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 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.     

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.     

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.     

Evidence that infectious stages of Tetracapsula bryosalmonae for rainbow trout Oncorhynchus mykiss are present throughout the year

Proliferative kidney disease (PKD) is a hyperplastic condition of the lymphoid tissue of salmonids infected with the spores of Tetracapsula bryosalmonae, a myxozoan parasite formerly designated PKX, which has recently been described as a parasite of several species of bryozoans. The occurrence of PKD is generally associated with seasonal increase in water temperature, with research indicating that transmission of the disease does not occur below 12 to 13 degrees C. This suggested that the infectious stages are absent from about November to March/April. Here we document the transmission of PKD at water temperatures and seasons previously considered to be non permissive for PKD infection. The exposure of naive rainbow trout Oncorhynchus mykiss (Walbaum) to PKD-infected water ranging from 8 to 13 degrees C during the Autumn, Winter and early Spring, resulted in the infection of kidney interstitium once the trout were transferred to 16 degrees C. In addition, cohabitation studies were conducted with the bryozoan host Fredericella sultana collected from a river at times of low seasonal temperatures because this bryozoan species overwinters as living colonies. Cohabitation of trout with colonies of F sultana in parasite-free city water at 16 degrees C, also led to renal lymphoid tissue infection with the parasite and even to nephromegaly. Our results provide evidence that the infectious stages of T bryosalmonae for rainbow trout were present in the water throughout the entire year and that the impact of temperature on the development of PKD is primarily a result of the kinetics of Tetracapsula multiplication in bryozoan and fish hosts.     

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.     

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.     

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.     

Severe mortality in wild Atlantic salmon Salmo salar due to proliferative kidney disease (PKD) caused by Tetracapsuloides bryosalmonae (myxozoa)

Extensive mortality in Atlantic salmon fry was reported in the River Aelva from 2002 to 2004. Dead fish were collected in late summer 2006, and live fish were sampled by electrofishing in September the same year. At autopsy and in histological sections, the fish kidneys were found to be pale and considerably enlarged. Proliferative lesions with characteristic PKX cells were seen in a majority of the fish. DNA from kidney samples of diseased fish was subjected to PCR and sequencing, and the amplified sequences matched those of Tetracapsuloides bryosalmonae. We concluded that this myxozoan transmitted from bryozoans was the main cause of the observed mortality in salmon fry in 2006. Results from quantitative electrofishing in 2005 and 2006, combined with the observed fry mortality from 2002 to 2004, show that the smolt production in the river is severely reduced and that T. bryosalmonae is the most likely explanation for this decline. The present study is the first to report a considerable negative population effect in wild Atlantic salmon due to proliferative kidney disease (PKD). It also represents the northernmost PKD outbreak in wild fish. The river is regulated for hydroelectric power purposes, causing reduced water flow and elevated summer temperatures, and the present PKD outbreak may serve as an example of increased disease vulnerability of northern fish populations in a warmer climate.     

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.     

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.     

Role of altitude and water temperature as regulating factors for the geographical distribution of Tetracapsuloides bryosalmonae infected fishes in Switzerland

The role of water temperature and altitude of the river sites on distribution, prevalence and infection intensity of Tetracapsuloides bryosalmonae infected salmonids was investigated. The study is based on a 6 year survey of salmonids, mainly brown trout, Salmo trutta from 2000 to 2006 at 287 sampling sites across Switzerland. Almost 7000 fishes were tested for the presence of T. bryosalmonae by histological and immunohistochemical techniques. A good correlation between altitude and water temperature in Swiss rivers could be demonstrated. Therefore, the relation between altitude and sites tested for T. bryosalmonae infected fishes was investigated since water temperature values from these sites were not available. Percentage of sites with positive (infected) fishes was identical for altitudes between 200 and 800 m above sea level (m a.s.l.), while above 800 m a.s.l. only three sites with infected fishes were recorded. Site‐specific prevalence values ranging from 0 to 100% and infection intensities from 0 to 6 were found over the whole altitude range from 200 to 800 m a.s.l., and no correlation with altitude was found. Conditions at altitudes >800 m may not be favourable for the establishment of the infection in fishes. With rising temperatures due to climatic changes, however, the prevalence of T. bryosalmonae infected fishes in altitudes >800 m a.s.l. could increase.     

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.     

Trypanosoma cruzi: genetic structure of populations and relevance of genetic variability to the pathogenesis of chagas disease

Chagas disease, caused by the protozoan Trypanosoma cruzi, has a variable clinical course, ranging from symptomless infection to severe chronic disease with cardiovascular or gastrointestinal involvement or, occasionally, overwhelming acute episodes. The factors influencing this clinical variability have not been elucidated, but it is likely that the genetic variability of both the host and the parasite are of importance. In this work we review the the genetic structure of T. cruzi populations and analyze the importance of genetic variation of the parasite in the pathogenesis of the disease under the light of the histotropic-clonal model.     

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.     

Disentangling the influence of parasite genotype, host genotype and maternal environment on different stages of bacterial infection in Daphnia magna

Individuals naturally vary in the severity of infectious disease when exposed to a parasite. Dissecting this variation into genetic and environmental components can reveal whether or not this variation depends on the host genotype, parasite genotype or a range of environmental conditions. Complicating this task, however, is that the symptoms of disease result from the combined effect of a series of events, from the initial encounter between a host and parasite, through to the activation of the host immune system and the exploitation of host resources. Here, we use the crustacean Daphnia magna and its parasite Pasteuria ramosa to show how disentangling genetic and environmental factors at different stages of infection improves our understanding of the processes shaping infectious disease. Using compatible host-parasite combinations, we experimentally exclude variation in the ability of a parasite to penetrate the host, from measures of parasite clearance, the reduction in host fecundity and the proliferation of the parasite. We show how parasite resistance consists of two components that vary in environmental sensitivity, how the maternal environment influences all measured aspects of the within-host infection process and how host-parasite interactions following the penetration of the parasite into the host have a distinct temporal component.     

A Major Genetic Locus in Trypanosoma brucei Is a Determinant of Host Pathology

The progression and variation of pathology during infections can be due to components from both host or pathogen, and/or the interaction between them. The influence of host genetic variation on disease pathology during infections with trypanosomes has been well studied in recent years, but the role of parasite genetic variation has not been extensively studied. We have shown that there is parasite strain-specific variation in the level of splenomegaly and hepatomegaly in infected mice and used a forward genetic approach to identify the parasite loci that determine this variation. This approach allowed us to dissect and identify the parasite loci that determine the complex phenotypes induced by infection. Using the available trypanosome genetic map, a major quantitative trait locus (QTL) was identified on T. brucei chromosome 3 (LOD = 7.2) that accounted for approximately two thirds of the variance observed in each of two correlated phenotypes, splenomegaly and hepatomegaly, in the infected mice (named TbOrg1). In addition, a second locus was identified that contributed to splenomegaly, hepatomegaly and reticulocytosis (TbOrg2). This is the first use of quantitative trait locus mapping in a diploid protozoan and shows that there are trypanosome genes that directly contribute to the progression of pathology during infections and, therefore, that parasite genetic variation can be a critical factor in disease outcome. The identification of parasite loci is a first step towards identifying the genes that are responsible for these important traits and shows the power of genetic analysis as a tool for dissecting complex quantitative phenotypic traits.     

Evolutionary epidemiology of schistosomiasis: linking parasite genetics with disease phenotype in humans

Here we assess the role of parasite genetic variation in host disease phenotype in human schistosomiasis by implementing concepts and techniques from environmental association analysis in evolutionary epidemiology. Schistosomiasis is a tropical disease that affects more than 200 million people worldwide and is caused by parasitic flatworms belonging to the genus Schistosoma. While the role of host genetics has been extensively studied and demonstrated, nothing is yet known on the contribution of parasite genetic variation to host disease phenotype in human schistosomiasis. In this study microsatellite genotypes of 1561 Schistosoma mansoni larvae collected from 44 human hosts in Senegal were linked to host characteristics such as age, gender, infection intensity, liver and bladder morbidity by means of multivariate regression methods (on each parasite locus separately). This revealed a highly significant association between allelic variation at the parasite locus L46951 and host infection intensity and bladder morbidity. Locus L46951 is located in the 3′ untranslated region of the cGMP-dependent protein kinase gene that is expressed in reproductive organs of adult schistosome worms and appears to be linked to egg production. This putative link between parasite genetic variation and schistosomiasis disease phenotype sets the stage for further functional research.     

The kinetics of Theileria parva infection and lymphocyte transformation in vitro

Theileriaparva is an intracellular protozoan parasite that causes a fatal lymphoproliferative disease of cattle known as East Coast Fever. The parasite infects host lymphocytes causing their transformation and uncontrolled proliferation. Infiltration of major organs with parasitized lymphoblasts results in most cases in death within 3 weeks. Although both T and B lymphocytes are susceptible to infection, the majority of cell lines arising from infection of peripheral blood mononuclear cells in vitro are of T cell lineage. To explore the basis of this phenotypic bias we have followed the very early stages of parasite development in vitro at the single cell level. Peripheral blood mononuclear cells were infected and stained for both surface phenotype and intracellular parasite antigen and analysed by flow cytometry. Although the parasite antigen was detected intracellularly as early as 6h p.i., our data indicate that parasite infection does not lead to cell transformation in all instances. Rather, specific cell types appear to undergo selection very early after infection and expansion of particular cell subsets results in survival and growth of only a small proportion of the cells originally parasitized.