Effect of water temperature on the development ...

International Journal for Parasitology 29 (1999) 627±641

Eect of water temperature on the development, release and survival of the triactinomyxon stage of Myxobolus cerebralis in its oligochaete host M. El-Matbouli a, b, *, T.S. McDowell a, D.B. Antonio a, K.B. Andree a, R.P. Hedrick a a Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA Institute of Zoology, Fish Biology and Fish Diseases, Faculty of Veterinary Medicine, University of Munich, Kaulbachstr. 37, 80539 Munich, Germany

b

Received 13 October 1998; received in revised form 21 December 1998; accepted 21 December 1998

Abstract 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 eect of temperature over a range of 5± 308C 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 158C to 258C or 308C. Transmission electron microscopic examinations of the tubi®cids held at 258C and 308C 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, tubi®cid worms held at 58C and 108C examined at the same time were heavily infected with many early developmental stages of triactinomyxon. At 158C, the optimal temperature for development, maturing and mature stages of the parasite were evident. Infected T. tubifex transferred from 158C to 208C stopped producing triactinomyxon spores after 15 days. However, 15 days at 208C was not sucient to destroy all developmental stages of the parasite. When the tubi®cid worms were returned to 158C, 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 308C for 3 weeks and shifted to 158C 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 158C. In natural aquatic systems, the triactinomyxon spores may survive and keep their infectivity for periods even longer than 15 days. # 1999 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. Keywords: Myxobolus cerebralis; Myxozoa; Oligochaete; Temperature; Tubifex tubifex; Whirling disease

* Corresponding author. Present address: Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA. Fax: 530-752-0414; e-mail: [email protected] 0020-7519/99/$20.00 # 1999 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 0 - 7 5 1 9 ( 9 9 ) 0 0 0 0 9 - 0

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M. El-Matbouli et al. / International Journal for Parasitology 29 (1999) 627±641

1. Introduction Whirling disease (WD) is an ecologically and economically devastating parasitic disease of salmonid ®sh caused by the myxozoan Myxobolus cerebralis. It is typically a chronic disease that can cause high mortalities in feral and hatcheryreared trout fry and ®ngerlings. Myxobolus cerebralis has a two-host life-cycle involving a salmonid ®sh and the oligochaete Tubifex tubifex [1, 2]. The spores of M. cerebralis can be released into the aquatic environment only after death and autolysis of infected ®sh or after consumption and excretion by predators [3, 4]. Free M. cerebralis spores are ingested by T. tubifex and develop intercellularly in their gut epithelium into the actinosporean triactinomyxon, which is the only infective stage for salmonid ®sh. This transformation of the parasite in the oligochaete host occurs within 3 months at water temperatures of 152 28C through four phases, which are schizogony, gametogony, gametogamy and sporogony [5]. The triactinomyxon spores reach the water either by excretion through the gut lumen or following the death of the infected tubi®cids. The waterborne triactinomyxon stage enters susceptible salmonid ®sh through the epithelial cells of skin, ®ns, gills, buccal cavity and oesophagus, then develops and transforms into M. cerebralis spores after about 4 months at 13.58C [6, 7]. Studies on the molecular phylogeny of Myxosporea using the ssrRNA gene sequences have resulted in techniques useful for identifying corresponding actinosporean and myxosporean stages in the life-cycle of M. cerebralis and for development of new diagnostic approaches [8]. Both PCR [9] and in situ hybridization (ISH) [10] assays can detect all developmental stages of M. cerebralis in both hosts. Recent results on the determination of the nuclear DNA concentration in the cells of M. cerebralis and its triactinomyxon stage have shown that both meiosis and copulation of gametes take place in the oligochaete host T. tubifex [11]. Therefore, the authors consider T. tubifex as the de®nitive host and the salmonid the intermediate host.

Data on the eects of temperature on the development of myxosporean and actinosporean spores in ®sh and oligochaetes, respectively, are rather scarce. Halliday [12] showed experimentally that high water temperatures increase the rate of development of M. cerebralis in rainbow trout. Spores of M. cerebralis were apparently fully formed in trout held at 178C by 52 days post-exposure to the infection, while they took 101 days and 120 days at 128C and 78C, respectively. Homan and Markiw [13] reported that M. cerebralis spores heated at 908C for 10 min and 708C for 100 min were nearly all stained with methylene blue, presumptive evidence that they were killed. El-Matbouli and Homann [4] showed experimentally that M. cerebralis spores survive freezing at ÿ208C for at least 2 months. Markiw [14] reported that the experimentally produced waterborne actinosporean stages of M. cerebralis were short-lived, persisting for only 3± 4 days at 12.58C and for less time at warmer temperatures. According to published data on whirling disease, the optimal temperature for experimental infection of T. tubifex with M. cerebralis is 15 228C [1, 2]. The present study was conducted to examine the eect of temperatures above and below the optimum of 158C on the development and release of the triactinomyxon stage of M. cerebralis from infected T. tubifex. Previously infected T. tubifex were also re-exposed to M. cerebralis spores to determine if they were still susceptible to a second infection. Furthermore, the eects of dierent water temperatures on the infectivity of the waterborne triactinomyxon stage of M. cerebralis for rainbow-trout fry were examined. 2. Materials and methods 2.1. Eect of dierent water temperatures on the release of triactinomyxon spores from T. tubifex Oligochaete cultures with about 90% T. tubifex were maintained in aquaria with slowly running water at 158C until the worms were separated and cleaned for exposure. Myxobolus cerebralis spores for infection of oligochaetes


were collected by the plankton centrifuge method described by O'Grodnick [15]. Tubi®cid worms (15 g) were exposed to M. cerebralis spores (300 spores per worm) and held in an incubator at 158C. Three months post-exposure, tubi®cid worms were isolated from the substrate and then distributed individually into 24-well plates ®lled with dechlorinated tap water. The plates were placed in an incubator at 158C overnight. Infected T. tubifex, which were identi®ed by release of triactinomyxon spores overnight, were collected and transferred to six-well plates prepared with a 2-mm layer of autoclaved sand as a substrate. Ten worms were placed in each of three wells per plate, then plates were transferred to incubators at either 5, 10, 15, 20, 25 or 308C. Triactinomyxon spores released into the water by T. tubifex held at each temperature were enumerated twice a week for 64 days. Water from each well at each temperature was collected separately. Duplicate aliquots of 200 ml were placed in a 60mm petri dish with a 2-mm grid. A 20 mm40 mm cover glass was placed on the water drop, and all the triactinomyxon in the dish were counted. The average number of the total parasites counted was used to calculate the total number of the parasites from each sample. 2.2. In¯uences of dierent temperatures on the development of triactinomyxon spores in T. tubifex The same procedures as in the previous experiment were repeated, using 120 instead of 30 infected worms per temperature. Seven worms, sampled daily for a time period of 10 days from each plate at the ®ve temperatures, were ®xed in 10% buered formalin, embedded in paran, and duplicate 3±4-mm-thick sections were stained with H & E and Giemsa for light microscopic examination. An additional three worms at each sampling period were ®xed in Karnovsky's ®xative (2.0% paraformaldehyde and 2.5% glutaraldehyde in 0.06 M-Sorensen's phosphate buer at pH 7.2) overnight at 48C and embedded in Epoxy resin for EM investigations. Fifteen days after beginning the experiment, the tubi®cid groups held at 20, 25 and 308C were

629

transferred to an incubator at 158C. The other three groups held at 5, 10 and 158C were maintained at these temperatures until termination of the experiment. The water from each group was changed once a week and examined microscopically for the presence of waterborne triactinomyxon spores until 5 months post-transfer. Seven months later, ®ve worms from the tubi®cid groups held at 5, 15, 20, 25 and 308C were sampled by the nested PCR as described by Andree et al. [9]. Also, ®ve worms from each group were ®xed in 10% buered formalin for 24 h and then transferred into 70% ethanol and processed for ISH as described by Antonio et al. [10]. 2.3. Re-infection of T. tubifex Six-hundred infected T. tubifex were separated from an experimentally exposed oligochaete population as described above. Four-hundred were placed in a 500-ml container with autoclaved sand as a substrate. The other 200 infected worms were transferred to a second 500-ml container. Both containers were held in a 308C incubator for 3 weeks. At the end of that time, both containers were held at room temperature for 5 h and then transferred to a 158C incubator. After 1 week, the container with 400 worms was exposed to isolated and enriched M. cerebralis spores (300 spores per worm). The second container with 200 T. tubifex remained without addition of M. cerebralis spores as a control. Beginning from 3 months post-exposure, the water from both containers was ®ltered once a week for the presence of waterborne triactinomyxon spores. Four months post-exposure 20 worms from each container were randomly separated and placed individually in 24-well plates ®lled with dechlorinated tap water in order to determine the percentage of re-infected T. tubifex by counting the worms releasing triactinomyxon spores in the water. At 7 months post-exposure, 10 T. tubifex from the exposed and unexposed containers were removed and then ®xed in 10% buered formalin and processed for histological examination.

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2.4. Eect of water temperature on the infectivity of waterborne triactinomyxon stages of M. cerebralis The waterborne triactinomyxon spores used in this experiment were harvested from two containers. Each container (10 L) contained sand as a substrate for 150 g of tubi®cid worms exposed to M. cerebralis spores (350 spores per worm) and held at 13.58C. For this study four 10-L aquaria were prepared with tap water and minimal air supply. No worms or substrate were present in these aquaria. One-million freshly harvested triactinomyxon spores were placed in each aquarium, then the aquaria were transferred to incubators at either 6, 10, 15, or 208C. A 50-ml water sample was taken daily from each aquarium and ®ltered through 10-mm-mesh screen. Triactinomyxon spores trapped on the ®lter from each aquarium were examined microscopically and the percentage of spores with morphological changes, such as discharged polar ®laments or empty shell valves, was documented as evidence of non-viability. After 15 days all aquaria were transferred to room temperature (13.0 2 2.08C). Five hours later, 10 rainbow-trout fry (Oncorhynchus mykiss) (2±4 cm) were placed

into each aquarium, without the addition of running water. After 24 h the water ¯ow was resumed to each aquarium. An additional aquarium with 10 rainbow-trout fry and water without triactinomyxon spores served as a negative control. Starting 50 days post-exposure, ®sh were checked daily for clinical signs. At 5 months post-exposure, ®sh from all four containers were necropsied for detection of M. cerebralis infection. Fresh mount and stained tissue sections of the cartilage present in the cranial regions was examined microscopically as described by ElMatbouli and Homann [2].

3. Results 3.1. Eect of dierent temperatures on the release of triactinomyxon spores from infected T. tubifex Twenty-four hours after transfer of infected T. tubifex into multi-well plates at 5, 10, 15, 25 and 308C, the waterborne triactinomyxon spores in each group were enumerated. The average number of triactinomyxon spores released by individual T. tubifex in each group after 24 h was 32 at

Fig. 1. Eect of temperature on release of the triactinomyxon stage of M. cerebralis from infected T. tubifex.


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Fig. 2. Histological cross-section of infected T. tubifex. L, Gut lumen; MP, mature pansporocysts; arrows, early pansporocysts; arrowheads, one-cell stage and binucleate cell stage. Toluidine blue stain, 4 days at 158C. Scale bar = 50 mm. Fig. 3. Cross-section through infected T. tubifex showing migration of non-mature pansporocyst into the gut lumen (arrow). Note the increased number of mucous cells (arrowheads) and the degenerated parasite stages (S); L, gut lumen. Toluidine blue stain, 1 day post-transfer to 158C. Scale bar = 50 mm.

632


Fig. 4. Pansporocyst (arrow) and early developmental stages (arrowheads) of triactinomyxons migrating out of the gut epithelial cells into the gut lumen of infected T. tubifex. Giemsa, 1 day post-transfer to 308C. Scale bar = 30 mm. Fig. 5. Cross-section through T. tubifex showing degenerated triactinomyxon stages (arrows). Toluidine blue stain, 2 days posttransfer to 308C. Scale bar = 50 mm.


633

Fig. 6. Gut epithelium of infected T. tubifex showing degenerated triactinomyxon developmental stages (arrows). Note that some one-cell stages are still intact (arrowheads). Toluidine blue stain, 3 days post-transfer to 258C. Scale bar = 30 mm. Fig. 7. Cross-section through infected T. tubifex showing gut epithelium recovering from infection with triactinomyxon. Toluidine blue stain, 10 days post-transfer to 258C. Scale bar = 30 mm. Fig. 8. Degenerated triactinomyxon developmental stages (arrows) and a high number of mucous cells (arrowheads) in infected T. tubifex held at 208C. Toluidine blue stain, 10 days post-transfer to 208C. Scale bar = 50 mm.

634


Fig. 9. Electronmicrograph of infected T. tubifex held at 58C showing uninucleate cell (arrow) and binucleate cell stages (arrowheads). N, Nuclei of the host gut epithelial cells. Scale bar = 2 mm. Fig. 10. Infected T. tubifex held at 208C showing normal early pansporocyst stage containing zygotes (arrows) and a pansporocyst with degenerated sporoblasts (arrowhead). After 3 days post-transfer to 208C. Scale bar = 5 mm.


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Fig. 11. Infected T. tubifex held at 208C showing degenerated pansporocysts (arrows) and vacuolated binucleate cell stages (arrowheads); L, gut lumen. At 7 days post-transfer to 208C. Scale bar = 2 mm. Fig. 12. Infected T. tubifex held at 258C showing vacuolated gut epithelium and degenerated stages of triactinomyxon (arrows). M, Mucous cells. At 3 days post-transfer to 258C. Scale bar = 5 mm.

636


Fig. 13. Infected T. tubifex held at 308C. Note that the parasite stages turned into labyrinth membranous clusters (arrows) between the gut epithelial cells of the host. R, Remnants of the parasite; M, mucous cells; N, host cell nuclei; L, gut lumen. At 2 days posttransfer to 308C. Scale bar = 2 mm. Fig. 14. Infected T. tubifex held at 308C showing electron-dense inclusions of degenerated parasite stages (arrows). At 3 days posttransfer to 308C. Scale bar = 2 mm.


58C, 20 at 108C, 160 at 158C, 42 at 208C, 432 at 258C and 200 at 308C. Tubifex worms held at 258C and 308C stopped releasing triactinomyxon spores by 4 days after transfer (Fig. 1). After 15 days the tubi®cid worms held at 208C also stopped releasing triactinomyxon spores. Tubifex tubifex held at 5, 10 and 158C produced triactinomyxon spores until the end of the experiment (64 days post-transfer). As shown in Fig. 1, an enormous peak of released triactinomyxon spores by the tubi®cid group held at 58C could be seen 22 days posttransfer. The amount of waterborne triactinomyxon spores increased from 70 to 2580 spores per worm. The reason for the sudden increase in the number of released spores was an accidental increase of the water temperature when the incubator was left open over two nights allowing room temperature air to enter the chamber. 3.2. In¯uences of dierent temperatures on the development of triactinomyxon stages in infected T. tubifex By light microscopy, all tubi®cid samples held at 5, 10 and 158C showed a normal development of the parasite stages in the gut epithelium (Fig. 2). Twenty-four hours after transfer of infected tubi®cids to 308C, histological examination of the tubi®cid samples showed that the majority of the mature and maturing pansporocysts were released into the gut lumen (Figs. 3 and 4). The early developmental stages between the gut epithelial cells coalesced into cell aggregates intensively stained with Giemsa (Fig. 5). The same destruction of the parasite stages could also be seen after 3 days in tubi®cid worms held at 258C (Fig. 6) and after 10 days in the tubi®cid worms held at 208C. At 10 days post-exposure, the gut epithelial cells of tubi®cids held at 308C and 258C completely recovered from the infection (Fig. 7). At that time, single, degenerated pansporocysts were observed occasionally in the samples held at 208C (Fig. 8). By EM, the complete development of the triactinomyxon stages in the gut epithelium of the tubi®cid worms held at 5, 10, and 158C were evident in all samples. Multi-nuclear schizogonic

637

stages, one-cell stages, binucleate-cell stages, early and mature pansporocysts were present in most samples of worms from all three temperature groups. It was noticeable that more early developmental stages rather than maturing pansporocysts were detected in the tubi®cid samples held at 58C (Fig. 9). The ®rst appearance of parasite destruction in worms held at 208C was seen 3 days post-transfer. Beside non-aected pansporocysts, the zygotes inside some pansporocysts had degenerated into clustered membranes with few ribosomes and organelle remnants (Fig. 10). Most of the one-cell stages were found intact between the host epithelial cells in all samples taken from the tubi®cid group held at 208C. The binucleate-cell stages and the early pansporocysts were mostly vacuolated and their cell membranes were detached (Fig. 11). During the ®rst 3 days post-transfer, progressive degeneration of all developmental stages of the parasite were seen in the samples held at 25 and 308C (Figs. 12±14). The parasite stages degenerated into electron-dense inclusions between the vacuolated gut epithelial cells. There was a marked increase in the number of mucous cells in the gut epithelium of the tubi®cid samples held at 25 and 308C. The remaining tubi®cid worms held at 258C and 308C for 2 weeks and then transferred into 158C did not release triactinomyxon spores through the end of the experiment (7 month post-transfer). Tubi®cid worms held for 2 weeks at 208C and then transferred to 158C began to release spores after 85 days post-transfer and stopped 35 days later. The other three groups held continuously at 5, 10 and 158C released triactinomyxon spores until the last water examination at 5 months post-transfer. Seven months after the beginning of the experiment all tubi®cid worms previously held at 208C for 2 weeks and those held continuously at 5 and 108C showed positive infection with triactinomyxons using the nested PCR test and ISH. The other tubi®cid samples from both groups, which were held for 15 days at 25 and 308C were negative for the triactinomyxon stage of M. cerebralis, as con®rmed by both PCR and ISH.

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Fig. 15. Survival of waterborne triactinomyxon spores of M. cerebralis held at dierent water temperatures. Non-viable triactinomyxons were evaluated by the extrusion of the polar ®laments and empty spores.

3.3. Reinfection of T. tubifex Sixty days after re-exposure of T. tubifex previously cured of infection by holding at 308C, to M. cerebralis spores, microscopic examination of the worms revealed positive infection with early developmental triactinomyxon stages. One-cell stages and binucleate-cell stages, as well as early pansporocyst formations, could be easily seen microscopically within live tubi®cid worms. A whitish coloration of most re-exposed worms indicated a high prevalence of infection. Three months after the beginning of the experiment, waterborne triactinomyxon spores could be ®ltered from the container with the worms reexposed to M. cerebralis spores. Filtration of the water from the control container (not re-exposed) did not show any waterborne triactinomyxons. Seventeen out of 20 (85%) worms randomly separated from the exposed group released triactinomyxon spores after holding overnight in multi-well plates. None of the 20 worms from the control group released triactinomyxon spores. A histological examination of the tubi®cid samples taken 7 months post-exposure revealed seven of

10 positive worms. All worms from the control container were free of infection. 3.4. Eect of water temperature on the infectivity of waterborne triactinomyxon stages of M. cerebralis In this experiment, survival as well as infectivity of waterborne triactinomyxon spores held at four dierent temperatures was studied. Triactinomyxon spores that had extruded their polar ®laments or empty shell valves were considered as non-viable. The daily prevalence of viable waterborne triactinomyxon spores for a period of 15 days post-transfer are shown in Fig. 15. Fifteen days after holding waterborne triactinomyxon spores at 68C and 108C, about 23% of the triactinomyxon spores either had discharged their polar ®laments or were void of sporoplasm cells. The other two groups held at 158C and 208C revealed 32% and 50% non-viable spores, respectively. The results of exposure of rainbow-trout fry to each temperature-treated triactinomyxon group

M. El-Matbouli et al. / International Journal for Parasitology 29 (1999) 627±641 Table 1 The in¯uence of dierent water temperatures on the infectivity of waterborne triactinomyxon spores of Myxobolus cerebralis for rainbow-trout fry

Black taila Whirling movementsa Mortality No. of ®sh with M. cerebralis sporesb Prevalence of infection (%)

68C

108C

158C

208C

2 2 1

3 4 2

1 2 Ð

Ð Ð Ð

5

6

4

2

50%

60%

40%

20%

a

The number of ®sh showing clinical signs characteristic of whirling disease recorded in each group (n = 10). Prior to exposure, triactinomyxon spores (1106) were held for 15 days at each temperature. b The number of infected ®sh (n = 10) with spores of M. cerebralis as evaluated 5 months post-exposure to triactinomyxons held at each water temperature.

aged for 15 days are shown in Table 1. Whirling disease symptoms such as black tail and whirling movement appeared in the groups of trout fry exposed to the triactinomyxon spores held for 15 days at 6, 10 and 158C. The native microscopic and the histological examination of the trout from all three groups at the end of the experiment (5 months post-exposure) showed 40± 60% infection with M. cerebralis. Rainbow-trout fry exposed to triactinomyxon spores that were held at 208C for 15 days did not develop any disease symptoms. Wet mount preparation as well as histological examination of two out of 10 ®sh were positive for M. cerebralis. All unexposed control ®sh tested negative for M. cerebralis.

4. Discussion The relationship between environmental temperatures and the development of myxozoan parasites (Myxosporea and Actinosporea) is not well characterised. One diculty in studying this relationship has been unresolved life-cycles of these parasites. A few reports have discussed the eects of summer increases in water temperature as causing outbreaks of myxosporean infections [16±18]. In the case of M. cerebralis infecting salmonid ®sh, understanding the in¯u-

639

ences of water temperature on the development of whirling disease is one of the essential requirements to develop control measures for this disease. The ability to complete the life-cycle of M. cerebralis in both hosts in the laboratory and therefore to produce and enrich both parasite spore stages has made it possible to perform the current study. The ®rst part of our study demonstrated that infected T. tubifex stopped releasing triactinomyxon spores 4 days after transfer to 258C or 308C and after 15 days at 208C. In contrast, T. tubifex held at 5, 10 and 158C continued to produce triactinomyxon spores until the end of the experiment (64 days post-transfer). This result indicates that among the temperatures tested, 158C is the optimal temperature for regular production of triactinomyxon spores in infected T. tubifex, a ®nding identical to that of Markiw [6] and ElMatbouli et al. [7]. Lower temperature seems to delay the development and maturation of the pansporocysts and triactinomyxon spores. It also appears that temperatures between 5 and 108C serve to extend the period of spore production. The results of the EM examination of tubi®cid samples in the second part of our study support this hypothesis, because more early developmental stages were seen in the gut epithelium of tubi®cid worms held at 5 and 108C compared with those worms continuously held at 158C. The fact that many one-cell stages survived between the gut epithelial cells in the tubi®cid worms held at 208C for 2 weeks may explain why these worms retained the ability to release triactinomyxon spores after transfer to 158C. However, worms held continuously for 70 days at 208C did not release any triactinomyxon spores after 15 days post-transfer and the histological examination did not show any parasite stages in their gut epithelium. In contrast, all developmental stages of the parasite in the tubi®cid samples held at 258C and 308C had completely degenerated as early as 4 days post-transfer. The mechanism by which higher water temperature aects the development of the triactinomyxon stages in T. tubifex may be due to a combination of factors, including the fact that temperature ranges for survival are dierent

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between the triactinomyxon (parasite) and the T. tubifex (host), and considering that the triactinomyxon develops intercellularly, the mature parasite stages may have an increased capability to migrate out of the gut epithelium compared with early developmental stages that then either die or degenerate. An explanation for the increased number of intestinal mucous cells in the worms held at 25 and 308C has not yet been determined. Re-infection of T. tubifex may explain the long period (over 1 year) of releasing triactinomyxon spores by experimentally infected worms as reported by Markiw [19] and El-Matbouli and Homann [5]. This ®nding has to be considered when developing control methods concerning the oligochaete host. Our results demonstrate that the waterborne triactinomyxon spores of M. cerebralis persisted for periods longer than 15 days at water temperatures up to 208C, although they had decreasing ability to infect rainbow trout with increasing water temperatures. More than 60% of the triactinomyxon spores held at 6, 10 or 158C survived and were able to infect 40±60% of exposed trout, as demonstrated by the development of clinical signs of whirling disease. Up to 50% of the triactinomyxon spores held at 208C for 15 days survived and, though sucient to infect 20% of the trout fry, these ®sh demonstrated only light infections with M. cerebralis and no disease signs. Our results dier from those presented by Markiw [14], who found that triactinomyxon spores of M. cerebralis are short-lived, persisting for only 5 days at 12.58C when evaluated by vital staining with ¯uorescein diacetate and propidium iodide. Furthermore, her studies showed that triactinomyxon spores were even less stable (3 days) when assayed by their infectivity for rainbow-trout fry. A possible explanation for the discrepancy between our results and those of Markiw [14] could be dierences in the experimental conditions. While Markiw [14] held the triactinomyxon spores in glass tubes at dierent temperatures, the spores in our work were held in a 10-L glass aquarium at a density of 1106. Concerning the longevity of other actinosporeans, Ratli [20] showed that water containing the infective agent of Ceratomyxa shasta, at that

time unknown (now known as an actinosporean and published by Bartholomew et al. [21]), kept its infectivity to ®sh for at least 7 days. According to Yokoyama et al. [22] the actinosporean Raabeia sp. survived for 25 days at 158C and 10 days at 258C. In their study, the authors de®ned longevity as the period from the release of the spores into the water until the sporoplasms were lost. The infectivity of this actinosporean to ®sh could not be determined, since at that time the ®sh host was not identi®ed [22]. In conclusion, development of the triactinomyxon spores in the oligochaete host is signi®cantly in¯uenced by water temperature. Water temperatures between 58C and 108C delay the development of the triactinomyxon stage of M. cerebralis, while temperatures between 108C and 158C provide an optimal environment for normal development and release of the parasite. Temperatures between 158C and 208C appear to accelerate the development of the parasite, increasing the number of released spores and decreasing the period of release. Water temperatures higher than 208C inhibit or are lethal to the triactinomyxon stages of M. cerebralis. The results reported here may aid in the management of whirling disease when water temperatures can be either manipulated or exploited for determining the best periods to avoid exposures to the infective stage of the parasite. The fact that reinfection of T. tubifex can occur contributes to understanding of the life-cycle of M. cerebralis. More than 60% of experimentally produced waterborne triactinomyxon spores of M. cerebralis can survive for 15 days at temperatures up to 158C and for less time at higher temperature. In natural aquatic environments at the same temperature, the triactinomyxon spores may survive and keep their infectivity for even longer than 15 days. Acknowledgements We would like to thank the Whirling Disease Foundation and the Turner Foundation, the National Fish and Wildlife Foundation, The US Fish and Wildlife Service, US Department of


Interior, Agreement Nos. 1445-RM09-97-0014 and AP96-002 and Trout Unlimited for supporting and funding this work. References [1] Wolf K, Markiw ME. Biology contravenes taxonomy in the myxozoa: new discoveries show alternation of invertebrate and vertebrate hosts. Science 1984;255:1449±52. [2] El-Matbouli M, Homann RW. Experimental transmission of two Myxobolus spp. developing bisporogeny via tubi®cid worms. Parasitol Res 1989;75:461±4. [3] Homan GL, Putz RE. Eect of freezing and aging on the spores of Myxobolus cerebralis, the causative agent of salmonid whirling disease. Progve Fish Cult 1971;33:95±8. [4] El-Matbouli M, Homann RW. Eect of freezing, aging and passage through the alimentary canal of predatory animals on the viability of Myxobolus cerebralis spores. J Aquat Anim Health 1991;3:260±2. [5] El-Matbouli M, Homann RW. Light and electron microscopic study on the chronological development of Myxobolus cerebralis in Tubifex tubifex to the actinosporean stage. Int J Parasitol 1998;28:195±217. [6] Markiw ME. Portal of entry for salmonid whirling disease in rainbow trout. Dis Aquat Organ 1989;6:7±10. [7] El-Matbouli M, Homann RW, Mandok C. Light and electron microscopic observations on the route of the triactinomyxon-sporoplasm of Myxobolus cerebralis from epidermis into rainbow trout (Oncorhynchus mykiss) cartilage. J Fish Biol 1995;46:919±35. [8] Andree KB, Gresoviac SJ, Hedrick RP. Small subunit ribosomal RNA sequences unite alternate actinosporean and myxosporean stages of M. cerebralis, the causative agent of whirling disease in salmonid ®sh. J Euk Microbiol 1997;44:208±15. [9] Andree KB, MacConnell E, Hedrick RP. A nested polymerase chain reaction for the detection of genomic DNA of M. cerebralis in rainbow trout (Oncorhynchus mykiss). Dis Aquat Organ 1998;34:145±54. [10] Antonio DB, Andree KB, McDowell TS, Hedrick RP. Detection of Myxobolus cerebralis in rainbow trout and oligochaete tissues using a nonradioactive in situ hybridization protocol. J Aquat Anim Health 1998;10:338±347.

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