Whirling disease revisited: pathogenesis, parasite biology and disease intervention.

Whirling disease (WD) is an ecologically and economically debilitating disease of rainbow trout Oncorhynchus mykiss caused by the actinosporean spores of the parasite Myxobolus cerebralis. M. cerebralis has a complex, 2-host life cycle alternating between salmonid fish and the oligochaete host Tubifex tubifex. The parasite alternates between 2 spore forms as transmission stages: an actinosporean triactinomyxon spore that is produced in the oligochaete host and a myxosporean spore that develops in the salmonid host. Waterborne triactinomyxon spores released from infected T. tubifex oligochaetes attach to the salmonid host by polar filament extrusion elicited by chemical (nucleoside) and mechanical (thigmotropy) stimuli-a process which is rapidly followed by active penetration of the sporoplasms into the fish epidermis. Upon penetration, sporoplasms multiply and migrate via peripheral nerves and the central nervous system to reach the cartilage where they form trophozoites which undergo further multiplication and subsequent sporogenesis. M. cerebralis myxospores are released into the aquatic environment when infected fish die and autolyse, or when they are consumed and excreted by predators. Myxospores released into the water are ingested by susceptible T. tubifex where they develop intercellularly in the intestine over a period of 3 mo through 4 developmental stages to give rise to mature actinospores. In this article, we review our current understanding of WD-the parasite and its alternate hosts, life cycle and development of the parasite in either host, disease distribution, susceptibility and resistance mechanisms in salmonid host and strategies involved in diagnosis, prevention and control of WD.

Biostatic activity of piscine serum and mucus on myxozoan fish infective stages.

Since the basis of host specificity in Myxozoa, i.e. the differential disposition and extinction of erroneously penetrated myxozoan infective stages in non-susceptible fish hosts, remains puzzling, we aimed to explore the role of the innate immune system in this issue. In a comparative incubation challenge of actinospore sporoplasms of the freshwater parasite species Myxobolus cerebralis, Henneguya nuesslini and Myxobolus pseudodispar to isolates of host and non-host muci and blood sera, we measured cellular disintegration proportions and times by means of a double staining viability assay utilizing fluorescent dyes. After their activation, emerging primary and secondary sporoplasm cells were evaluated microscopically for physical integrity and onset of cell death due to exposure. Impairment by any mucus used was not detected up to 100 min of exposure. All parasites showed significantly increased cellular breakdown in non-susceptible host serum compared to the respective substrates from susceptible host fish. Except for M. cerebralis, the serum of the susceptible host was considerably less effective over time. In this species, both the primary and the secondary cells were affected in much shorter times than in the other two representatives. Inhibition of protease activity did not affect carp serum effect on M. cerebralis stages. We suggest the active components to be complement or complement induced factors since heat inactivation and withdrawal of bivalent metal ions lowered serum activity significantly. The study marks the first in vitro viability challenge of activated myxozoan transmission stages with teleost derived immune factors.

Differences in viability and reactivity of actinospores of three myxozoan species upon ageing.

Little is known about the viability of myxozoan actinospore stages after harvest from laboratory cultures of infected oligochaete worms. The viability and reactivity of actinospores of three myxozoan species was evaluated after short-term storage at 4 degrees C and 12 degrees C. Two methods of determining actinospore viability were compared: differential fluorescent staining and direct microscopic observation of morphological indicators of spore integrity. Spore reactivity was quantified by measuring polar filament discharge rates in a micro-assay with fish mucus substrate and mechanical stimulation by vibration. The age-dependent viability of the three species showed clear differences. Myxobolus cerebralis actinospores had the shortest effective life span whereas Henneguya nuesslini actinospores survived significantly longer. Storage at lower temperatures yielded higher viability in all species. Myxobolus pseudodispar actinospores were significantly robust up to 12 degrees C when assessed by staining, but showed similar viability characteristics as H. nuesslini when analyzed morphologically. Evaluation of spore viability by fluorescent staining correlated with morphological assessment, although fewer viable actinospores were usually detected microscopically. Polar filament discharge activity of morphologically intact actinospores did not significantly decrease until the third day of storage compared to freshly harvested samples. The results indicate that durability and reactivity trends during storage of actinospores differ among myxozoan species.

Parental genetic diversity of brown trout (Salmo trutta m. fario) brood stock affects offspring susceptibility to whirling disease.

BACKGROUND: Whirling disease, caused by the myxozoan parasite Myxobolus cerebralis, has high economical and ecological importance worldwide. Susceptibility to the disease varies considerably among salmonid species. In brown trout (Salmo trutta) the infection is usually subclinical with low mortality, which increases the risk of parasite dissemination, especially when farm fish are used for stocking natural habitats. The influence of intraspecific genetic differences (especially the level of homozygosity) on susceptibility is unknown. Therefore, we examined the possible correlations between parental genetic diversity and offspring susceptibility of brown trout stocks to whirling disease. METHODS: Two brown trout brood stocks from a German and a Hungarian fish farm were genetically characterized using microsatellite and lineage-specific genetic markers. The individual inbreeding coefficient f and pairwise relatedness factor r were estimated based on eight microsatellite markers. Brood stock populations were divided into groups according to low and high f and r value estimates and subjected to selective fertilization. The offspring from these separate groups were exposed to M. cerebralis actinospores, and the infection prevalence and intensity was measured and statistically analysed. RESULTS: The analysis of phylogeographic lineage heritage revealed high heterogeneity in the Hungarian brood stock since > 50% of individuals were Atlantic-Danubian hybrids, while only pure Atlantic-descending specimens were detected in the German population. Based on f msat and r msat estimations, classified non-inbred (NIB), inbred (IB) and a group of closely related fish (REL) were created. The susceptibility of their offspring varied considerably. Although there was no significant difference in the prevalence of M. cerebralis infection, the mean intensity of infection differed significantly between NIB and IB groups. In REL and IB groups, a high variability was observed in infection intensity. No external clinical signs were observed in the exposed brown trout groups. CONCLUSIONS: Our findings indicate that the allelic diversity of brown trout brood stock may constitute a significant factor in disease susceptibility, i.e. the intensity of parasite infection in the subsequent generation.