New cell motility model observed in parasitic cnidarian Sphaerospora molnari (Myxozoa:Myxosporea) blood stages in fish.

Cellular motility is essential for microscopic parasites, it is used to reach the host, migrate through tissues, or evade host immune reactions. Many cells employ an evolutionary conserved motor protein- actin, to crawl or glide along a substrate. We describe the peculiar movement of Sphaerospora molnari, a myxozoan parasite with proliferating blood stages in its host, common carp. Myxozoa are highly adapted parasitic cnidarians alternately infecting vertebrates and invertebrates. S. molnari blood stages (SMBS) have developed a unique "dancing" behaviour, using the external membrane as a motility effector to rotate and move the cell. SMBS movement is exceptionally fast compared to other myxozoans, non-directional and constant. The movement is based on two cytoplasmic actins that are highly divergent from those of other metazoans. We produced a specific polyclonal actin antibody for the staining and immunolabelling of S. molnari's microfilaments since we found that neither commercial antibodies nor phalloidin recognised the protein or microfilaments. We show the in situ localization of this actin in the parasite and discuss the importance of this motility for evasion from the cellular host immune response in vitro. This new type of motility holds key insights into the evolution of cellular motility and associated proteins.

Detection of wide genetic diversity and several novel strains among non-avium nontuberculous mycobacteria isolated from farmed and wild animals in Hungary.

AIMS: Besides Mycobacterium avium numerous nontuberculous Mycobacterium (NTM) species exist, which pose constant health risk to both humans and animals. The aim of our study was to identify non-avium NTM isolates from veterinary origin in Hungary, and to detect the occurrence of rifampicin resistance among them. METHODS AND RESULTS: Two hundred and twenty-five strains isolated between 2006 and 2013 from domestic and wild animals and veterinary important samples were identified on the basis of partial DNA sequences of different structural or coding genes, besides commercial kits and multiplex PCR. From 14 different sources, 28 NTM strains and 8 hitherto unidentified strain types were detected. Mycobacterium nonchromogenicum was the most frequently occurring strain (25·78%). Besides, new hosts and mycobacteria-related pathological symptoms were detected. Noticeable rifampicin resistance (42·76%) was found among 159 strains from six different host species. Furthermore, we described the problematics of strain-misidentifications using commercial kits. CONCLUSIONS: Our study identified the most common non-avium NTM strains in Hungary, and provided account of their occurrence, host range, and pathogenicity. The detected high rifampicin resistance among the strains isolated mainly from fallow and red deer clearly shows that more attention should be paid to the examination of wild animals especially to those ones which may have contact or shared territory with farmed animals. SIGNIFICANCE AND IMPACT OF THE STUDY: In domestic animal husbandry the maintenance of tuberculosis free status is of primary importance. As immunological cross-reactions due to NTM hamper the diagnosis of bovine tuberculosis, the precise identification of NTM strains would be essential in the veterinary diagnostics, especially for potentially zoonotic strains. This is the first study investigating the strain diversity of non-avium NTM in Hungary.

Inosine-arginine salt as a promising agent for in vitro activation of waterborne fish-pathogenic myxozoan actinospores.

Mucus-derived nucleosides serve as key host cues for myxozoan actinospore fish host recognition, but to date their use for experimental actinospore activation in the laboratory or application in disease prevention has not progressed very far. One obstacle has been the low solubility of pure inosine and guanosine. To overcome this, we used inosine-arginine salt (ino-arg), which incorporates both high activation properties and high solubility. We tested its efficacy both in microassays directly observing reactions of actinospores of 2 distantly related myxozoan species, Myxobolus cerebralis and M. pseudodispar in comparison to inosine, as well as its actinospore-inactivation properties by premature polar capsule discharge in an infection experiment. Ino-arg was considerably more effective in eliciting polar capsule discharge and sporoplasm emission at much lower concentrations than pure inosine and, in contrast to the latter, remained dissolved in aqueous solution. Ino-arg exposure of M. pseudodispar actinospores resulted in polar capsule discharge and sporoplasm emission before host contact and subsequently in a lower infection rate in roach Rutilus rutilus.

Molecular characterization of Sphaerospora molnari (Myxozoa), the agent of gill sphaerosporosis in common carp Cyprinus carpio carpio.

Sphaerospora molnari Lom, Dyková, Pavlásková and Grupcheva, 1983 often causes severe infections in the gills and skin of common carp fingerlings Cyprinus carpio carpio in Central Europe. Although most Sphaerospora spp. are coelozoic and affect the excretory system of fish, S. molnari develops mature spores in the epithelia of gill filaments, making it a rare representative of histozoic freshwater species within the genus. On the basis of a partial 18S rDNA sequence assigned as belonging to S. molnari, previous phylogenetic studies located the species within the Myxobolus clade. In the present study, S. molnari isolates from Hungary and the Czech Republic were characterized based on morphology, DNA sequence analysis and phylogenetic comparison. The obtained 3714 bp final consensus 18S rDNA sequence of the parasite showed several, sometimes extremely long inserts in the variable regions of the gene and differed considerably from the one published in GenBank in 2002. In situ hybridization confirmed the validity of the obtained DNA sequence and detected pre-sporogonic blood stages in the interstitium and blood vessels of the kidney. Phylogenetic analysis showed that S. molnari clusters within the Sphaerospora sensu stricto clade with a high support, revealing it as the first known histozoic member of the Sphaerospora subclade comprising parasites of freshwater fish.

Morphology, seasonality and phylogeny of Zschokkella soleae sp. n. (Myxozoa, Myxosporea) parasite of Solea solea (L.) (Pleuronectiformes, Soleidae) from Ghar El Melh Lagoon, Tunisia.

A new Myxosporea, Zschokkella soleae sp. n., was found in the gall bladder and the bile of common sole, Solea solea (L.), from Ghar El Melh Lagoon in north-east Tunisia. This is the first record for the presence of Zschokkella species in Tunisian waters. The parasite's plasmodia are polysporic with variable size and shape. Some plasmodia appeared attached to the gall bladder epithelium while others were found free in bile. Mature spores are ellipsoidal in frontal view 13.8±0.38 µm long and 10.86±0.40 µm wide with two equal size spherical polar capsules 3.6±0.43 µm in size. The prevalence of infection seems to correlate with host size and changes over the year with maximum percentage in summer. Based on the 18S rDNA sequence data, Z. soleae sp. n. is readily distinguishable from other myxozoan DNA sequences in GenBank. Phylogenetically, the new species is placed in the freshwater Myxidium clade including several Zschokkella spp. infecting the gall bladder. Morphology, histology as well as DNA sequence analysis indicate that the examined species differs from all previously described Zschokkella species.

'Who's who' in renal sphaerosporids (Bivalvulida: Myxozoa) from common carp, Prussian carp and goldfish--molecular identification of cryptic species, blood stages and new members of Sphaerospora sensu stricto.

Myxozoans are a group of diverse, spore-forming metazoan microparasites bound to aquatic environments. Sphaerospora dykovae (previously S. renicola) causes renal sphaerosporosis and acute swim bladder inflammation (SBI) in juvenile Cyprinus carpio carpio, in central Europe. A morphologically similar species with comparably low pathogenicity, S. angulata has been described from C. c. carpio, Carassius auratus auratus and Carassius gibelio. To clarify uncertainties and ambiguities in taxon identification in these hosts we decided to re-investigate differences in spore morphology using a statistical approach, in combination with SSU and LSU rDNA sequence analyses. We found that developing spores of S. angulata and S. dykovae cannot be distinguished morphologically and designed a duplex PCR assay for the cryptic species that demonstrated S. dykovae is specific to C. c. carpio, whereas S. angulata infects C. a. auratus and C. gibelio. The molecular identification of myxozoan blood stages in common carp and goldfish, which had previously been ascribed to Sphaerospora spp. showed that approximately 75% of blood stages were from non-sphaerosporid coelozoic species infecting these cyprinids and more than 10% were from an alien species, Myxobilatus gasterostei, developing in sticklebacks. We hereby report non-selective myxozoan host invasion and multi-species infections, whose role in SBI still requires clarification.

Comparison of the Myxobolus fauna of common barbel from Hungary and Iberian barbel from Portugal.

We compared Myxobolus infection of common barbel Barbus barbus from the Danube River in Hungary with that in Iberian barbel Luciobarbus bocagei from the Este River in Portugal. In Hungary, we recorded 5 known Myxobolus species (M. branchialis, M. caudatus, M. musculi, M. squamae, and M. tauricus) and described M. branchilateralis sp. n. In Portugal we recorded 6 Myxobolus species (M. branchialis, M. branchilateralis sp. n., M. cutanei, M. musculi, M. pfeifferi, and M. tauricus). Species found in the 2 habitats had similar spore morphology and only slight differences were observed in spore shape or measurements. All species showed a specific tissue tropism and had a definite site selection. M. branchialis was recorded from the lamellae of the gills, large plasmodia of M. branchilateralis sp. n. developed at both sides of hemibranchia, M. squamae infected the scales, plasmodia of M. caudatus infected the scales and the fins, and M. tauricus were found in the fins and pin bones. In the muscle, 3 species, M. musculi, M. pfeifferi and M. tauricus were found; however they were found in distinct locations. Plasmodia of M. musculi developed intracellularly in muscle cells, plasmodia of M. tauricus were found in the dense connective tissue of the pin bones, whereas M. pfeifferi formed plasmodia in the connective tissue of the intramuscular septa. This latter species was often found in the cartilaginous gill arch as well. Comparative morphological and phylogenetic studies, as well as 18S rDNA sequences, revealed differences between the Myxobolus fauna of the 2 barbel species originating from different geographic regions.

The susceptibility of diverse species of cultured oligochaetes to the fish parasite Myxobolus pseudodispar Gorbunova (Myxozoa).

This study provides detailed information on the invertebrate hosts of Myxobolus pseudodispar (Myxozoa) and explores the susceptibility range of several species and analyses the relevance of the species composition of an oligochaete population. Our findings demonstrate that the oligochaete host range of M. pseudodispar is similarly wide as the number of vertebrate host species. Besides Tubifex tubifex and Limnodrilus hoffmeisteri, Psammoryctides barbatus and Psammoryctides moravicus were also found to be susceptible invertebrate hosts. The genetic characterization of the mitochondrial 16S rDNA of T. tubifex sensu lato revealed that lineages I, II and III are susceptible to M. pseudodispar, whereas T. tubifex lineage VI seems to be non-susceptible. T. tubifex lineage V and L. hoffmeisteri specimens were positive in a M. pseudodispar-specific PCR, but in most cases, the release of mature actinospores could not be detected. Hence, these non-susceptible oligochaetes likely serve as `biological filters` as they remove myxospores from the sediment without producing actinospores. Together with the phylogenetic analysis of the susceptible and non-susceptible oligochaete hosts on the basis of mt 16S rDNA sequences, the route of the development of M. pseudodispar in the oligochaete hosts was tracked by in situ hybridization. According to our findings, the gut epithelia seem to be a portal of entry of the sporoplasms, where the development of the parasite also takes place. The basal lamina seems to be involved in the migration of the parasite, and the worm's cellular immune response is activated by the infection.

Myxobolus erythrophthalmi sp. n. and Myxobolus shaharomae sp. n. (Myxozoa: Myxobolidae) from the internal organs of rudd, Scardinius erythrophthalmus (L.), and bleak, Alburnus alburnus (L.).

During a survey of myxosporean parasites of cyprinid fish in Hungary, infections caused by unknown Myxobolus spp. were found in the internal organs of rudd, Scardinius erythrophthalmus, and bleak, Alburnus alburnus. Small plasmodia developed in blood vessels of the kidney, liver, testes and intestinal wall. The parasites were studied on the basis of spore morphology and by histological and molecular methods. In most cases, plasmodia were surrounded by host tissue without a host reaction; however, in advanced cases, a connective tissue capsule was seen around plasmodia. Spores collected from the two fish species differed from each other and from the known Myxobolus spp. both in their morphology and 18S rDNA sequences. The two species, described as M. erythrophthalmi sp. n. from rudd and M. shaharomae sp. n. from bleak, are characterized by a specific histotropism to blood vessels, while the organ specificity involves the kidney and for the latter species, most internal organs.

Differentially expressed parasite genes involved in host recognition and invasion of the triactinomyxon stage of Myxobolus cerebralis (Myxozoa).

The host recognition and invasion process of Myxobolus cerebralis actinospores (triactinomyxon, TAM) was studied on a genetic level. A small-scale in vitro assay was developed to activate a large number of TAMs simultaneously, and to monitor the host invasion in the absence of live fish. The transcriptomes of non-activated and in vitro-activated TAMs were compared by suppressive subtractive hybridization (SSH) to identify parasite genes involved in the host invasion process. Differential screening and a subsequent BLAST search revealed 15 of 452 SSH-library clones expressed differently in activated TAMs. None of the 15 transcripts obtained has previously been identified from M. cerebralis. Quantitative real-time PCR was used to examine the relative expression profile of 8 selected transcripts upon TAM activation and after penetration of the host. Four of these were found to be up-regulated in activated TAMs, while expression was relatively low in non-activated TAMs and in infected fish tissue, indicating that they are relevant genes during host recognition or subsequent host invasion of M. cerebralis TAMs.

Myxozoan transmission via actinospores: new insights into mechanisms and adaptations for host invasion.

Various mechanisms that enable and improve transmission success of myxozoan actinospore stages towards fish hosts are described, based upon a combination of experimental data and functional analysis of morphological characters. For this purpose, laboratory-reared actinospores of Myxobolus cerebralis, Myxobolus parviformis, Henneguya nuesslini and Myxobolus pseudodispar were employed to exemplarily investigate aspects of host attachment and invasion. The process of polar filament discharge of M. cerebralis actinospores was analysed, showing that full discharge occurs in less than 10 msec. Additionally, a mechanism that rapidly contracts the discharged filament after discharge is described for the first time. Its purpose is most likely to bring the actinospore apex rapidly into intimate contact with the surface of the host. Unlike M. cerebralis, M. parviformis actinospores did not discharge polar filaments after mechanical and chemical stimulation, suggesting a different mode of triggering. For H. nuesslini actinospores, experimental results indicated that polar filament discharge is independent of external calcium-ion concentration but is influenced by osmolality. After attachment of an actinospore and prior to penetration into the host, an ensheathed unit ('endospore'), containing the sporoplasm, was emitted from the valves in a manner which varied from species to species. Experimentally induced sporoplasm emission was time-dependent and was found to be independent of polar filament discharge in H. nuesslini. Remarkably, it could be concluded that the sporoplasm is able to recognize host-stimuli while still within the intact spore. An updated summary of the sequential course of events during host recognition and invasion by actinospores is given.

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.

Comparative morphological and molecular studies on Myxobolus spp. infecting chub from the river Danube, Hungary, and description of M. muellericus sp. n.

During a survey on fishes from the River Danube, the occurrence of 8 Myxobolus species (Myxozoa: Myxobolidae) was registered in chub Leuciscus cephalus L. Most species had a specific location within the fish host. M. cycloides was found in the wall of the swimbladder; the branched plasmodia of M. dujardini were located typically in the epithelium of the non-lamellar part of gill filaments; the plasmodia of M. ellipsoides infected fins between 2 fin rays; M. muelleri and Myxobolus sp. 2 formed large elongated plasmodia in the afferent gill artery of filaments, while the round cysts of M. muellericus sp. n. filled the capillary network of the gill lamellae. Intramuscular plasmodia of M. pseudodispar proved to be the most common, although large cysts of Myxobolus sp. 1 were also frequently found in the intestinal wall. Despite similarities of some species in spore morphology, 18S rDNA sequences showed clear differences between the species examined.

Description of a new synactinomyxon type from the River Sousa, Portugal.

Actinospore infection of oligochaetes collected from the mud of 2 freshwater biotopes in Portugal was studied. Using the 'cell-well plate method', a new synactinomyxon type was found in 2 specimens (1.3%) of the examined Tubifex tubifex oligochaetes from the River Sousa north of Porto, Portugal. In Criodrilus lacuum and Dero digitata specimens collected from the same river, no actinosporeans were released during the 12 wk observation period. Infected oligochaetes were only found immediately post-collection, and no further actinosporean release was recorded in Tubifex specimens kept alive for several weeks. Actinospore infection showed high intensity in oligochaetes in both positive cases. No actinosporean stages of myxosporeans have as yet been described from Portugal. On the basis of spore morphology and 18S rDNA sequence data, the synactinomyxon type presented in this paper differs from those already known and described in the literature.

The life cycle of Henneguya nuesslini Schuberg & Schroder, 1905 (Myxozoa) involves a triactinomyxon-type actinospore.

The life cycle of the histozoic myxozoan parasite Henneguya nuesslini was investigated in two salmonid host species. Naive brown trout, Salmo trutta, and brook trout, Salvelinus fontinalis, were experimentally infected in two trials by triactinomyxon type actinospores from naturally infected Tubifex tubifex. In exposed common carp, Cyprinus carpio, no myxospore production was detected. The parasite formed cysts with mature myxospores in the connective tissue of the fish 102 days post-exposure. The morphology of both actinosporean and myxosporean stages was described by light microscopy and a 1417-bp fragment of the 18S rDNA gene was sequenced. Sequence analysis confirmed the absolute congruence of the two developmental stages and assisted in determining species identity. Host range, tissue specificity and myxospore measurements provided sufficiently distinctive features to confirm species validity and were thus crucial for identification. The triactinomyxon spores had 16 secondary germ cells, unique dimensions, a very opaque sporoplasm matrix and three conspicuously protruding, pyriform polar capsules. This is the first record of a Henneguya sp. life cycle with a triactinomyxon-type actinospore, which suggests a close relationship with the Myxobolus group and a polyphyletic origin of the genus Henneguya.

Development of Myxobolus macrocapsularis (Myxosporea: Myxobolidae) in an oligochaete alternate host, Tubifex tubifex.

The development of Myxobolus macrocapsularis Reuss, 1906, a myxosporean parasite of the gills of common bream Abramis brama L., was studied in experimentally infected oligochaetes. In 3 experiments uninfected Tubifex tubifex Muller and Limnodrilus hoffmeisteri (Claparéde) were exposed to mature myxospores of M. macrocapsularis. In all experiments, typical triactinospores developed in T. tubifex specimens but no infection was found in L. hoffmeisteri. Triactinospores were released from oligochaetes 66 to 99 d after initial exposure. At that time pansporocysts containing 8 triactinospores were located in the gut epithelium of experimental oligochaetes, but free actinosporean stages were also found in the gut lumen of the oligochaetes. Each triactinospore had 3 pyriform polar capsules and a barrel-shaped sporoplasm with 32 secondary cells. The spore body joined the 3 caudal projections with a stout style.

Identification of fish-parasitic Myxobolus (Myxosporea) species using a combined PCR-RFLP method.

Polymerase chain reaction (PCR) with primers specific for the family Myxobolidae was used to amplify a part of the 18S ribosomal RNA gene of Myxobolus species. The length of the amplified fragments was approximately 1600 base pairs. Six Myxobolus species identified on the basis of morphological features were compared using a combined PCR-RFLP method. The cleavage patterns generated by 2 frequent cutter restriction enzymes (HinfI and MspI) were suitable for the differentiation of the examined Myxobolus species.

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.