Kudoa hexapunctata n. sp. (Myxozoa: Multivalvulida) from the somatic muscle of Pacific bluefin tuna Thunnus orientalis and re-description of K. neothunni in yellowfin tuna T. albacares

Since Kudoa septempunctata in olive flounder (Paralichthys olivaceus) was indicated to cause food poisoning in humans, other Kudoa species are suspected to have pathogenic potential. Recently, a myxosporean possibly associated with food poisoning in humans consuming raw Pacific bluefin tuna, Thunnus orientalis, was identified as Kudoa neothunni. This is a known causative myxosporean of post-harvest myoliquefaction in yellowfin tuna Thunnus albacares. Regardless of the significant differences in the 28S rDNA sequence and the pathological character (with/without myoliquefaction) between the two T. orientalis and T. albacares isolates, they were considered intraspecific variants of K. neothunni. However, the light and low-vacuum electron microscopic observations in the present study revealed that there were two morphotypes; pointed- and round-type spores, which were significantly differentiated by the ratio of suture width to spore width. Furthermore, the two morphotypes were genetically distinguishable by the 28S rDNA sequence analysis. This morphological and molecular evidence validates that the two Kudoa types are separate species, and thus the pointed- and round-types are referred to as K. neothunni and Kudoa hexapunctata n. sp., respectively. K. neothunni was detected solely from T. albacares, whereas K. hexapunctata n. sp. was found not only from T. orientalis but also from T. albacares.     

Detection and characterization of Kudoa thunni from uncooked yellowfin tuna (Thunnus albacares) in Southeast Asia

Myxosporean parasites Kudoa spp. have been reported in several marine fish species worldwide. However, little is known about the contamination of these parasites in raw fish in Southeast Asia, where the consumption demand of uncooked fish is increasing. In 2019, the occurrence of several cases of raw yellowfin tuna (Thunnus albacares) obtained from retail shops with the presence of unknown white, nodular cysts within the musculature have raised public health concerns for the consumption of raw marine fish in Vietnam. Microscopic examination revealed numerous myxospores with the quadratic shape of the Kudoidae. Morphologically, stained spores detected in this study are suspected to Kudoa thunni. To confirm the suspected Kudoa species, further examination of the 18S small-subunit (SSU) was conducted and the results of nucleotide sequence analysis obtained from nodular cysts revealed 99.18–100% identity to that of Kudoa thunni sequences available in GenBank. Detection of K. thunni infection in tuna in Southeast Asia highlights the need for appropriate surveillance and control measures to ensure high quality standards and safety on raw fish production and consumption.     

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.     

Discovery of intermediate hosts for two species of blood flukes Cardicola orientalis and Cardicola forsteri (Trematoda: Aporocotylidae) infecting Pacific bluefin tuna in Japan

Fish blood flukes (Aporocotylidae) are important pathogens of farmed finfish around the world. Among them, Cardicola spp. infecting farmed tuna are considered to be serious threats to tuna farming and have received tremendous attention. We conducted periodical samplings at a tuna farming site in Japan between January and May, 2015 to determine the life cycle of Cardicola spp. We collected over 4700 terebellid polychaetes from ropes, floats and frames of tuna culture cages and found nearly 400 infected worms. Sporocysts and cercariae found in Nicolea gracilibranchis were genetically identified as Cardicola orientalis by 28S and ITS2 ribosomal DNA sequences. This was the first discovery of the intermediate host for this parasite species. Infection prevalence and the abundance of N. gracilibranchis significantly varied between sampling points and the highest number of infected terebellids were collected from ropes. We also demonstrated morphologically and molecularly that asexual stages found in a single Amphitrite sp. (Terebellidae) and adult worms isolated from farmed juvenile tuna were Cardicola forsteri. This is the first report of C. forsteri in Pacific bluefin tuna (PBT) Thunnus orientalis in Japan. Our results demonstrated that all three species of Cardicola orientalis, C. forsteri and Cardicola opisthorchis exist in Japanese farmed PBTs and that they all use terebellid polychaetes as the intermediate hosts.     

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.     

Kudoa prunusi n. sp. (Myxozoa: Multivalvulida) from the brain of Pacific bluefin tuna Thunnus orientalis (Temminck & Schlegel, 1844) cultured in Japan

Kudoa prunusi n. sp. (Myxozoa; Multivalvulida) is described from the brain of Pacific bluefin tuna Thunnus orientalis cultured in Japan. Numerous white cysts, up to 0.5mm in size, were found on and in the brain. Spores having typically five spore valves and five polar capsules resembled a five-petal cherry blossom in apical view and were conical shape with a round bottom in side view. Average spore size was 9.63 (8.5-10.3) μm in width and 7.50 (6.7-8.6) μm in length. The spore dimensions of K. prunusi overlapped with those of Kudoa yasunagai ex Sillago ciliata having five to six spore valves, but they were clearly distinct in spore shape, 18S rDNA and 28S rDNA sequences (0.3% and 1.7% differences, respectively). Phylogenetic analysis of 18S rDNA revealed that K. prunusi grouped with the brain-infecting multivalvulid species, K. yasunagai, K. chaetodoni, K. lethrini and K. neurophila, rather than five-valved Kudoa spp. Combined with morphological, molecular and biological differences, K. prunusi was proven to be a new species.     

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.     

Intra-specific variation of Kudoa spp. (Myxosporea: Multivalvulida) from apogonid fishes (Perciformes), including the description of two new species, K. cheilodipteri n. sp. and K. cookii n. sp., from Australian waters

Kudoa spp. from the musculature and intestinal mucosa of species of the teleost family Apogonidae were examined for their taxonomic identity. Two novel species are characterised: Kudoa cheilodipteri n. sp. from the musculature of Cheilodipterus quinquelineatus Cuvier, Ostorhinchus cyanosoma (Bleeker) and O. aureus (Lacépède); and Kudoa cookii n. sp. from the submucosa of the intestines of O. cookii (Macleay) only. Both species are characterised using morphology, small subunit ribosomal DNA (SSU rDNA), large subunit ribosomal DNA (LSU rDNA), and biological characters. Three new host records, O. cyanosoma, O. aureus and Apogon doederleini, and associated geographical, morphological and genetic data are also provided for Kudoa whippsi Burger & Adlard, 2010. Morphological and molecular intra-specific variation of all isolates assigned to K. whippsi is also examined. Phylogenetic analyses further support the idea that tissue tropism is a distinguishing character between morphologically similar species; species reported here display close relatedness to morphologically similar species infecting the same tissue within their hosts.     

The Species and Origin of Shark Fins in Taiwan’s Fishing Ports,Markets, and Customs Detention: A DNA Barcoding Analysis

The increasing consumption of shark products, along with the shark’s fishing vulnerabilities, has led to the decrease in certain shark populations. In this study we used a DNA barcoding method to identify the species of shark landings at fishing ports, shark fin products in retail stores, and shark fins detained by Taiwan customs. In total we identified 23, 24, and 14 species from 231 fishing landings, 316 fin products, and 113 detained shark fins, respectively. All the three sample sources were dominated by Prionace glauca, which accounted for more than 30% of the collected samples. Over 60% of the species identified in the fin products also appeared in the port landings, suggesting the domestic-dominance of shark fin products in Taiwan. However, international trade also contributes a certain proportion of the fin product markets, as four species identified from the shark fin products are not found in Taiwan’s waters, and some domestic-available species were also found in the customs-detained sample. In addition to the species identification, we also found geographical differentiation in the cox1 gene of the common thresher sharks (Alopias vulpinus), the pelagic thresher shark (A. pelagicus), the smooth hammerhead shark (Sphyrna zygaena), and the scalloped hammerhead shark (S. lewini). This result might allow fishing authorities to more effectively trace the origins as well as enforce the management and conservation of these sharks.     

Two multivalvulid myxozoans causing postmortem myoliquefaction: Kudoa megacapsula n. sp. from red barracuda (Sphyraena pinguis) and Kudoa thyrsites from splendid alfonso (Beryx splendens)

Postmortem myoliquefaction associated with multivalvulid myxozoans was found in fillets of red barracuda (Sphyraena pinguis) and splendid alfonso (Beryx splendens), which were imported to Japan from China and South Africa, respectively. Morphological examinations of the myxozoans from the somatic muscle of red barracuda revealed that spores (30.3-44.7 microm in maximum thickness) had 4 distinct winglike valves, in which 1 extremely large (12.7 x 5.8 microm), 2 small, and 1 vestigial polar capsule were present. The small subunit ribosomal DNA sequence analysis showed that the myxozoan cluster within a clade was composed of Kudoa thyrsites, Kudoa minithyrsites, and Kudoa lateolabracis, all having stellate spores with 1 polar capsule larger than the other 3. On the basis of these characteristics, we describe this parasite as Kudoa megacapsula n. sp. Morphological and molecular analyses of the myxozoan from splendid alfonso identified it as K. thyrsites, which has been described from many marine fishes. To our knowledge, this is the first record of K. thyrsites in splendid alfonso.     

SSU rDNA analysis of Kudoa rosenbuschi (Myxosporea) from the Argentinean hake Merluccius hubbsi

The cloning and sequencing of the small subunit (SSU) ribosomal DNA gene from Kudoa rosenbuschi (myxosporean species associated with post-mortem myoliquefaction process in the Argentinean hake Merluccius hubbsi) is reported. The SSU rDNA was found to contain 1740 bp with a single polymorphic site with either a C or T at position 221. The sequence data obtained in this study and those known sequences of Kudoa species deposited in the GenBank were all analyzed to construct a phylogenetic tree. Nucleotide sequences showed the highest degree of identity with K. funduli, followed by K. miniauriculata, K. clupeidae and K. dianae. Phylogenetic analysis placed K. rosenbuschi in the same branch of K. clupeidae and K. funduli, and showed it to be closely related to K. dianae, K. paniformis and K. miniauriculata.     

Tracking Microbial Contamination in Retail Environments Using Fluorescent Powder - A Retail Delicatessen Environment Example

Cross contamination of foodborne pathogens in the retail environment is a significant public health issue contributing to an increased risk for foodborne illness. Ready-to-eat (RTE) processed foods such as deli meats, cheese, and in some cases fresh produce, have been involved in foodborne disease outbreaks due to contamination with pathogens such as Listeria monocytogenes. With respect to L. monocytogenes, deli slicers are often the main source of cross contamination. The goal of this study was to use a fluorescent compound to simulate bacterial contamination and track this contamination in a retail setting. A mock deli kitchen was designed to simulate the retail environment. Deli meat was inoculated with the fluorescent compound and volunteers were recruited to complete a set of tasks similar to those expected of a food retail employee. The volunteers were instructed to slice, package, and store the meat in a deli refrigerator. The potential cross contamination was tracked in the mock retail environment by swabbing specific areas and measuring the optical density of the swabbed area with a spectrophotometer. The results indicated that the refrigerator (i.e. deli case) grip and various areas on the slicer had the highest risk for cross contamination. The results of this study may be used to develop more focused training material for retail employees. In addition, similar methodologies could also be used to track microbial contamination in food production environments (e.g. small farms), hospitals, nursing homes, cruise ships, and hotels.     

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.     

Digital PCR Validates 8q Dosage as Prognostic Tool in Uveal Melanoma

Background

Uveal melanoma (UM) development and progression is correlated with specific molecular changes. Recurrent mutations in GNAQ and GNA11 initiate UM development while tumour progression is correlated with monosomy of chromosome 3 and gain of chromosome 8q. Hence, molecular analysis of UM is useful for diagnosis and prognosis. The aim of this study is to evaluate the use of digital PCR (dPCR) for molecular analysis of UM.

Methods

A series of 66 UM was analysed with dPCR for three hotspot mutations in GNAQ/GNA11 with mutation specific probes. The status of chromosomes 3 and 8 were analysed with genomic probes. The results of dPCR analysis were cross-validated with Sanger sequencing, SNP array analysis, and karyotyping.

Results

Using dPCR, we were able to reconstitute the molecular profile of 66 enucleated UM. With digital PCR, GNAQ/GNA11 mutations were detected in 60 of the 66 UM. Sanger sequencing revealed three rare variants, and, combined, these assays revealed GNAQ/GNA11 mutations in 95% of UM. Monosomy 3 was present in 43 and chromosome 8 aberrations in 52 of the 66 UM. Survival analysis showed that increasing 8q copy numbers were positively correlated with metastasis risk.

Conclusion

Molecular analysis with dPCR is fast and sensitive. Just like the recurrent genomic aberrations of chromosome 3 and 8, hotspot mutations in GNAQ and GNA11 are effectively detected in heterogeneous samples. Increased sensitivity contributes to the number of mutations and chromosomal aberrations detected. Moreover, quantification of copy number with dPCR validated 8q dosage as a sensitive prognostic tool in UM, of which implementation in disease prediction models will further improve prognostication.     

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.     

Kudoa iwatai (Myxosporea: Multivalvulida) in wild and cultured fish in the Red Sea: redescription and molecular phylogeny

Gilt-head sea bream, Sparus aurata L., the Mediterranean's most important mariculture species, has been cultured for the last 30 yr in Eilat (Israeli Red Sea). Kudoa sp. was the first myxosporean parasite reported from this species. In recent years, an increase in prevalence in both land-based and sea-cage facilities in Eilat has been observed. Infections with the same Kudoa species appeared in cultured European sea bass Dicentrarchus labrax (L.) and grey mullet Mugil cephalus in the same farms, as well as in 10 species of wild Red Sea reef fish, indicating that Kudoa sp. is not fastidious with regard to its host. All affected species displayed 1- to 2-mm (up to 5 mm) whitish, spherical, or oval polysporous plasmodia. The parasite established multiple site infections, most commonly in the muscles and intracranial adipose tissue of the brain and eye periphery. Other sites were subcutaneous adipose tissue, nerve axons, mouth, eye, mesenteries, peritoneum, swim bladder, intestinal musculature, heart, pericardium, kidney, and ovary. On the basis of spore morphology, the parasite was identified as Kudoa iwatai Egusa and Shiomitsu, 1983. Ultrastructural features were comparable to those of previously studied Kudoa species. The 18S rDNA from 7 Red Sea isolates was sequenced and compared with the sequence of the same gene from K. iwatai isolated from cultured red sea bream, Pagrus major, in Japan. The phylogenetic position of K. iwatai within the genus was determined using sequence analysis of all related taxa available in GenBank. The 3 isolates of K. iwatai clustered together on a newly formed, highly supported clade. The Red Sea strain of K. iwatai is apparently native to the region. In the absence of records of this Kudoa sp. from the extensive Mediterranean sea bream and sea bass production industries, introduction with its Mediterranean hosts seems unlikely. Therefore, we conclude that K. iwatai is an Indo-Pacific species that, in the Red Sea, has extended its host range to include the allochthonous gilt-head sea bream, European sea bass, and grey mullet.     

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.