Jerri L. Bartholomew

Recent advances in our knowledge of the Myxozoa

Abstract In the last few years two factors have helped to significantly advance our understanding of the Myxozoa. First, the phenomenal increase in fin fish aquaculture in the 1990s has lead to the increased importance of these parasites; in turn this has lead to intensified research efforts, which have increased knowledge of the development, diagnosis, and pathogenesis of myxozoans. The hallmark discovery in the 1980s that the life cycle of Myxobolus cerebralis requires development of an actinosporean stage in the oligochaete, Tubifex tubifex, led to the elucidation of the life cycles of several other myxozoans. Also, the life cycle and taxonomy of the enigmatic PKX myxozoan has been resolved: it is the alternate stage of the unusual myxozoan, Tetracapsula bryosalmonae, from bryozoans. The 18S rDNA gene of many species has been sequenced, and here we add 22 new sequences to the data set. Phylogenetic analyses using all these sequences indicate that:1) the Myxozoa are closely related to Cnidaria (also supported by morphological data); 2) marine taxa at the genus level branch separately from genera that usually infect freshwater fishes; 3) taxa cluster more by development and tissue location than by spore morphology; 4) the tetracapsulids branched off early in myxozoan evolution, perhaps reflected by their having bryozoan, rather than annelid hosts; 5) the morphology of actinosporeans offers little information for determining their myxosporean counterparts (assuming that they exist); and 6) the marine actinosporeans from Australia appear to form a clade within the platysporinid myxosporeans. Ribosomal DNA sequences have also enabled development of diagnostic tests for myxozoans. PCR and in situ hybridisation tests based on rDNA sequences have been developed for Myxobolus cerebralis, Ceratomyxa shasta, Kudoa spp., and Tetracapsula bryosalmonae (PKX). Lectin-based and antibody tests have also been developed for certain myxozoans, such as PKX and C. shasta. We also review important diseases caused by myxozoans, which are emerging or re-emerging. Epizootics of whirling disease in wild rainbow trout (Oncorhynchus mykiss) have recently been reported throughout the Rocky Mountain states of the USA. With a dramatic increase in aquaculture of fishes using marine netpens, several marine myxozoans have been recognized or elevated in status as pathological agents. Kudoa thyrsites infections have caused severe post-harvest myoliquefaction in pen-reared Atlantic salmon (Salmo salar), and Ceratomyxa spp., Sphaerospora spp., and Myxidium leei cause disease in pen-reared sea bass (Dicentrarchus labrax) and sea bream species (family Sparidae) in Mediterranean countries.

IgT, a primitive immunoglobulin class specialized in mucosal immunity

Teleost fish are the most primitive bony vertebrates that contain immunoglobulins. In contrast to mammals and birds, these species are devoid of immunoglobulin A (IgA) or a functional equivalent. This observation suggests that specialization of immunoglobulin isotypes into mucosal and systemic responses took place during tetrapod evolution. Challenging that paradigm, here we show that IgT, an immunoglobulin isotype of unknown function, acts like a mucosal antibody. We detected responses of rainbow trout IgT to an intestinal parasite only in the gut, whereas IgM responses were confined to the serum. IgT coated most intestinal bacteria. As IgT and IgA are phylogenetically distant immunoglobulins, their specialization into mucosal responses probably occurred independently by a process of convergent evolution.

Status and opportunities for genomics research with rainbow trout

The rainbow trout (Oncorhynchus mykiss) is one of the most widely studied of model fish species. Extensive basic biological information has been collected for this species, which because of their large size relative to other model fish species are particularly suitable for studies requiring ample quantities of specific cells and tissue types. Rainbow trout have been widely utilized for research in carcinogenesis, toxicology, comparative immunology, disease ecology, physiology and nutrition. They are distinctive in having evolved from a relatively recent tetraploid event, resulting in a high incidence of duplicated genes. Natural populations are available and have been well characterized for chromosomal, protein, molecular and quantitative genetic variation. Their ease of culture, and experimental and aquacultural significance has led to the development of clonal lines and the widespread application of transgenic technology to this species. Numerous microsatellites have been isolated and two relatively detailed genetic maps have been developed. Extensive sequencing of expressed sequence tags has begun and four BAC libraries have been developed. The development and analysis of additional genomic sequence data will provide distinctive opportunities to address problems in areas such as evolution of the immune system and duplicate genes.

The life cycle of Ceratomyxa shasta, a myxosporean parasite of salmonids, requires a freshwater polychaete as an alternate host.

The actinosporean life stage of Ceratomyxa shasta, a myxozoan parasite of salmonids, and the annelid worm that serves as its alternate host were identified in laboratory transmission experiments and their roles were confirmed using molecular techniques. Infection by the parasite occurred in susceptible fish that were either exposed to or force fed the freshwater polychaete, Manayunkia speciosa, infected with the actinosporean. These observations were confirmed using the polymerase chain reaction with primers designed from the C. shasta 18S rDNA sequence. DNA was amplified from polychaetes harboring the actinosporean that caused infection in the fish but not from uninfected polychaetes. Amplified DNA from an infected polychaete was sequenced and its homology with the 18S rDNA sequence of C. shasta spores verified the proposed life cycle. Ultrastructural examination of the actinosporean in the polychaete showed developmental stages in the epidermis rather than within the intestinal epithelium as described for other myxozoans. The methods described will be useful in identifying alternate hosts and morphologically diverse life stages in the complex life cycles of other myxosporea and in understanding the relationships between these parasites and their hosts.

Host Resistance to Infection by the Myxosporean Parasite Ceratomyxa shasta: A Review

Abstract The capacity of certain salmonids to resist infection and disease caused by the myxosporean Ceratomyxa shasta is well known. Observations of differential mortality among strains suggest that intraspecies differences in susceptibility developed as a result of selective pressure in waters where the parasite is enzootic. Research efforts have identified resistant strains for use in stocking programs and examined the heritability of the trait. This paper reviews the research on resistance of salmonids to C. shasta, the conclusions drawn from these studies, and how this information has been applied.

INVOLVEMENT OF MANAYUNKIA SPECIOSA (ANNELIDA: POLYCHAETA: SABELLIDAE) IN THE LIFE CYCLE OF PARVICAPSULA MINIBICORNIS, A MYXOZOAN PARASITE OF PACIFIC SALMON

A coelomic myxozoan infection was detected in freshwater polychaetes, Manayunkia speciosa from the Klamath River, Oregon/California, a site enzootic for the myxozoan parasites Ceratomyxa shasta and Parvicapsula minibicornis. The tetractinomyxon type actinospores had a near-spherical spore body 7.9 × 7.1 μm, with 3 spherical, protruding polar capsules, no valve cell processes, and a binucleate sporoplasm. Parvicapsula minibicornis-specific primers Parvi1f and Parvi2r amplified DNA from infected polychaetes in a polymerase chain reaction (PCR) assay. The small subunit 18S rRNA gene of the spores was sequenced (GenBank DQ231038) and was a 99.7% match with the sequence for P. minibicornis myxospore stage in GenBank (AF201375). Chinook salmon (Oncorhynchus tshawytscha) exposed to a dose of 1,000 actinospores per fish tested PCR positive for P. minibicornis at 14 wk postinfection and presporogonic stages were detected in the kidney tubules by histology at 20 wk. This life cycle is 1 of only about 30 known from more than 1,350 myxozoan species, and only the second known from a freshwater polychaete.

Spatial and Temporal Occurrence of the Salmonid Parasite Ceratomyxa shasta in the Oregon–California Klamath River Basin

Abstract The parasite Ceratomyxa shasta has been implicated as a significant source of salmonid mortality in the lower Klamath River, California (i.e., below Iron Gate dam). A study of the prevalence of C. shasta and its geographic and temporal distribution throughout the Klamath River basin was conducted to determine when and where juvenile salmonids encounter lethal parasite doses. Susceptible rainbow trout Oncorhynchus mykiss were exposed to C. shasta 3–4 d at seven locations in the Klamath River between Beaver Creek and Keno Reservoir in April, June, July, September, and November 2003. Individuals from a Klamath River strain of fall Chinook salmon O. tshawytscha were held in three locations in the upper Klamath River in April, June, and July. In June 2004, rainbow trout were exposed to the parasite for 4 d at 18 locations from Klamath Lake to the mouth of the Klamath River, including several major spawning tributaries; one exposure occurred in the lower Klamath River. Rainbow trout mortality due to in...

Density of the Waterborne Parasite Ceratomyxa shasta and Its Biological Effects on Salmon

ABSTRACT The myxozoan parasite Ceratomyxa shasta is a significant pathogen of juvenile salmonids in the Pacific Northwest of North America and is limiting recovery of Chinook (Oncorhynchus tshawytscha) and coho (O. kisutch) salmon populations in the Klamath River. We conducted a 5-year monitoring program that comprised concurrent sentinel fish exposures and water sampling across 212 river kilometers of the Klamath River. We used percent mortality and degree-days to death to measure disease severity in fish. We analyzed water samples using quantitative PCR and Sanger sequencing, to determine total parasite density and relative abundance of C. shasta genotypes, which differ in their pathogenicity to salmonids. We detected the parasite throughout the study zone, but parasite density and genetic composition fluctuated spatially and temporally. Chinook and coho mortality increased with density of their specific parasite genotype, but mortality-density thresholds and time to death differed. A lethality threshold of 40% mortality was reached with 10 spores liter−1 for Chinook but only 5 spores liter−1 for coho. Parasite density did not affect degree-days to death for Chinook but was negatively correlated for coho, and there was wider variation among coho individuals. These differences likely reflect the different life histories and genetic heterogeneity of the salmon populations. Direct quantification of the density of host-specific parasite genotypes in water samples offers a management tool for predicting host population-level impacts.

DETECTION OF MYXOZOAN PARASITES IN OLIGOCHAETES IMPORTED AS FOOD FOR ORNAMENTAL FISH

To determine the potential for dissemination of myxozoan parasites by transfer of their alternate oligochaete hosts, shipments of tubificid worms obtained from an overseas commercial aquarium supplier were screened for actinospore stages of myxozoan parasites. At least 7 different triactinomyxon types were identified. The morphological characteristics of the actinospores recovered from these tubificids shared characteristics with triactinomyxons characterized in other surveys, particularly from eastern Europe. Analysis of the screened samples by polymerase chain reaction and comparison of morphological data indicated that these actinospores did not correspond to the triactinospore of Myxobolus cerebralis. Although identification of these triactinomyxon types was beyond the scope of this study, these data suggest that the unregulated import–export and exchange of live organisms for ornamental fish food may result in accidental introduction or dissemination of myxozoan parasites.

Effects of water flow on the infection dynamics of Myxobolus cerebralis

Myxobolus cerebralis, the myxozoan parasite responsible for whirling disease in salmonid fishes, has a complex life-cycle involving an invertebrate host and 2 spore stages. Water flow rate is an environmental variable thought to affect the establishment and propagation of M. cerebralis; however, experimental data that separates flow effects from those of other variables are scarce. To compare how this parameter affected parasite infection dynamics and the invertebrate and vertebrate hosts, dead, infected fish were introduced into a naive habitat with susceptible hosts under 2 experimental flow regimes: slow (0 x 02 cm/s) and fast (2 x 0 cm/s). Throughout the 1-year study, uninfected fry were held in both systems, the outflows were screened weekly for spores and the annelid populations were monitored. We found clear differences in prevalence of infection in the worms, prevalence and severity of infection in the fish, and host survival. Both flows provided environments in which M. cerebralis could complete its life-cycle; however, both the parasite and its invertebrate host proliferated to a greater extent in the slow flow environment over the 1-year study period. This finding is of significance for aquatic systems where the flow rate can be manipulated, and should be incorporated into risk analysis assessments.