Surveillance of densoviruses and mesomycetozoans inhabiting grossly normal tissues of three Aotearoa New Zealand asteroid species.

Asteroid wasting events and mass mortality have occurred for over a century. We currently lack a fundamental understanding of the microbial ecology of asteroid disease, with disease investigations hindered by sparse information about the microorganisms associated with grossly normal specimens. We surveilled viruses and protists associated with grossly normal specimens of three asteroid species (Patiriella regularis, Stichaster australis, Coscinasterias muricata) on the North Island / Te Ika-a-Maui, Aotearoa New Zealand, using metagenomes prepared from virus and ribosome-sized material. We discovered several densovirus-like genome fragments in our RNA and DNA metagenomic libraries. Subsequent survey of their prevalence within populations by quantitative PCR (qPCR) demonstrated their occurrence in only a few (13%) specimens (n = 36). Survey of large and small subunit rRNAs in metagenomes revealed the presence of a mesomycete (most closely matching Ichthyosporea sp.). Survey of large subunit prevalence and load by qPCR revealed that it is widely detectable (80%) and present predominately in body wall tissues across all 3 species of asteroid. Our results raise interesting questions about the roles of these microbiome constituents in host ecology and pathogenesis under changing ocean conditions.

Infection of the Carpathian brook lamprey (Eudontomyzon danfordi Regan, 1911) with a dermocystid parasite in the Tisza River Basin, Hungary.

The Carpathian brook lamprey (Eudontomyzon danfordi Regan, 1911) is an endemic protected species of Cephalaspidomorphi in the Carpathian Basin. No parasites have become known from these jawless vertebrates to date. Here, the authors describe an infection from a single specimen manifesting in protuberant skin cysts 7-10 mm in diameter, scattered on the body surface. Similar dermal infection was observed in 25 of the 274 lampreys recorded in the population survey. Skin cysts filled with round spore-like structures of a dermocystid parasite were found. These particles measured 8-14 µm in diameter and had an about 0.5 µm thick wall, and containing mainly a granular mass and a relatively scarce plasma. No hyphae were recorded. Despite conspicuous morphological changes in the skin, no inflammatory reactions were found. The molecular analysis of 18S rDNA showed similarity to dermocystid species of several fish species but differed from them approximately by 2%. This is the first record of a dermocystid parasite infecting a jawless vertebrate.

Ichthyophonus sp. Infection in Opaleye (Girella nigricans).

Over a 3-year-period, 17 wild-caught opaleye (Girella nigricans) housed in a public display aquarium were found dead without premonitory signs. Grossly, 4 animals had pinpoint brown or black foci on coelomic adipose tissue. Histologically, liver, spleen, heart, and posterior kidney had mesomycetozoan granulomas in all cases; other organs were less commonly infected. Four opaleye had goiter; additional substantial lesions were not identified. Granulomas surrounded melanized debris, leukocytes, and mesomycetozoa represented by folded membranes (collapsed schizont walls), intact schizonts (50- to >200 µm in diameter with a multilaminate membrane), plasmodia (budding from schizonts or free in tissue), or rarely germinal tubes (budding from schizonts). Ichthyophonus was grown from fresh tissues in tissue explant broth cultures of the heart, liver, and/or spleen. Polymerase chain reaction using 18S ribosomal DNA primers amplified a 1730-bp region, and the DNA sequence was most similar to Ichthyophonus hoferi, which is often associated with freshwater aquaculture fish.

Dermocystidium sp. in the gills of farmed Oreochromis niloticus in Brazil.

The genus Dermocystidium is very comprehensive in the host and site of infection, however this is the first report of the occurrence of Dermocystidium sp. in the gills of Nile tilapia. This study was carried out in a fish farming located in the state of Santa Catarina, Brazil. No mortalities were reported in the facility studied and the animals were clinically healthy. During the histopathological analysis of the gills, 8.33% of the fish presented spores of Dermocystidium sp. in the gill tissue. The spores reported herein had a mean length and width of 6.206 x 5.233 µm and a refractile body diameter of 1.965 µm and were studied by histopathology and Transmission Electron Microscopy. This study highlights the importance of a new branchial pathogen in farmed tilapia, as well as to its pathogenic potential, considering the outbreaks of mortalities associated with other fish species.

Analytical and diagnostic performance of a qPCR assay for Ichthyophonus spp. compared to the tissue culture 'gold standard'.

Parasites of the genus Ichthyophonus infect many fish species and have a non-uniform distribution within host tissues. Due in part to this uneven distribution, the comparative sensitivity and accuracy of using molecular-based detection methods versus culture to estimate parasite prevalence is under debate. We evaluated the analytical and diagnostic performance of an existing qPCR assay in comparison to the 'gold standard' culture method using Pacific herring Clupea pallasii with known exposure history. We determined that the assay is suitable for use in this host, and diagnostic specificity was consistently high (>98%) in both heart and liver tissues. Diagnostic sensitivity could not be fully assessed due to low infection rates, but our results suggest that qPCR is not as sensitive as culture under all circumstances. Diagnostic sensitivity of qPCR relative to culture is likely affected by the amount of sample processed. The prevalence values estimated by the 2 methods were not significantly different when sample amounts were equal (heart tissue), but when the assayed sample amounts were unequal (liver tissue), the culture method detected a significantly higher prevalence of the parasite than qPCR. Further, culture of liver also detected significantly more Ichthyophonus infections than culture of heart, suggesting that the density and distribution of parasites in tissues also plays a role in assay sensitivity. This sensitivity issue would be most problematic for fish with light infections. Although qPCR does not detect the presence of a live organism, DNA-based pathogen detection methods provide the opportunity for alternate testing strategies when culture is not possible.

Ichthyophonus in sport-caught groundfishes from southcentral Alaska.

This report of Ichthyophonus in common sport-caught fishes throughout the marine waters of southcentral Alaska represents the first documentation of natural Ichthyophonus infections in lingcod Ophiodon elongates and yelloweye rockfish Sebastes ruberrimus. In addition, the known geographic range of Ichthyophonus in black rockfish S. melanops has been expanded northward to include southcentral Alaska. Among all species surveyed, the infection prevalence was highest (35%, n = 334) in Pacific halibut Hippoglossus stenolepis. There were no gross indications of high-level infections or clinically diseased individuals. These results support the hypothesis that under typical conditions Ichthyophonus can occur at high infection prevalence accompanied with low-level infection among a variety of fishes throughout the eastern North Pacific Ocean, including southcentral Alaska.

High-Prevalence and Low-Intensity Ichthyophonus Infections in Pacific Halibut.

Ichthyophonus occurred at high prevalence but low intensity in Pacific Halibut Hippoglossus stenolepis throughout the West Coast of North America, ranging from coastal Oregon to the Bering Sea. Infection prevalence in adults was variable on spatial and temporal scales, with the lowest prevalence typically occurring on the edges of the geographic range and highest prevalence consistently occurring inside Prince William Sound, Alaska (58-77%). Additionally, intra-annual differences occurred at Albatross-Portlock, Alaska (71% versus 32% within 2012), and interannual differences occurred along coastal Oregon (50% in 2012 versus 12% in 2015). The infection prevalence was influenced by host age, increasing from 3% or less among the youngest cohorts (age ≤ 6) to 39-54% among age-9-17 cohorts, then decreasing to 27% among the oldest (age-18+) cohorts. There was little indication of significant disease impacts to Pacific Halibut, as the intensity of infection was uniformly low and length at age was similar between infected and uninfected cohorts. These results suggest that Ichthyophonus in Pacific Halibut currently represents a stable parasite-host paradigm in the North Pacific.

Origin and invasion of the emerging infectious pathogen Sphaerothecum destruens.

Non-native species are often linked to the introduction of novel pathogens with detrimental effects on native biodiversity. Since Sphaerothecum destruens was first discovered as a fish pathogen in the United Kingdom, it has been identified as a potential threat to European fish biodiversity. Despite this parasite's emergence and associated disease risk, there is still a poor understanding of its origin in Europe. Here, we provide the first evidence to support the hypothesis that S. destruens was accidentally introduced to Europe from China along with its reservoir host Pseudorasbora parva via the aquaculture trade. This is the first study to confirm the presence of S. destruens in China, and it has expanded the confirmed range of S. destruens to additional locations in Europe. The demographic analysis of S. destruens and its host P. parva in their native and invasive range further supported the close association of both species. This research has direct significance and management implications for S. destruens in Europe as a non-native parasite.

Dermocystidium sp. Infection in Blue Ridge Sculpin Captured in Maryland.

Raised pale cysts were observed on Blue Ridge Sculpin Cottus caeruleomentum during stream fish community surveys in Catoctin Mountain Park, Maryland. When examined histologically, preserved sculpin exhibited multiple cysts containing spherical endospores with a refractile central body characteristic of Dermocystidium spp. Cysts were not observed on the gills or internally. The portion of the watershed in which affected sculpin were observed contained lower than expected numbers of sculpin, raising concerns about the population effects of this infection. A nearby stream lacked sculpin even though they are common in this region, further suggesting the possibility of regional effects. This is the first report of a Dermocystidium infecting any fish species in the eastern United States. Received October 16, 2015; accepted February 14, 2016.

Two New Species of Marine Saprotrophic Sphaeroformids in the Mesomycetozoea Isolated From the Sub-Arctic Bering Sea.

The genus Sphaeroforma previously encompassed organisms isolated exclusively from animal symbionts in marine systems. The first saprotrophic sphaeroformids (Mesomycetozoea) isolated from non-animal hosts are described here. Sphaeroforma sirkka and S. napiecek are also the first species in the genus possessing endogenous DNA-containing motile propagules and central vacuoles, traits that have previously guided morphological differentiation of sphaeroformids from the genus Creolimax. Phylogenetic analysis of DNA sequences from the 18S rRNA and the ITS1-5.8S--ITS2 loci firmly place S. sirkka and S. napiecek within Sphaeroforma, extending the number of known species to six within this genus. The discovery of these species increases the geographical range, cellular variation and life history complexity of the sphaeroformids.

Neurotropic mesomycetozoean-like infection in larvae of the southern toad Anaxyrus terrestris in Florida, USA.

As part of a state-wide multispecies survey of amphibian diseases, sampling was conducted at Archbold Biological Station, Venus, Florida, USA, on 15 April 2011. Gross examination of southern toad (Anaxyrus terrestris) larvae was unremarkable, but infections by a mesomycetozoean-like organism were observed in longitudinally sectioned routine haematoxylin and eosin-stained histologic slides. In 100% of the sectioned specimens examined (n = 5), a high density of the organism, representing several developmental stages, was found in the central nervous system, mainly in the spinal cord, brain, retina and optic nerve. No host inflammatory responses were found to be associated with the parasitic infection. Free, mature schizonts were occasionally found in the gill chamber and, more commonly, in the dorsal roof area. No organisms were found in other organs examined histologically, i.e. liver, kidney, heart, alimentary tract, exocrine pancreas and skeletal muscles. Presumptive mesomycetozoean ichthyophonids in anurans are usually reported to be pathogenic, especially affecting skeletal muscle tissue and causing death. To our knowledge, this is the first report of a similar organism infecting primarily the central nervous system in an amphibian.

Evidence of threat to European economy and biodiversity following the introduction of an alien pathogen on the fungal-animal boundary.

Recent years have seen a global and rapid resurgence of fungal diseases with direct impact on biodiversity and local extinctions of amphibian, coral, or bat populations. Despite similar evidence of population extinction in European fish populations and the associated risk of food aquaculture due to the emerging rosette agent Sphaerothecum destruens, an emerging infectious eukaryotic intracellular pathogen on the fungal-animal boundary, our understanding of current threats remained limited. Long-term monitoring of population decline for the 8-year post-introduction of the fungal pathogen was coupled with seasonal molecular analyses of the 18S rDNA and histological work of native fish species organs. A phylogenetic relationship between the existing EU and US strains using the ribosomal internal transcribed spacer sequences was also carried out. Here, we provide evidence that this emerging parasite has now been introduced via Pseudorasbora parva to sea bass farms, an industry that represents over 400 M€€ annually in a Mediterranean region that is already economically vulnerable. We also provide for the first time evidence linking S. destruens to disease and severe declines in International Union for Conservation of Nature threatened European endemic freshwater fishes (i.e. 80% to 90 % mortalities). Our findings are thus of major economic and conservation importance.

Ichthyophonus-infected walleye pollock Theragra chalcogramma (Pallas) in the eastern Bering Sea: a potential reservoir of infections in the North Pacific.

In 2003, the Alaska walleye pollock industry reported product quality issues attributed to an unspecified parasite in fish muscle. Using molecular and histological methods, we identified the parasite in Bering Sea pollock as Ichthyophonus. Infected pollock were identified throughout the study area, and prevalence was greater in adults than in juveniles. This study not only provides the first documented report of Ichthyophonus in any fish species captured in the Bering Sea, but also reveals that the parasite has been present in this region for nearly 20 years and is not a recent introduction. Sequence analysis of 18S rDNA from Ichthyophonus in pollock revealed that consensus sequences were identical to published parasite sequences from Pacific herring and Yukon River Chinook salmon. Results from this study suggest potential for Ichthyophonus exposures from infected pollock via two trophic pathways; feeding on whole fish as prey and scavenging on industry-discharged offal. Considering the notable Ichthyophonus levels in pollock, the low host specificity of the parasite and the role of this host as a central prey item in the Bering Sea, pollock likely serve as a key Ichthyophonus reservoir for other susceptible hosts in the North Pacific.

PCR testing for diagnosis of Ichthyophonus hoferi: comment on Hamazaki et al. (2013).

It is our opinion that Hamazaki et al. (2013; Dis Aquat Org 105:21-25) overstate the usefulness of PCR as a field diagnostic technique and underestimate the accuracy and utility of in vitro explant culture. In order for field diagnostic studies to be meaningful they should accurately and dependably identify the infected individuals within a population, both subclinical and clinical cases. Although explant culture, like most techniques, can miss some infected individuals, 'false positives' are impossible, unlike for cPCR based methodologies.

PCR testing for diagnosis of Ichthyophonus hoferi: reply to LaPatra & Kocan (2013).

LaPatra & Kocan (2013) critiqued our paper Hamazaki et al. (2013; Dis Aquat Org 105:21-25) for data not supporting the conclusions of 'PCR testing is as accurate as culture…', but they neither pointed out what part of our data did not support our conclusion, nor did they provide any contrary scientific evidence supporting their argument that PCR testing is less accurate than culture. In the absence of any contradictory data, we stand by our data and our conclusion: PCR test is as suitable as culture as a diagnostic and field surveillance tool.

PCR testing can be as accurate as culture for diagnosis of Ichthyophonus hoferi in Yukon River Chinook salmon Oncorhynchus tshawytscha .

We evaluated the comparability of culture and PCR tests for detecting Ichthyophonus in Yukon River Chinook salmon Oncorhynchus tshawytscha from field samples collected at 3 locations (Emmonak, Chena, and Salcha, Alaska, USA) in 2004, 2005, and 2006. Assuming diagnosis by culture as the 'true' infection status, we calculated the sensitivity (correctly identifying fish positive for Ichthyophonus), specificity (correctly identifying fish negative for Ichthyophonus), and accuracy (correctly identifying both positive and negative fish) of PCR. Regardless of sampling locations and years, sensitivity, specificity, and accuracy exceeded 90%. Estimates of infection prevalence by PCR were similar to those by culture, except for Salcha 2005, where prevalence by PCR was significantly higher than that by culture (p < 0.0001). These results show that the PCR test is comparable to the culture test for diagnosing Ichthyophonus infection.

Development and validation of a quantitative PCR assay for Ichthyophonus spp.

Members of the genus Ichthyophonus are trophically transmitted, cosmopolitan parasites that affect numerous fish species worldwide. A quantitative PCR (qPCR) assay specific for genus Ichthyophonus 18S ribosomal DNA was developed for parasite detection and surveillance. The new assay was tested for precision, repeatability, reproducibility, and both analytical sensitivity and specificity. Diagnostic sensitivity and specificity were estimated using tissue samples from a wild population of walleye pollock Theragra chalcogramma. Ichthyophonus sp. presence in tissue samples was determined by qPCR, conventional PCR (cPCR), and histology. Parasite prevalence estimates varied depending upon the detection method employed and tissue type tested. qPCR identified the greatest number of Ichthyophonus sp.-positive cases when applied to walleye pollock skeletal muscle. The qPCR assay proved sensitive and specific for Ichthyophonus spp. DNA, but like cPCR, is only a proxy for infection. When compared to cPCR, qPCR possesses added benefits of parasite DNA quantification and a 100-fold increase in analytical sensitivity. Because this novel assay is specific for known members of the genus, it is likely appropriate for detecting Ichthyophonus spp. DNA in various hosts from multiple regions. However, species-level identification and isotype variability would require DNA sequencing. In addition to distribution and prevalence applications, this assay could be modified and adapted for use with zooplankton or environmental samples. Such applications could aid in investigating alternate routes of transmission and life history strategies typical to members of the genus Ichthyophonus.

Isolation and culture of Sphaerothecum destruens from Sunbleak (Leucaspius delineatus) in the UK and pathogenicity experiments in Atlantic salmon (Salmo salar).

The sunbleak (Leucaspius delineatus), a cyprinid fish native to continental Europe and now established in the UK, is experiencing population decline which appears to be linked to the spread of the invasive Asian cyprinid (Pseudorasbora parva). A population of sunbleak in the UK has previously been identified as infected with S. destruens at low prevalence. Because Sphaerothaecum destruens has, on occasion, caused severe disease in cultured and wild salmonids the aim of this work was to establish laboratory cultures of S. destruens from sunbleak in the UK and use these cultures in challenge experiments to determine if the UK isolate of S. destruens from cyprinid species is a potential threat to Atlantic salmon (Salmo salar). The first isolation and culture of S. destruens in the UK and from a cyprinid species is described. Cultured S. destruens spores from sunbleak are infective to EPC, CHSE and FHM cells, replicating most rapidly in FHM and EPC cells. Spores can be induced to zoosporulate in water forming motile, uni-flagellated zoospores. Challenge experiments indicated the spores are able to replicate and disperse in Atlantic salmon and are associated with increased mortality (up to 90%) when injected intraperitonealy.