While investigating the parasite fauna of wild coho salmon. Oncorhynchus kisutch (Walbaum, 1792), histological examination provided evidence of a new species of Myxobolus (Myxozoa: Myxosporea) infecting nerves of skeletal muscle. Spores were morphologically similar to those of the intramuscular Myxobolus insidiosus Wyatt and Pratt, 1963, both having pyriform spores with clavate polar capsules. However, the former developed exclusively in the nerves of skeletal muscle rather than in myocytes. We examined both species of Myxobolus derived from coho salmon; Chinook salmon, Oncorhynchus tshawytscha (Walbaum, 1792); cutthroat trout, Oncorhynchus clarkii (Richardson, 1836); and rainbow trout Oncorhynchus mykiss (Walbaum, 1792) from freshwater in Oregon. Spore morphology, small subunit ribosomal RNA gene (rDNA) sequences, and site of infection were compared. Myxobolus arcticus Pugachev and Khokhlov, 1979 has pyriform spores, infects the central nervous system of many salmonids, and is found in the Pacific Northwest. It was therefore included in the analyses to rule out conspecificity with the new species. Together, these data show that the Myxobolus sp. from peripheral nerves in the skeletal musculature of coho salmon, rainbow trout, and cutthroat trout is a new species, described herein as Myxobolus fryeri n. sp.

Flavobacterium psychrophilum, the etiological agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), causes significant economic losses in salmonid aquaculture, particularly in rainbow trout (Oncorhynchus mykiss). Prior studies have used multilocus sequence typing (MLST) to examine genetic heterogeneity withinF. psychrophilum. At present, however, its population structure in North America is incompletely understood, as only 107 isolates have been genotyped. Herein, MLST was used to investigate the genetic diversity of an additional 314 North AmericanF. psychrophilumisolates that were recovered from ten fish host species from 20 U.S. states and 1 Canadian province over nearly four decades. These isolates were placed into 66 sequence types (STs), 47 of which were novel, increasing the number of clonal complexes (CCs) in North America from 7 to 12. Newly identified CCs were diverse in terms of host association, distribution, and association with disease. The largestF. psychrophilumCC identified was CC-ST10, within which 10 novel genotypes were discovered, most of which came fromO. mykissexperiencing BCWD. This discovery, among others, provides evidence for the hypothesis that ST10 (i.e., the founding ST of CC-ST10) originated in North America. Furthermore, ST275 (in CC-ST10) was recovered from wild/feral adult steelhead and marks the first recovery of CC-ST10 from wild/feral fish in North America. Analyses also revealed that at the allele level, the diversification ofF. psychrophilumin North America is driven three times more frequently by recombination than random nucleic acid mutation, possibly indicating how new phenotypes emerge within this species.IMPORTANCEFlavobacterium psychrophilumis the causative agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), both of which cause substantial losses in farmed fish populations worldwide. To better prevent and control BCWD and RTFS outbreaks, we sought to characterize the genetic diversity of several hundredF. psychrophilumisolates that were recovered from diseased fish across North America. Results highlighted multipleF. psychrophilumgenetic strains that appear to play an important role in disease events in North American aquaculture facilities and suggest that the practice of trading fish eggs has led to the continental and transcontinental spread of this bacterium. The knowledge generated herein will be invaluable toward guiding the development of future disease prevention techniques.

Pseudoloma neurophilia (Microsporidia) is the most common pathogen found in zebrafish Danio rerio research facilities. The parasite is associated with marked emaciation. Zebrafish laboratories usually disinfect eggs to prevent transmission of pathogens, typically with chlorine at 25 to 50 ppm for 10 min. The ability of chlorine to kill spores of P. neurophilia and 2 other microsporidia, Glugea anomala and Encephalitozoon cuniculi, was evaluated using 2 viability stains. SYTOX ® Green was used to visualize dead spores, and live spores were identified by their ability to extrude polar tubes in Fungi-Fluor™ solution following UV exposure. Results with both stains were similar at various chlorine concentrations for P. neurophilia and G. anomala, but Fungi-Fluor was not useful for E. cuniculi, due to the much smaller spore size. Using the SYTOX stain, we found that 5 ppm chlorine for 10 min causes 100% death in spores of E. cuniculi, which was similar to findings in other studies. In contrast, the spores of P. neurophilia and G. anomala were much more resistant to chlorine, requiring >100 or 1500 ppm chlorine, respectively, to achieve > 95% spore death. Repeating chlorine exposures with spores of P. neurophilia using solutions adjusted to pH 7 increased the efficacy of 100 ppm chlorine, achieving > 99% spore inactivation. We corroborated our viability staining results with experimental exposures of zebrafish fry, achieving heavy infections in fry at 5 to 7 d postexposure in fish fed spores treated at 50 ppm (pH 9). Some fish still became infected with spores exposed to 100 ppm chlorine (pH 9.5). This study demonstrates that spores of certain fish microsporidia are highly resistant to chlorine, and indicates that the egg disinfection protocols presently used by most zebrafish research facilities will not prevent transmission of P. neurophilia to progeny.KEY WORDS: SYTOX Green · Fungi-Fluor · Pseudoloma neurophilia · Glugea anomala · Encephalitozoon cuniculi · Chlorine toxicity · Viability staining Resale or republication not permitted without written consent of the publisher

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Mycobacterial infections in laboratory zebrafish (Danio rerio) are common and widespread in research colonies. Mycobacteria within free living amoebae have been shown to be transmission vectors for mycobacteriosis. Paramecium caudatum are commonly used as a first food for zebrafish, and we investigated this ciliate’s potential to serve as a vector of Mycobacterium marinum and M. chelonae. The ability of live P. caudatum to transmit these mycobacteria to larval, juvenile and adult zebrafish was evaluated. Infections were defined by histologic observation of granulomas containing acid-fast bacteria in extraintestinal locations. In both experiments, fish fed paramecia containing mycobacteria became infected at a higher incidence than controls. Larvae (exposed at 4 days post hatch) fed paramecia with M. marinum exhibited an incidence of 30% (24/80) and juveniles (exposed at 21 days post hatch) showed 31% incidence (14/45). Adult fish fed a gelatin food matrix containing mycobacteria within paramecia or mycobacteria alone for 2 wk resulted in infections when examined 8 wk after exposure as follows: M. marinum OSU 214 47% (21/45), M. marinum CH 47% (9/19), M. chelonae 38% (5/13). In contrast, fish feed mycobacteria alone in this diet did not become infected, except for 2 fish (5%) in the M. marinum OSU 214 low dose group. These results demonstrate that Paramecium caudatum can act as a vector for mycobacteria. This provides a useful animal model for evaluation of natural mycobacterial infections and demonstrates the possibility of mycobacterial transmission in zebrafish facilities via contaminated paramecia cultures.