest that the mechanisms which are responsible for the resistance of carp to secondary infections with Goussia carpelli were not affected by hydrocortisole, dexamethasone or X-ray treatment.

Changes of intestinal epithelial structure and cell turnover in carp Cyprinus carpio infected with Goussia carpelli (Protozoa: Apicomplexa).

Epithelial cell turnover in the intestine of common carp Cyprinus carpio infected with the coccidian parasite Goussia carpelli (Leger & Stankovitch, 1921) was investigated during laboratory infection using histological and electron microscopical techniques. During the development of the parasite an increased number of mitotic enterocytes, identified by bromodeoxyuridine (BRDU) uptake, were observed at the base of infected mucosal folds. During the merogonic and gamogonic development of the parasite, severe damage to infected epithelium occurred, and concomitantly BRDU-positive cells spread along the mucosal folds. These cells exhibited immature characteristics, including a squamous to cuboidal shape, nuclear apolarity, a high number of ribosomes, and short or reduced microvilli. Contact with adjoining cells was formed by tight junctions and desmosomes, indicating the epithelial origin of these cells. These cells covered gut segments with damaged epithelium within a few days, suggesting a high regenerative capacity of the carp intestine, and this could explain the mild clinical symptoms in fish affected by G. carpelli-coccidiosis. Our study for the first time describes epithelial cell responses to injuries caused by enteric protozoa in piscine hosts.

A study of sequential histopathology of Trypanoplasma borreli (Protozoa: Kinetoplastida) in susceptible common carp Cyprinus carpio.

The tissue response of common carp Cyprinus carpio to the kinetoplastid blood parasite Trypanoplasma borreli Laveran & Mesnil, 1901 was investigated during a laboratory infection of a highly susceptible carp line. With the development of the parasitaemia an increased proliferation of the lymphoid renal interstitial tissue was induced, which resulted in a progressive depression and deterioration of renal tubules. In heavily infected carp at Days 20 to 28 post inoculation (PI), a tubulonephrosis, a glomerulitis caused by a massive accumulation of leukocytes in glomerular capillaries, and large numbers of trypanoplasms in blood vessels and renal interstitium were observed. Corresponding with rising T. borreli numbers in the peripheral blood, splenic lymphocytes showed increasing proliferation rates, and the capillaries of the liver, gills, heart and intestine were infiltrated with lymphocytes and trypanoplasms. In heavily infected carp, congestion of liver sinusoids, focal necroses of hepatic tissue, extensive accumulations of erythrocytes in the spleen and in the blood marked anaemia were observed. These carp often showed abdominal distension, exophthalmus and swimming disorders described as 'sleeping sickness of carp'. Proliferation of cells from the interstitial lymphoid tissue of the kidney, which bears a close resemblance to the bone marrow of higher vertebrates, is considered a normal immune response of fish to antigen challenge. We here describe the unique case of a severe but ineffective immune reaction which results in the destruction of excretory renal structures. This has to be considered a severe disturbance of osmoregulation in affected carp, which, together with a decrease in oxygen uptake due to anaemia, is likely a major cause of death in these carp.

Cytopathological observations on renal tubule epithelium cells in common carp Cyprinus carpio under Trypanoplasma borreli (Protozoa: Kinetoplastida) infection.

Cytological alterations in renal tubule epithelium cells of carp Cyprinus carpio infected with the blood flagellate Trypanoplasma borreli Laveran & Mesnil, 1901 were investigated during the course of a laboratory infection of a highly susceptible carp line. With the development of the parasitaemia, a hyperplasia of the interstitial renal tissue was induced, which resulted in a tubulus necrosis. Cytological changes were already seen in tubulus epithelium cells on Day 7 post injection (PI) of the parasite. The basilar invaginations of the cells fragmented and a swelling of mitochondria was noted. With increasing parasitaemia, on Days 14 and 21 PI, these changes progressed up to the loss of the basilar invagination and high amplitude swellings of mitochondria and deterioration of their internal membrane structures. Cells of the distal tubule segment reacted earlier and more rapidly than cells of the proximal tubule. The cytological alterations suggested a loss of function of the epithelum cells, which most likely resulted in impaired ionic and osmotic regulation of T. borreli-infected fishes. Our findings indicate that in response to the proliferation of the interstitial renal tissue cell structures of the renal tubule cells are altered quickly and in a progressive manner.

Some immune parameters in carp cyprinus Carpio susceptible and resistant to the haemoflagellate Trypanoplasma borreli.

The present study addresses aspects of the (specific) immune response of carp to the haemoflagellate Trypanoplasma borreli. Sera of resistant carp contained antibodies, which agglutinated the flagellates in vitro. When flagellates were incubated in fish sera from resistant carp, binding of antibodies to flagellates could be demonstrated by flow cytometry, and T. borreli were effectively killed. Heat-treatment of the sera prevented killing, indicating that complement activation is important for the control of a T. borreli infection. Sera of carp that were highly susceptible to infection with T. borreli contained no antibodies capable of binding to or killing the parasite. Furthermore, specific antibodies were not generated after experimental infection. This lack of antibody production in susceptible carp is not due to a general unresponsiveness of lymphoid cells, since peripheral blood leukocytes (PBL) from susceptible and resistant carp responded to mitogenic stimuli in vitro with lymphocyte proliferation in a similar manner. However, viable flagellates were significantly less able to stimulate proliferation of PBL from susceptible carp. In vitro-produced culture supernatants of freshly isolated PBL from both carp lines (but not those of head kidney cells) differentially modulated the mitogen-induced proliferation of PBL from susceptible and resistant carp. The supernatants enhanced the proliferation of leukocytes obtained from individuals from the same carp line, but suppressed the mitogen-induced proliferation of PBL from the other line tested. This indicates that lymphoid cells from susceptible and resistant carp differ in their spectrum of spontaneously produced immunomodulatory mediators. Whether this is decisive for a T. borreli-specific and successful immune response is discussed.

In vitro cultivation of Trypanoplasma borreli (protozoa: kinetoplastida), a parasite from the blood of common carp Cyprinus carpio.

An in vitro culture system was developed for Trypanoplasma borreli, a pathogenic flagellate from the blood of European cyprinids. Trypanoplasms multiplied rapidly in a mixture of Hanks' balanced salt solution (HBSS, 45%), L15 (22.5%), Earle's minimum essential medium (MEM, 22.5%) and 10% distilled water, which was supplemented with 5 to 10% heat-inactivated pooled carp serum. In medium supplemented with fetal bovine serum, multiplication of T. borreli seemed to be inhibited. Cultures initiated with less than 100 000 T. borreli per ml culture medium did not survive, and a substantial multiplication of trypanoplasms was found at inocula beginning with 630 000 flagellates ml(-1). Trypanoplasms multiplied at 15, 20 and 25 degrees C. In cultures incubated at 4 degrees C the trypanoplasms remained viable but the number of flagellates did not increase. Trypanoplasms from in vitro cultures retained their infectivity for carp for at least 90 d (5 passages). The trypanoplasms survived in culture over a period of up to 5 mo (10 passages). The established culture system allows the propagation of high numbers of fish-infective trypanoplasms, which are required to study parasite-host relationships in carp.

Flow cytometric analysis of proliferative responses of carp Cyprinus carpio peripheral blood leukocytes to mitogens and to the hemoflagellate Trypanoplasma borreli.

The activation of carp peripheral blood leukocytes (PBL) was analysed radiometrically and by means of flow cytometry (FCM) in order to compare the results obtained with both methods. The qualitative and quantitative FCM analyses of cellular morphology and viability resulted in a further characterisation of proliferative responses of carp PBL to Trypanoplasma borreli in vivo and in vitro. The lymphocyte population of PBL from T. borreli-infected carp exhibited a marked shift in forward scattered light (FSC; cell size). When PBL from healthy carp were stimulated with mitogens in vitro, a lymphoid population with increased FSC profiles was also observed. The number of these cells coincided to ratios of 3H-thymidine incorporation, recorded from corresponding cultures. Thus, it was concluded that the increase in size of stimulated lymphocytes could be due to blastogenic transformation. The advantage of the FCM procedure is that activation and proliferation of carp lymphocytes can be monitored without labelling the cells. Cocultures of mitogen-stimulated carp PBL and T. borreli revealed the ability of the parasite to suppress lymphocyte proliferation in vitro.

The role of tubificid oligochaetes in the transmission of Goussia carpelli.

Tubifex tubifex and Limnodrilus hoffmeisteri, fed intestinal tissue of carp containing oocysts of Goussia carpelli, produced infections in laboratory-reared carp. Sporocysts ingested by the tubificids released the sporozoites that were found to be motile in the intestinal contents and incorporated in intestinal cells of the oligochaetes. Tubificids remained infective for carp at least up to 57 days postexposure (PE). Tubificids were also able to transmit G. carpelli to carp that had recovered from a previous infection, whereas attempts of direct transmission of G. carpelli among these carp failed. Direct transmission to uninfected carp by fecal contamination was possible. Organisms from the pond plankton, benthic Limnadia sp., and chironomid larvae did not transmit G. carpelli to carp.

The parasitemia of cloned Trypanoplasma borreli Laveran and Mesnil, 1901, in laboratory-infected common carp (Cyprinus carpio L.).

The course of parasitemia of cloned Trypanoplasma borreli in laboratory-infected common carp was investigated. In 25-42-g carp kept at 20 C, the prepatent period was 8 days; after a phase of exponential growth, the parasitemia peaked at day 39 postinjection (PI) at a level of about 10(3) T. borreli/microliters blood. This maximum was followed by a chronic phase of about 6 wk with large numbers of T. borreli. At 20 wk PI, T. borreli was absent in infected carp. In 2.2-g carp kept at 20 C, the prepatent period was 4 days only, and the parasitemia peaked at day 23 PI. At 30 C, T. borreli was present in the blood only for 12 wk, and the number of T. borreli did not exceed 162 trypanoplasms/microliters blood. Carp kept at 8 and 15 C showed retarded development of parasitemia. The prepatent period lasted longer and the generation time was increased, but the level of parasitemia was not affected. Carp, inoculated at 8 C and then warmed to 20 C on days 27 and 55 PI, developed a parasitemia of 10(4) flagellates/microliters blood and showed high mortalities. During the prepatent period, T. borreli was found in the muscle tissue of the inoculation area but in no other tissue. In the kidney, T. borreli was found 27 hr PI, whereas in the circulating blood it was manifest at day 3 PI. At the same time it was manifest in the liver and spleen.

Development of Trypanoplasma borreli (Mastigophora: Kinetoplastida) in the leech vector Piscicola geometra and its infectivity for the common carp, Cyprinus carpio.

The development of Trypanoplasma borreli in the crop of the leech vector Piscicola geometra was characterized by significant changes in morphology. Immediately after ingestion by the leech, stumpy-shaped T. borreli predominated and numerous dividing specimens were found. This led to long and slender trypanoplasms near the end of the infection. The infection was terminated with complete digestion of the blood stored in the crop of the leech. The longest period of infection observed was 11 days. Trypanoplasma borreli was found only in the crop of the leech. At any time during the infection, T. borreli isolated from P. geometra cause a parasitemia when inoculated into parasite-free carp. There was no difference in morphology or infectivity among T. borreli isolated from various crop regions of P. geometra.

Piscine gill epithelial cell necrosis due to Mycoplasma mobile strain 163 K: comparison of in-vivo and in-vitro infection.

This paper describes experiments with Mycoplasma mobile 163 K in tench inoculated via the gills, skin, peritoneal cavity or whole body surface and kept at two different temperatures (20 and 25 degrees C). Gill tissues from experimentally infected tench and rainbow-trout gill tissue explants infected in vitro were compared by transmission electron microscopy, revealing that M. mobile was capable of producing gill epithelial cell necrosis in both, but that it was much more severe in the explants. M. mobile was found attached to chloride cells in the tench and between necrotic epithelial cells in the trout gill explants. M. mobile was recovered from the gills for up to 28 days after inoculation, from the skin and swim bladder for up to 14 days, and from the hind gut, kidneys and spleen for up to 8 days. There was no significant difference between the results at 20 and 25 degrees C.

Some nematodes and acanthocephalans from exotic ornamental freshwater fishes imported into Germany.

Five species of adult nematodes, unidentifiable nematode larvae, and three species of acanthocephalans, were found in freshwater ornamental fishes newly imported into Germany from Brazil, Colombia, Indonesia, Malaysia, Nigeria, Peru, Sri Lanka and Thailand. The following species were identified: Adult Nematoda: Pseudocapillaria tomentosa, Capillariidae gen. sp., Dichelyne hartwichi sp. n., Procamallanus (Spirocamallanus) pintoi and Spinitectus allaeri; Acanthocephala: Pseudogorgorhynchus arii gen. et sp. n., Neoechinorhynchus sp. and Pallisentis sp. The nematode Dichelyne hartwichi sp. n. (male only) from the intestine of Chelonodon fluviatilis (Hamilton) from Thailand is characterised mainly by the presence of minute cuticular spines on the tail tip, length of spicules (510 microns) and arrangement of caudal papillae. The acanthocephalan Pseudogorgorhynchus arii sp. n. from the intestine of Ariopsis seemanni (Günther) from Colombia represents a new genus Pseudogorgorhynchus gen. n., differing from other genera of the Rhadinorhynchidae mainly in possessing a small proboscis armed with markedly few (18) hooks arranged in six spiral rows. Spinitectus macheirus Boomker et Puylaert, 1994 and Spinitectus moraveci Boomker et Puylaert, 1994 are considered junior synonyms of Spinitectus allaeri Campana-Rouget, 1961.