Persistence of Piscirickettsia salmonis and detection of serum antibodies to the bacterium in white seabass Atractoscion nobilis following experimental exposure.

White seabass Atractoscion nobilis surviving experimental exposure to Piscirickettsia salmonis harbored the bacterium for periods up to at least 123 d post injection (dpi). Intraperitoneal injections of juvenile white seabass with 1.26 x 10(2) TCID50 P. salmonis fish(-1) resulted in a 29% cumulative mortality over a 27 d period. Both molecular and histologic methods provided evidence for persistence of the bacterium in fish sampled sequentially from the surviving population. Throughout the period of acute mortality, the bacterium was detected in all impression smears of liver tissue stained with Giemsa and was reisolated in cell cultures from all dead fish sampled. Polymerase chain reaction (PCR) assays detected P. salmonis-specific DNA in 13.3 to 50% of the fish sampled at time points between 28 and 123 dpi, while cell culture reisolation was largely ineffective in detecting the bacterium. An enzyme-linked immunosorbent assay (ELISA) detected serum anti-P. salmonis antibodies in 48 of 59 white seabass exposed to P. salmonis but not in fish which were not exposed to the bacterium. At the end of the 4 mo experiment, microscopic lesions consisting of single to multiple and coalescing granulomas were found in liver and kidney tissues of 9 of 10 fish examined from the exposure group, while no lesions were detected in the fish from the control group. Immunohistochemical staining with anti-P. salmonis polyclonal antibodies detected bacterial antigens in some but not all granulomas examined from the exposure group at 4 mo. This study demonstrates that P. salmonis may persist among white seabass following infection, and thus provide a potential reservoir of infection contributing to transmission both within and between fish species in the marine environment.

Effects of temperature on infectivity and of commercial freezing on survival of the North American strain of viral hemorrhagic septicemia virus (VHSV).

Temperature affected the growth of the North American strain of viral hemorrhagic septicemia virus (VHSV) in experimentally infected cell cultures and in Pacific sardine Sardinops sagax. In addition, commercial freezing significantly reduced the infectivity of VHSV in tissues of experimentally infected sardine. Isolates of VHSV representing the geographic range of North American VHSV replicated in the EPC (Epithelioma papulosum cyprini) cell line at 10, 15 and 20 degrees C, but the more northern isolates from British Columbia, Canada, demonstrated significantly reduced growth at 20 degrees C compared to VHSV from more southern locations (p <0.001). An injection challenge of Pacific sardine with VHSV from California resulted in 66.7% mortality at a seawater temperature of 13 degrees C compared to 6.7% at 20 degrees C. Commercial blast-freezing of sardine experimentally infected with VHSV reduced median concentrations of virus in the kidney and spleen from 5.25 x 10(6) to 5.5 x 10(3) pfu (plaque-forming units) g(-1). Decreased growth of the California isolate of VHSV at higher temperatures following experimental infection of the sardine and reduced virus survival following commercial freezing of infected sardine are factors that would lessen the risk of transmission of VHSV through frozen baitfishes.

Genetic characterization and experimental pathogenesis of Piscirickettsia salmonis isolated from white seabass Atractoscion nobilis.

An intracellular bacterium originally isolated from hatchery-reared juvenile white seabass Atractoscion nobilis in southern California, USA, was identified by sequences of the small and large subunit ribosomal (16S and 23S) DNA and the internal transcribed spacer (ITS) as Piscirickettsia salmonis. Considering all rDNA sequences compared, the white seabass isolate (WSB-98) had a 96.3 to 98.7% homology with 4 previously described strains of P. salmonis isolated from salmon in Chile, Norway, and British Columbia, Canada. Experimental infections induced by intraperitoneal injections of juvenile white seabass with WSB-98 resulted in disease and mortality similar to that observed in P. salmonis infections in salmon. After 60 d, the cumulative mortality among P. salmonis-injected white seabass was 82 and 40%, respectively, following a high (1.99 x 10(4) TCID50) or low (3.98 x 10(2) TCID50) dose-challenge with WSB-98. The bacterium was recovered by isolation in cell culture or was observed in stains from tissues of injected white seabass but not from control fish. There were no external signs of infection. Internally, the most common gross lesion was a mottled appearance of the liver, sometimes with distinct nodules. Microscopic lesions were evident in both the capsule and parenchyma of the liver and were characterized by multifocal necrosis, often with infiltration of mononuclear leukocytes. Macrophages filled with bacteria were present at tissue sites exhibiting focal necrosis. Foreign body-type granulomas were prevalent in livers of experimentally infected white seabass, but not in control fish. Similar granulomatous lesions were observed in the spleen, kidney, intestine and gills, but these organs were considered secondary sites of infection, with significantly fewer and less severe histologic lesions compared to the liver. The results from this study clearly indicate that infections with P. salmonis are not restricted to salmonid fishes and that the bacterium can cause a disease similar to piscirickettsiosis in nonsalmonid hosts.

Molecular and bioassay-based detection of Toxoplasma gondii oocyst uptake by mussels (Mytilus galloprovincialis).

Toxoplasma gondii is associated with morbidity and mortality in a variety of marine mammals, including fatal meningoencephalitis in the southern sea otter (Enhydra lutris nereis). The source(s) of T. gondii infection and routes of transmission in the marine environment are unknown. We hypothesise that filter-feeding marine bivalve shellfish serve as paratenic hosts by assimilation and concentration of infective T. gondii oocysts and their subsequent predation by southern sea otters is a source of infection for these animals. We developed a TaqMan PCR assay for detection of T. gondii ssrRNA and evaluated its usefulness for the detection of T. gondii in experimentally exposed mussels (Mytilus galloprovincialis) under laboratory conditions. Toxoplasma gondii-specific ssrRNA was detected in mussels as long as 21 days post-exposure to T. gondii oocysts. Parasite ssrRNA was most often detected in digestive gland homogenate (31 of 35, i.e. 89%) compared with haemolymph or gill homogenates. Parasite infectivity was confirmed using a mouse bioassay. Infections were detected in mice inoculated with any one of the mussel sample preparations (haemolymph, gill, or digestive gland), but only digestive gland samples remained bioassay-positive for at least 3 days post-exposure. For each time point, the total proportion of mice inoculated with each of the different tissues from T. gondii-exposed mussels was similar to the proportion of exposed mussels from the same treatment groups that were positive via TaqMan PCR. The TaqMan PCR assay described here is now being tested in field sampling of free-living invertebrate prey species from high-risk coastal locations where T. gondii infections are prevalent in southern sea otters.

Development of PCR-based methods for detection of Sphaerothecum destruens in fish tissues.

Single-round and nested polymerase chain reaction (PCR) tests were developed for amplification of a 434 bp fragment of the small subunit ribosomal RNA (18S rRNA) gene from Sphaerothecum destruens, previously known as the rosette agent, an intracellular parasite of salmonid fishes. Both tests have successfully amplified S. destruens-specific DNA from different isolates of S. destruens but not from related organisms. The limits of detection using the nested PCR test were 1 pg for purified S. destruens genomic DNA and 0.1 fg for plasmid DNA. We conducted 2 experimental transmission studies, consisting of injection or waterborne exposure of juvenile winter-run Chinook salmon Oncorhynchus tshawytscha to spore stages of the parasite. In the injection study, parasite DNA was detected in 100% of kidney samples from exposed fish (n = 83) at 1 and 3 mo post-exposure using nested PCR, versus 98% using microscopic analysis of Gram-stained impression smears made from the kidney. Following waterborne exposure, fish were sampled over the course of a year. From each fish, samples of gill, liver, posterior intestine and kidney were analyzed. S. destruens-specific DNA was detected most often in gill and kidney over the course of the experiment, and 71% (64/90) of the exposed fish were identified as positive for S. destruens using the nested PCR test, versus 16% (14/90) using microscopic analysis of Gram-stained kidney smears. Natural infections in captive broodstock of adult winter-run Chinook salmon, originally diagnosed by examination of Gram-stained kidney smears, were confirmed using the nested PCR test in all fish examined (15/15). Further, the nested test amplified parasite-specific DNA from other tissues in these fish with varying frequencies. This report introduces the first DNA-based detection method for S. destruens, to be used alone as a diagnostic tool or in conjunction with histologic tests for confirmatory identification of the parasite.

Introduced pathogens and native freshwater biodiversity: a case study of Sphaerothecum destruens.

A recent threat to European fish diversity was attributed to the association between an intracellular parasite, Sphaerothecum destruens, and a healthy freshwater fish carrier, the invasive Pseudorasbora parva originating from China. The pathogen was found to be responsible for the decline and local extinction of the European endangered cyprinid Leucaspius delineatus and high mortalities in stocks of Chinook and Atlantic salmon in the USA. Here, we show that the emerging S. destruens is also a threat to a wider range of freshwater fish than originally suspected such as bream, common carp, and roach. This is a true generalist as an analysis of susceptible hosts shows that S. destruens is not limited to a phylogenetically narrow host spectrum. This disease agent is a threat to fish biodiversity as it can amplify within multiple hosts and cause high mortalities.

Identification of a rosette-like agent as Sphaerothecum destruens, a multi-host fish pathogen.

A recent threat to European fish diversity was attributed to an infectious pathogen, a rosette-like intracellular parasite carried by invasive cyprinids. Here we show that the emerging rosette-like agent is Sphaerothecum destruens, originally found to be responsible for disease outbreaks in salmon in the United States. Sequencing of the ribosomal internal transcribed spacer (ITS) DNA highlights some level of geographical isolation. Unlike the situation in the United States, its occurrence in invasive fishes presents a risk of spread from wild invasive populations to sympatric populations of susceptible native fish and as such represents a risk for fisheries, as movement of fish for stocking purposes is common practice.

Observations on the life stages of Sphaerothecum destruens n. g., n. sp., a mesomycetozoean fish pathogen formerly referred to as the rosette agent [correction].

The rosette agent is an obligate intracellular parasite that causes morbidity and mortality in salmonid fish. In laboratory cultures, the spore stage (2-6 microm diam.) replicates in a salmonid cell line by sequential asexual division, giving rise to daughter cells. If infected cell cultures are transferred to distilled water, the spore stage undergoes internal division to give rise to at least 5 cells each of which develops into a uniflagellated zoospore with a body of approximately 2 microm and a flagellum approximately 10 microm long. Zoosporulation does not occur in cell culture medium alone, artificial seawater, or phosphate-buffered saline. This parasite is currently classified as a member of the Class Mesomycetozoea (formerly Ichthyosporea) based on phylogenetic analyses of the small subunit ribosomal DNA of three different isolates from fish. Given these new morphological observations combined with the available molecular phylogenetic data on other mesomycetozoeans, we propose to classify the rosette agent as Sphaerothecum destruens, n. g., n. sp. This new genus has unique features including (1) intracellular development of spore stages in various organs eliciting a host granulomatous response; and (2) the differentiation of mature spores into multiple, flagellated zoospores. Taken together, these characteristics clearly distinguish it from the closely related genera Dermocystidium and Rhinosporidium.