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.

Inability to demonstrate fish-to-fish transmission of Ichthyophonus from laboratory infected Pacific herring Clupea pallasii to naïve conspecifics.

The parasite Ichthyophonus is enzootic in many marine fish populations of the northern Atlantic and Pacific Oceans. Forage fishes are a likely source of infection for higher trophic level predators; however, the processes that maintain Ichthyophonus in forage fish populations (primarily clupeids) are not well understood. Lack of an identified intermediate host has led to the convenient hypothesis that the parasite can be maintained within populations of schooling fishes by waterborne fish-to-fish transmission. To test this hypothesis we established Ichthyophonus infections in Age-1 and young-of-the-year (YOY) Pacific herring Clupea pallasii (Valenciennes) via intraperitoneal (IP) injection and cohabitated these donors with naïve conspecifics (sentinels) in the laboratory. IP injections established infection in 75 to 84% of donor herring, and this exposure led to clinical disease and mortality in the YOY cohort. However, after cohabitation for 113 d no infections were detected in naïve sentinels. These data do not preclude the possibility of fish-to-fish transmission, but they do suggest that other transmission processes are necessary to maintain Ichthyophonus in wild Pacific herring populations.

Development of a polymerase chain reaction for the detection of abalone herpesvirus infection based on the DNA polymerase gene.

A 5781-base pair (bp) fragment of genomic DNA from the Taiwanese abalone herpesvirus was obtained and showed 99% (5767/5779) homology in the nucleotide sequence and 99% (1923/1926) in the amino acid sequence with the DNA polymerase gene of the abalone herpesvirus strain Victoria/AUS/2007. Homology of the amino acid sequence with the DNA polymerase of ostreid herpesvirus 1 was 30% (563/1856). In this study, a PCR-based procedure for detecting herpesvirus infection of abalone, Haliotis diversicolor supertexta, in Taiwan was developed. The method employed primer sets targeting the viral DNA polymerase gene, and was able to amplify DNA fragments of the expected size from infected samples. Primer sets of 40f and 146r were designed for amplification of an expected PCR product of 606 bp. Combining the new PCR protocol with histopathology, this assay can serve as a reliable diagnostic for herpesvirus infections in abalone.

Pathology of cultured paua Haliotis iris infected with a novel haplosporidian parasite, with some observations on the course of disease.

Mortalities among juvenile paua Haliotis iris Martyn 1784 in a commercial culture facility were reported in April 2000. Histology of moribund paua showed heavy systemic infections of a uni- to multi-nucleate stage of a novel organism later confirmed by transmission electron microscopy (TEM) and molecular studies to be a haplosporidian. Multinucleate plasmodia up to 25 microm diameter with up to 17 nuclei were detectable in wet preparations of hemolymph from heavily infected paua. The presence of the haplosporidian in the affected facility was associated with mortalities of slow growing 'runt' paua during the summer months. Total mortalities in affected raceways 6 mo after mortalities began were between 82.5 and 90%. Heavily infected paua exhibited behavioural abnormalities including lethargy, loss of righting reflex, and were easily detached from surfaces. Some heavily infected paua exhibited oedema and pale lesions in the foot and mantle, but no reliable gross signs of disease were noted. Light infections of the haplosporidian were also found in apparently healthy paua from the facility. Histology indicated that the early stages of infection were characterised by small numbers of plasmodia in the connective tissue surrounding the gut, amongst glial cells adjacent to nerves in the mantle and foot and within gill lamellae. In heavy infections, large numbers of small plasmodia (mean size 5.5 x 7 microm in histological sections) were present in the hemolymph, gills, heart, kidneys, mantle, foot, epipodium and connective tissue of the digestive gland. Infections were not transferred horizontally at 14 and 19 degrees C after cohabiting heavily infected paua with uninfected paua for 3 mo in aquaria, or 3 mo after injecting healthy paua with hemolymph containing haplosporidian plasmodia. This may indicate that the prepatent period for disease is longer than 3 mo, that disease is not expressed below 20 degrees C, or that an intermediate host is required for transmission. Spore formation was not observed in juvenile paua but sporocyst-like bodies containing putative spores were observed amongst haplosporidian plasmodia in the right kidney of poorly performing adult paua collected from the wild.

Detection of 'Candidatus Xenohaliotis californiensis' (Rickettsiales-like prokaryote) inclusions in tissue squashes of abalone (Haliotis spp.) gastrointestinal epithelium using a nucleic acid fluorochrome.

Rickettsiales-like prokaryotes appear to be etiologic agents of a number of newly described diseases of fish and shellfish. 'Candidatus Xenohaliotis californiensis' is a Rickettsiales-like prokaryote responsible for withering syndrome, a fatal disease of wild and farmed Eastern Pacific abalone, Haliotis spp. The bacterium proliferates in gastrointestinal epithelial cells, forming large intracytoplasmic inclusions. We describe a method of rapidly detecting and assessing the intensity of 'Candidatus Xenohaliotis californiensis' infections in abalone gastrointestinal tissue using the nucleic acid-specific fluorochrome Hoechst 33258. In excised tissue pieces dried onto slides, rehydrated in the Hoechst stain and viewed with ultraviolet light, the large bacterial inclusions were strongly fluorescent and could be easily distinguished from smaller host cell nuclei. This provided a rapid, inexpensive alternative to paraffin section microscopy or molecular techniques, allowing detection of the pathogen within minutes of tissue excision. Comparison of the fluorochrome method with conventional histological analysis for the ability to detect inclusions in 109 samples was 90% accurate, with discrepancies due to false negative diagnosis of low-level infections. An alternative nucleic acid-specific fluorochrome, propidium iodide, showed a staining pattern identical to that of Hoechst 33258. These methods should prove useful for the rapid detection of inclusion-forming Rickettsiales-like prokaryotes in tissues from many host species.

'Candidatus Xenohaliotis californiensis', a newly described pathogen of abalone, Haliotis spp., along the west coast of North America.

Withering syndrome is a fatal disease of wild and cultured abalone, Haliotis spp., that inhabit the west coast of North America. The aetiological agent of withering syndrome has recently been identified as a member of the family Rickettsiaceae in the order Rickettsiales. Using a combination of morphological, serological, life history and genomic (16S rDNA) characterization, we have identified this bacterium as a unique taxon and propose the provisional status of 'Candidatus Xenohaliotis californiensis'. The Gram-negative, obligate intracellular pleomorphic bacterium is found within membrane-bound vacuoles in the cytoplasm of abalone gastrointestinal epithelial cells. The bacterium is not cultivable on synthetic media or in fish cell lines (e.g. CHSE-214) and may be controlled by tetracyclines (oxytetracycline) but not by chloramphenicol, clarithromycin or sarafloxicin. Phylogenetic analysis based on the 16S rDNA of 'Candidatus Xenohaliotis californiensis' places it in the alpha-subclass of the class Proteobacteria but not to the four recognized subtaxa of the alpha-Proteobacteria (alpha-1, alpha-2, alpha-3 and alpha-4). The bacterium can be detected in tissue squashes stained with propidium iodide, microscopic examination of stained tissue sections, PCR or in situ hybridization. 'Candidatus Xenohaliotis californiensis' can be differentiated from other closely related alpha-Proteobacteria by its unique 16S rDNA sequence.

Increased Virulence in an Introduced Pathogen: Haplosporidium nelsoni (MSX) in the Eastern Oyster Crassostrea virginica.

The protistan parasite Haplosporidium nelsoni has caused extensive mortality in the eastern oyster Crassostrea virginica along the mid-Atlantic coast of the United States since 1957. The origin of H. nelsoni has remained unresolved. Molecular diagnostic tools were used to examine the hypothesis that a haplosporidian parasite in the Pacific oyster C. gigas is H. nelsoni. A DNA probe specific for H. nelsoni reacted positively in in situ hybridizations with haplosporidian plasmodia from C. gigas collected in Korea, Japan, and California. Primers that specifically amplify H. nelsoni DNA in the polymerase chain reaction amplified product from Californian C. gigas infected with the haplosporidian parasite. The DNA sequence of the 565-base pair amplified product was identical to the H. nelsoni sequence except for a single nucleotide transition, a similarity of 99.8%. These results are conclusive evidence that the parasite in C. gigas is H. nelsoni and strongly support previous speculation that the parasite was introduced into Californian populations of C. gigas from Japan. Results also support previous speculation that H. nelsoni was introduced from the Pacific Ocean to C. virginica on the East Coast of the United States, likely with known importations of C. gigas. These results document greatly increased virulence in a naive host-parasite association and reinforce potential dangers of intentional, but improper, introductions of exotic marine organisms for aquaculture or resource restoration.

Nocardia crassostreae sp. nov., the causal agent of nocardiosis in Pacific oysters.

Seven strains of bacteria were isolated from Pacific oysters, Crassostrea gigas, with a focal or systemic disease. The strains were aerobic, Gram-positive, acid-fast, produced a mycelium which fragmented into irregular rod-like elements, had a peptidoglycan containing meso-diaminopimelic acid, arabinose and galactose as major sugars, mycolic acids with 46-58 carbon atoms and G + C-rich DNA. All of these properties are consistent with the classification of the organisms in the genus Nocardia. A partial sequence of the 16S rRNA gene of isolate NB4H was determined following isolation and cloning of the PCR-amplified gene. The sequence was aligned with those of representative mycolic-acid-containing taxa and a phylogenetic tree was generated using the neighbour-joining method. It was evident from the phylogenetic tree that the three strains tested, RB1, OB3P and NB4H, were identical and belonged to the Nocardia otitidiscaviarum rRNA sub-group. The biochemical, chemical, morphological and physiological properties of the isolates were also essentially identical and served to distinguish them from representative nocardiae. It is, therefore, proposed that the strains isolated from the diseased Pacific oysters be assigned to a new species, Nocardia crassostreae. The type strain is NB4H (= ATCC 700418).

Haplosporidiosis of the Pacific oyster, Crassostrea gigas.

Haplosporidan parasites were observed in 10/100 spat and 1/171 adult Pacific oysters, Crassostrea gigas, reared in Matsushima Bay, Japan. Eight of the infected spat contained mild to severe plasmodial infections. The multinucleated plasmodia were 6-12 microm x 7-15 microm and were associated with an infiltration of hemocytes that occurred throughout the vesicular connective tissues of all infected oysters. Two oysters, one adult and one spat, contained advanced sporogonic infections. These were characterized by the presence of sporocysts and immature and mature operculated spores that measured 5.6-6.0 microm x 6.0-8.0 microm and were found exclusively within the digestive tubule epithelium. Electron microscopic examination revealed that mature spores contained a hinge operculum, striated and layered wall, spherule, single nucleus, and haplosporosome formative regions. Parasite morphology and infection pattern closely resemble that of Haplosporidium nelsoni, a pathogen of American oysters (C. virginica).

Use of articaine local anesthesia in children under 4 years of age--a retrospective report.

A retrospective survey reports the use of articaine hydrochloride as an anesthetic in children under 4 years of age. Data was collected by a record audit in two pediatric dentistry offices. Articaine anesthetic was administered to 211 patients, 29 having additional administrations of the agent. In some instances, the dosages exceeded the recommended concentrations for older children. No adverse systemic adverse reactions were noted on the charts or known to the clinicians. The present report provides initial evidence for the use of articaine in children under 4 years of age.