Transmission of Tetracapsuloides bryosalmonae (Myxozoa: Malacosporea) to Fredericella sultana (Bryozoa: Phylactolaemata) by various fish species

Tetracapsuloides bryosalmonae is a myxozoan parasite of salmonids and freshwater bryozoans, which causes proliferative kidney disease (PKD) in the fish host. To test which fish species are able to transmit T. bryosalmonae to bryozoans, an infection experiment was conducted with 5 PKD-sensitive fish species from different genera. Rainbow trout Oncorhynchus mykiss, brown trout Salmo trutta, brook trout Salvelinus fontinalis, grayling Thymallus thymallus and northern pike Esox lucius were cohabitated with T. bryosalmonae-infected Fredericella sultana colonies and then subsequently cohabitated with statoblast-reared parasite free Bryozoa. Statoblasts from infected colonies were tested by PCR to detect cryptic stages of T. bryosalmonae, which may indicate vertical transmission of the parasite. In this study, brown trout and brook trout were able to infect Bryozoa, while there was no evidence that rainbow trout and grayling were able to do so. Few interstitial kidney stages of the parasite were detected by immunohistochemistry in brown trout and brook trout, while rainbow trout and grayling showed marked proliferation of renal interstitial tissue and macrophages with numerous parasite cells. Intraluminal stages in the kidney tubules were only detected in brown trout and rainbow trout. In contrast to previous observations, pike was not susceptible to PKD in these trials according to the results of T. bryosalmonae-specific PCR. No DNA of T. bryosalmonae was detected in any statoblast.     

Severe, chronic proliferative kidney disease (PKD) induced in rainbow trout Oncorhynchus mykiss held at a constant 18 degrees C

Proliferative kidney disease (PKD), caused by the myxozoan parasite Tetracapsuloides bryosalmonae, is well documented as a seasonal disease of rainbow trout Oncorhynchus mykiss. Water temperatures influence the course of the infection both within the fish and the invertebrate host, the recovery of fish from the disease being accelerated with decreasing water temperatures. During this study, groups of rainbow trout were held at a constant temperature (18 degrees C) for a sustained period of time following initial exposure to T. bryosalmonae. While the majority of these fish had recovered from the clinical disease after 9 mo, 10% remained infected, showing clinical signs of disease. A histological study revealed that the majority exhibited very high parasite loads and unusually severe symptoms of PKD. This demonstrates that while most rainbow trout can recover from PKD independent of water temperature, there exists a sub-population that cannot.     

Infection of bryozoans by Tetracapsuloides bryosalmonae at sites endemic for salmonid proliferative kidney disease

Laboratory-reared colonies of the bryozoans Fredericella sultana and Plumatella fungosa were placed upstream of 2 fish farms endemic for salmonid proliferative kidney disease (PKD) to assess rates of infection of bryozoans by Tetracapsuloides bryosalmonae, the causative agent of PKD. Colonies were deployed in the field for 8 trial periods of 2 wk each throughout the summer of 2001. Following each trial, bryozoan colonies were maintained in laboratory culture for 28 d and were regularly monitored for infection by searching for sac stages of T. bryosalmonae. Infections were never identified by observations of sac stages, however positive PCR results and sequencing of cultured material confirmed that cryptic infections were present in colonies of both species deployed at one site. The possibility that PCR results reflected contamination of surfaces of bryozoans can be excluded, given the short period of spore viability of T. bryosalmonae. Highest rates of infection occurred when 4 of 23 colonies of F. sultana and 1 of 12 colonies of P. fungosa were infected during the period 10 to 24 July. No infections were detected from mid-August to late October at this site. None of the colonies at the other site became infected throughout the period of study. Our data provide the first estimates of infection rates of bryozoans by T. bryosalmonae. Additionally, they provide evidence that a cryptic stage can be maintained within bryozoan hosts for a period of 4 to 6 wk.     

Temperature-driven proliferation of Tetracapsuloides bryosalmonae in bryozoan hosts portends salmonid declines

Proliferative kidney disease (PKD) is an emerging disease of salmonid fishes. It is provoked by temperature and caused by infective spores of the myxozoan parasite Tetracapsuloides bryosalmonae, which develops in freshwater bryozoans. We investigated the link between PKD and temperature by determining whether temperature influences the proliferation of T. bryosalmonae in the bryozoan host Fredericella sultana. Herein we show that increased temperatures drive the proliferation of T. bryosalmonae in bryozoans by provoking, accelerating and prolonging the production of infective spores from cryptic stages. Based on these results we predict that PKD outbreaks will increase further in magnitude and severity in wild and farmed salmonids as a result of climate-driven enhanced proliferation in invertebrate hosts, and urge for early implementation of management strategies to reduce future salmonid declines.     

Proliferative, presaccular stages of Tetracapsuloides bryosalmonae (Myxozoa: Malacosporea) within the invertebrate host Fredericella sultana (Bryozoa: Phylactolaemata)

Proliferative kidney disease (PKD), caused by the malacosporean parasite, Tetracapsuloides bryosalmonae, is a major disease of salmonid culture both in western Europe and North America. The fish are infected from spores that develop within freshwater bryozoans and are released into the water column. Although sporogenesis has been studied in the bryozoan host and occurs within sacs, the formation of these sacs from presaccular stages has only been hypothesized. Examination of infected bryozoans by using a range of techniques identified proliferating, presaccular amoeboid stages of T. bryosalmonae on the body wall of the bryozoan Fredericella sultana. These stages possessed unique electron-dense bodies and were observed as aggregating within the bryozoan metacoel, differentiating to form spore sacs. Spore sac growth was associated with the assimilation of the presaccular parasites rather than through cryptomitosis of sac mural cells. This sac formation through aggregation and assimilation suggests an intriguing mechanism by which T. bryosalmonae can cross-fertilize.     

Kidney pathology and parasite intensity in rainbow trout Oncorhynchus mykiss surviving proliferative kidney disease: time course and influence of temperature

Proliferative kidney disease (PKD) is an endoparasitic disease of salmonids caused by the myxozoan parasite Tetracapsuloides bryosalmonae. We recently described the development of the disease from initial infection until manifestation of clinical disease signs in rainbow trout held at 2 water temperatures, 12 and 18°C. The aim of the present study is to investigate whether (1) infected fish surviving the clinical phase would recover from renal pathological changes, (2) whether they would be able to reduce the parasite load in the kidneys, and (3) whether water temperatures would influence renal recovery and parasite clearance. At 18°C, fish showed a gradual recovery of normal kidney morphology which was associated with a decline in parasite numbers and infection prevalence. Fish kept at 12°C initially showed an enhancement of kidney lesions before recovery of normal kidney morphology took place. The decrease in renal parasite load was retarded compared to 18°C. The results from the present study provide evidence that rainbow trout surviving the clinical phase of PKD are able to (1) fully restore renal structure, and (2) significantly reduce renal parasite loads, although 100% clearance was not achieved within the experimental period of this study. Water temperature influences the rate but not the outcome of the recovery process.     

Diversity and systematics of the Malacosporea (Myxozoa)

Abstract. Myxozoans belonging to the recently described class Malacosporea parasitize freshwater bryozoans during at least part of their life cycle. There are at present only two species described in this class: Buddenbrockia plumatellae and Tetracapsuloides bryosalmonae . The former can exist as vermiform and sac-like stages in bryozoan hosts. The latter, in addition to forming sac-like stages in bryozoans, is the causative agent of salmonid proliferative kidney disease (PKD). We undertook molecular and ultrastructural investigations of new malacosporean material to further resolve malacosporean diversity and systematics. Phylogenetic analyses of 18S rDNA sequences provided evidence for two new putative species belonging to the genus Buddenbrockia , revealing a two-fold increase in the diversity of malacosporeans known to date. One new malacosporean is a vermiform parasite infecting the bryozoan Fredericella sultana and the other occurs as sac-like stages in the rare bryozoan, Lophopus crystallinus . Both bryozoans represent new hosts for the genus Buddenbrockia . Our results have established that the malacosporean which infected F. sultana was not a vermiform stage of T. bryosalmonae , although it was collected from a site endemic for PKD. Ultrastructural investigation of new material of B . plumatellae revealed the presence of numerous external tubes associated with developing polar capsules, confirming that the absence of external tubes should no longer be considered as a character of the class Malacosporea.     

Seasonal occurrence of the infectious stage of proliferative kidney disease (PKD) and resistance of rainbow trout, Salmo gairdneri Richardson, to reinfection

The seasonality of proliferative kidney disease (PKD) in rainbow trout, Salmo gairdneri Richardson, at the American River Hatchery, California was found to be primarily dependent on the presence or absence of the infectious stage in the water supply. This was determined by introducing sentinel trout into the hatchery water supply on a monthly basis, followed by their transfer to the laboratory for subsequent holding in 18° C, pathogen-free water for one month prior to examination. These exposures demonstrated that infections were obtained from April through October at ambient temperatures 12–20° C. Trout which had recovered from clinical infections were found to be resistant to reinfection. Resistance was induced by active infection and not just previous exposure to the infectious stage. Trout surviving PKD were also found to harbour later sporogonic stages of the parasite for at least one year following initial infection, but fully formed spores, as judged by well-developed valves, were not observed.     

The phylogeography of salmonid proliferative kidney disease in Europe and North America

Salmonid proliferative kidney disease (PKD) is caused by the myxozoan Tetracapsuloides bryosalmonae. Given the serious and apparently growing impact of PKD on farmed and wild salmonids, we undertook a phylogeographic study to gain insights into the history of genealogical lineages of T. bryosalmonae in Europe and North America, and to determine if the global expansion of rainbow trout farming has spread the disease. Phylogenetic analyses of internal transcribed spacer 1 sequences revealed a clade composed of all North American sequences plus a subset of Italian and French sequences. High genetic diversity in North America and the absence of genotypes diagnostic of the North American clade in the rest of Europe imply that southern Europe was colonized by immigration from North America; however, sequence divergence suggests that this colonization substantially pre-dated fisheries activities. Furthermore, the lack of southern European lineages in the rest of Europe, despite widespread rainbow trout farming, indicates that T. bryosalmonae is not transported through fisheries activities. This result strikingly contrasts with the commonness of fisheries-related introductions of other pathogens and parasites and indicates that fishes may be dead-end hosts. Our results also demonstrate that European strains of T. bryosalmonae infect and induce PKD in rainbow trout introduced to Europe.     

Castrating parasites and colonial hosts

Trajectories of life-history traits such as growth and reproduction generally level off with age and increasing size. However, colonial animals may exhibit indefinite, exponential growth via modular iteration thus providing a long-lived host source for parasite exploitation. In addition, modular iteration entails a lack of germ line sequestration. Castration of such hosts by parasites may therefore be impermanent or precluded, unlike the general case for unitary animal hosts. Despite these intriguing correlates of coloniality, patterns of colonial host exploitation have not been well studied. We examined these patterns by characterizing the responses of a myxozoan endoparasite, Tetracapsuloides bryosalmonae, and its colonial bryozoan host, Fredericella sultana, to 3 different resource levels. We show that (1) the development of infectious stages nearly always castrates colonies regardless of host condition, (2) castration reduces partial mortality and (3) development of transmission stages is resource-mediated. Unlike familiar castrator-host systems, this system appears to be characterized by periodic rather than permanent castration. Periodic castration may be permitted by 2 key life history traits: developmental cycling of the parasite between quiescent (covert infections) and virulent infectious stages (overt infections) and the absence of germ line sequestration which allows host reproduction in between bouts of castration.     

PKX, the causative agent of proliferative kidney disease (PKD) in Pacific salmonid fishes and its affinities with the Myxozoa

Proliferative kidney disease (PKD), caused by an unclassified protozoan (PKX), is reported from Pacific salmon, Oncorhynchus tshawytscha (Walbaum) and O. kisutch (Walbaum), and steelhead trout, Salmo gairdneri Richardson, held at the Mad River Hatchery in California, USA. The cumulative mortality attributed to the disease was 95, 13, and 18% respectively. The mortalities were greatest at mean water temperatures of 12-14 degrees C during July 1983. The ultrastructure of the PKX organism and its associated pathology during clinical disease in all three species were consistent with those of the parasite in rainbow trout (Salmo gairdneri) as described in European outbreaks. Significant mortalities did not occur after August, at which time the parasite could no longer be detected in the salmon species. The steelhead continued to exhibit parasites in the kidney interstitium and epithelium and lumens of the tubules. Myxosporidan trophozoites and developing spores were also observed in the lumens of the kidney tubules of these fish. Although a mixed infection with another parasite may have occurred, evidence suggests that the myxosporidans are later stages of PKX. They were only observed in fish exposed to water with the infective stage and were particularly prominent in recovering fish. The PKX organism is similar to UBO, an unclassified protozoan of carp suspected to be an early stage of Sphaerospora renicola Dykovà & Lom. Both parasites infect the blood and kidney, divide by endogeny, and are released by disintegration of the primary mother cell. The intraluminal myxosporean forms show similarities to Sphaerospora spp. in that they are monosporous and sporoblasts are formed within pseudoplasmodia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of malacosporean life cycles through transmission studies

Myxozoans, belonging to the recently described Class Malacosporea, parasitise freshwater bryozoans during at least part of their life cycle, but no complete malacosporean life cycle is known to date. One of the 2 described malacosporeans is Tetracapsuloides bryosalmonae, the causative agent of salmonid proliferative kidney disease. The other is Buddenbrockia plumatellae, so far only found in freshwater bryozoans. Our investigations evaluated malacosporean life cycles, focusing on transmission from fish to bryozoan and from bryozoan to bryozoan. We exposed bryozoans to possible infection from: stages of T. bryosalmonae in fish kidney and released in fish urine; spores of T. bryosalmonae that had developed in bryozoan hosts; and spores and sac stages of B. plumatellae that had developed in bryozoans. Infections were never observed by microscopic examination of post-exposure, cultured bryozoans and none were detected by PCR after culture. Our consistent negative results are compelling: trials incorporated a broad range of parasite stages and potential hosts, and failure of transmission across trials cannot be ascribed to low spore concentrations or immature infective stages. The absence of evidence for bryozoan to bryozoan transmissions for both malacosporeans strongly indicates that such transmission is precluded in malacosporean life cycles. Overall, our results imply that there may be another malacosporean host which remains unidentified, although transmission from fish to bryozoans requires further investigation. However, the highly clonal life history of freshwater bryozoans is likely to allow both long-term persistence and spread of infection within bryozoan populations, precluding the requirement for regular transmission from an alternate host.     

Transmission of freshwater myxozoans during the asexual propagation of invertebrate hosts

The phylum Myxozoa contains over 1350 species almost all of which are considered to be obligate parasites of aquatic animals. The phylum is composed of two classes, the Myxosporea and the Malacosporea, species of which are important pathogens responsible for severe economic losses in cultured fisheries. The life cycles of freshwater Myxozoa are believed to involve horizontal, indirect transmission, involving an invertebrate (oligochaetes or bryozoans) and a vertebrate host (fish or amphibians). Here, we describe myxozoan propagation through the fragmentation of invertebrate hosts to form new infected individuals. The two hosts examined are an oligochaete Lumbriculus variegatus infected with an unidentified myxosporean (Triactinomyxon sp.) and the bryozoan Fredericella sultana infected with the malacosporean Tetracapsuloides bryosalmonae which causes proliferative kidney disease, a major constraint of the European rainbow trout industry. Such intra-clonal propagation is a novel form of vertical transmission that is likely to be widespread within the Myxozoa and could form an important method by which some of these parasites maintain and proliferate within the aquatic environment.     

The effects of infection by Tetracapsuloides bryosalmonae (Myxozoa) and temperature on Fredericella sultana (Bryozoa)

The myxozoan, Tetracapsuloides bryosalmonae, exploits freshwater bryozoans as definitive hosts, occurring as cryptic stages in bryozoan colonies during covert infections and as spore-forming sacs during overt infections. Spores released from sacs are infective to salmonid fish, causing the devastating Proliferative Kidney Disease (PKD). We undertook laboratory studies using mesocosm systems running at 10, 14 and 20 °C to determine how infection by T. bryosalmonae and water temperature influence fitness of one of its most important bryozoan hosts, Fredericella sultana, over a period of 4 weeks. The effects of infection were context-dependent and often undetectable. Covert infections appear to pose very low energetic costs. Thus, we found that growth of covertly infected F. sultana colonies was similar to that of uninfected colonies regardless of temperature, as was the propensity to produce dormant resting stages (statoblasts). Production of statoblasts, however, was associated with decreased growth. Overt infections imposed greater effects on correlates of host fitness by: (i) reducing growth rates at the two higher temperatures; (ii) increasing mortality rates at the highest temperature; (iii) inhibiting statoblast production. Our results indicate that parasitism should have a relatively small effect on host fitness in the field as the negative effects of infection were mainly expressed in environmentally extreme conditions (20 °C for 4 weeks). The generally low virulence of T. bryosalmonae is similar to that recently demonstrated for another myxozoan endoparasite of freshwater bryozoans. The unique opportunity for extensive vertical transmission in these colonial invertebrate hosts couples the reproductive interests of host and parasite and may well give rise to the low virulence that characterises these systems. Our study implies that climate change can be expected to exacerbate PKD outbreaks and increase the geographic range of PKD as a result of the combined responses of T. bryosalmonae and its bryozoan hosts to higher temperatures.     

Fish infected with Sphaerospora spp. Thélohan (Myxosporea) from waters enzootic for proliferative kidney disease of salmonids

Sphaerospores were found among three species of fish examined from waters known to be enzootic for proliferative kidney disease (PKD) of salmonids. They were detected in the renal tubules of both hatchery-reared rainbow trout (Salmo gairdneri) exposed to the infectious stage of PKD and in chubs (Gila bicolor) in the headwaters of a hatchery where PKD is enzootic. Sticklebacks (Gasterosteus aculeatus) collected near net pens where Pacific salmon had experienced a PKD epizootic were also found to harbor sphaerospores in the lumen of the kidney tubules. The latter two host species contained developmental stages of a myxosporidan in the blood and in the lumen of the kidney tubules which are similar to those of PKX, the causative agent of PKD in salmonid fish. The sphaerospores observed in the rainbow trout are the first to be observed in this species. The similarity to previously observed developmental stages, rarity, and presence of these sphaerospores in salmonid fish from a hatchery where PKD is enzootic suggest that they are the most mature stage of the PKX myxosporidan yet observed.     

Fish Infected with Sphaerospora spp. Thélohan (Myxosporea) from Waters Enzootic for Proliferative Kidney Disease of Salmonids1

ABSTRACT. Sphaerospores were found among three species of fish examined from waters known to be enzootic for proliferative kidney disease (PKD) of salmonids. They were detected in the renal tubules of both hatchery-reared rainbow trout (Salmo gairdneri) exposed to the infectious stage of PKD and in chubs (Gila bicolor) in the headwaters of a hatchery where PKD is enzootic. Sticklebacks (Gasterosteus aculeatus) collected near net pens where Pacific salmon had experienced a PKD epizootic were also found to harbor sphaerospores in the lumen of the kidney tubules. The latter two host species contained developmental stages of a myxosporidan in the blood and in the lumen of the kidney tubules which are similar to those of PKX, the causative agent of PKD in salmonid fish. The sphaerospores observed in the rainbow trout are the first to be observed in this species. The similarity to previously observed developmental stages, rarity, and presence of these sphaerospores in salmonid fish from a hatchery where PKD is enzootic suggest that they are the most mature stage of the PKX myxosporidan yet observed.     

PKX,the Causative Agent of Proliferative Kidney Disease (PKD) in Pacific Salmonid Fishes and Its Affinities with the Myxozoa1

Proliferative kidney disease (PKD), caused by an unclassified protozoan (PKX), is reported from Pacific salmon, Oncorhynchus tshawytscha (Walbaum) and O. kisutch (Walbaum), and steelhead trout, Salmo gairdneri Richardson, held at the Mad River Hatchery in California, USA. The cumulative mortality attributed to the disease was 95, 13, and 18% respectively. The mortalities were greatest at mean water temperatures of 12-14°C during July 1983. The ultrastructure of the PKX organism and its associated pathology during clinical disease in all three species were consistent with those of the parasite in rainbow trout (Salmo gairdneri) as described in European outbreaks. Significant mortalities did not occur after August, at which time the parasite could no longer be detected in the salmon species. The steelhead continued to exhibit parasites in the kidney interstitium and epithelium and lumens of the tubules. Myxosporidan trophozoites and developing spores were also observed in the lumens of the kidney tubules of these fish. Although a mixed infection with another parasite may have occurred, evidence suggests that the myxosporidans are later stages of PKX. They were only observed in fish exposed to water with the infective stage and were particularly prominent in recovering fish. The PKX organism is similar to UBO, an unclassified protozoan of carp suspected to be an early stage of Sphaerospora renicola Dyková & Lom. Both parasites infect the blood and kidney, divide by endogeny, and are released by disintegration of the primary mother cell. The intraluminal myxosporean forms show similarities to Sphaerospora spp. in that they are monosporous and sporoblasts are formed within pseudoplasmodia. It is possible that PKX migrates to the lumen of the kidney tubule and subsequently sporulates. If the myxosporean forms are later stages of PKX, then it would belong to the phlyum Myxozoa.     

A new class and order of myxozoans to accommodate parasites of bryozoans with ultrastructural observations on Tetracapsula bryosalmonae (PKX organism)

Tetracapsula bryosalmonae, formerly PKX organism, is a myxozoan parasite that causes proliferative kidney disease in salmonid fish. Its primary hosts, in which it undergoes a sexual phase, are phylactolaemate bryozoans. It develops in the bryozoan coelomic cavity as freely floating sacs which contain two types of cells, stellate cells and sporoplasmogenic cells, which become organised as spores. Eight stellate cells differentiate as four capsulogenic cells and four valve cells which surround a single sporoplasmogenic cell. The sporoplasmogenic cell undergoes meiosis and cytoplasmic fission to produce two sporoplasms with haploid nuclei. Sporoplasms contain secondary cells. The unusual development supports previously obtained data from 18S rDNA sequences, indicating that species of Tetracapsula form a clade. It diverged early in the evolution of the Myxozoa, before the radiation that gave rise to the better known genera belonging to the two orders in the single class Myxosporea. The genus Tetracapsula as seen in bryozoans shares some of the characters unique to the myxosporean phase and others typical of the actinosporean phase of genera belonging to the class Myxosporea. However, it exhibits other features which are not found in either phase. A new class Malacosporea and order Malacovalvulida are proposed to accommodate the family Saccosporidae and genus Tetracapsula. Special features of the new class are the sac-like proliferative body, valve cells not covering the exit point of the polar filament, lack of a stopper-like structure sealing the exit, maintenance of valve cell integrity even at spore maturity, absence of hardened spore walls and unique structure of sporoplasmosomes in the sporoplasms.     

Sporogony of Tetracapsuloides bryosalmonae in the brown trout Salmo trutta and the role of the tertiary cell during the vertebrate phase of myxozoan life cycles

Tetracapsuloides bryosalmonae is the myxozoan that causes the commercially and ecologically important proliferative kidney disease of salmonid fish species. Immunohistochemistry and electron microscopy were used to examine the development of this parasite within the kidney of the brown trout Salmo trutta. The main replicative phase of T. bryosalmonae is a cell doublet composed of a primary cell and a single secondary cell. Engulfment of one secondary cell by another to form a secondary-tertiary doublet (S-T doublet) heralded the onset of sporogony whereupon the parasite migrated to the kidney tubule lumen. Within the tubule, the parasite transformed into a pseudoplasmodium and anchored to the tubule epithelial cells via pseudopodial extensions. Within each pseudoplasmodium developed a single spore, composed of 4 valve cells, 2 polar capsules and 1 sporoplasm. The pseudoplasmodia formed clusters suggesting that large numbers of spores develop within the fish. This examination of T. bryosalmonae suggests that the main replicative phase of freshwater myxozoans within vertebrates is via direct replication of cell doublets rather than through the rupturing of extrasporogonic stages, while tertiary cell formation relates only to sporogony. Taken in conjunction with existing phylogenetic data, 5 distinct sporogonial sequences are identified for the Myxozoa.