Triactinomyxon Spores Research Articles

Description, life cycle, and development of the myxozoan Myxobolus rasmusseni n. sp. in fathead minnows, Pimephales promelas: A possible emerging pathogen in southern Alberta, Canada

Morphological, gene sequence, host tissue tropism, and life cycle characteristics were utilized to describe the myxozoan, Myxobolus rasmusseni n. sp. from fathead minnow, Pimephales promelas, collected from reservoirs in southern Alberta. Results from serial histological sections of whole heads showed that myxospores were contained within irregular-shaped and sized coelozoic capsules (=plasmodia). Clusters of membrane-bound, myxospore-filled plasmodia filled the head cavities of juvenile fathead minnows, leading to the development of large, white, disfiguring lesions in mid to late summer. Bilateral exopthalmia (pop-eye disease) was a common outcome of M. rasmusseni n. sp. development. BLASTn search of a 1974 bp sequence of the 18S rDNA gene isolated from myxospores indicated that M. rasmusseni n. sp. was distinct from other coelozoic and histozoic Myxobolus spp. cataloged in GenBank. 18S rDNA gene sequences from triactinomyxon spores released from the oligochaete Tubifex were 100% identical to sequences from myxospores collected from syntopic fathead minnows. Results from a longitudinal survey of the 2020 cohort of fathead minnows showed that young-of-the-year are exposed at 1–5 mo and that 60–90% of these had developed myxospore-filled lesions approximately one year later. Data regarding potential sources and timing of M. rasmusseni n. sp. emergence in fathead minnow populations are needed.

Effect of substrate on the proliferation of Myxobolus cerebralis in the mitochondrial lineages of the Tubifex tubifex host

The study goal was to examine the effects of sand and mud on the propagation of Myxobolus cerebralis, the whirling disease agent, in four mitochondrial 16S ribosomal DNA lineages (I, III, V, VI) of its oligochaete host, Tubifex tubifex (Tt). In all the lineage groups held continuously in either substrate (non-shifted) or transferred from sand to mud (shifted), substrate influenced parasite proliferation only in lineage III. Sporogenesis and release of triactinomyxon spores (TAMs) were more prevalent in lineage III Tt in mud compared to sand. Low-infection prevalence and lack of parasite development in lineage I is associated with the greater number of resistant worms and were not affected by substrate type. Substrate did not impact Tt from lineages V and VI that failed to develop any parasite stages in either substrate even after shifting from sand to mud. The relationship between the microbial community in the substrate and parasite proliferation in lineage III was described but not analyzed due to small sample size. Substrate-associated bacteria were hypothesized as essential dietary source for the oligochaete host feeding selectively on fine (mud)-microflora. Progeny was produced by all lineage groups shifted to mud with disparate survival profiles in lineage V and VI and high mortalities in lineage III. Our study demonstrates that substrate type can alter parasite proliferation in lineage III. Conversely, parasite development and infectivity were not altered in lineage V and VI that are refractory to the parasite nor among the more resistant phenotypes (I), regardless of substrate type.

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.

Identification and characterization of one novel type of Triactinospomyxon with short spore axis

This study identifies and characterizes one novel type of triactinospomyxon in oligochaete specimen of Branchiura sowerbyi Beddard collected from a fish pond used for rearing gibel carp located in Caidian Experimental Station of the Institute of Hydrobiology. It is nominated as Triactinospomyxon caidianensis type. The spore is of characteristic triactinomyxon "anchor" shape and possesses a spore body with sporoplasm containing 32 germ cells, 3 polar capsules, and 3 caudal processes. Compared with other triactinomyxon spores described previously, T. caidianensis type has a short spore axis with 76.5 μm in length and a very short style with 38.9 μm in length. Molecular analyses on 18S rDNA sequences indicate that the novel T. caidianensis type is most closely related to Triactinomyxon sp SA-2005, Antonactinomyxon sp KAB-2001, and Synactinomyxon sp KAB-2001 with 80.33% to 81.92% identities. On the basis of spore morphology and genetic data, the T. caidianensis type presented in this paper differs from those already known and described in the literatures.

Susceptibility of rainbow trout ( Oncorhynchus mykiss) and carp ( Cyprinus carpio ) to Myxobolus cerebralis , the agent of whirling disease

agglomerated) in the tail fin of carp. The nested-PCR results showed that only M. cerebralis exists in 15% of the caudal fin samples in the rainbow trout 60 days p.e , although in the single-round PCR test, all the samples were tested negative. Our results indicated that the nested-PCR-method is more sensitive to detect early stages after 12 h in the rainbow trout and carps in comparison to the singleround PCR test and histological examination. The infection of rainbow trout with M. cerebralis -spores was confirmed by clinical symptoms and histological lesions in the cartilaginous areas of the skull, while in all the infected carps, it was not possible to divide the triactinomyxon-spores (TAM) in the epidermis at 12 h p.e ; thus, it penetrates deep into the dermis in order to reach the brain.

Prevalence of Myxobolus cerebralis Infections Among Genetic Lineages of Tubifex tubifex at Three Locations in the Madison River, Montana

Host biodiversity can impact disease risk and influence the transmission of parasitic disease. Stream sediment-dwelling worms, Tubifex tubifex (Clitellata: Oligochaeta), are the definitive host of the parasite Myxobolus cerebralis (Myxozoa: Myxosporea), which causes whirling disease in salmonid fishes. Genetic diversity of T. tubifex is correlated with host susceptibility to M. cerebralis , and mitochondrial Lineage III is generally shown to be more likely to be infected and produce the triactinomyxon (TAM) spores than other lineages. We determined the mitochondrial lineage, relative abundance, and prevalence of infection of T. tubifex collected at 3 sites in the Madison River, Montana, where previous study had shown variation in whirling disease prevalence and severity in caged trout fry. We also compared visual identification of TAMs released from cultured worms with a molecular genetic assay (diagnostic polymerase chain reaction [PCR]) for parasite detection of both infected and uninfected worms. We estimated that mitochondrial Lineage III was most abundant at the site previously shown to have high fish disease and was also most likely to be infected. The 2 techniques for detecting parasite infection did not always agree, and the likelihood of PCR (+) and spore (-) was not significantly different from PCR (-) and spore (+). Differences in the relative infection prevalence for these 2 lineages may explain the wide range of infection in natural streams.

Expression of immune-regulatory genes, arginase-2 and inducible nitric oxide synthase (iNOS), in two rainbow trout (Oncorhynchus mykiss) strains following exposure to Myxobolus cerebralis

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.

Characterization of glycans in the developmental stages of Myxobolus cerebralis (Myxozoa), the causative agent of whirling disease

Glycans and sugar-binding molecules (lectins) form an interactive recognition system, which may enable parasitic organisms to adhere to host cells and migrate into target tissues. The aim of the present study was to analyse surface-associated glycans in the developmental stages of Myxobolus cerebralis (Hofer), the causative agent of whirling disease. A panel of biotin-labelled plant lectins was used to detect a broad spectrum of glycan motifs with high specificity. Binding sites were detected histochemically in the tissue sections of infected rainbow trout, Oncorhynchus mykiss (Walbaum), and infected Tubifex tubifex (Müller), and were characterized by light, fluorescence and transmission electron microscopy. With mannose-specific lectins [Lens culinaris agglutinin, Pisum sativum agglutinin, Canavalia ensiformis agglutinin (LCA, PSA, CanA)] mannose-containing glycans were detected in all the developmental stages and host tissues. No binding sites for galactose-specific lectins were present in M. cerebralis spores but reactivity with host tissues occurred. Diversity in glycans was detected by N-acetyl-D-galactosamine-specific lectins in sporoplasm cells of M. cerebralis and triactinomyxon spores. In the group of lectins with monosaccharide-specificity for N-acetyl-D-glucosamine (GlcNAc), the reactivity of Datura stramonium agglutinin (DSA), Lycopersicon esculentum agglutinin (LEA) and Solanum tuberosum agglutinin (STA) was restricted to polar capsules whereas Griffonia simplicifolia agglutinin II (GSA II) also bound to sporoplasm cells of stages in the fish host but not in those present in infected T. tubifex. Moreover, Triticum vulgaris (wheat germ) agglutinin (WGA) and succinylated WGA indicated the presence of N-acetyl-D-glucosamine polymers in polar capsules. No specificity for spores was observed concerning 'bisected'N-glycans and no reactivity in parasitic stages was observed with the fucose-binding lectin Ulex europaeus agglutinin (UEA) I, Sambucus nigra agglutinin (SNA) (specific for alpha2,6-sialylated glycans) and Maackia amurensis agglutinin (MAAI) (specific for alpha2,3-sialylated glycans). Arachis hypogaea (peanut) agglutinin (PNA), Erythrina cristagalli agglutinin (ECA), GSA I, Sophora japonica agglutinin (SJA), Dolichos biflorus agglutinin (DBA) and GSA II detected reactive sites solely confined to the developmental stages of M. cerebralis and were not reactive in the fish host. These parasite-specific glycans may play a role in the adhesion process of the parasite to fish epidermis prior to infection, but may provide protection to the host by activating the complement system, or stimulating an adaptive immune response as putative antigens.

Construction and screening of a cDNA library from the triactinomyxon spores ofMyxobolus cerebralis, the causative agent of salmonid Whirling Diseases

The ZAP Express cDNA library was constructed using mRNA extracted from the triactinomyxon spores of Myxobolus cerebralis. First-strand cDNA was synthesized using Moloney Murine leukaemia virus reverse transcriptase. Following second-strand cDNA synthesis, the double-stranded cDNA was digested with Xho I restriction enzyme, cDNA fragments less than 400 bp were removed and the remaining cDNA was ligated with the lambda ZAP Express vector. The recombinants were packaged in vitro using Gigapack III gold packaging extract. The primary cDNA library titre contained 0.5 x 10(6) clones, with 97% recombinant and only 3% non-recombinant clones. The cDNA library was then screened using the anti-triactinomyxon antibodies. Positive clones were selected and re-screened twice more to give a final selection of 526 clones. One clone (46-5) was selected randomly and subjected to in vivo excision of the pBK-CMV phagemid from the ZAP express vector. The sequence of the entire clone was obtained using rapid amplification of the cDNA ends. A search of the clone sequence against GenBank revealed that it related to ribosomal protein L23 and it had a high percentage similarity to this protein from different species. A conserved domain for ribosomal protein L23 was also identified in the clone sequence.

Characterisation of carbohydrate-binding sites in developmental stages of Myxobolus cerebralis

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.

The life cycle of Henneguya nuesslini Schuberg & Schröder, 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.

Molecular methods clarify morphometric variation in triactinomyxon spores (Myxozoa) released from different oligochaete hosts.

Thirty-nine freshwater tubificid oligochaetes were isolated, each of which harboured a triactinomyxon infection. Spore characteristics include the typical triactinomyxon anchor shape, eight germ cells within the sporoplasm and three unequal (two long and one shorter) caudal processes with square tips. Despite morphological similarities between the spores from the different hosts, their morphometrical data varied considerably; significantly, the ranges of dimensions of the smallest and largest exemplars were mutually exclusive. In order to ascertain the true number of spore types present, molecular methods were employed. Samples of waterborne spores, including the smallest and largest representatives, were selected from 11 host oligochaetes (all Tubifex tubifex Müller) and a nested PCR-RFLP 'riboprint' analysis performed. The small subunit ribosomal DNA gene (18S rDNA) was targetted and amplified through two rounds of PCR, then digested with the restriction enzymes Dde I and Hha I. The resultant major cleavage patterns produced by both enzymes indicated a single triactinomyxon form; however, the pattern of several less intense bands varied between the samples. From a subset of five samples drawn from across the full spectrum of spore sizes, a 327 bp region near the 5' was sequenced and was identical for all five samples. Comparison of this 327 bp region with that of 12 other triactinomyxons in GenBank showed 68.7-96.9% similarity (at least 9 base differences). A further 469 bp generated for each of the smallest, largest and mid-range (= reference) spore samples was identical also. The reference sample was sequenced further to yield 1,554 bp of 18S rDNA (GenBank accession number AY162270); comparison with other Myxozoa indicated this sequence was novel. The morphometrics of our triactinomyxon did not correlate with any published description. The morphometrical variation exhibited by spores of the triactinomyxon type in this study raises questions about the validity of using morphometrical data to distinguish spore types and suggests that there could be taxonomic redundancy in the diversity of actinosporeans recorded in the literature. The additional information provided by molecular data in this study was pivotal in the clarification of morphometrical variation exhibited by morphologically similar triactinomyxon spores released from different oligochaete hosts.

Real-time quantitative polymerase chain reaction (QPCR) to identify Myxobolus cerebralis in rainbow trout Oncorhynchus mykiss

This study describes the development of a TaqMan real-time quantitative polymerase chain reaction (QPCR) technique using the heat-shock protein 70 (Hsp 70) and 18S ribosomal DNA (18S rDNA) sequences to identify Myxobolus cerebralis and attempt to quantify infection severity within rainbow trout fry Oncorhynchus mykiss. Rainbow trout for this study were exposed to M. cerebralis under natural river conditions and examined for infection by histology, polymerase chain reaction (PCR) and QPCR analysis at 900 Celsius temperature units (CTUs) following exposure. Detection sensitivity by QPCR was shown to be equal to traditional PCR but greater than histopathology. Primer/probe combinations developed for this study were capable of specifically detecting M. cerebralis DNA in infected fish tissue and single triactinomyxon (TAM) spores with a sensitivity of 12.5 and 6.3 pg microl(-1) of DNA for the Hsp 70 and 18S rDNA sequences, respectively. A strong relationship between QPCR and infection severity was found for the Hsp 70 probe when parasite copy number and histology scores of 0-4 were compared (R2 = 0.96, p = 0.003). However, a reduction in copy number was observed at higher histology scores for the 18S probe (scores of 4 and 5) and the Hsp 70 probe (score of 5). The results of this study demonstrate that QPCR analysis is an effective tool for detecting M. cerebralis in fish tissue and may provide a relative indication of infection severity.

Effects of water temperature and substrate type on spore production and release in eastern Tubifex tubifex worms infected with Myxobolus cerebralis.

Eastern Tubifex tubifex worms were exposed to Myxobolus cerebralis spores at 9, 13, 17, and 20 C in 1-L jars that contained sand, mud, or leaf litter as substrata. Beginning 60 days after exposure, water from each jar was filtered daily and examined for the presence of waterborne triactinomyxon spores (TAMs). On discovering a single TAM from an experimental jar, 48 T. tubifex worms from that jar were placed individually into 24-well plates. Spores released from individual infected T. tubifex worms were quantified to determine the first day of TAM release from infected worms, the infection rate, the total number of TAMs released per worm, and the duration of release. No TAMs were found in any of the jars incubated at 20 C or in uninfected, control worms at any temperature. The total number of TAMs released by infected worms in mud and sand was highest at 13 C compared with other temperatures. Infection rates among individual worms increased with temperature between 9 and 17 C. Higher temperatures (up to 17 C) induced earlier TAM releases among infected worms, and substratum did not influence this production parameter. The average duration of TAM release decreased as the temperature increased from 9 to 17 C, and there was a significant effect of substratum in the groups maintained at 13 and 17 C. In all temperature treatments between 9 and 17 C, the duration of release was least in the worms maintained in leaf litter, as was the total number of TAMs released during the experimental period and the median number of TAMs per production day.

Description of two new actinosporean types from a brook of Fuji Mountain, Honshu, and from Chitose River, Hokkaido, Japan.

Actinospore infection of oligochaetes living in the mud of 3 freshwater biotopes in Japan was studied. Using the cell-well plate method, a new aurantiactinomyxon type was found in 0.77% of the examined Tubifex tubifex oligochaete specimens from a brook near Yamanashi Prefectural Fisheries Experimental Station on Fuji Mountain. In 0.14% of Lumbriculus variagetus collected from Chitose River, near Chitose Salmon Hatchery, a new siedleckiella type was found, while at the same time 8.1% of the Lumbriculus spp. oligochaetes released triactinomyxons of Myxobolus arcticus. Of the examined Rhyacodrilus komarovi oligochaetes collected from the Mena River system, Hokkaido, 0.2, 0.6, 0.5 and 0.8% were infected with echinactinomyxon, neoactinomyxum and 2 types of triactinomyxon spores, respectively, and described in our previous paper. The oligochaetes released actinospores for several weeks. Actinospore infection showed high intensity in positive oligochaetes in the case of all the actinosporean types. Two of the actinospore types (aurantiactinomyxon and siedleckiella) presented here have not been previously described.

Occurrence of actinosporean stages of myxosporeans in an inflow brook of a salmon hatchery in the Mena River System, Hokkaido, Japan.

Actinospore infection of oligochaetes living in the mud and on the roots of vegetation in an inflow brook of a Hokkaido salmon hatchery was studied within the framework of a Japanese-Hungarian research program. Two triactinomyxon types, 1 echinactinomyxon, and 1 neoactinomyxum type were isolated from the oligochaete Rhyacodrilus komarovi Timm, 1990 collected during the survey. The aurantiactinomyxons were recorded over a period of 3 mo starting from the day after oligochaete collection. The oligochaetes released actinospores for several weeks from the first day of the study. Spore excretion of individual oligochaetes was not synchronous. Of the oligochaetes examined, 0.7, 7, 3 and 3%, were infected with the echinactinomyxon, neoactinomyxum and the 2 types of triactinomyxon spores, respectively. Actinospore infection was intense in the positive oligochaetes in all 4 types. Of the 4 actinospore types presented here, 3 are described for the first time.

Effects of whirling disease on selected hematological parameters in rainbow trout.

Hematological responses to whirling disease in rainbow trout (Oncorhynchus mykiss) were investigated. Two-mo-old fingerling rainbow trout were exposed to cultured triactinomyxon spores of Myxobolus cerebralis at 9,000 spores/fish in December, 1997. Twenty-four wks post-exposure, fish were taken from infected and uninfected groups for peripheral blood and cranial tissue sampling. Histological observations on cranial tissues confirmed M. cerebralis infection in all exposed fish. Differences in hematological parameters between the two groups included significantly lower total leukocyte and small lymphocyte counts for the infected fish. No effects on hematocrit, plasma protein concentration, or other differential leukocyte counts were noted.

Ultraviolet irradiation inactivates the waterborne infective stages of Myxobolus cerebralis: a treatment for hatchery water supplies.

The effects of ultraviolet (UV) irradiation on the viability of the waterborne triactinomyxon stages of Myxobolus cerebralis were evaluated by vital staining and the infectivity for juvenile rainbow trout Oncorhynchus mykiss. A dose of 1300 mWs cm-2 was required to inactivate 100% of the triactinomyxons held under a static collimated beam of UV as determined by vital staining. Juvenile rainbow trout were protected from infections with M. cerebralis when exposed to 14,000 or 1400 triactinomyxon spores per fish that had been treated with the collimating beam apparatus (1300 mWs cm-2). Among all fish receiving UV-treated triactinomyxons, none had clinical signs of whirling disease, or evidence of microscopic lesions or spores of M. cerebralis after 5 mo at water temperatures of 15 degrees C. In contrast, 100% of the fish receiving the higher dose of untreated triactinomyxons developed clinical signs of whirling disease and both microscopic signs of infection and spores were detected in all of the high and low dose trout receiving untreated triactinomyxon exposures. Two additional trials evaluated the Cryptosporidium Inactivation Device (CID) for its ability to treat flow-through 15 degrees C well water to which triactinomyxons were added over a 2 wk period. CID treatments of a cumulative dose exceeding 64,000 triactinomyxons per fish protected juvenile rainbow from infections with M. cerebralis. Rainbow trout controls receiving the same number of untreated triactinomyxons developed both microscopic lesions and cranial spore concentrations up to 10(4.6) per 1/2 head, although no signs of clinical whirling disease were observed. UV (126 mWs cm-2, collimated beam apparatus) was also effective in killing Flavobacterium psychrophilum, the agent causing salmonid bacterial coldwater disease, as demonstrated by the inability of bacterial cells to grow on artificial media following UV treatment.

Effect of water temperature on the development, release and survival of the triactinomyxon stage of Myxobolus cerebralis in its oligochaete host

The development of the triactinomyxon stage of Myxobolus cerebralis and release of mature spores from Tubifex tubifex were shown to be temperature dependent. In the present work, the effect of temperature over a range of 5–30°C on the development and release of the triactinomyxon stages of M. cerebralis was studied. Infected T. tubifex stopped releasing triactinomyxon spores 4 days after transfer from 15°C to 25°C or 30°C. Transmission electron microscopic examinations of the tubificids held at 25°C and 30°C for 3 days showed that all developmental stages degenerated and transformed to electron-dense clusters between the gut epithelial cells of T. tubifex. In contrast, tubificid worms held at 5°C and 10°C examined at the same time were heavily infected with many early developmental stages of triactinomyxon. At 15°C, the optimal temperature for development, maturing and mature stages of the parasite were evident. Infected T. tubifex transferred from 15°C to 20°C stopped producing triactinomyxon spores after 15 days. However, 15 days at 20°C was not sufficient to destroy all developmental stages of the parasite. When the tubificid worms were returned to 15°C, the one-cell stages and the binucleate-cell stages resumed normal growth. It was also demonstrated that T. tubifex cured of infection by holding at 30°C for 3 weeks and shifted to 15°C could be re-infected with M. cerebralis spores. The waterborne triactinomyxon spores of M. cerebralis did not appear to be as short-lived as previously reported. More than 60% of experimentally produced waterborne triactinomyxon spores survived and maintained their infectivity for rainbow trout for 15 days at water temperatures up to 15°C. In natural aquatic systems, the triactinomyxon spores may survive and keep their infectivity for periods even longer than 15 days.

Whirling disease: host specificity and interaction between the actinosporean stage of Myxobolus cerebralis and rainbow trout Oncorhynchus mykiss.

Scanning electron microscopic studies were conducted on rainbow trout Oncorhynchus mykiss in the first 60 min after their exposure to the triactinomyxon spores of Myxobolus cerebralis. The results demonstrated that as early as 1 min post exposure the whole process, from the attachment of the triactinomyxon spores to the complete penetration of their sporoplasm germs, had occurred. The triactinomyxon spores sought out the secretory openings of mucous cells of the epidermis, the respiratory epithelium and the buccal cavity of trout and used them as portals of entry. Exposure experiments of the triactinomyxon spores of M. cerebralis to non-salmonid fish, such as goldfish Carassius auratus, carp Cyprinus carpio, nose Chondrostoma nasus, medaka Oryzias latipes, guppy Poecilia reticulata and also the amphibian tadpole Rana pipiens as well as to rainbow trout fry indicated a specificity for salmonids. Attempts to activate the triactinomyxon spores by exposure to mucus prepared from cyprinid and salmonid fish showed no significant differences from those conducted in tap water. The results suggest that the simultaneous presence of both mechano- and chemotactic stimuli was required for finding the salmonid fish host.