Digenean Holostephanus (Trematoda: Digenea: Cyathocotylidae) metacercariae in common carp (Cyprinus carpio Linnaeus, 1758) muscle: zoonotic potential and sensitivity to physico-chemical treatments.

Metacercariae of various species within the genus Holostephanus Szidat, 1936 (Trematoda: Digenea: Cyathocotylidae) occur in muscles of both farmed and wild fish, including common carp (Cyprinus carpio Linnaeus, 1758). The life cycle includes a snail as first intermediate host, fish as second intermediate host and birds or mammals as final hosts. We studied the zoonotic potential and the viability of Holostephanus metacercariae from common carp following exposure to various physical and chemical treatments. Muscle tissue samples of common carp specimens from a fish farm in the north-eastern part of Hungary were examined and metacercariae recovered. The zoonotic potential was evaluated experimentally by using small mammals as models (albino mice, n = 2; and Syrian hamsters, n = 4) infected per os with Holostephanus cysts. Parallelly, Metagonimus metacercariae were used as positive controls. We could not confirm the zoonotic potential of Holostephanus metacercariae as they did not survive in the mammalian intestine whereas Metagonimus metacercariae developed to the adult stage. We assessed the viability of metacercariae isolated from common carp specimens during exposure to different physical treatments (temperatures of -18°C, +20°C, +40°C and +60°C) and chemical agents (5% and 10% acetic acid and 10% sodium chloride (NaCl)). Metacercariae lost viability by freezing at -18°C (2 h), heating at 60°C (20 min), incubation in 5% and 10% acetic acid (5 min) and 10% NaCl (2 h). These methods served as models to investigate the effectiveness of food preparation techniques (such as cold and hot smoking, freezing, salting and pickling) on the survival of metacercariae.

Differential survival of 3rd stage larvae of Contracaecum rudolphii type B infecting common bream (Abramis brama) and common carp (Cyprinus carpio).

The main fish host reaction to an infection with third stage anisakid nematode larvae is a response in which host immune cells (macrophages, granulocytes, lymphocytes) in affected internal organs initially are attracted to the parasite whereafter fibroblasts may enclose the parasite forming granuloma. Generally, the reaction is non-lethal to the parasite which may survive for years in the fish host retaining infectivity to the final host. This may also apply for the anisakid nematode Contracaecum rudolphii (having the adult stage in cormorants, using copepods as first intermediate/paratenic host and zooplankton feeding fish as paratenic hosts). The present study has shown that most Contracaecum rudolphii larvae survive in bream (Abramis brama) (from Lake Balaton, Hungary) whereas the majority of the nematode larvae die in Cyprinus carpio (from Lake Hévíz, directly connected to Lake Balaton). Both cyprinid host species interacted with the nematode larvae through establishing a marked cellular encapsulation around them but with different effects. The differential survival in common carp and bream may theoretically be explained by ecological factors, such as the environmental temperature which either directly or indirectly affect the development of nematode larvae, and/or intrinsic host factors, such as differential immune responses and host genetics.

Histopathological changes on the gills of asp (Aspius aspius) and European catfish (Silurus glanis) caused by Lamproglena pulchella and a Lamproglena sp. (Copepoda: Lernaeidae), respectively.

In a parasitology survey of Hungarian fishes, heavy infections of parasitic copepods Lamproglena pulchella and a Lamproglena sp. were found in the gills of the asp and the European catfish, respectively. Individuals of both fish species were emaciated and infected with hundreds of Lamproglena. Copepods located close to the tip of gill filaments and formed a depression at the attachment sites. In histological sections, cell degenerations and local haemorrhages were present adjacent to the maxillipeds and where the maxillary claws pierced the gill tissue. Around maxillae and in the midgut of the Lamproglena, damaged piscine blood cells and remains of the gill tissue were observed. Host reaction was expressed by proliferation of epithelioid cells, increase in both number and size of goblet and mast cells and formation of giant cells.

Malformations of the gill filaments of the ruffe Gymnocephalus cernuus (L.) (Pisces) caused by echinostomatid metacercariae.

In parasite surveys of fishes from Lake Balaton and its tributaries in Hungary, infections with metacercariae of a species of the digenean genus Echinochasmus (Trematoda: Echinostomatidae) were found in seven species of fish. In ruffe, Gymnocephalus cernuus, malformations of the gill filaments apparently caused by these infections were observed. These malformations were in the form of bifurcations of the filaments at about their mid-length. At the point where the filaments bifurcate, an Echinochasmus metacercaria was always embedded in the cartilaginous ray of the gill filament. All specimens of the ruffe were found to be infected by these metacercariae, and each ruffe specimen was infected by 30-300 metacercariae. Such a bifurcation was found in all of the ruffe specimens, but, apart from these gill malformations, the metacercariae produced only local changes in the cartilage. In the other six infected fish species, only local signs were observed in the cartilage. Experimental infections of chicks with metacercariae resulted in the finding of the sexual adult (marita) of an unidentified species of Echinochasmus. ITS sequences of the adult and metacercaria corresponded with each other, and also with a cercaria isolated from a gravel snail (Lithoglyphus naticoides), with a 99.5-100% similarity.

An evaluation of indices of gross pathology associated with the nematode Anguillicoloides crassus in eels.

This study compares two alternative indices for quantifying the gross pathology of the swimbladder of eels, Anguilla anguilla (L.), infected with the nematode Anguillicoloides crassus. Two observers recorded twice the scores obtained by the two indices on the same set of 71 wild caught eels (from elver to silver eels, French Mediterranean lagoons). The Length Ratio Index (LRI), performed better than the Swimbladder Degenerative Index (SDI), in three of four predefined criteria of decision. First, the LRI better correlated with an estimate of the swimbladder volume reduction, a functional consequence of the infection (representativeness). Also, the LRI was less prone to subjectivity (inter-observer variability) and more precise (intra-observer variability), although less easy to generate (time needed for measurement/assessment). Using a sub-sample of 32 unaffected eels (showing minor if any swimbladder damage and no living worms at autopsy), we ascertained a linear relationship between the swimbladder length and the total body length, a prerequisite of isometric growth, to definitively accept the new ratio index as a valid alternative to the SDI. Also, because the LRI can be recorded on live specimens with radio-imagery (non-invasive method), we recommend its use, and provide a graph of correspondence between the SDI scores, the LRI scores and the estimated proportion of gas loss in the swimbladder.

Evidence of the American Myxobolus dechtiari was introduced along with its host Lepomis gibbosus in Europe: Molecular and histological data.

The American pumpkinseed Lepomis gibbosus, was introduced to Europe more than one hundred years ago. Currently it is a common fish in European freshwaters but relatively few specific parasites infect this fish in this new habitat. In Europe only a single species, Myxobolus dechtiari seems to represent the American myxosporean fauna of centrarchid fishes. M. dechtiari was found in both Portugal and Hungary. This species forms plasmodia with elongated shape inside the cartilaginous rays of gill filaments. In the advanced stage of infection, after disruption of plasmodia, small groups of myxospores remain enclosed in the cartilaginous gill rays causing distortions in the filaments. Myxospores were ellipsoidal in frontal view and lemon-shape in sutural, length 12.5 ± 0.46 (12-13.4) µm, width 10 ± 0.37 (9.6-10.4) µm, and thickness 7.4 ± 0.37 (7-8) µm; the polar capsules were pyriform, equal in size, length 5.6 ± 0.21 (5.3-6) µm, width 3.2 ± 0.16 (3-3.6) µm; Seven to eight polar tube coils were arranged perpendicularly to the capsule length. There was a small, round, 0.4 ± 0.1 (0.3-05) (N = 50) intercapsular appendix in the spores. The small subunit ribosomal DNA (ssrDNA) of M. dechtiari differed from other myxozoans sequenced to date. Phylogenetic analysis of the ssrDNA gene sequence placed this species in a clade including actinospores and Myxobolus species: Raabeia type1, Triactinomyxon sp., and Myxobolus osburni infecting the same host fish. The focus of our study was to prove that the pumpkinseed, a fish originated from North-America introduced one of its myxosporean parasite to Europe. Emphasis was put on to demonstrate the unique feature of this parasite causing infection in the cartilaginous gill rays.

Molecular Genetic Studies on Myxobolus cylindricus and Henneguya mystasi (Myxosporea: Myxobolidae) Infecting Two Indian Fish Species, Channa gachua and Mystus vittatus, Respectively.

INTRODUCTION: Myxozoan infections of Indian freshwater fishes are relatively well studied, but their validity is supported with molecular and phylogenetic data only for a few species. OBJECTIVES: The primary objective was to present molecular data for two Myxozoan species, Myxobolus cylindricus and Henneguya gachua collected from Indian freshwater fishes, the dwarf snakehead Channa gachua and the striped dwarf catfish Mystus vittatus, respectively. METHODS: Various organs of 56 C. gachua and 48 M. vittatus were dissected. Myxozoan plasmodia with mature spores were collected from the gills under a dissecting microscope. Spores obtained from plasmodia from infected hosts were fixed in 80% ethanol in vials and sent for further morphological and molecular examinations to Hungary. The 18S rDNA gene of Myxobolus and Henneguya spp. was amplified using nested polymerase chain reaction (PCR). Phylogenetic analysis was performed using maximum likelihood (ML) and Bayesian inference (BI) methods. RESULTS: Morphological characteristics of M. cylindricus and H. mystasi spores corresponded to the original descriptions made by Sarkar, Mazumdar and Pramanik, 1985 and Haldar, Samal, and Mukhopadhyay, 1997, respectively. Phylogenetic analysis of the 18S rDNA gene revealed that the sequences of M. cylindricus differed from those of most Indian Myxobolus sp., known mostly from cyprinid fishes and formed a subgroup with Myxobolus neurophilus, a parasite of a perciform host, and with Henneguya chaudhuryi, a species belonging to a different genus but described from a closely related channid host. It was also closely related to another Henneguya species, H. lesteri, described from Sillago analis, a coastal fish. Henneguya mystasi had the closest similarity to Henneguya bicaudi, a species described from an Indian cyprinid fish and to Henneguya pellucida reported from a characid fish known from South America. CONCLUSION: Molecular data received by us gives a solid basis for further identification of these myxozoans, the pathogenicity of which probably plays an economic role at culturing the hosts.

Infection of the heart of the common bream, Abramis brama (L.), with Myxobolus s.l. dogieli (Myxozoa, Myxobolidae).

Myxobolus dogieli Bykhovskaya-Pavlovskaya & Bykhovski, 1940 is regarded as a site specific myxosporean, infecting the heart of cyprinid fish. During a survey of the myxosporean fauna of Lake Balaton fish, heart myxobolosis was found in the common bream, Abramis brama, with heavy infection of the ventricle and the bulbus arteriosus in some infected bream. Developing and mature plasmodia were mostly in the connective tissue of the subepicardium and subendocardium. Plasmodia developing in the subendocardium protruded into the lumen of the heart, while plasmodia developing in the subepicardium protruded over the epicardium forming large sausage-like outgrowths. Plasmodia with mature spores were found in the summer. The shape and size of the spores corresponded to those of the original description. Phylogenetic analysis based on the 18S rDNA sequence of M. dogieli showed that this species fit well in the genus Myxobolus. As no molecular data are available on spores from the type host, common carp, the species studied by us is temporarily designated as Myxobolus s.l. dogieli.

Prevention of Alzheimer's disease.

The already considerable public health burden of Alzheimer's disease will likely worsen as populations around the world age. As a result, there is considerable motivation to develop effective strategies for preventing the disease. A wide variety of such strategies are under investigation and include pharmaceuticals, nutriceuticals, diet, physical activity and cognitive activity. We review here the most promising candidates and the epidemiologic evidence for their efficacy. Although none of these have yet to be definitively shown to prevent Alzheimer's disease, further research should help to clarify what role they may play in reducing the burden of this disease.

Description of Myxobolus gayerae sp. n. and re-description of M. leuciscini infecting European chub from the Hungarian stretch of the river Danube.

Myxobolus gayerae sp. n. and M. leuciscini González-Lanza & Alvarez-Pellitero, 1985 (Myxozoa: Myxobolidae) have been described and re-described from European chub Leuciscus cephalus L. from the Hungarian stretch of the river Danube. The ellipsoidal plasmodia of M. gayerae sp. n. were found in the mucosa of the intestinal wall, whereas the large, elongated plasmodia of M. leuciscini infected the afferent arteries of the gill filaments. The spores of M. gayerae sp. n. are relatively large, slightly oval and almost rectangular in shape. On the basis of spore morphology and 18S rDNA sequences, the most similar species was M. cycloides Gurley, 1893, but the 2 species differed in host and tissue tropism as well as in the size of the spores. The spores of M. leuciscini from L. cephalus, having no intercapsular appendix or occasionally a very small one, showed a high morphological similarity to spores collected from L. cephalus cabeda, Chondrostoma polylepis and Rutilus arcasi in Spain and described as M. leuciscini González-Lanza & Alvarez-Pellitero, 1985.

Myxobolus infections of common carp (Cyprinus carpio) in Syrian fish farms.

During a survey on Myxobolus infection of pond-cultured common carp in Syria three Myxobolus spp. were found. Myxobolus dispar infected the gill arteries, forming large elongated plasmodia in the gill filaments. The plasmodia of M basilamellaris were located in the gill arches at the base of the filaments. Elongated filiform plasmodia of M. encephalicus were found in the blood vessels of the brain. Despite the common occurrence of the above parasites, no disease symptoms were observed in the infected fish specimens. This is the first report on myxosporean infection of fish from Syrian waters.

Myxozoan pathogens in cultured Malaysian fishes. I. Myxozoan infections of the sutchi catfish Pangasius hypophthalmus in freshwater cage cultures.

Cage-cultured sutchi catfish Pangasius hypophthalmus (Sauvage, 1878), a favourite food fish in Southeast Asia, proved to be infected by 6 myxozoan species. Three species belonged to the genus Hennegoides (H. berlandi, H. malayensis, and H. pangasii), 1 to Henneguya (H. shariffi) and 2 to Myxobolus (M. baskai, and M. pangasii). Five myxozoans infected the gills and 1 was found on the spleen. Myxozoans infecting the gills were characterised by a specific site selection. H. shariffi sp. n. and H. berlandi sp. n. formed plasmodia in the multi-layered epithelium of the gill filaments. Of the 2 vascular species H. pangasii sp. n. developed in the gill arteries, while M. baskai sp. n. infected the capillary network of the gill lamellae. Plasmodia of H. malayensis sp. n. were found inside the cartilaginous gill rays of the filaments. Large plasmodia of M. pangasii sp. n. were located in a groove of the spleen but they affected only the serosa layer covering the spleen.

Myxozoan pathogens in cultured Malaysian fishes. II. Myxozoan infections of redtail catfish Hemibagrus nemurus in freshwater cage cultures.

Cage-cultured Asian redtail catfish Hemibagrus nemurus (Valenciennes, 1840), a popular food fish in Southeast Asia, proved to be infected by 3 myxozoan species. All the 3 species belonged to the genus Henneguya: 2 were identified as H. mystusia Sarkar, 1985 and H. hemibagri Tchang et Ma, 1993, while the other was described as H. basifilamentalis sp. n. All plasmodia were found in the gills and were characterised by a specific site selection. H. mystusia formed plasmodia in the multi-layered epithelium between the gill lamellae and in the non-lamellar edge of the gill filaments, while H. hemibagri developed in the capillary network of the lamellae. H. basifilamentalis sp. n. had large oval plasmodia located deep among the filaments just above the gill arch.

Longitudinal change in hippocampal volume as a function of apolipoprotein E genotype.

The epsilon4 allele of the apolipoprotein E (APOE) gene confers an increased risk for the development of AD. The authors compared longitudinal rates of change in hippocampal volume as a function of APOE genotype in nondemented elderly individuals. Rate of volumetric loss was significantly greater among epsilon4+ compared with epsilon4- individuals. These results indicate that individuals positive for the APOE epsilon4 allele may show a greater rate of hippocampal atrophy than their epsilon4- counterparts, even in the absence of a diagnosis of AD.

Quantitative MR volumetry in Alzheimer's disease. Topographic markers and the effects of sex and education.

We determined topographic selectivity and diagnostic utility of brain atrophy in probable Alzheimer's disease (AD) and correlations with demographic factors such as age, sex, and education. Computerized imaging analysis techniques were applied to MR images in 32 patients with probable AD and 20 age- and sex-matched normal control subjects using tissue segmentation and three-dimensional surface rendering to obtain individualized lobar volumes, corrected for head size by a residualization technique. Group differences emerged in gray and white matter compartments particularly in parietal and temporal lobes. Logistic regression demonstrated that larger parietal and temporal ventricular CSF compartments and smaller temporal gray matter predicted AD group membership with an area under the receiver operating characteristic curve of 0.92. On multiple regression analysis using age, sex, education, duration, and severity of cognitive decline to predict regional atrophy in the AD subjects, sex consistently entered the model for the frontal, temporal, and parietal ventricular compartments. In the parietal region, for example, sex accounted for 27% of the variance in the parietal CSF compartment and years of education accounted for an additional 15%, with women showing less ventricular enlargement and individuals with more years of education showing more ventricular enlargement in this region. Topographic selectivity of atrophic changes can be detected using quantitative volumetry and can differentiate AD from normal aging. Differential effects of sex and years of education can also be detected by these methods. Quantification of tissue volumes in vulnerable regions offers the potential for monitoring longitudinal change in response to treatment.

Memory impairments associated with hippocampal versus parahippocampal-gyrus atrophy: an MR volumetry study in Alzheimer's disease.

Delayed memory impairments and medial temporal-lobe atrophy are considered to be cardinal features of Alzheimer's disease (AD). The goal of the present magnetic resonance (MR) volumetry study was to investigate the relationship between both features. We determined MR-derived estimates of hippocampal and parahippocampal volume in a sample of 27 AD patients and in a group of 26 healthy control subjects (NCs) of comparable age and education. We examined the performance of the two groups on immediate and delayed recall trials of an auditory-verbal list-learning task (CVLT), a visual non-verbal memory task (Visual Reproduction of the WMS-R), and a screening procedure that provides an estimate of overall cognitive functioning (DRS). Volumes of the hippocampus and the parahippocampal gyrus were significantly smaller in AD patients than in NCs. AD patients were impaired in their overall level of cognitive functioning and showed memory deficits under immediate and delayed recall conditions. The association between medial temporal-lobe atrophy and cognitive impairments in AD was found to be highly specific: Hippocampal volume correlated positively with delayed but not immediate recall of the verbal auditory list learning task. In contrast, parahippocampal gyrus volume, specifically in the right hemisphere, was positively related to delayed but not immediate recall of the non-verbal visual memory task. In NCs, there was a trend towards a negative association between hippocampal volumes and delayed verbal recall. Our results suggest that hippocampal and parahippocampal gyrus atrophy in AD are related to distinct aspects of the patients' memory impairments. Our findings have implications for current discussions regarding contributions of the hippocampus and the parahippocampal gyrus to memory in the intact human brain.

Radiodiagnostic method for studying swimbladder inflammation caused by Anguillicola crassus (Nematoda: Dracunculoidea).

The nematode Anguillicola crassus appeared in Europe about 15 yr ago, and has subsequently become recognised as a cause of major pathological lesions in the swimbladder of the European eel Anguilla anguilla. The radiographic method reported in this paper showed be a useful complement to diagnostic methods that have so far been based exclusively on fish dissection. Using this method, the infection of the swimbladder and the severity of its pathological changes can be assessed in a reliable manner without causing damage to the fish. By analysing radiographs of 45 eels from Lake Balaton, 5 stages of swimbladder lesions were distinguished. The method enables the examiner to draw conclusions on the air, worm and exudate content of the swimbladder; however, data on thickening of the swimbladder wall can be obtained only indirectly. The radiograph also provides information on the air and worm content of the pneumatic duct. Results obtained by radiography showed good agreement with dissection findings. Radiographic results are compared with pathological findings obtained from fish dissections separately for each severity grade of infection.

Experimental detection of the actinospores of Myxobolus pseudodispar (Myxosporea: Myxobolidae) in oligochaete alternate hosts.

The development of Myxobolus pseudodispar Gorbunova, 1936, an intracellular myxosporean muscle parasite of the roach Rutilus rutilus L., was studied in experimentally infected oligochaetes. In one experiment, uninfected Tubifex tubifex Müller and Limnodrilus hoffmeisteri (Claparéde) were exposed to mature spores of M. pseudodispar. Triactinomyxon spores developed both in T. tubifex and L. hoffmeisteri specimens. Triactinospores were first released from the oligochaetes 76 d after initial exposure. At that time, pansporocysts containing 8 triactinospores were located in the gut epithelium of experimentally infected oligochaetes, but free actinosporean stages were also found in their gut lumen. Each triactinospore had 3 pyriform polar capsules and an elongated cylindrical sporoplasm with 8 secondary cells. The spore body joined the 3 caudal projections with a relatively long style. One of the 3 caudal projections was shorter than the other two. The total length of the triactinospore was on average 206.5 microns.

Comparative study of X-ray computerised tomography and conventional X-ray methods in diagnosis of swimbladder infection in eels caused by Anguillicola crassus.

To date, swimbladder lesions due to Anguillicola crassus infection of the European eel Anguilla anguilla have so far been studied only by conventional X-ray methods. This is the first study to report the use of computerised tomography (CT) for studying lesions induced by anguillicolosis. Of 50 eels caught by electrofishery from Lake Balaton, Hungary, in autumn 2002 and pre-selected by a conventional X-ray method, 22 specimens were examined with a Siemens Somatom Plus S40 spiral CT scanner. Tomograms, radiographs and photographs of 5 of these, showing anguillicolosis-induced swimbladder lesions of varying severity, are presented. Computerised tomograms provide information on the inner structure, air content and wall thickness of the swimbladder as well as on the number of worms it contains. When the swimbladder is not severely affected or not completely filled with worms, computerised tomography provides adequate data on the shape of the swimbladder, thickness of the swimbladder wall and the location of worms in the lumen. However, in more severe cases, i.e. when the swimbladder is tightly packed with worms or contains no air as a result of wall-thickening, this method fails to determine the number and location of helminths or the thickness of the swimbladder wall.

Relationships among members of the genus Myxobolus (Myxozoa: Bilvalvidae) based on small subunit ribosomal DNA sequences.

Sequences representing approximately 1,700 base pairs of the 18S rRNA gene from 10 different species in the genus Myxobolus were found to group them into 3 clusters that showed little correlation with spore morphology and size or host specificity, criteria currently used for both higher and lower taxonomic placements in the Myxozoa. Of the phenotypic criteria examined, tissue tropism was most correlated with the rRNA groupings observed. Spores of similar size and shape (Myxobolus cerebralis vs. Myxobolus squamalis) were distantly related in some instances, whereas spores with divergent morphology and size were sometimes found to be closely related (M. cerebralis and Myxobolus insidiosus). These initial investigations into the phylogenetic relationships of putative members of the genus Myxobolus clearly indicate the potential limitations of groupings based on size and morphological properties of the spores and host species infected. We propose that 18S rRNA gene sequences, combined with information on tissue tropism, host species infected, and developmental cycles in the fish and alternate host (when and if known) be given greater consideration in taxonomic placements of myxosporeans.