Developmental stages of Amphibiocystidium sp., a parasite from the Italian stream frog (Rana italica).

Amphibian parasites of the genus Amphibiocystidium are members of the class Ichthyosporea (=Mesomycetozoea), within the order Dermocystida. Most of the species in the Dermocystida fail to grow in ordinary culture media, so their life cycle has only been partially constructed by studies in host tissues. However, to date, there have been few reports on the life cycle of Amphibiocystidium parasites with respect to the developmental life stages of both Dermocystidium and Rhinosporidium parasites. In this study, we provide light and electron microscopic findings of developmental phenotypes of Amphibiocystidium sp., a parasite previously characterized in the Italian stream frog (Rana italica), which has caused an ongoing infection in a natural population of Central Italy. These phenotypes exhibited distinct morphological characteristics that were similar to A. ranae from the skin of R. temporaria, but showed histochemical properties particularly comparable with those of maturing phenotypes of Rhinosporidium seeberi, and compatible with fungal-like parasites. Therefore, for Amphibiocystidium sp. phenotypes, we suggest adopting the terminology used for maturing stages of R. seeberi, such as juvenile sporangia, early mature sporangia and mature sporangia. The characterization of these developmental stages will be useful to increase the understanding of the life cycle of parasites of the genus Amphibiocystidium and of the interactions with their amphibian hosts.

Characterization of Dermotheca sp. Infection in a midwestern state-endangered salamander (Ambystoma platineum) and a co-occurring common species (Ambystoma texanum).

Ichthyosporean parasites (order Dermocystida) can cause morbidity and mortality in amphibians, but their ecology and epidemiology remain understudied. We investigated the prevalence, gross and histologic appearance, and molecular phylogeny of a novel dermocystid in the state-endangered silvery salamander (Ambystoma platineum) and the co-occurring, non-threatened small-mouthed salamander (Ambystoma texanum) from Illinois. Silvery salamanders (N = 610) were sampled at six ephemeral wetlands from 2016 to 2018. Beginning in 2017, 1-3 mm raised, white skin nodules were identified in 24 silvery salamanders and two small-mouthed salamanders from five wetlands (prevalence = 0-11.1%). Skin biopsy histology (N = 4) was consistent with dermocystid sporangia, and necropsies (N = 3) identified infrequent hepatic sporangia. Parasitic 18S rRNA sequences (N = 5) from both salamander species were identical, and phylogenetic analysis revealed a close relationship to Dermotheca viridescens. Dermocystids were not identified in museum specimens from the same wetlands (N = 125) dating back to 1973. This is the first report of Dermotheca sp. affecting caudates in the Midwestern United States. Future research is needed to determine the effects of this pathogen on individual and population health, and to assess whether this organism poses a threat to the conservation of ambystomatid salamanders.

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.

Transmission of a Freshwater Isolate of Ichthyophonus (Clade C) to Two Marine Fish Species.

The ingestion of infected prey is the most recognizable mode of transmission for Ichthyophonus, but because this mode of transmission is unidirectional from small prey fish to larger predators, it cannot sustain the parasite within or among populations nor does it explain transmission to planktivores. Recently, waterborne transmission was demonstrated in cultured Rainbow Trout Oncorhynchus mykiss, which could explain how the parasite is transmitted without piscivory. However, it is possible that this is an adaptation to aquaculture conditions, and may not occur among wild fish. To address this question, experiments were conducted to determine if a freshwater isolate of Ichthyophonus is infectious and pathogenic to marine species, as well as if transmission is possible between different marine species. Pacific Staghorn Sculpins Leptocottus armatus were fed a freshwater isolate of Ichthyophonus (clade C) and then housed with susceptible sentinel Rock Soles Lepidopsetta bilineata. Ninety two percent of the orally exposed sculpins and 30% of the sentinel soles were Ichthyophonus-positive at the end of the study, with 0% infected controls. These results demonstrate that a freshwater isolate of Ichthyophonus is infectious and pathogenic to marine species and can be transmitted in seawater in the absence of piscivory. It also provides a plausible mechanism for transmission to small prey fish and planktivores, as well as within a population of piscivores when infected prey is not available.

The rise of the rosette agent in Europe: An epidemiological enigma.

International biodiversity assessments often overlook the role of emerging infectious pathogens in the decline of freshwater fish populations despite the many examples of emerging diseases in other more visible taxa on a global scale. Whilst the introduction of the rosette agent Sphaerothecum destruens in Europe remained an epidemiological enigma, recent findings have shown that this parasite arrived in Europe with the introduction of the healthy carrier Pseudorasbora parva from China nearly 60 years ago and its emergence went unnoticed for over 45 years despite its severe impact on European fish biodiversity. Recent reports on the host and pathogen phylogeny point towards an ancient host-pathogen co-evolution with direct implications on disease risk. Here, we postulate that the observed rapid population decline of native fish species following their infection with virulent strains of S. destruens has underpinned the rapid establishment of P. parva populations during the invasion process. We reviewed the existing evidence supporting the claim of an S. destruens' emergence worldwide and also suggest that the origin of the US strains is to be found among contaminated Asian Oncorhynchus tshawytscha living in sympatry with native Asian P. parva population. Finally, several important preventative steps are suggested as a way to manage the impact of S. destruens on local fish communities.

Metamorphosis of Ichthyophonus Schizonts Transiting the Gastrointestinal Tract of Experimentally Exposed Rainbow Trout.

Other than the initial infectious cell, schizonts are the only stage of the parasite Ichthyophonus sp. that has been identified in the tissues of a living host, and they are known to initiate new infections when ingested by a suitable host. However, after feeding Ichthyophonus-infected tissue to Rainbow Trout Oncorhynchus mykiss, we observed that once infection was initiated, some schizonts proceeded to develop into several other morphologic forms indistinguishable from those previously described from recently deceased hosts, decomposing infected corpses, and in vitro culture. It appeared that not all schizonts participated in the infection process; some initiated infection, as expected, while others passed into the intestines, where they morphed into multiple cell types (e.g., schizonts, some with partially digested or ruptured capsules, ameboid plasmodia, merozoites, hyphenated cells, and empty capsules). Some of these cells were viable when cultured, but none was infectious to naïve Rainbow Trout when administered by gavage. We posit that (1) not all tissue schizonts are programmed to perform the same function or (2) not all respond similarly to their environment. After consumption by a piscivore, those schizonts that do not initiate an infection do not die but rather metamorphose into different cell types as they transit the gastrointestinal tract and are ultimately released back into the aquatic environment through defecation. The fate of these cells after exiting the host is presently unknown, but they likely represent a segment of the Ichthyophonus life cycle.

Phylogenetic and environmental DNA insights into emerging aquatic parasites: implications for risk management.

Species translocation leads to disease emergence in native species of considerable economic importance. Generalist parasites are more likely to be transported, become established and infect new hosts, thus their risk needs to be evaluated. Freshwater systems are particularly at risk from parasite introductions due to the frequency of fish movements, lack of international legislative controls for non-listed pathogens and inherent difficulties with monitoring disease introductions in wild fish populations. Here we used one of the world's most invasive freshwater fish, the topmouth gudgeon, Pseudorasbora parva, to demonstrate the risk posed by an emergent generalist parasite, Sphaerothecum destruens. Pseudorasbora parva has spread to 32 countries from its native range in China through the aquaculture trade and has introduced S. destruens to at least five of these. We systematically investigated the spread of S. destruens through Great Britain and its establishment in native fish communities through a combination of phylogenetic studies of the host and parasite and a novel environmental DNA detection assay. Molecular approaches confirmed that S. destruens is present in 50% of the P. parva communities tested and was also detected in resident native fish communities but in the absence of notable histopathological changes. We identified specific P. parva haplotypes associated with S. destruens and evaluated the risk of disease emergence from this cryptic fish parasite. We provide a framework that can be applied to any aquatic pathogen to enhance detection and help mitigate future disease risks in wild fish populations.

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.

Effect of Ichthyophonus on blood plasma chemistry of spawning Chinook salmon and their resulting offspring in a Yukon River tributary.

Ichthyophonus is a protozoan parasite of Alaska Chinook salmon Oncorhynchus tshawytscha. In this study, we determined whether spawning Chinook salmon in the Yukon River drainage exhibited a measurable stress response (i.e. elevated plasma cortisol concentrations) and detectable changes in selected blood plasma chemistry parameters when infected with Ichthyophonus. The resulting alevin were also analyzed for any differences in blood plasma chemistry caused by parental infection with Ichthyophonus. In 2010, 2011, and 2012, spawning adult Chinook salmon were collected from the Salcha River, Alaska, USA, and the prevalence of Ichthyophonus in these fish was 7.8, 6.3, and 8.3%, respectively. Fish with no clinical signs of Ichthyophonus and Ichthyophonus-positive parents were cross-fertilized to investigate potential second-generation effects as a result of Ichthyophonus infection. We found no significant difference in cortisol concentrations in blood plasma between Ichthyophonus-positive and -negative adults or between alevin from Ichthyophonus-positive and -negative parents. There were no significant differences in blood plasma parameters (e.g. alanine aminotransferase, creatine kinase, glucose) of Ichthyophonus-negative and -positive adults, with the exception of aspartate aminotransferase, which was significantly higher in plasma of Ichthyophonus-negative adults. All clinical chemistry parameters for alevin resulting from both Ichthyophonus-negative and -positive parents were not significantly different. Based on this study, which has a limited sample size and low prevalence of Ichthyophonus, offspring of Chinook salmon appear to suffer no disadvantage as a result of Ichthyophonus infection in their parents on the Salcha River.

Pathological and phylogenetic characterization of Amphibiothecum sp. infection in an isolated amphibian (Lissotriton helveticus) population on the island of Rum (Scotland).

Outbreaks of cutaneous infectious disease in amphibians are increasingly being attributed to an overlooked group of fungal-like pathogens, the Dermocystids. During the last 10 years on the Isle of Rum, Scotland, palmate newts (Lissotriton helveticus) have been reportedly afflicted by unusual skin lesions. Here we present pathological and molecular findings confirming that the pathogen associated with these lesions is a novel organism of the order Dermocystida, and represents the first formally reported, and potentially lethal, case of amphibian Dermocystid infection in the UK. Whilst the gross pathology and the parasite cyst morphology were synonymous to those described in a study from infected L. helveticus in France, we observed a more extreme clinical outcome on Rum involving severe subcutaneous oedema. Phylogenetic topologies supported synonymy between Dermocystid sequences from Rum and France and as well as their distinction from Amphibiocystidium spp. Phylogenetic analysis also suggested that the amphibian-infecting Dermocystids are not monophyletic. We conclude that the L. helveticus-infecting pathogen represents a single, novel species; Amphibiothecum meredithae.

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.

The consequences of reservoir host eradication on disease epidemiology in animal communities.

Non-native species have often been linked with introduction of novel pathogens that spill over into native communities, and the amplification of the prevalence of native parasites. In the case of introduced generalist pathogens, their disease epidemiology in the extant communities remains poorly understood. Here, Sphaerothecum destruens, a generalist fungal-like fish pathogen with bi-modal transmission (direct and environmental) was used to characterise the biological drivers responsible for disease emergence in temperate fish communities. A range of biotic factors relating to both the pathogen and the surrounding host communities were used in a novel susceptible-exposed-infectious-recovered (SEIR) model to test how these factors affected disease epidemiology. These included: (i) pathogen prevalence in an introduced reservoir host (Pseudorasbora parva); (ii) the impact of reservoir host eradication and its timing and (iii) the density of potential hosts in surrounding communities and their connectedness. These were modelled across 23 combinations and indicated that the spill-over of pathogen propagules via environmental transmission resulted in rapid establishment in adjacent fish communities (<1 year). Although disease dynamics were initially driven by environmental transmission in these communities, once sufficient numbers of native hosts were infected, the disease dynamics were driven by intra-species transmission. Subsequent eradication of the introduced host, irrespective of its timing (after one, two or three years), had limited impact on the long-term disease dynamics among local fish communities. These outputs reinforced the importance of rapid detection and eradication of non-native species, in particular when such species are identified as healthy reservoirs of a generalist pathogen.

Associated disease risk from the introduced generalist pathogen Sphaerothecum destruens: management and policy implications.

The rosette agent Sphaerothecum destruens is a novel pathogen, which is currently believed to have been introduced into Europe along with the introduction of the invasive fish topmouth gudgeon Pseudorasbora parva (Temminck & Schlegel, 1846). Its close association with P. parva and its wide host species range and associated host mortalities, highlight this parasite as a potential source of disease emergence in European fish species. Here, using a meta-analysis of the reported S. destruens prevalence across all reported susceptible hosts species; we calculated host-specificity providing support that S. destruens is a true generalist. We have applied all the available information on S. destruens and host-range to an established framework for risk-assessing non-native parasites to evaluate the risks posed by S. destruens and discuss the next steps to manage and prevent disease emergence of this generalist parasite.

Infected Donor Biomass and Active Feeding Increase Waterborne Transmission of Ichthyophonus sp. to Rainbow Trout Sentinels.

The precise nature of Ichthyophonus sp. transmission among wild fishes has eluded description for over a century. Transmission among piscivores is direct, via ingestion of infected prey, but there is also evidence for waterborne transmission between infected and uninfected individuals. Transmission among planktivores is believed to be via a waterborne infectious cell, but definitive proof of this mechanism has not been forthcoming. To explore possible mechanisms of transmission we used Rainbow Trout Oncorhynchus mykiss as a model system and examined the consequence of housing infected donor fish with uninfected (sentinel) fish, without physical contact. We examined two variables linked to transmission: (1) feeding and nonfeeding sentinel fish, and (2) biomass of infected donor fish. Specific-pathogen free sentinel trout were placed in fine-mesh baskets suspended in tanks containing varying numbers of larger Ichthyophonus-infected donor fish and held for 10 weeks, during which time they were examined by in vitro explant culture for the presence of Ichthyophonus. Treatment groups consisted of fed and unfed sentinels housed with infected donors of increasing biomass. After 10 weeks infection prevalence in fed sentinels was significantly higher than in unfed sentinels, and Ichthyophonus was detected earlier in fed fish than in unfed fish. There was no correlation between infection prevalence and donor biomass in fed sentinels, but there was a strong correlation between infection prevalence and increasing donor biomass in unfed sentinels. These data suggest that Ichthyophonus is maintained in wild fish populations by two distinct mechanisms: (1) waterborne infectious cells ingested directly from the water by planktivores, and (2) both infected prey and waterborne infectious cells ingested by piscivores. Received November 13, 2015; accepted February 13, 2016.

The parasite Ichthyophonus sp. in Pacific herring from the coastal NE Pacific.

The protistan parasite Ichthyophonus occurred in populations of Pacific herring Clupea pallasii Valenciennes throughout coastal areas of the NE Pacific, ranging from Puget Sound, WA north to the Gulf of Alaska, AK. Infection prevalence in local Pacific herring stocks varied seasonally and annually, and a general pattern of increasing prevalence with host size and/or age persisted throughout the NE Pacific. An exception to this zoographic pattern occurred among a group of juvenile, age 1+ year Pacific herring from Cordova Harbor, AK in June 2010, which demonstrated an unusually high infection prevalence of 35%. Reasons for this anomaly were hypothesized to involve anthropogenic influences that resulted in locally elevated infection pressures. Interannual declines in infection prevalence from some populations (e.g. Lower Cook Inlet, AK; from 20-32% in 2007 to 0-3% during 2009-13) or from the largest size cohorts of other populations (e.g. Sitka Sound, AK; from 62.5% in 2007 to 19.6% in 2013) were likely a reflection of selective mortality among the infected cohorts. All available information for Ichthyophonus in the NE Pacific, including broad geographic range, low host specificity and presence in archived Pacific herring tissue samples dating to the 1980s, indicate a long-standing host-pathogen relationship.

Persistence of external signs in Pacific herring Clupea pallasii Valenciennes with ichthyophoniasis.

The progression of external signs of Ichthyophonus infection in Pacific herring Clupea pallasii Valenciennes was highly variable and asynchronous after intraperitoneal injection with pure parasite preparations; however, external signs generally persisted through the end of the study (429 days post-exposure). Observed signs included papules, erosions and ulcers. The prevalence of external signs plateaued 35 days post-exposure and persisted in 73-79% of exposed individuals through the end of the first experiment (147 days post-exposure). Among a second group of infected herring, external signs completely resolved in only 10% of the fish after 429 days. The onset of mortality preceded the appearance of external signs. Histological examination of infected skin and skeletal muscle tissues indicated an apparent affinity of the parasite for host red muscle. Host responses consisted primarily of granulomatous inflammation, fibrosis and necrosis in the skeletal muscle and other tissues. The persistence and asynchrony of external signs and host response indicated that they were neither a precursor to host mortality nor did they provide reliable metrics for hindcasting on the date of exposure. However, the long-term persistence of clinical signs in Pacific herring may be useful in ascertaining the population-level impacts of ichthyophoniasis in regularly observed populations.

Infecting Pacific Herring with Ichthyophonus sp. in the Laboratory.

The protistan parasite Ichthyophonus sp. occurs in coastal populations of Pacific Herring Clupea pallasii throughout the northeast Pacific region, but the route(s) by which these planktivorous fish become infected is unknown. Several methods for establishing Ichthyophonus infections in laboratory challenges were examined. Infections were most effectively established after intraperitoneal (IP) injections with suspended parasite isolates from culture or after repeated feedings with infected fish tissues. Among groups that were offered the infected tissues, infection prevalence was greater after multiple feedings (65%) than after a single feeding (5%). Additionally, among groups that were exposed to parasite suspensions prepared from culture isolates, infection prevalence was greater after exposure by IP injection (74%) than after exposure via gastric intubation (12%); the flushing of parasite suspensions over the gills did not lead to infections in any of the experimental fish. Although the consumption of infected fish tissues is unlikely to be the primary route of Ichthyophonus sp. transmission in wild populations of Pacific Herring, this route may contribute to abnormally high infection prevalence in areas where juveniles have access to infected offal.