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.

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.

The alternate role of direct and environmental transmission in fungal infectious disease in wildlife: threats for biodiversity conservation.

Emerging fungal pathogens have substantial consequences for infected hosts, as revealed by the global decline of amphibian species from the chytrid fungus. According to the "curse of the Pharaoh" hypothesis, free-living infectious stages typical of fungal pathogens lengthen the timespan of transmission. Free-living infectious stages whose lifespan exceeds the infection time of their hosts are not constrained by virulence, enabling them to persist at high levels and continue transmitting to further sensitive hosts. Using the only Mesomycetozoea fungal species that can be cultured, Sphaerothecum destruens, we obtained tractable data on infectivity and pathogen life cycle for the first time. Here, based on the outcomes of a set of infectious trials and combined with an epidemiological model, we show a high level of dependence on direct transmission in crowded, confined environments and establish that incubation rate and length of infection dictate the epidemic dynamics of fungal disease. The spread of Mesomycetozoea in the wild raise ecological concerns for a range of susceptible species including birds, amphibians and mammals. Our results shed light on the risks associated with farming conditions and highlight the additional risk posed by invasive species that are highly abundant and can act as infectious reservoir hosts.

Impact of Ichthyophonus infection on spawning success of Yukon River Chinook salmon Oncorhynchus tshawytscha.

We examined the impacts of Ichthyophonus infection on spawning success of Yukon River Chinook salmon Oncorhynchus tshawytscha at spawning grounds of the Chena and Salcha Rivers, Alaska, USA. During the period 2005 to 2006, 1281 salmon carcasses (628 male, 652 female) were collected throughout the spawning season and from the entire spawning reaches of the Chena and Salcha Rivers. For each fish, infection status was determined by culture method and visual inspection of lesions of heart tissue as uninfected (culture negative), infected without lesions (culture positive with no visible lesions), and infected with lesions (culture positive with visible lesions), and spawning status was determined by visually inspecting the percentage of gametes remaining as full-spawned (<10%), partial-spawned (10-50%), and unspawned (>50%). Among the 3 groups, the proportion of full-spawned (i.e. spawning success) females was lower for those infected without lesions (69%) than those uninfected (87%) and infected with lesions (86%), but this did not apply to males (uninfected 42%, infected without lesions 38%, infected with lesions 41%). At the population level, the combined (infected and uninfected) proportion of female spawning success was 86%, compared to 87% when all females were assumed uninfected. These data suggest that while Ichthyophonus infection slightly reduces spawning success of infected females, its impact on the spawning population as a whole appears minimal.

Sphaerothecum destruens pathology in cyprinids.

Sphaerothecum destruens is a significant intracellular parasite of fish which has caused disease and mortalities in cultured north American Chinook salmon Oncorhynchus tshawytscha and Atlantic salmon Salmo salar. Several hosts for S. destruens have been identified within the Salmonidae family, and the histopathology of the infection can differ between hosts. Recently, S. destruens has been associated with the most invasive cyprinid species in Europe, topmouth gudgeon Pseudorasbora parva. Accurate disease identification based on thorough descriptions of clinical signs and histopathology in this new range of hosts is thus paramount to support further epizootiological studies. In this study, the associated histopathology of S. destruens infection is described along with its pathogenesis in the endangered cyprinid sunbleak Leucaspius delineatus. Histological examination of 100 L. delineatus in a wild population in the south of England revealed the presence of S. destruens infections, with a prevalence of 5% with S. destruens, suggesting an over-dispersed distribution within the L. delineatus sample. Clinical signs of the infection were absent, but histological examination revealed the presence of both disseminated and nodular lesions in several organs.

Dermocystid infection and associated skin lesions in free-living palmate newts (Lissotriton helveticus) from Southern France.

Since the early 1900s, mesomycetozoan parasites have been reported in both European anuran and caudate species. These reports have primarily been descriptive, which has made assessing the impact of these parasites on host populations difficult. Anecdotal reports of dermocystidium-like parasites are becoming widespread across Europe, possibly indicating that these mesomycetozoan parasites are increasing in distribution and/or abundance. This highlights the need for further investigations into the occurrence, pathogenesis and effects on host health of these parasitic infections for free-living amphibian populations, particularly those which are already stressed or threatened by other factors. Here we report the results of pathological, microbiological and molecular investigations used to characterize unidentified skin lesions in palmate newts (Lissotriton helveticus) from Larzac, France. We confirm that the lesions are the result of infection with a novel dermocystidium-like parasite, which is related to Amphybiocystidium ranae. We also show that the same parasite is distributed across several newt breeding sites. The lesions that result from infection with this parasite range from single or few vesicular or nodular cutaneous lesions to multiple coalescing skin ulcers with extensive hemorrhages. The latter have not been previously described in amphibians due to mesomycetozoan parasitic infection. Dermocystid DNA was detected only in newts that showed lesions, providing comparative evidence of the parasite's pathogenicity. We discuss the potential significance of the presence of this pathogen in the context of the population health of palmate newts.

Release of infectious cells from epidermal ulcers in Ichthyophonus sp.-infected Pacific herring (Clupea pallasii): evidence for multiple mechanisms of transmission.

A common clinical sign of ichthyophoniasis in herring and trout is "sandpaper" skin, a roughening of the epidermis characterized by the appearance of small papules, followed by ulceration and sloughing of the epithelium; early investigators hypothesized that these ulcers might be a means of transmitting the parasite, Ichthyophonus sp., without the necessity of ingesting an infected host. We examined the cells associated with the epidermal lesions and confirmed that they were viable Ichthyophonus sp. cells that were readily released from the skin into the mucous layer and ultimately into the aquatic environment. The released cells were infectious when injected into the body cavity of specific-pathogen-free herring. Our hypothesis is that different mechanisms of transmission occur in carnivorous and planktivorous hosts: Planktonic feeders become infected by ingestion of ulcer-derived cells, while carnivores become infected by ingestion of whole infected fish.

Infection of spotted salamanders (Ambystoma maculatum) with Ichthyophonus-like organisms in Virginia.

Ichthyophonus-like organisms were found in two free-ranging adult spotted salamanders (Ambystoma maculatum) captured within two different vernal ponds in the Virginia Commonwealth University Rice Center for Environmental Life Sciences in Charles City County, Virginia. Histopathologic examination of necropsied specimens revealed large spores, often enclosed by granulomas. These enclosed spores resembled those caused by the fish pathogen Ichthyophonus hoeferi. One salamander displayed an externally visible large swelling beneath the jaws. The other lacked macroscopic abnormalities, but histologic sections of ventral muscle revealed early-stage Ichthyophonus-like organisms and minimal granulomatous reactions. This is the first report of Ichthyophonus-like infection of Ambystoma maculatum in Virginia.

In vitro leukocyte-encapsulation model in rainbow trout (Oncorhynchus mykiss).

We developed an in vitro model to study the cellular and molecular mechanisms of granulomatous inflammation in response to invading pathogens. Ichthyophonus hoferi was used as a target for encapsulation by cultivated leukocytes from the kidney of the rainbow trout (Oncorhynchus mykiss). The encapsulation process was observed over 1 week. The leukocytes were identified as either macrophages in the inner layer, or neutrophils and lymphocytes in the outer layer. The encapsulation response was inhibited by treatment with heat, but not formalin or methanol. The recognition of heat-unstable molecules on the pathogen surface could induce encapsulation. Increased expression of pro-inflammatory cytokines, such as interleukin (IL)-1beta, IL-8 and tumor necrosis factor-alpha2, was observed during encapsulation. These cytokines might play crucial roles in the encapsulation process. In particular, IL-8, which was expressed at a late phase, might recruit specific cell populations, such as the lymphocytes comprising the outer cellular layer around the target.

Widespread infection of the Eastern red-spotted newt (Notophthalmus viridescens) by a new species of Amphibiocystidium, a genus of fungus-like mesomycetozoan parasites not previously reported in North America.

Given the worldwide decline of amphibian populations due to emerging infectious diseases, it is imperative that we identify and address the causative agents. Many of the pathogens recently implicated in amphibian mortality and morbidity have been fungal or members of a poorly understood group of fungus-like protists, the mesomycetozoans. One mesomycetozoan, Amphibiocystidium ranae, is known to infect several European amphibian species and was associated with a recent decline of frogs in Italy. Here we present the first report of an Amphibiocystidium sp. in a North American amphibian, the Eastern red-spotted newt (Notophthalmus viridescens), and characterize it as the new species A. viridescens in the order Dermocystida based on morphological, geographical and phylogenetic evidence. We also describe the widespread and seasonal distribution of this parasite in red-spotted newt populations and provide evidence of mortality due to infection.