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.

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.

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.

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.

Proposed changes to the nomenclature of Ichthyophonus sp. life stages and structures.

Much of the terminology describing Ichthyophonus sp. life stages and structures can be traced to the mistaken classification of this organism as a fungus. This misidentification led early investigators to use mycological terms for the structures they observed; while some terminology is not so easily explained, it appears to have been co-opted from the fields of botany and bacteriology. The purpose of this exercise is to attempt to standardize the terminology associated with Ichthyophonus and to bring it into agreement with terminology currently used to define similar life stages of other protists. The proposed changes are (1) spore/macrospore/mother spore to "schizont," (2) microspore/endospore to "merozoite," and (3) pseudohyphae to "hyphae" or "germ tube."

Reproductive and transgenerational effects of methylmercury or Aroclor 1268 on Fundulus heteroclitus.

This research determined the potential for methylmercury or Aroclor 1268 to disrupt reproduction and sexual differentiation in Fundulus heteroclitus. The research determined whether fish that are exposed to mercury or Aroclor 1268 survive and successfully reproduce; whether offspring of exposed fish hatch, survive, produce eggs, and fertilize them; and whether the second-generation offspring of exposed fish hatch and survive. Fundulus heteroclitus were exposed to mercury or Aroclor 1268 via contaminated food. Endpoints evaluated included survival, growth, fecundity, fertilization success, hatch success, larval survival, sex ratios, and the prevalence of gonadal abnormalities. In general, polychlorinated biphenyls were highly bioavailable and accumulated well through feeding. The only statistically significant effect observed as a result of treatment with Aroclor 1268 was an increase in growth in the offspring of exposed fish. Mercury was accumulated in a dose-dependent fashion via food exposures. Exposure to mercury in food increased mortality in male F. heteroclitus, which possibly occurred as a result of behavioral alterations. Increased mortality was observed at body burdens of 0.2 to 0.47 microgram/g. Offspring of F. heteroclitus fed mercury-contaminated food were less able to successfully reproduce, with reduced fertilization success observed at egg concentrations of 0.01 to 0.63 microgram/g, which corresponds with parent whole-body concentrations of 1.1 to 1.2 micrograms/g. Offspring of exposed fish also had altered sex ratios, with treatment at moderate concentrations producing fewer females and treatment at the highest concentration producing more females than expected. Alterations in sex ratios were observed at concentrations of less than 0.01 microgram/g in eggs or between 0.44 and 1.1 micrograms/g in parents. Offspring of mercury-exposed fish also had increased growth in moderate treatments, when egg concentrations were less than 0.02 microgram/g, or when parent whole bodies contained 0.2 to 0.47 microgram/g. In summary, exposure to mercury reduced male survival, reduced the ability of offspring to successfully reproduce, and altered sex ratios in offspring. Both direct effects on exposed fish and transgenerational effects were observed.

Survival of the North American strain of viral hemorrhagic septicemia virus (VHSV) in filtered seawater and seawater containing ovarian fluid, crude oil and serum-enriched culture medium.

The North American strain of viral hemorrhagic septicemia virus (NA-VHSV) could be recovered for up to 40 h in natural filtered seawater (27 ppt) with a 50% loss of infectivity after approximately 10 h at 15 degrees C. Addition of 10 ppb North Slope crude oil to the seawater had no effect on virus survival. However, when various concentrations of teleost ovarian fluid were added to seawater, virus could be recovered after 72 h at 0.01% ovarian fluid and after 96 h at 1.0%. When cell culture medium supplemented with 10% fetal bovine serum was added to the seawater, 100% of the virus could be recovered for the first 15 d and 60% of the virus remained after 36 d. These findings quantify NA-VHSV infectivity in natural seawater and demonstrate that ovarian fluid, which occurs naturally during spawning events, significantly prolongs the survival and infectivity of the virus. The extended stabilization of virus in culture medium supplemented with serum allows for low titer field samples to be collected and transported in an unfrozen state without significant loss of virus titer.

Epizootiology of viral hemorrhagic septicemia virus in Pacific herring from the spawn-on-kelp fishery in Prince William Sound, Alaska, USA.

Both the prevalence and tissue titer of viral hemorrhagic septicemia virus (VHSV) increased in Pacific herring Clupea pallasi following their introduction into net pens (pounds) used in the closed pound spawn-on-kelp (SOK) fishery in Prince William Sound, Alaska. VHSV was also found in water samples from inside and outside the SOK pounds after herring had been confined for several days; however, water samples taken near wild free-ranging, spawning herring either failed to test positive or tested weakly positive for virus. Little or no virus was found in tissue samples from free-ranging, spawning herring captured from the vicinity of the pounds, nor did the prevalence of VHSV increase following spawning as it did in impounded herring. The data indicated that increased prevalences of VHSV were correlated with confinement of herring for the closed pound SOK fishery and that infection was spread within the pounds through waterborne exposure to virus particles originating from impounded fish. In addition, pounds containing predominantly young fish had higher prevalences of VHSV, suggesting that older fish may be partially immune, perhaps as a result of previous infection with the virus. Operation of SOK pounds during spawning seasons in which young herring predominate may amplify the disease and possibly exacerbate the population fluctuations observed in wild herring stocks.

Pathogenicity of Ichthyophonus hoferi for laboratory-reared Pacific herring Clupea pallasi and its early appearance in wild Puget Sound herring.

Laboratory-reared pathogen-free Pacific herring were exposed to pure cultures of Ichthyophonus hoferi, and reproduced the disease seen in naturally infected fish--thus fulfilling Koch's Postulates. Pathogen-free herring used in this study were reared from artificially spawned eggs incubated in filtered, UV-sterilized seawater, eliminating the variables associated with multiple infections, which are common in wild herring. Wild free-ranging herring were captured monthly from June through October by dip net from 'herring balls' located in the northern Puget Sound. I. hoferi infections were identified in these fish soon after metamorphoses, about 4 mo post-hatch. The prevalence increased from 5 to 6% in 0-yr fish to 24% in 1-yr-old fish to 50 to 70% in fish over 2 yr old, with no associated increase in mortality. The route of natural transmission to wild herring was not determined, but carnivorous fish became infected and died when they were experimentally fed tissues infected with the organism. In vitro culture of tissues was the most sensitive method for identifying both clinical and subclinical infections.

Toxicity of weathered coal tar for shortnose sturgeon (Acipenser brevirostrum) embryos and larvae.

Weathered coal tar collected from the Connecticut River near Holyoke, Massachusetts, was toxic to shortnose sturgeon embryos and larvae in whole sediment flow-through and elutriate static-renewal laboratory exposures. Sterile laboratory sand and clean Connecticut River sand, collected upstream from the coal tar deposits, produced no significant difference in toxicity to sturgeon embryos-larvae, while coal tar-contaminated sediment produced over 95% embryo-larval mortality. Hydrocarbon transfer and subsequent toxicity appeared to be via direct contact of the embryos with contaminated sediment, rather than via exposure to soluble hydrocarbons. This conclusion was supported by exposure of embryos and larvae to elutriates (e.g., water soluble extract) of coal-tar sediments, that resulted in embryo and larval mortality at low molecular weight PAH concentrations-0.47 mg/L, higher than would occur naturally. No decrease in petroleum hydrocarbon concentration was observed in sediments exposed to flowing water for 14 d, supporting the contention that soluble hydrocarbons were not responsible for the observed toxicity in whole sediment exposures under the conditions employed in this study.

Steroidal alkaloid toxicity to fish embryos.

Embryos of two species of fish were evaluated for their suitability as model systems for steroidal alkaloid toxicity, the Japanese rice fish, medaka (Oryzius latipes) and the rainbow trout (Oncorhynchus mykiss). Additionally, the equine neurotoxic sesquiterpene lactone repin, was also tested. A PROBIT program was used to evaluate the EC1, EC50 and EC99 as well as the associated confidence limits. The steroidal alkaloids tested were the Solanum potato glycoalkaloids alpha-chaconine, alpha-solanine, the aglyclones solanidine and solasodine and the Veratrum alkaloid, jervine. Embryo mortality, likely due to structural or functional abnormalities in the early development stages of the embryo, were the only response observed in both species. The rainbow trout exhibited a toxic response to chaconine, solasidine, repin and solanine but the medaka embryos were only affected by the compounds, chaconine and solanine. Rainbow trout may indeed serve as a good lower vertebrate model for studying the toxicity of steroidal alkaloids.

Sister chromatid exchange analysis in cultured peripheral blood leukocytes of the coldwater marine fish, Pacific staghorn sculpin (Leptocottus armatus): a feasible system for assessing genotoxic marine pollutants.

The genotoxicity of environmental contaminants and test compounds to aquatic and marine fish has primarily been assessed by in vivo techniques that require sacrifice of the test organism for analysis. The major objective of this research was to develop an in vitro sister chromatid exchange (SCE) assay which would utilize cultured peripheral blood leukocytes (PBLs) of a coldwater marine fish species. Use of PBLs in cytogenetic genotoxicity tests has several advantages, the major one being that the experimental fish need not be sacrificed for sample collection. In addition, this nondestructive method of tissue collection permits the investigator to take multiple samples from a single individual and thereby allows the use of an individual as its own control and to monitor its SCE frequency over time. A suitable in vitro culture method for fish PBLs was a prerequisite for cytogenetic analysis of this tissue. The in vitro culture conditions necessary to provide a sufficient number of dividing cells for performance of the SCE assay were established in our laboratory for the PBLs of the Pacific staghorn sculpin (Leptocottus armatus), a common bottom-dwelling Puget Sound fish. The major components of this culture system are heparinized whole blood, fetal bovine serum-supplemented enriched tissue culture medium (RPMI 1640), purified protein derivative of tuberculin as a mitogen, and an incubation temperature of 13.5 degrees C. This in vitro PBL culture system is unique because it involves cultured blood cells from a coldwater marine fish species. Using this culture method, SCE induction was investigated in Pacific staghorn sculpin PBLs which had been exposed in vitro to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a known direct-acting inducer of SCEs. Cultured cells exposed in vitro responded to MNNG in a dose-related manner in regard to SCE induction, and the frequency of "outlier" cells increased at the higher concentrations of MNNG. With further development, this technique may be adaptable for use with in vivo genotoxicity studies and provide information concerning the induction and persistence of chemically induced SCEs in fish. This PBL/SCE assay may also be a feasible assessment tool for detecting exposure of marine fish to genotoxic environmental contaminants in laboratory and field situations.

Sequestration and release of polycyclic aromatic hydrocarbons by vertebrate cells in vitro.

Vertebrate fibroblasts grown in vitro and exposed to various concentrations of the mutagen/carcinogen benzo(a)pyrene (B(a)P) internalized the compound and recrystallized it in lysosomes by 6-18 h postexposure. This phenomenon occurred when B(a)P at or above 10 micrograms/ml was introduced to the culture medium in a solvent such as DMSO or acetone but not when introduced dissolved in serum. Likewise, high fetal bovine serum concentrations in the culture medium (greater than 20%) as well as human serum (10%) inhibited crystal formation, presumably owing to lipid competition for the compound. Electron-microscopic observations of the cells during the uptake and crystal forming periods revealed that the cell membrane became altered within 3 h of exposure to B(a)P. This was followed by a return to normal of the membrane and the appearance of vesicles within the cell by 6-8 h. The vesicles then became filled with crystals which continued to grow while in the presence of B(a)P and disappeared when the cells were exposed to B(a)P-free culture medium or serum lipids. Introduction of B(a)P into culture medium containing delipidated serum resulted in crystal formation indistinguishable from that which occurred when whole serum was present. Crystals were not formed when total lipoprotein and the lipoprotein components VLDL, LDL, HDL2, and HDL3 were used as solvents for B(a)P. The process of crystal formation was inhibited by the addition of 10(-3) M KCN, and removal of the crystals was dependent on the concentration of lipoprotein in the culture medium.

Anaphase aberrations: a measure of genotoxicity in mutagen-treated fish cells.

Rainbow trout gonad cells (RTG-2) were cultured for various lengths of time in the presence of several classes of known mutagenic chemicals and several related compounds that possessed no known mutagenic/carcinogenic activity. During the course of exposure the cells were examined for the presence of abnormalities in the chromosome arrangement of anaphase figures during mitosis. Untreated and solvent-treated (dimethylsulfoxide-treated) cells exhibited a background abnormality rate of 12% with only minor chromosomal defects being observed. This was also true for those cells exposed to naphthol and anthracene, two chemicals with no proven mutagenic or carcinogenic activity. Conversely, significant increases in the frequency of anaphase aberrations were produced in cells treated with N-methyl-N'-nitro-N-nitrosoguanidine, benzo(a)pyrene, 9-aminoacridine and mitomycin-C. These abnormalities were also far more complex and extensive than those observed in the control and nonmutagen-treated cells. Many species of fish have extremely small and numerous chromosomes, making resolution of chromosome defects such as sister chromatid exchange and deletions more difficult than in most mammalian diploid cells, which generally have larger and fewer chromosomes. Examination of cells during anaphase eliminates the need to observe each chromosome separately as well as the need to produce well-spread metaphase chromosomes. Since the sensitivity of anaphase aberrations to known mutagenic/carcinogenic compounds appears to be quite high in trout cells and since hundreds of suitable cells are available for analysis, this may be an appropriate alternative or addition to some of the more standard chromosome macrolesion tests developed in mammalian systems.

In vitro effect of some mutagens/carcinogens on cultured fish cells.

Fibroblast cells derived from juvenile bluegill sunfish were tested for their response the effects of known mutagens/carcinogens. The cells grew well and cloned in Eagle's minimal essential medium which contained fetal bovine serum. Growth rate was temperature dependent and increased steadily as temperature was raised from 15 to 33 degrees C. Direct mutagens and promutagens were toxic to the cells, and fluorescence spectroscopy revealed the capability of metabolizing at least one promutagen, benzo(a)pyrene, to water-soluble intermediates. Ouabain resistant mutants were produced by exposing the cells to N-methyl-N-nitro-nitrosoguanidine (MNNG) or to benzo(a)pyrene (B(a)P). The mutant clones were resistant to 100-fold increases in concentration of the selective agent ouabain, over that which was lethal to wild type cells. Spontaneous mutation frequency to ouabain in mass cultures averaged 1.2 X 10(-6) for all experiments. A fluctuation test confirmed an expected random occurrence of spontaneous mutations. Visible crystals formed within the cells when high concentrations of B(a)P (greater than 5 micrograms/ml were added to the tissue culture medium. Crystal formation resulted in no apparent cell damage but did reduce the growth rate of the cultures. The crystals were rhombic, resembling those described for pure native B(a)P, and gradually disappeared when the cells were exposed to mutagen-free medium which contained serum.

Duck plague virus replication in muscovy duck fibroblast cells.

Duck embryo fibroblast cell cultures from seven species of ducks were compared for virus yield, plaque quality, and sensitivity to infection by the duck plague herpesvirus (duck virus enteritis). Muscovy duck and wood duck cells gave the best results for virus yield and plaque quality, but muscovies were considered superior because they are more available than wood ducks. Pintails and lesser scaup gave the poorest results, and pekin duck, black duck, and redhead duck were intermediate. A growth curve for the virus, determined in muscovy cells, had a latent period of six hours and a maximum new virus titer reached at 36 hours. Because of their superior plaque production and ability to replicate the virus, muscovy embryo fibroblasts (MCE) are recommended for diagnostic and research work.