Pathological changes of stomach in ringed seal (Pusa hispida) from Arviat (North Canada) caused by anisakid nematodes.

This is a report of lesions associated with the nematodes (Anisakidae) from the stomach of ringed seals (Pusa hispida). On thirty one examined ringed seals from Arviat, thirteen were infected by 1 to 24 anisakids. Identification of nematodes from their stomachs showed two species; Contracaecum osculatum (sensu lato) (79 specimens) and Pseudoterranova decipiens (sensu lato) (11 specimens). In the gastric sections with parasites, larvae and adults of nematodes were present mainly in the fundic portion of the stomach. The anterior parts of the nematodes were embedded in mucosa and submucosa. Anisakids were associated with ulcerous gastric lesions and raised inflammatory areas in the stomachs. The histological examination of a sample taken from the tissue surrounding anisakids revealed the presence of more or less confluent focal necrotic areas. Most small petechial hemorrhages were located in the mucosal layer of the gastric wall and were surrounded by inflammatory mononuclear cells such as lymphocytes, histiocytes, eosinophils and fibroblasts. The Anisakidae larvae in the stomach caused atrophy of glands, hemorrhaging and eosinophilic infiltrations that consequently healed, creating fibrotic scars.

Selected mitochondrial genes as species markers of the Arctic Contracaecum osculatum complex.

This study, aimed at testing the hypothesis that some mitochondrial genes can serve as species-specific markers, involved a comparison of the sequence variance of selected mitochondrial DNA genes of the Arctic Contracaecum osculatum species (C. osculatum A, C. osculatum B and C. osculatum C). We compared differences between five complete (ND2, CYTB, ND3, ND4L and ND6) and three partial (CO1, CO3 and ND5) protein-coding genes. The total length of the sequence of each of the 13 specimens was 4830 bp. The sample consisted of C. osculatum L3 larvae collected from Reinhardtius hippoglossoides and Gadus ogac from the Barents Sea and Davis Strait. The K2P distance values between the species ranged within 0.06-0.12, the intraspecific variability (0.01-0.03) proving 3-6 times lower. The lowest interspecific divergence was observed between C. osculatum A and C. osculatum B, whereas the highest intraspecific diversity was typical of C. osculatum C. Among the C. osculatum species studied, the highest nucleotide diversity was recorded in the CYTB, CO3 and ND5 genes. These genes may be useful in species identification of the very closely related Contracaecum sibling species.

Development of larval Contracaecum rudolphii Hartwich, 1964 (Ascaridida: Anisakidae) in experimentally infected goldfish (Carassius auratus L., 1758).

Laboratory-bred goldfish were experimentally infected with Contracaecum rudolphii, either directly (with nematode larvae) or indirectly via infected zooplankton. Intensity and prevalence of infection were markedly higher in fish exposed to the infected zooplankton than in goldfish exposed to nematode larvae. When transmitted to fish via the zooplankton, larvae developed much faster and, after 8 weeks, became firmly encysted in the intestine wall. The digestive tract of larvae isolated from the intestine wall showed a well-developed ventriculus, ventricular appendix and intestinal caecum. The mouth was surrounded by three lips. When free-living larvae induced infection, they rapidly penetrated the intestine wall and migrated to internal organs (e.g. liver). The primordial lips of those larvae were poorly visible, the ventricular appendix was short, and the intestinal caecum was absent. During the 10-week experiment, no encysted larvae were found in fish infected with free-living larvae.

Glycogen catabolism enzymes and protein fractions in the third and fourth larval stages of Anisakis simplex.

Extracts of Anisakis simplex third (L3) and fourth (L4) larval stages were assayed for protein content and activity and properties of alpha-amylase, glucoamylase and glycogen phosphorylase. Protein content in L4 was twice that in L3. SDS-PAGE applied to both larval stages revealed 22 protein fractions in each, including five stage-specific fractions in each larval stage. The L3 extracts contained three amylase isoenzymes: alpha 1, alpha 2 and alpha 3; their molecular weights were 64, 29 and 21 kDa, respectively. Only one amylase isoenzyme (64 kDa) was found in the L4 extracts. Glycogen in L3 was found to be broken down mostly by hydrolysis because of low glycogen phosphorylase activity. The alpha-amylase activity in L4 was higher than that in L3 by half and the glycogen phosphorylase activity was ten times higher. In addition, the same enzymes isolated from L3 and L4 were found to differ in their properties. These differences could be manifestations of metabolic adaptations of A. simplex larvae to host switch from fish (L3) to mammals (L4), i.e. adaptations to a new habitat.

Trehalose catabolism enzymes in L3 and L4 larvae of Anisakis simplex.

The presence of trehalase and trehalose phosphorylase in L3 and L4 larvae of Anisakis simplex was demonstrated. The activity of trehalase and trehalose phosphorylase in L3 larvae was 6 and 10 times higher, respectively, than in L4 larvae. This suggests that trehalose metabolism is more important for L3 than LA larvae. Trehalases of L3 and L4 differ in their characteristics. The enzyme of L3 was present mainly in the lysosomes and cytosol, whereas in L4 the highest enzyme activity was measured in the lysosomal fraction. Trehalase activity was increased by 29% in L3 and 55% in L4 with the addition of Mg2+ (0.1 mmol). Tris inhibited trehalase in L3 larvae by 42% and in L4 by 25%. The enzymes differed in their reaction to EDTA, CaCl2, ZnCl2, and CH2ICOOH (all 0.1 mmol). High activity of trehalase from L3 larvae was measured within the pH range of 5.0 to 6.5, with an optimum pH of 6.1. The trehalase was a thermally tolerant enzyme from 25 C to 60 C. The enzyme lost half of its activity after preincubation without substrate above 75 C. The paper also discusses the similarities and differences in characteristics of trehalase from A. simplex larvae and presents the comparison to enzymes from other nematodes.

Stickleback as a potential paratenic host in the Anisakis simplex life cycle in the Baltic Sea: results of experimental infection.

Anisakis simplex stage 3 larvae found in herring were used to experimentally infect 155 sticklebacks and 6 nine-spined sticklebacks, caught in the Gdynia marina (Gulf of Gdansk). The larvae were observed in the fish body cavity as early as 24 h after infection, only the sticklebacks being affected. Altogether, 50 live and 2 dead larvae were found in the stickleback body cavity. The infection prevalence and mean intensity were 13.6% and 2.4 larvae, respectively, from 1 to 8 larvae per fish being recorded. It is concluded that the stickleback may serve as a paratenic host for Anisakis simplex.

[The enzymes of carbohydrates metabolism from Cysttidicola farionis (Cystidicolidae)]. / Enzymy katabolizmu wwglowodanów z Cysttidicola farionis (Cystidicolidae).

The content of glycogen, glucose and trehalose was measured in larvae and adults of Cystidicola farionis, the parasite isolated from the swim bladder of Osmerus eperlanus from Vistula Lagoon. Activity of glycogen phosphorylase, alpha-amylase, glucoamylase, maltase, trehalase, and trehalose phosphorylase were measured. The highest activity was recorded for alpha-amylase 10.07 +/- 0.97 mu/mg and 7.47 +/- 0.24 mu/mg, next maltase 1.34 +/- 0.63 micromol/mg and 2.06 +/- 1.65 micronol/mg respectively for larvae and adults. The activity of glucoamylase was nearly the same for adults and larvae (about 0.20 micromol/mg). The trehalase activity was higher at adults (0.49 +/- 0.42 micromol/mg) than at larvae (0.18 +/- 0.12 micromol/mg). The activity of glycogen phosphorylase was much higher at larvae (3.58 +/- 1.49 micromol/mg) than at adults parasite (0.10 +/- 0.02 micromol/mg). The trehalose phosphorylase was present in both stages of parasite, but its activity was low. The content of glycogen and glucose was two-times higher in the adults' body than in larvae.

Influence of the pH of the cultivation medium on survival and development of stage III larvae of Anisakis simplex.

The cultivation was done on Eagle's media as well as Eagle's media enriched with the supplement of 10 and 20% of bovine serum. In the media without serum the larvae lived significantly shorter and did not get through molting. They lived the longest and came through molting the fastest in the media with pH 2.0 supplemented with 20% SB. In the media with the pH 4.0 and 7.3 the larvae lived significantly shorter and only a small proportion reached stage IV.

[The comparison of properties of alpha-amylase from the third and fourth stage of Anisakis simplex larvae]. / Porównanie wlasnosci alpha-amylazy z larw trzeciego i czwartego stadium Anisakis simplex.

alpha-Amylase is present in the third (L3) and in the fourth-stage (L4) of larvae from Anisakis simplex. The enzymes from both sources differ in some of their properties. alpha-Amylase from L3 showed a maximum at pH 7.8, enzyme from L4 stage at pH 6.5. The alpha-amylase from L3 was mainly lysosomal enzyme. The enzyme from L4 was located in the microsomal fraction. The L3 alpha-amylase showed the inhibition by EDTA and by -SH reagent iodoacetic acid. These agents did not change the activity of L4 enzyme. Both izoenzymes were unaffected by calcium and magnesium ions. Generally the alpha-amylase from L4 stage had higher activity (3.71 u/mg) than L3 one (2.29 u/mg).

A PCR-RFLP assay for identification of Anisakis simplex from different geographical regions.

Anisakis simplex, a nematode from the family Anisakidae, is a parasite of fish and mammals. It is a casual agent of a human disease called anisakiosis. We found that the assay based on PCR amplification of the ITS-1-5.8 S-ITS-2 fragment of rDNA and subsequent restriction fragment length polymorphism, previously described on the basis of A. simplex isolated solely from one geographical region, can be used as a general test for identification of this worm species. The restriction patterns analysed for four restriction enzymes were found to be identical in the case of all A. simplex individuals isolated from as different geographical regions as Baltic Sea, Norwegian Sea, Bering Sea and Sea of Okchotsk. Moreover, our results support the previously proposed hypothesis, based on the studies of isoenzymes, that there is a remarkable genetic homogeneity within A. simplex from different geographical regions.

The occurrence of the nematodes anisakis simplex pathogenic to man in pike-perch from the Vistula Lagoon, Poland.

Of the 400 pike-perches examined, 12% were infected with L3 Anisakis simplex. The infected fish were over 45 cm long. The hypothesis submitted was that the pike-perches may constitute the source of infection in accidental hosts, i.e. in man.

Bioaccumulation of selected trace elements in lung nematodes, Pseudalius inflexus, of harbor porpoise (Phocoena phocoena) in a Polish zone of the Baltic Sea.

Concentrations of Cd, Pb, Cu, Zn, Cr, Co, Ni, Mn and Fe were determined by AAS in lung nematodes and their host organ of harbor porpoise in the Polish zone of the Baltic Sea. Correlation coefficients computed for all element pairs indicate significant co-associations between concentrations of Zn and Mn as well as Mn and Fe in P. inflexus and the host organ, and for the pair Cu-Cr in the parasite. Based on both concentration and discrimination factors it is well documented that the metals studied, especially Fe, Mn and Zn are bioaccumulated in P. inflexus with respect to the host lung, showing significant inter-specimen variations.

Lead and cadmium content of two cestodes, Monobothrium wageneri and Bothriocephalus scorpii, and their fish hosts.

The adult cestodes Monobothrium wageneri and Bothriocephalus scorpii from the intestines of their respective final hosts, tench (Tinca tinca) caught in the river Ruhr, Germany, and turbot (Scophthalmus maximus) collected from two sampling sites on the coast of Gdansk, Poland, were analyzed for lead and cadmium by atomic absorption spectrometry. Both cestode species contained significantly higher cadmium contents than did the muscle, liver, and intestine of their fish hosts. Whereas M. wageneri also contained several times more lead than did the organs of tench, B. scorpii showed nearly the same lead burden as did the liver and intestine of turbot. Posterior sections of B. scorpii comprising gravid proglottids contained significantly higher concentrations of lead and cadmium than did the anterior proglottids.

Leukocyte parameters in the course of pig infection with III larval stage of Anisakis simplex.

Experimental anisakiosis covered 6 pigs infected with III larval stage of A. simplex. Changes in leukocytes were analysed. The blood showed an increased number of eosinophiles after 5th day. The number of High-affinity and EAC rosettes decreased after 8th day.

Haematological and morphological changes in experimental anisakiasis in pigs.

The aim of this paper is to establish the haematological and organic changes in halothane-sensitive and halothane-resistant pigs in the course of experimental anisakiasis. Experiments were carried out on two groups of pigs (3 animals each). The pigs from the first group were given fifty A. simplex B larvae, the pigs from the second one received ten larvae and then again fifteen larvae each after the 5th and 6th days. The number of leucocytes, neutrophils, lymphocytes, monocytes and eosinophils was greater--different in both series, but similar in halothane (stress)-resistance and -sensitive pigs. In the case of sensitive pigs much greater reactive changes were found in the stomach submucosa than in that of resistant pigs. In this group of pigs nematode larvae have also been traced in the submucosa of the same organ.

Infestation dynamics of external parasites of saithe (Pollachius virens) from the North and Norwegian Seas.

An attempt has been made to examine the saithe's external parasites with respect to host age and the reproduction time of Clavella adunca in the North Sea. 1835 specimens of Pollachius virens caught in 1976-1980 and 1986 were examined. They were infested with Diclidophora denticulata, Udonella caligorum (Monogenea) and Clavella adunca, Lepeophtheirus pollachius, Caligus elongatus (Copepoda). The large number of adult females of C. adunca in winter and spring, the juvenile forms in summer and forms of 4th developmental phase autumn suggest that the reproduction cycle runs over the year in the North Sea with the peak between the late spring and early summer. The intensity values of infestation are highest for sexually mature fish but fall in older ones.

Electrophoretic identification of Anisakis sp. larvae (Ascaridida: Anisakidae) from Clupea harengus L. in Baltic Sea.

On the basis of electrophoretic studies carried out on 15 gene-enzyme systems, 80 Anisakis sp. larvae from the herring Clupea harengus, fished in South Baltic Sea and Gdanisk Bay, were identified as Anisakis simplex B. This is the first record of Anisakis simplex B in the Baltic Sea. The spawning migration of the herrings from the North Sea to the Baltic Sea and the distribution of A. simplex B are briefly discussed.