Cerebralis Myxospores Research Articles

Morphological and molecular confirmation of Myxobolus cerebralis myxospores infecting wild‑caught and cultured trout in North Carolina (SE USA).

We used microscopy and molecular biology to provide the first documentation of infections of Myxobolus cerebralis (Myxozoa: Myxobolidae), the etiological agent of whirling disease, in trout (Salmonidae) from North Carolina (USA) river basins. A total of 1085 rainbow trout Oncorhynchus mykiss, 696 brown trout Salmo trutta, and 319 brook trout Salvelinus fontinalis from 43 localities across 9 river basins were screened. Myxospores were observed microscopically in pepsin-trypsin digested heads of rainbow and brown trout from the Watauga River Basin. Those infections were confirmed using the prescribed nested polymerase chain reaction (PCR; 18S rDNA), which also detected infections in rainbow, brown, and brook trout from the French Broad River Basin and the Yadkin Pee-Dee River Basin. Myxospores were 9.0-10.0 µm (mean ± SD = 9.6 ± 0.4; N = 119) long, 8.0-10.0 µm (8.8 ± 0.6; 104) wide, and 6.0-7.5 µm (6.9 ± 0.5; 15) thick and had polar capsules 4.0-6.0 µm (5.0 ± 0.5; 104) long, 2.5-3.5 µm (3.1 ± 0.3; 104) wide, and with 5 or 6 polar filament coils. Myxospores from these hosts and rivers were morphologically indistinguishable and molecularly identical, indicating conspecificity, and the resulting 18S rDNA and ITS-1 sequences derived from these myxospores were 99.5-100% and 99.3-99.8% similar, respectively, to published GenBank sequences ascribed to M. cerebralis. This report comprises the first taxonomic circumscription and molecular confirmation of M. cerebralis in the southeastern USA south of Virginia.

Accelerated deactivation of Myxobolus cerebralis myxospores by susceptible and non-susceptible Tubifex tubifex.

In the 1990s, the Tubifex tubifex aquatic oligochaete species complex was parsed into 6 separate lineages differing in susceptibility to Myxobolus cerebralis, the myxozoan parasite that can cause whirling disease (WD). Lineage III T. tubifex oligochaetes are highly susceptible to M. cerebralis infection. Lineage I, IV, V and VI oligochaetes are highly resistant or refractory to infection and may function as biological filters by deactivating M. cerebralis myxospores. We designed a 2-phased laboratory experiment using triactinomyxon (TAM) production as the response variable to test that hypothesis. A separate study conducted concurrently demonstrated that M. cerebralis myxospores held in sand and water at temperatures ≤15°C degrade rapidly, becoming almost completely non-viable after 180 d. Those results provided the baseline to assess deactivation of M. cerebralis myxospores by replicates of mixed lineage (I, III, V and VI) and refractory lineage (V and VI) oligochaetes. TAM production was zero among 7 of 8 Lineage V and Lineage VI T. tubifex oligochaete groups exposed to 12500 M. cerebralis myxospores for 15, 45, 90 and 135 d. Among 4 mixed lineage exposure groups, TAM production averaged 14641 compared with 2202495 among 12 groups of Lineage III oligochaetes. Among the 6 unexposed Lineage III experimental groups seeded into original Phase 1 substrates for the 45, 90 and 135 d treatments during the Phase 2 portion of the study, TAM production was reduced by 98.9, 99.9 and 99.9%, respectively, compared with the average for the 15 d exposure groups. These results are congruent with the hypothesis that Lineage V and Lineage VI T. tubifex oligochaetes can deactivate and destroy M. cerebralis myxospores.

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.

Effect of cadmium on the susceptibility of Tubifex tubifex to Myxobolus cerebralis (Myxozoa), the causative agent of whirling disease

Environmental pollutants alter a wide range of host-parasite interactions in various ways. In some cases, pollution leads to a significant increase in parasite abundance, causing epidemics of parasitic diseases. In other cases, toxicants restrict the transmission success of parasites, resulting in reduction of their abundance. However, very little is known regarding whether and to what extent aquatic pollution affects myxozoan obligate parasites commonly found in fish. We investigated the effect of cadmium (Cd) on the aquatic oligochaete Tubifex tubifex infected with the myxozoan Myxobolus cerebralis. The oligochaetes were experimentally exposed to M. cerebralis myxospores and kept in various concentrations of Cd for 4 mo. Neither survival nor reproduction of the worms was affected by the metal, but infection prevalence and numbers of triactinosmyxon spores produced by individual worms were higher in the Cd-exposed group than the unexposed control. A comparative assay of a lethal Cd concentration (LC50) on infected and non-infected T. tubifex revealed that infected worms are more resistant to the acute toxicity of Cd, probably because uptake of Cd was reduced by the infection. These results suggest that the abundance of M. cerebralis likely increases in polluted waters and escalates the risk of whirling disease in the respective area.

Adherence of Myxobolus cerebralis Myxospores to Waders: Implications for Disease Dissemination

AbstractThe vectors involved in the spread of whirling disease, which is caused by Myxobolus cerebralis, are only partly understood. However, the parasite has rapidly become established in many regions, suggesting that it is easily disseminated. We gained insight into transport vectors by examining the surface porosity of common wading equipment materials and the adherence of M. cerebralis myxospores to them. Interstitial spaces within rubber, felt, lightweight nylon, and neoprene were measured on scanning electron microscope images. Myxospores were applied to each material, the material was rinsed, and the myxospores recovered to assess adherence. The mean interstitial space size of rubber was the smallest (2.0 μm), whereas that of felt was the largest (31.3 μm). The highest recovery rates were from rubber and the glass control. Percent myxospore recovery varied by material, the recovery from felt being lower than that from all other materials. The potential for felt to carry even small numbers of myxospores suggests that the introduction of M. cerebralis by felt‐soled wading boots is possible.

Variability in Triactinomyxon Production from Tubifex tubifex Populations from the Same Mitochondrial DNA Lineage Infected with Myxobolus cerebralis, the Causative Agent of Whirling Disease in Salmonids

Myxobolus cerebralis, the causative agent of whirling disease, infects both salmonid fish and an aquatic oligochaete, Tubifex tubifex. Although M. cerebralis has been detected in river drainages throughout the United States, disease severity among wild fish populations has been highly variable. Tubifex tubifex populations have been genetically characterized using sequences from the 16S mitochondrial DNA (mtDNA) gene, the 18S ribosomal RNA gene, the internal transcribed spacer region 1 (ITS1), and randomly amplified polymorphic DNA (RAPD). Our earlier work indicated that large differences in compatibility between the parasite and populations of T. tubifex may play a substantial role in the distribution of whirling disease and resulting mortality in different watersheds. In the present study, we examined 4 laboratory populations of T. tubifex belonging to 16S mtDNA lineage III and 1 population belonging to 16S mtDNA lineage I for triactinomyxon (TAM) production after infection with M. cerebralis myxospores. All 4 16S mtDNA lineage III populations produced TAMs, but statistically significant differences in TAM production were observed. Most individuals in the 16S mtDNA lineage III-infected populations produced TAMs. The 16S mtDNA lineage I population produced few TAMs. Further genetic characterization of the 16S mtDNA lineage III populations with RAPD markers indicated that populations producing similar levels of TAMs had more genetic similarity.

Myxobolus cerebralis infection patterns in Yellowstone cutthroat trout after natural exposure

Salmonid species and sub-species exhibit a range of susceptibility to Myxobolus cerebralis infection. Little is known about lesion severity and location, or time required for M. cerebralis myxospores to develop in Yellowstone cutthroat trout Oncorhynchus clarki bouvieri. In 2002 we performed three 10 d exposures of Yellowstone cutthroat trout fry in Pelican Creek, an M. cerebralis-positive tributary to Yellowstone Lake. At 90 and 150 d post-exposure we examined the fish for clinical signs, for infection prevalence, and by histology to determine M. cerebralis infection location and severity of lesions. The most prevalent clinical signs in Yellowstone cutthroat were whirling behavior and skeletal deformities, especially at 90 d post-exposure. Prevalence of infection and severity of cartilage lesions were not statistically different between fish held for 90 or 150 d post-exposure. Histopathology was most severe in cartilage of the cranium and the lower jaw, whereas cartilage of the nares and gill arches was seldom damaged. This study suggests that Yellowstone cutthroat trout are highly vulnerable to M. cerebralis and that current population declines in the Yellowstone Lake basin may, in part, result from whirling disease. Our results answer important questions in fish health and will aid in the development of diagnostic tools and management efforts against this pathogen in native cutthroat trout and other vulnerable salmonids.

Water Temperature Affects a Host–Parasite Interaction: Tubifex tubifex and Myxobolus cerebralis

AbstractThe aquatic oligochaete worm Tubifex tubifex is an obligate host of Myxobolus cerebralis, the causative agent of salmonid whirling disease. Tubifex tubifex becomes infected by ingesting M. cerebralis myxospores and releases triactinomyxons (TAMs) into the water that then infect salmonids. In a laboratory experiment, we examined how water temperature influenced changes in oligochaete population size and biomass and the release of TAMs in three genetically identified strains of T. tubifex when exposed and not exposed to M. cerebralis myxospores. Increases in population size and biomass were greatest at 8.0°C for the Gallatin River strain, greatest at 15.0°C for the Madison River strain, and similar at both temperatures for the Mt. Whitney strain. After exposure to M. cerebralis myxospores, Mt. Whitney and Madison River T. tubifex both released TAMs at 15°C beginning on day 88 and at 8.0°C on day 169 (Mt. Whitney strain) and day 182 (Madison River strain); thus, between 1,320 and 1,456 degree‐days were required for M. cerebralis to develop. Gallatin River T. tubifex did not release TAMs. We found no evidence that M. cerebralis developed differently in strains of T. tubifex with different temperature requirements; therefore, it appears that temperature‐driven parasite development rates are a primary determinant of the timing of TAM release.

Lectin blot studies on proteins of Myxobolus cerebralis, the causative agent of whirling disease

It is known that Myxobolus cerebralis antigens, both surficial and secreted, are key modulators for, or targets of, host immune system compounds. We undertook SDS-PAGE glycoprotein characterisation of M. cerebralis developmental stages isolated from infected rainbow trout and Western blot analyses using selected biotin-labelled plant lectins (GSA-I, PHA-E, SJA, GSA-II) and anti-triactinomyxon polyclonal antibodies. Glycoproteins were isolated with lectin-affinity chromatography, and prominent bands were characterised by matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI/MS). We identified glycoproteins of M. cerebralis myxospores that contained carbohydrate motifs reactive with Phaseolus vulgaris erythroagglutinin (proteins 20 to 209 kDa, PHA-E), Sophora japonica agglutinin (proteins 7 to 70 kDa, SJA), Griffonia simplicifolia Agglutinin I (proteins 10 to 209 kDa, GSA-I) and G. simplicifolia Agglutinin II (proteins 5 to 40 kDa, GSA-II). Mcgp33, a glycoprotein isolated by lectin-affinity chromatography, was reactive with SJA (about 33 kDa). Antiserum produced against M. cerebralis triactinomyxons was found to have differences in the antigenicity of isolated glycoproteins from both M. cerebralis myxospores and actinospores. We also demonstrated modified antigen expression, especially involving the glycoprotein Mcgp33, in different developmental stages of M. cerebralis.

The Toxicity of Bayluscide and TFM to Tubifex tubifex: Implications for Chemical Control of the Oligochaete Host of Myxobolus cerebralis, the Causative Agent of Whirling Disease

AbstractStandard 24‐h toxicity tests were performed to determine the acute toxicity of Bayluscide (5,2′‐dichloro‐4′‐nitrosalicylanilide) and 3‐trifluoromethyl‐4‐nitrophenol (TFM) to two strains of the oligochaete worm Tubifex tubifex exposed and unexposed to Myxobolus cerebralis, the causative agent of whirling disease. In addition, microcosm trials were conducted to test the effect of three treatment levels of each chemical on the density of T. tubifex in an earthen hatchery pond. In the toxicity tests, worms that were susceptible to M. cerebralis infection were more sensitive to both chemicals than worms that were resistant to the parasite. The toxicity differences of exposed and unexposed worms within a lineage were not as great as those between lineages. Among worms that were susceptible to M. cerebralis, the concentrations of TFM that were lethal to 50% of the test organisms (LC50s) were 6.3 mg/L for worms that had been exposed to M. cerebralis myxospores and 7.3 mg/L for unexposed worms; for resistant worms, the corresponding LC50s were 8.2 and 7.7 mg/L. Among susceptible worms, the LC50s of Bayluscide were 0.060 mg/L for organisms exposed to M. cerebralis myxospores and 0.062 mg/L for unexposed organisms; resistant worms had LC50s of 0.096 and 0.106 mg/L. In the microcosm trials, Bayluscide treatments reduced worm densities by 73–82%, while the TFM treatments reduced worm densities by 34–88%. Given its rapid degradation, relatively selective toxicity, lower cost, and regulatory status, Bayluscide appears to be the best candidate for further study as a chemical control of the oligochaete host of the parasite that causes whirling disease in salmonid fishes.

Validation of a single round polymerase chain reaction assay for identification of Myxobolus cerebralis myxospores

Validation of a single round PCR-based assay to confirm as Myxobolus cerebralis myxospores obtained from pepsin-trypsin digest preparations is described. The assay is a modification of a PCR assay published previously, based on the amplification of a segment of the gene encoding the 18S ribosomal subunit of M. cerebralis. The sensitivity, specificity and upper and lower detection limits were determined using known M. cerebralis and non-M. cerebralis myxospores and M. cerebralis-free fish. The sensitivity of PCR confirmation was 100% (95% confidence interval of 83.2-100%). The specificity was 100% (95% confidence interval of 87.2-100%). The upper detection limit was approximately 100,000 myxospores per reaction; the lower detection limit was approximately 50 myxospores per reaction. Given the high sensitivity and specificity of the assay, substitution of this assay for histologic confirmation of M. cerebralis infection is encouraged.