First report of three Kudoa species from eastern Australia: Kudoa thyrsites from mahi mahi (Coryphaena hippurus), Kudoa amamiensis and Kudoa minithyrsites n. sp. from sweeper (Pempheris ypsilychnus)

Fish species around the world are parasitized by myxozoans of the genus Kudoa, several of which infect and cause damage of commercial importance. In particular, Kudoa thyrsites and Kudoa amamiensis infect certain cultured fish species causing damage to muscle tissue, making the fish unmarketable. Kudoa thyrsites has a broad host and geographic range infecting over 35 different fish species worldwide, while K. amamiensis has only been reported from a few species in Japanese waters. Through morphological and molecular analyses we have confirmed the presence of both of these parasites in eastern Australian waters. In addition, a novel Kudoa species was identified, having stellate spores, with one polar capsule larger than the other three. The SSU rDNA sequence of this parasite was 1.5% different from K. thyrsites and is an outlier from K. thyrsites representatives in a phylogenetic analysis. Furthermore, the spores of this parasite are distinctly smaller than those of K. thyrsites, and thus it is described as Kudoa minithyrsites n. sp. Although the potential effects of K. minithyrsites n. sp. on its fish hosts are unknown, both K. thyrsites and K. amamiensis are associated with flesh quality problems in some cultured species and may be potential threats to an expanding aquaculture industry in Australia.     

Two unusual myxozoans,Kudoa quadricornis n. sp. (Multivalvulida) from the muscle of goldspotted trevally (Carangoides fulvoguttatus) and Kudoa permulticapsula n. sp. (Multivalvulida) from the muscle of Spanish mackerel (Scomberomorus commerson) from the Great Barrier Reef,Australia

Two unusual myxozoan parasites are described from the somatic muscle of 2 reef fishes from Australia's Great Barrier Reef. Kudoa quadricornis n. sp. from the somatic muscle of Carangoides fulvoguttatus is morphologically consistent with other Kudoa sp., having 4 polar capsules and 4 shell valves. Kudoa quadricornis n. sp. is unique in that it has a pyriform spore body with a greater length than width (7.82-9.95 and 5.94-8.66 microm, respectively) and distinct posterolateral projections. Spores of Kudoa permulticapsula n. sp. observed within pseudocysts of the somatic muscle tissue of Scomberomorus commerson are different from those of all other myxozoans. The ovoid spores (length, 4.69-6.65 microm; width, 8.42-9.92 microm; thickness, 6.36-8.33 microm) contain 13 polar capsules with an equal number of shell valves. Phylogenetic analysis using small subunit ribosomal DNA sequences of K. quadricornis n. sp. and K. permulticapsula n. sp. showed that these parasites cluster within a clade comprised of Kudoa species. This brings into question the division of parasites of the Multivalvulida into genera based solely on polar capsule numbers.     

Analysis of Kudoa thyrsites (Myxozoa: Myxosporea) spore antigens using monoclonal antibodies.

A method employing Percoll gradient centrifugation was developed to purify Kudoa thyrsites spores from somatic muscle tissue of Atlantic salmon Salmo salar. Highly purified spores were then used to immunize inbred BALB/c mice for derivation of hybridomas secreting Kudoa-specific monoclonal antibodies (mAbs). Analysis of mAbs by immunofluorescence microscopy and flow cytometry showed that several were specific for antigens on the surface of K. thyrsites spores whereas other mAbs reacted with polar capsules or with polar filaments of spores of K. thyrsites, K. paniformis and K. crumena. Immunoblots on spore lysates using the surface-binding mAbs showed a broad band of 46 to > 220 kDa, whereas mAbs specific for antigens of polar capsules and polar filaments detected sharper bands of various molecular masses, depending on the Kudoa species. The dominant epitope of the K. thyrsites spore surface antigen was shown to be carbohydrate as determined by its sensitivity to treatment with anhydrous trifluoromethane sulfonic acid and by its resistance to treatment with Proteinase K. Immunofluorescence microscopy using the K. thyrsites-specific mAbs on isolated, intact, permeabilized plasmodia and on thin sections of somatic muscle tissue containing plasmodia revealed intense labeling of spores both within the spore-producing plasmodia and in the flesh of infected Atlantic salmon. As few as 100 spores were detected by immunoblotting, indicating that these mAbs have potential for use in developing a field-based diagnostic test.     

Systemic infection of Kudoa lutjanus n. sp. (Myxozoa: Myxosporea) in red snapper Lutjanus erythropterus from Taiwan

A new species of Kudoa lutjanus n. sp. (Myxosporea) is described from the brain and internal organs of cultured red snapper Lutjanus erythropterus from Taiwan. The fish, 260 to 390 g in weight, exhibited anorexia and poor appetite and swam in the surface water during outbreaks. Cumulative mortality was about 1% during a period of 3 wk. The red snapper exhibited numerous creamy-white pseudocysts, 0.003 to 0.65 cm (n = 100) in diameter, in the eye, swim bladder, muscle and other internal organs, but especially in the brain. The number of pseudocysts per infected fish was not correlated with fish size or condition. Mature spores were quadrate in apical view and suboval in side view, measuring 8.2 +/- 0.59 microm in width and 7.3 +/- 0.53 microm in length. The 4 valves were equal in size, each with 1 polar capsule. Polar capsules were pyriform in shape, measuring 3.62 +/- 0.49 microm in length and 2.2 +/- 0.49 microm in width. Mild inflammatory responses or liquefaction of host tissue were associated with K. lutjanus n. sp. infection. The junction of shell valves appeared as overlapping, straight lines. The polar filament formed 2 to 3 coils. A general PCR (polymerase chain reaction) primer for Kudoa amplified the small subunit (SSU) rDNA sequences, and the amplified gene was sequenced. It was evident from the phylogenetic tree that the 3 strains tested, AOD93020M, AOD93028M and AOD93028B, were identical and belonged to the Kudoa SS rRNA subgroup. The evolutionary tree showed that these strains form a unique clade, at a distance from other Kudoa species and myxosporeans. The spore's morphological and ultrastructural characteristics, as well as the SS rDNA properties of the isolates, were also essentially identical and served to distinguish them from representative Kudoa. It is, therefore, proposed that the strains isolated from the diseased red snapper be assigned to a new species.     

Characterization of Kudoa monodactyli n. sp. (Myxosporea: Multivalvulida) from the Muscle of Monodactylus argenteus (Teleostei: Monodactylidae) from Moreton Bay, Queensland, Australia

Kudoa monodactyli n. sp. is described from the somatic musculature of Monodactylus argenteus from several localities in southern Queensland, Australia. This is the first record of a myxozoan parasite from the family Monodactylidae. The spores typically have five polar capsules, making this species similar to the four other five-valved Kudoa species (K. neurophila, K. muscularis, K. shulmani, K. cutanea) that have been described to date. However, morphometric measurements particularly of spore length and width make the species from M. argenteus distinct from the other species. Comparison of the small subunit ribosomal DNA sequence of this species with its congeners for which sequence data are available, provides further evidence of novelty. Kudoa monodactyli n. sp. displays 38 (of 1,554) nucleotide differences compared with rDNA sequence of Kudoa neurophila, which on phylogenetic analysis places these species in clades exclusive of each other. Phylogenetic analyses also provide evidence that the number of valves per spore in this genus is an imperfect indicator of relatedness.     

Multivalvulid myxozoans from eastern Australia: three new species of Kudoa from scombrid and labrid fishes of the Great Barrier Reef, Queensland, Australia

Three new species of Kudoa, each having 6 polar capsules, are described from the somatic muscle of fishes collected on the Great Barrier Reef, Queensland, Australia. Kudoa grammatorcyni n. sp. was observed in the shark mackerel Grammatorcynus bicarinatus. Spores are stellate in apical view, width (all measurements in microm) 8.62 (8.03-8.95); thickness 8.14 (7.63-8.68); suture width 7.7 (7.24-8.16); length 6.54 (6.32-6.71); polar capsule length 3.68 (3.55-3.82); polar capsule width 1.72 (1.65-1.84). Kudoa scomberomori n. sp. is described from the Spanish mackerel Scomberomorus commerson. Spores are stellate in apical view, width 7.56 (6.84-8.16); thickness 6.79 (6.18-7.63); suture width 5.92 (5.26-6.32); length 5.43 (5.00-6.18); polar capsule length 3.24 (3.03-3.55); polar capsule width 1.37 (1.25-1.51). Kudoa thalassomi n. sp. is described from the moon wrasse Thalassoma lunare. Spores are stellate in apical view, width 10.66 (9.47-11.84); thickness 9.37 (8.55-10.79); suture width 7.98 (6.84-8.82); length 6.65 (6.18-7.11); polar capsule length 4.92 (4.74-5.00); polar capsule width 2.12 (2.04-2.24). All 3 species differ in spore morphology from the 1 previously described myxozoan with 6 polar capsules, Hexacapsula neothunni from yellowfin tuna Neothunnus macropterus, which has since been reassigned to Kudoa.     

Supplemental diagnosis of Kudoa funduli (Myxozoa) parasitizing Fundulus heteroclitus (Cyprinodontidae) from coastal northeastern North America

The diagnosis of Kudoa funduli (Hahn, 1915) Meglitsch, 1948 (Myxozoa), is supplemented through study of new material collected from Fundulus heteroclitus (Cyprinodontidae) in coastal waters of Nova Scotia, Canada, and Connecticut. Plasmodia normally develop intracellularly in striated muscle of the flank and head, eventually rupturing and releasing spores. Spores disperse along adjacent epimysium, sometimes as far as the skin surface. Some plasmodia develop extracellularly within the bony cavities of vertebrae. Formalin-fixed spores viewed with a light microscope possess rounded edges, an inconspicuous apical region, thin sutural ridges, measure 6.6-7.4 microm wide, 4.3-5 microm thick, and 5.1-5.4 microm long, and have 4 equally sized polar capsules, 1.7-2.3 microm length by 1.4-1.7 microm width. Scanning electron microscopy revealed that spores are almost stellate, with inconspicuous uplifted tips, and that, within intracellular plasmodia, are embedded in an extensive honeycomb-like matrix. Prevalence of infection of K. funduli was 100% in host populations sampled in both Nova Scotia and Connecticut. Molecular sequence data of the 18S ribosomal DNA (737 base pairs) reveal that K. funduli is a valid species and a member of a clade that includes Kudoa dianae Dyková, Avila, and Fiala, 2002, Kudoa miniauriculata Whitaker, Kent, and Sakanari, 1996, and Kudoa paniformis Kabata and Whitaker, 1981.     

Light- and electron-microscope description of Kudoa paralichthys n. sp. (Myxozoa,Myxosporea) from the brain of cultured olive flounder Paralichthys olivaceus in Korea

A new Myxosporea, Kudoa paralichthys n. sp., is described from the brain of cultured olive flounder Paralichthys olivaceus in South Korea. Mature spores were quadrate in apical view, measuring 5.19 +/- 0.54 microm in length, 8.23 +/- 0.50 microm in width, and 6.87 +/- 0.45 microm in thickness. Four valves were equal in size, each with 1 polar capsule. Polar capsules were pyriform in shape, measuring 2.2 +/- 0.22 microm in length and 1.2 +/- 0.14 microm in breadth. The sporoplasm consisted of a larger outer cell completely surrounding a smaller inner one, and had cytoplasmic projections. The junctions of shell valves were L-shaped. The sutural planes converged at the anterior ends of the spores and were associated with 4 small apex prominences in the central meeting point of the spores.     

Phylogeography of the cosmopolitan marine parasite Kudoa thyrsites (Myxozoa: Myxosporea)

Kudoa thyrsites (Myxozoa: Multivalvulida) is a cosmopolitan marine parasite of fishes associated with post-mortem tissue degradation. Financial losses incurred as a result of these infections are of concern to commercial fisheries. There is conflicting evidence whether K. thyrsites represents a cryptic species complex. Myxospore morphology is very similar for K. thyrsites across its range, but preliminary genetic analyses show some differences. Kudoa thyrsites and the morphologically similar Kudoa histolytica were examined from hosts in British Columbia, Canada, Oregon, USA, Chile, England, South Africa, Australia, and Japan. We compared myxospore morphology and DNA sequences of heat shock protein 70 and the small subunit, large subunit, and internal transcribed spacer 1 of the ribosomal DNA. There was some morphological variation between regional representatives, inconsistent with genetic analyses. Phylogenetically, major separations correlated to four broad geographic regions: Japan, Australia, eastern Pacific, and eastern Atlantic. Within these regions there was little additional genetic structure. These data are evidence for regional subdivision of K. thyrsites suggesting global transplantation of fishes has yet to homogenize these distinctions. Within regions, parasite gene flow appears to be high between host species, suggesting little host specificity and minimal cryptic speciation. Our data also indicate that K. histolytica is not a valid species, as it was morphologically and genetically indistinguishable from K. thyrsites.     

Kudoa cerebralis sp. n. (Myxosporidea,Chloromyxidae) from the Striped Bass,Morone saxatilis (Walbaum)*

Kudoa cerebralis sp. n. is described from connective tissue of the nervous system in the striped bass, Morone saxatilis (Walbaum), from the southern Chesapeake Bay area. This is the first time the genus Kudoa has been found in association with the nervous system. The polar view mean diameter of the spores was 7.0 μm and the polar capsule mean length was 3.7 μm.     

Epitopes associated with mature spores not recognized on Kudoa thyrsites from recently infected Atlantic salmon smolts

Atlantic salmon Salmo salar skeletal muscle was examined for Kudoa thyrsites by polymerase chain reaction (PCR) and positive fish were further examined by in situ hybridization (ISH) and immunohistochemistry (IHC). The infection was detected in 42% of salmon by PCR following a 60 d exposure to infective seawater at a temperature of 10 degrees C (= 600 degree-days, degreeD). The parasite was detected by ISH in skeletal and cardiac muscle but not in gill, kidney, spleen, liver, stomach, intestine, pyloric caeca and skin. None of 4 monoclonal antibodies (2F4, 4H2, 1H2, 3E8) raised against mature K. thyrsites spores reacted with the stages identified by ISH following a 600 degreeD exposure, but they did react with ISH-identified stages following a 1600 degreeD exposure. In contrast, a polyclonal antibody reacted with K. thyrsites stages in salmon with both 600 and 1600 degreeD exposures, suggesting that the parasite observed in 600 degreeD infections represents an antigenically distinct developmental stage of K. thyrsites.     

Myxobolus albi n. sp. (Myxozoa) from the Gills of the Common Goby Pomatoschistus microps Krøyer (Teleostei: Gobiidae)

ABSTRACT. A recent investigation into the myxozoan fauna of common gobies, Pomatoschistus microps , from the Forth Estuary in Scotland, revealed numerous myxosporean cysts within the gill cartilage. They were composed of polysporous plasmodia containing myxobolid spores that were morphologically different from the other known species of Myxobolus and from the myxosporeans previously recorded from this host (i.e. the ceratomyxid Ellipsomyxa gobii , infecting the gall bladder, and the kudoid Kudoa camarguensis , infecting the muscle tissues). Spores were ovoid, 9.4 × 9.1 μm with a thickness of 6.6 μm, with two pyriform polar capsules, the polar filaments of which had four to five turns. Molecular analysis of the parasite's small subunit rDNA region, based upon a contiguous sequence of 1,558 base pairs, discriminated it from other myxosporean species that have been characterized so far. A comparison of the spore morphology and the molecular sequences determined for this new isolate with other myxozoans described to date, confirmed its identity as a previously unknown myxobolid supporting the proposal that this isolate be elevated to the species level as a new species within the genus Myxobolus . A phylogenetic analysis places this new myxobolid, Myxobolus albi n. sp., in a basal position of a clade containing the majority of Henneguya spp. sequenced to date and various Myxobolus spp.     

A new genus Gadimyxa with three new species (Myxozoa, Parvicapsulidae) parasitic in marine fish (Gadidae) and the two-host life cycle of Gadimyxa atlantica n. sp

The myxozoans Gadimyxa atlantica n. sp. and G. sphaerica n. sp., and G. arctica n. sp. (Myxozoa, Parvicapsulidae), are described from Gadus morhua L. and Arctogadus glacialis (Peters) (Gadidae), respectively. They develop coelozoic in bisporic plasmodia in the urinary systems. Two morphological forms of spores were found in all 3 species, i.e., wide and (sub)spherical forms. Both spore types are bilaterally symmetrical along the suture line. The wide spores, semicircular in frontal view and elliptical in apical view, have 2 spherical polar capsules, which open in the sutural or median plane mid on the flat side of the spore. Mean widths of the wide spores of G. atlantica, G. sphaerica, and G. arctica are 7.5, 10.0, and 10.0 microm, respectively. The older, more thick-walled, (sub)spherical spores with binucleate sporoplasm are 8.0, 5.3, and 7.3 microm in mean width, respectively. The mean diameters of the polar capsules of (sub)spherical spores are 2.4, 1.7, and 2.2 microm, respectively. The (sub)spherical forms of Gadimyxa are most similar to Ortholinea within the Ortholineidae, but they differ in the development of the spores and in the arrangement of the polar capsules. The polychaetes Spirorbis spp. (Spirorbidae) act as invertebrate hosts of G. atlantica. The previously described actinospores of the tetractinomyxon type develop to myxospores in Gadus morhua within 8 wk. This is the second known myxozoan 2-host life cycle in the marine environment. Phylogenetic analyses based on partial small subunit rDNA sequences places Gadimyxa spp. among Parvicapsula spp. in the Parvicapsulidae.     

Kudoa hypoepicardialis n. sp. (Myxozoa: Kudoidae) and associated lesions from the heart of seven perciform fishes in the northern Gulf of Mexico

Kudoa hypoepicardialis n. sp. infects the space between the epicardium and the compact myocardium and, in intense infections, the pericardial chamber of man-of-war fish (Nomeus gronovii) (Nomeidae) (the type host), blue runner (Caranx crysos) (Carangidae), Warsaw grouper (Epinephelus nigritus) (Serranidae), Atlantic tripletail (Lobotes surinamensis) (Lobotidae), northern red snapper (Lutjanus campechanus) (Lutjanidae), black drum (Pogonias cromis) (Sciaenidae), and bluefish (Pomatomus saltatrix) (Pomatomidae) in the northern Gulf of Mexico. This is the first report of a Kudoa sp. from the heart of a fish in the Gulf of Mexico, and of these hosts, only the bluefish was previously identified as a host for a species of Kudoa. Spores of the new species varied slightly in size among these hosts but were regarded as conspecific based on their nearly identical (99.9%) small-subunit (SSU) ribosomal DNA (rDNA) sequence. The new species differs both from the 4 nominal species of Kudoa reported from fishes in the Gulf of Mexico and from K. pericardialis, an allopatric species that infects the pericardial cavity, by the combination of having a large spore, a small polar capsule, and a polar filament with a single coil. The new species is morphologically and genetically most similar to K. shiomitsui, an allopatric species that infects the heart and pericardial cavity, but is distinguished from it based on a 4.2% difference in the SSU rDNA sequence. Heart lesions primarily were restricted to the vicinity of plasmodia and included a layer of fibrinous inflammation characterized by lymphocytes, macrophages, and granulomas as well as epithelioid encapsulations around plasmodia. Heavily infected hosts had melanin-like deposits and adipose cells beneath the epicardium. and the epicardium was discontinuous and apparently breached by plasmodia in some regions. Cardiac muscle, gill, liver, spleen, intestine, and kidney were normal.     

Life history, pathology, and description of Kudoa ovivora n. sp. (Myxozoa, Myxosporea): an ovarian parasite of Caribbean labroid fishes

We describe Kudoa ovivora n. sp. from ovaries of bluehead wrasse, Thalassoma bifasciatum, and record its presence in 6 species (Labroidei) collected in the San Blas Islands. Panama. Kudoa ovivora spores are quadrate with rounded edges in apical view, oval-shaped with apical valve extensions in side view (mean spore dimensions: length 6.5 microns, width 7.7 microns, thickness 6.9 microns; mean polar capsule dimensions: length 2.1 microns, width 1.5 microns). This is the first Kudoa species from gonads of fishes. Prevalence of infection varied among labrids (Thalassoma bifasciatum, Halichoeres bivittatus, Halichoeres garnoti, Halichoeres poevi), with T. bifasciatum exhibiting the greatest prevalence. Density of infection, measured as percent infected eggs, also varied among species with highest densities occurring in H. garnoti. Kudoa ovivora may not require an intermediate host because fishes fed infected tissue developed more infections than unfed fish. Infected eggs are inviable and larger and heavier than uninfected eggs. Infected eggs contain more organic and inorganic material, indicating that K. ovivora increases resource allocation to eggs. Therefore, infected females may have reduced growth, fecundity, and/or spawning activity. Because males were uninfected and all identified hosts are protogynous sequential hermaphrodites, further studies of K. ovivora may provide new insights on the costs/benefits of sex change.     

Tetracapsula renicola n. sp. (Myxozoa : Saccosporidae); the PKX myxozoan--the cause of proliferative kidney disease of salmonid fishes

Proliferative kidney disease (PKD) of salmonid fishes is caused by the extrasporogonic stage of an enigmatic myxozoan, referred to as PKX. Sporogenesis occurs in the renal tubules, resulting in monosporous pseudoplasmodia. The spores are ovoid with indistinguishable valves and measure 12 microm in length and 7 microm in width. Two spherical polar capsules (2 microm diameter) with 4 coils occur at the anterior end of the spore. Prominent capsulogenic cell nuclei posterior to the polar capsules are evident in histological sections stained with hematoxylin and eosin. Regardless of the true nature of the valves (indistinguishable or absent), this myxozoan is morphologically distinct from all other described members of the phylum Myxozoa. Comparisons of small subunit rDNA sequences of PKX with other myxozoans demonstrated that it branches from all other members of the myxosporeans from fish examined thus far, including representatives of the phenotypically most closely related genera, Sphaerospora and Parvicapsula. Recent reports, based on rDNA comparisons, indicate that the alternate stage of PKX occurs in bryozoans, and that PKX clusters in a clade with Tetracapsula bryozoides. Our analyses and those of others, along with phenotypic observations, indicate that salmonids are the primary myxosporean hosts for PKX, that the cryptic spores of PKX in salmonids are the fully formed myxospores as they occur in the fish host, and that PKX represents distinct species that we previously place in the genus Tetracapsula in the family Saccosporidae. The latter 2 taxa were described based on stages from bryozoans, and the myxosporean stage in fish of the type species, T. bryozoides, has not been identified (if it exists). Thus, more complete resolution of the life cycle of both PKX and T. bryozoides, as well as more genetic data, are required to determine the precise relationship of these organisms.     

Recent Advances in Our Knowledge of the Myxozoa

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.     

Identification, characterization and deduced amino acid sequence of the dominant protease from Kudoa paniformis and K. thyrsites: a unique cytoplasmic cysteine protease

Kudoa paniformis and Kudoa thyrsites (Myxozoa: Myxosporea) infections are associated with severe proteolysis of host muscle tissue post-mortem. The present study was undertaken to identify and characterize the protease responsible for myoliquefaction and determine mechanisms controlling protease function in vivo. N-terminal sequence analysis of partially purified protease from hake muscle infected with K. paniformis and K. thyrsites revealed a 23 amino acid sequence that aligned with cysteine proteases. Enzyme inhibition assays confirmed the presence of an essential active site cysteine residue. Using the above K. paniformis amino acid sequence data, a corresponding cDNA sequence from K. thyrsites plasmodia was elucidated revealing a cathepsin L proenzyme (Kth-CL). The translated amino acid sequence lacked a signal sequence characteristic of lysosomal and secreted proteins suggesting a unique cytoplasmic location. Only the proenzyme form of Kth-CL was present in Atlantic salmon muscle anti-mortem but this form became processed in vivo when infected muscle was stored at 4 degrees C. The proenzyme of Kth-CL showed uninhibited activity at pH 6.0, negligible activity at pH 6.5 and no measurable activity at pH 7.0 whilst the processed protease showed stability and function over a broad pH range (pH 4.5-8.8). The pH dependent processing and function of Kth-CL was consistent with histidine residues in the proregion playing a critical role in the regulation of Kth-CL.     

Diagnostic polymerase chain reaction assay to detect Kudoa neurophila (Myxozoa: Multivalvulida) in a marine finfish hatchery

A single-round polymerase chain reaction (PCR) diagnostic assay was developed from a small subunit ribosomal DNA (SSU rDNA) gene sequence to detect the myxozoan parasite Kudoa neurophila, the causative agent of myxozoan disease in the hatchery reared marine finfish, striped trumpeter Latris lineata (Forster). The assay was developed for use as a disease control management tool in a hatchery system specifically designed to research and produce marine finfish such as striped trumpeter juveniles for aquaculture. The assay is sufficiently species specific and sensitive enough to detect a small fragment of the parasite's SSU rDNA. At the lower limits of detection, the test is consistently positive to an estimated 0.1 spore or 60 fg of parasite DNA per 25 microl PCR reaction in serial dilution and positive to an estimated 0.1 spore in 25 mg of infected fish CNS tissue (4 spores g(-1). Specifically, the test is capable of detecting early stages of the life cycle within the fish host and consequently diagnosing an infection not normally detected using traditional histological techniques. The test is also effective for screening water supplies and prey species cultures throughout the hatchery system to determine bio-security efficacy, to assist in identification of an alternate or other primary fish host, to indicate the location of potential disease reservoirs, and to enable a targetted approach to disease prevention.