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Glycans and lectins (carbohydrate-binding molecules) form a mutual recognition system, which enables parasitic organisms to attach themselves to the host cells and/or take part in the migration of their developmental stages into the target tissue. The aim of the present study was to identify and characterise the potential binding activity of glycoconjugates in different developmental stages of Myxobolus cerebralis, the causative agent of whirling disease in salmonids. The binding patterns of 13 biotinylated neoglycoconjugates were histochemically examined in thin-sections of infected rainbow trout (Oncorhynchus mykiss) and oligochaetes (Tubifex tubifex), as well as isolated waterborne triactinomyxon spores. A distinct structure-selective and developmental stage-regulated expression of certain classes of carbohydrate binding was observed. In triactinomyxon spores, the expression of carbohydrate binding activity for alpha-l-Fuc-BSA-biotin, alpha-d-GalNAc-BSA-biotin, beta-d-GlcNAc-BSA-biotin, Lac-BSA-biotin and ASF-biotin was up-regulated in the polar capsules; the shell valves showed no activity. In the gut of T. tubifex, polar capsules of the parasite showed strong positive reaction only for beta-d-GlcNAc-BSA-biotin. In fish cartilage, polar capsules were negative, but the spore shell valves showed a broad range of carbohydrate binding activity. No activity was detected for either alpha6- or alpha3-linked N-acetyl-d-neuraminic acid to galactose. An adhesion assay was performed on GlycoWell plates and Myxobolus spores were found to specifically adhere to matrices containing residues of lactose, fucose, galactose, N-acetyl-d-galactosamine and N-acetyl-d-glucosamine. This is the first study to identify lectin activity in a myxozoan parasite; activity that is likely to play a role in the recognition systems involved in host specificity and the processes of spore attachment and invasion.

A systematic inventory of actinosporeans and oligochaetes conducted over 3 years at a trout fish farm in Bavaria, Germany, allowed the identification of 12 actinosporeans from five collective groups: four Triactinomyxon (Triactinomyxon nov. types 1-4), two Raabeia (Raabeia nov. types 1, 2), two Echinactinomyxon ( E. radiatum, Echinactinomyxon nov. type 1), two Aurantiactinomyxon ( A. pavinsis, Aurantiactinomyxon nov. type 1) and two Neoactinomyxum (Neoactinomyxum nov. types 1, 2). Nine forms are novel but can be placed within existing collective groups. All 12 forms were detected in the laboratory in aquarium water associated with farm sediment. However, only four of these could be linked with an oligochaete host. Three families of oligochaetes were identified from the sediment: Tubificidae, represented by eight species, Lumbriculidae with one species and Naididae with two species. Only tubificid oligochaetes were found to host actinosporeans.

Tetracapsuloides bryosalmonae Canning et al., 1999 (Myxozoa) is the causative agent of proliferative kidney disease in various species of salmonids in Europe and North America. We have shown previously that the development and distribution of the European strain of T. bryosalmonae differs in the kidney of brown trout (Salmo trutta) Linnaeus, 1758 and rainbow trout (Oncorhynchus mykiss) Walbaum, 1792, and that intra-luminal sporogonic stages were found in brown trout but not in rainbow trout. We have now compared transcriptomes from kidneys of brown trout and rainbow trout infected with T. bryosalmonae using suppressive subtractive hybridization (SSH). The differentially expressed transcripts produced by SSH were cloned, transformed, and tested by colony PCR. Differential expression screening of PCR products was validated using dot blot, and positive clones having different signal intensities were sequenced. Differential screening and a subsequent NCBI-BLAST analysis of expressed sequence tags revealed nine clones expressed differently between both fish species. These differentially expressed genes were validated by quantitative real-time PCR of kidney samples from both fish species at different time points of infection. Expression of anti-inflammatory (TSC22 domain family protein 3) and cell proliferation (Prothymin alpha) genes were upregulated significantly in brown trout but downregulated in rainbow trout. The expression of humoral immune response (immunoglobulin mu) and endocytic pathway (Ras-related protein Rab-11b) genes were significantly upregulated in rainbow trout but downregulated in brown trout. This study suggests that differential expression of host anti-inflammatory, humoral immune and endocytic pathway responses, cell proliferation, and cell growth processes do not inhibit the development of intra-luminal sporogonic stages of the European strain of T. bryosalmonae in brown trout but may suppress it in rainbow trout.

The present endeavor was conducted to evaluate the role of activated macrophage in the susceptibility of two different rainbow trout (Oncorhynchus mykiss) strains, a susceptible American (T) and a more resistant German (H), to infection with Myxobolus cerebralis. Arginase-2 and inducible nitric oxide synthase (iNOS) genes were used as references to the alternative and classical pathway of macrophage activation. The expression level of both genes was measured using quantitative real-time polymerase chain reaction. The expression level of arginase-2 was significantly upregulated in strain T at 2 h and 8 days post exposure in the strain H. In case of iNOS, the expression level was significantly upregulated from 24 h to 8 days p.e. in strain T and only in 8 days p.e. in strain H. During this study also, the influence of nitric oxide (NO) on the viability of the triactinomyxon spores (TAMs) of M. cerebralis was evaluated using the NO-donor S-nitroso-N-acetyl-penicillamine (SNAP). Rising final concentrations of SNAP from 0.25 to 1 mM at 2, 4, and 24 h resulted in increasing numbers of propidium iodide-positive TAMs detected. The results of this study suggest an inability of strain T to react with an effective immune response against infection with M. cerebralis. Furthermore, the TAMs of M. cerebralis react with significant decrease of viable spores to rising concentration of SNAP and longer incubation, but there is also evidence for some resistance to NO activity.

Myxobolus pavlovskii from the gills of silver carp (Hypophthalmichthys molitrix) were used in attempts to transmit the infection under laboratory conditions. Spores of M. pavlovskii were placed in aquaria that contained sterilized sand and had been filled with tap water at 15-16 degrees C. Furthermore, oligochaetes (90% Tubifex tubifex) were added and examined by wet mounts and histology. Hexactinomyxon spores developed after about 3 months only in T. tubifex that had been exposed to M. pavlovskii. Myxosporea-free silver carp developed cysts in their gills that contained M. pavlovskii at 120 days after contact with Hexactinomyxon spores, proving that M. pavlovskii also needs an oligochaete intermediate host for its development.

The myxosporean and actinosporean spores of Myxobolus cerebralis develop through many stages in their respective hosts, salmonid fishes and a tubificid oligochaete. Using a modified, non-radioactive in situ hybridization protocol, the parasite, which exhibits radically different structural forms during its development in each host, could be specifically detected in paraffin-embedded tissues of both fish and oligochaetes. Our study aims to demonstrate the application of the technique for detection of early stages of M. cerebralis in both hosts.

Myxobolus cotti (Myxozoa: Myxosporea) is described as found in the central nervous system of the bullhead (Cottus gobio) caught in the Alpine lake Königssee and in a brook in the Bavarian Forest, Federal Republic of Germany (El-Matbouli and Hoffmann 1987). Aggregations of spores and polysporoblastic trophozoites compressed and replaced large areas of the white and grey matter of the brain and spinal cord. These aggregations may be surrounded by a thin, connective tissue capsule; in a few cases they were associated with loose infiltrates of glial cells. Neither conspicuous tissue reactions nor inflammatory responses were evident. No other organs were seen to be infected with M. cotti. Mature spores are oval, with a tapering anterior end, and the pyriform polar capsules are nearly equal in size. Fresh spores measured 8.9-15.1 microns in length (mean, 12.4 microns) and 8-12.4 microns in width (mean, 9.6 microns); polar capsules were 4.3-9 microns long (mean, 6.4 microns) and 2-3.8 microns wide (mean, 2.9 microns). Light microscopy, the ultrastructure of pansporoblasts, sporogenesis and mature spores are described.

Expression of immune-regulatory genes that code for cyclooxigenase-2 (COX-2), transforming growth factor beta (TGF-beta), and two isoforms of interleukin-1beta (IL-1beta1 and IL-1beta2) was studied in susceptible and non-susceptible rainbow trout strains for 200 days after exposure to Myxobolus cerebralis. Expression of COX-2, IL-1beta1, and IL-1beta2 increased 5 min post exposure (p.e.) and was always more elevated in the susceptible strain than in the non-susceptible strain. In both strains, expression of COX-2 returned to the control level within a few hours p.e. Expression of IL-1beta1 and IL-1beta2 showed two elevated waves in both strains until 4 days p.e. Expression of TGF-beta in the non-susceptible strain was elevated at nearly all sampling points, but was decreased in the susceptible strain until up-regulation between 4 and 20 days p.e.; TGF-beta was the only gene where the expression in the non-susceptible strain was more elevated than in the susceptible strain. Rainbow trout of the non-susceptible strain appeared to resist infection by M. cerebralis with only minor transcriptional regulation of the genes investigated. Increased transcriptions of genes in the susceptible strain may be the result of an inability to antagonize the infection.

A new molecular diagnostic assay was developed for detection of Tetracapsuloides bryosalmonae the causative agent of proliferative kidney disease (PKD) in salmonid fish using a loop-mediated isothermal amplification method (LAMP). The PKD-LAMP assay amplifies the T. bryosalmonae DNA extracted from infected kidney, under constant temperature of 65 degrees C within 1 h. The required equipment for DNA amplification is only a water bath. The amplification products were detected visually by using SYBR green I dye, which turns green in the presence of amplified products and remains orange in its absence, and by electrophoresis without any difference in the sensitivity of both methods. The developed PKD-LAMP assay demonstrated an exceptionally higher sensitivity than the conventional PCR. PKD-LAMP assay was found to be 100-fold more sensitive than the PCR assay. The developed assay is simple, rapid, cost-effective, specific and highly sensitive. The assay is also characterized by its field applicability, as it does not require the use of sophisticated equipment or skilled personnel.

Myxobolus cerebralis (Myxozoa: Myxosporea) has a complex two-host life cycle, which begins when waterborne triactinomyxon spores released from the infected oligochaete Tubifex tubifex contact a susceptible trout. Upon contact the triactinomyxon spores attach to the fish and release their sporoplasm cells into the epidermis. At approximately 50 days postinfection, sporogenesis begins, resulting in a large number of M. cerebralis spores in the cartilage of infected fish 6 weeks later. The spores of M. cerebralis can be released from infected fish only after the fish die or are eaten by predators. In both cases, spores released into the aquatic environment can be ingested by oligochaete worms of the species T. tubifex and then develop into the actinosporean triactinomyxon stage in the intestine within about 3 months. The triactinomyxon is the only stage infectious for salmonid fish. We determined the DNA concentration in sporoplasm cells, capsulogenic cells, and valvogenic cells of M. cerebralis spore stages from the trout and of triactinomyxon spore stages from T. tubifex. DNA was visualized using the DNA-specific fluorescent stain DAPI. Our results demonstrate that meiosis occurs only once in the developmental cycle of M. cerebralis in contrast to the previously published hypothesis. This takes place within the pansporocyst found in T. tubifex. Thereafter, the sporoplasm cells of the triactinomyxon spores in T. tubifex and M. cerebralis in trout are diploid.

Spores of Myxobolus cotti El-Matbouli and Hoffmann 1987 and M. cerebralis Hofer 1903 (fresh or after 5 months in mud) are taken in by tubifex worms and develop in their gut epithelium cells into actinosporea of the genus Triactinomyxon. Triactinomyxon deriving from M. cotti differ distinctly from those derived from M. cerebralis in morphology and the number of sporozoites in the epispore. It could be shown that Triactinomyxon spores infect fish either via the water (bullhead, rainbow trout) or by feeding of infected tubifex (rainbow trout), developing into Myxobolus spores in central nervous tissue (bullhead) or cartilage (rainbow trout). Fresh or ripened spores of M. cotti and M. cerebralis were not infectious for bullheads or rainbow trout, respectively. The results of our experiments confirm the hypothesis that the life cycle of M. cotti and M. cerebralis includes an intermediate host and a metamorphosis into actinosporea of the genus Triactinomyxon.