Pseudocapillaria tomentosa, Mycoplasma spp., and Intestinal Lesions in Experimentally Infected Zebrafish Danio rerio.

Intestinal neoplasms and preneoplastic lesions are common in zebrafish research facilities. Previous studies have demonstrated that these neoplasms are caused by a transmissible agent, and two candidate agents have been implicated: a Mycoplasma sp. related to Mycoplasma penetrans and the intestinal parasitic nematode, Pseudocapillaria tomentosa, and both agents are common in zebrafish facilities. To elucidate the role of these two agents in the occurrence and severity of neoplasia and other intestinal lesions, we conducted two experimental inoculation studies. Exposed fish were examined at various time points over an 8-month period for intestinal histopathologic changes and the burden of Mycoplasma and nematodes. Fish exposed to Mycoplasma sp. isolated from zebrafish were associated with preneoplastic lesions. Fish exposed to the nematode alone or with the Mycoplasma isolate developed severe lesions and neoplasms. Both inflammation and neoplasm scores were associated with an increase in Mycoplasma burden. These results support the conclusions that P. tomentosa is a strong promoter of intestinal neoplasms in zebrafish and that Mycoplasma alone can also cause intestinal lesions and accelerate cancer development in the context of nematode infection.

Further evaluation of the efficacy of emamectin benzoate for treating Pseudocapillaria tomentosa (Dujardin 1843) in zebrafish Danio rerio (Hamilton 1822).

Pseudocapillaria tomentosa is a pathogenic nematode parasite, causing emaciation and severe inflammatory lesions in the intestines in zebrafish Danio rerio (Hamilton 1822). Emamectin benzoate is commercially available analogue of ivermectin used for treating salmon for sea lice, under the brand name SLICE® , and we have used this for treating zebrafish with the P. tomentosa. Here, SLICE® , 0.2 per cent active emamectin benzoate, was used for oral treatments at 0.35 mg emamectin benzoate/kg fish/day for 14 days starting at 7 days post-exposure (dpe). Another experiment entailed initiating treatment during clinical disease (starting at 28 dpe). Early treatment was very effective, but delaying treatment was less so, presumably due to inappetence in clinically affected fish. We evaluated emamectin benzoate delivered in water, using Lice-Solve™ (mectinsol; 1.4% active emamectin benzoate) in two experiments. Application of four 24-hr treatments, space over 7 days was initiated at 28 dpe at either 0.168 or 0.56 mg emamectin benzoate/L/bath, and both treatments completely eradicated infections. This was 3 or 10 times manufacture's recommended dose, but was not associated with clinical or histological side effects.

Viability of Pseudocapillaria tomentosa Eggs Exposed to Heat, Ultraviolet Light, Chlorine, Iodine, and Desiccation.

Pseudocapillaria tomentosa is an important pathogen in zebrafish facilities. We investigated heat, ultraviolet (UV) light, chlorine, iodine, and dessciation for killing the parasite's eggs. Eggs released with feces larvate in about 5-10 days, and treatments were evaluated by exposing fresh eggs and subsequently comparing larvation to untreated eggs as an indication of survival. Collectively, untreated eggs in all trials showed high levels of survival. Eggs were exposed to elevated temperatures (40°C, 45°C and 50°C) for 1, 8, or 24 h, which resulted in substantial reduction in viability of eggs. UV radiation was effective, with no larvation at 50-300 mWs/cm2 and <2% at 20 mWs/cm2. Three chlorine products (JT Baker, Clorox®, and Bi-Mart) were tested at 25, 50, 100, 500, and 3,000 ppm (pH 7.0-7.3) with 10 min exposure. All were effective at 500 or 1,000 ppm. There was variability between three products and trials at lower concentrations, but overall chlorine was not very effective at 25-100 ppm except for Bi-Mart brand at 100 ppm. Povidone-iodine was not effective at 25 or 50 ppm for 10 min, but was effective at 200 ppm for 1 h. Desiccation was effective, and no eggs larvated after 2 h drying.

A longitudinal assessment of host-microbe-parasite interactions resolves the zebrafish gut microbiome's link to Pseudocapillaria tomentosa infection and pathology.

BACKGROUND: Helminth parasites represent a significant threat to the health of human and animal populations, and there is a growing need for tools to treat, diagnose, and prevent these infections. Recent work has turned to the gut microbiome as a utilitarian agent in this regard; components of the microbiome may interact with parasites to influence their success in the gut, meaning that the microbiome may encode new anthelmintic drugs. Moreover, parasite infections may restructure the microbiome's composition in consistent ways, implying that the microbiome may be useful for diagnosing infection. The innovation of these utilities requires foundational knowledge about how parasitic infection, as well as its ultimate success in the gut and impact on the host, relates to the gut microbiome. In particular, we currently possess limited insight into how the microbiome, host pathology, and parasite burden covary during infection. Identifying interactions between these parameters may uncover novel putative methods of disrupting parasite success. RESULTS: To identify interactions between parasite success and the microbiome, we quantified longitudinal associations between an intestinal helminth of zebrafish, Pseudocapillaria tomentosa, and the gut microbiome in 210 4-month-old 5D line zebrafish. Parasite burden and parasite-associated pathology varied in severity throughout the experiment in parasite-exposed fish, with intestinal pathologic changes becoming severe at late time points. Parasite exposure, burden, and intestinal lesions were correlated with gut microbial diversity. Robust generalized linear regression identified several individual taxa whose abundance predicted parasite burden, suggesting that gut microbiota may influence P. tomentosa success. Numerous associations between taxon abundance, burden, and gut pathologic changes were also observed, indicating that the magnitude of microbiome disruption during infection varies with infection severity. Finally, a random forest classifier accurately predicted a fish's exposure to the parasite based on the abundance of gut phylotypes, which underscores the potential for using the gut microbiome to diagnose intestinal parasite infection. CONCLUSIONS: These experiments demonstrate that P. tomentosa infection disrupts zebrafish gut microbiome composition and identifies potential interactions between the gut microbiota and parasite success. The microbiome may also provide a diagnostic that would enable non-destructive passive sampling for P. tomentosa and other intestinal pathogens in zebrafish facilities.

Pseudocapillaria tomentosa in laboratory zebrafish Danio rerio: patterns of infection and dose response.

Parasites in wild populations almost always exhibit aggregation (overdispersion), in which relatively few hosts are infected with high numbers of the parasites. This pattern of infection has also been observed in laboratory studies, where many of the sources of natural variation are removed. Pseudocapillaria tomentosa (Nematoda) is common in zebrafish (Danio rerio) facilities. We describe here patterns of infections in zebrafish experimentally infected with larvated P. tomentosa eggs in various trials with defined numbers of eggs. One trial with eggs delivered in a gelatin diet is also included. Fish were exposed at 25, 75, and 200 eggs fish-1, and the minimal infectious dose was estimated to be 1.5 eggs fish-1. The ID50 (50% infective dose) was calculated to be 17.5 eggs fish-1. We also included data from a trial and 2 previously published experiments with undefined doses in which zebrafish were exposed to infectious water and detritus from a tank that previously contained infected fish. All doses resulted in a high prevalence of infection (>70%), except at the 25 eggs fish-1 dose, where the prevalence was 43-46%. Mean abundance of worms corresponded to dose, from 0.57 worms fish-1 at 25 eggs fish-1 to 7 worms fish-1 at 200 eggs fish-1. Variance to mean ratios (V/M) and the k parameters showed aggregation across the 8 separate trials, including the gelatin diet. Aggregation increased with increased parasite abundance. Given the consistent observation of aggregation across our experiments, the zebrafish/P. tomentosa system provides a potentially robust, high-throughput model to investigate factors that influence differences in host susceptibility within defined populations.

Survival of Bacterial and Parasitic Pathogens from Zebrafish (Danio rerio) After Cryopreservation and Thawing.

Cryopreservation is a common method used to preserve the sperm of various animal species, and it is widely used with zebrafish (Danio rerio). As with other animals, there is a possibility of paternal pathogen transmission through sperm. We evaluated the ability of five common and important pathogens of zebrafish to survive cryopreservation as used with zebrafish sperm and freezing without cryopreservant. We evaluated Mycobacterium chelonae, Mycobacterium marinum, and Edwardsiella ictaluri, each originally isolated from zebrafish, eggs of Pseuodocapillaria tomentosa, and spores of Pseudoloma neurophilia. Each mycobacterial isolate showed relatively minimal reduction in survival after freezing and thawing, particularly when subjected to cryopreservation. E. ictaluri also showed survival after cryopreservation, but exhibited a several log reduction after freezing at -80°C without cryopreservant. With P. neurophilia, two separate experiments conducted 3 years apart yielded very similar results, showing some, but reduced, survival of spores by using three different viability assays: SYTOX stain, Fungi-Fluor stain, and presence of a spore vacuole. Eggs of P. tomentosa showed no survival based on larvation of eggs when subjected to either freezing method. Given that four of the five pathogens exhibited survival after cryopreservation, we recommend that sperm samples or donor male zebrafish fish be tested for pathogens when sperm are to be stored by using cryopreservation.

Draft Genome Sequence of Pseudomonas sp. Strain DrBHI1 (Phylum Proteobacteria).

Here, we report the draft genome sequence of Pseudomonas sp. strain DrBHI1. The total assembly length is 5,649,751 bp in 146 contigs. This strain was isolated from zebrafish (Danio rerio) feces.

Expansion of the Known Host Range of the Microsporidium, Pseudoloma neurophilia.

The microsporidium, Pseudoloma neurophilia, is the most common infectious organism found in laboratory zebrafish colonies. Many currently used zebrafish lines originally came from pet store fish, and the initial description of P. neurophilia came from zebrafish obtained from a retail pet store. However, as P. neurophilia has not been described from wild-caught zebrafish, whether P. neurophilia is a natural pathogen of zebrafish is an open question. The pooling of fish of different species in the aquarium fish trade is common and a generalist parasite could be transmitted to novel hosts in this scenario. We determined that P. neurophilia can infect seven species of fishes from five families by cohabitation with infected zebrafish: Betta splendens, Xiphophorus maculatus, Devario aequipinnatus, Pimephales promelas, Oryzias latipes, Carassius auratus and Paracheirodon innesi. Infections in these fishes were histologically similar to those of zebrafish. We include a case report of a laboratory population of fathead minnows with naturally acquired P. neurophilia infections. With such a broad host range, including several fish families, other laboratory fishes should be screened routinely for this and other microsporidian parasites.

Effects of Subclinical Mycobacterium chelonae Infections on Fecundity and Embryo Survival in Zebrafish.

Mycobacteriosis is the second most common infectious disease in zebrafish research colonies, and most often this is caused by Mycobacterium chelonae. The infection is characterized by multiple granulomas in the kidney, coelomic cavity, particularly the ovary. However, most fish still appear clinically normal. Developmental genetics remain a primary area of research with the zebrafish model, and hence, an important use of adult zebrafish is as brood fish to produce embryos. We investigated the effects of experimentally induced M. chelonae infections on fecundity. A total of 480 5D wild-type zebrafish were divided into four groups: controls, males infected, females infected, and both sexes. Exposed fish developed high prevalence of infection, including many females with ovarian infections. Fish were then first subjected to four separate group spawns with four replicate tanks/group. Then, a third of the fish were subjected to pairwise spawns, representing 20 pairs/group, and then the pairs were evaluated by histopathology. Overall, the group and pairwise spawns resulted numerous eggs and viable embryos. However, we found no statistical correlations between infection status and number of eggs or viability. In contrast to Egg Associated Inflammation and Fibroplasia, lesions in infected ovaries were more localized, with large regions of the ovary appearing normal.

Pseudoloma neurophilia Infection Combined with Gamma Irradiation Causes Increased Mortality in Adult Zebrafish (Danio rerio) Compared to Infection or Irradiation Alone: New Implications for Studies Involving Immunosuppression.

Gamma irradiation is commonly used as a bone marrow suppressant in studies of the immune system and hematopoiesis, most commonly in mammals. With the rising utility and popularity of the zebrafish (Danio rerio), gamma irradiation is being used for similar studies in this species. Pseudoloma neurophilia, a microparasite and common contaminant of zebrafish facilities, generally produces subclinical disease. However, like other microsporidia, P. neurophilia is a disease of opportunity and can produce florid infections with high morbidity and mortality, secondary to stress or immune suppression. In this study, we exposed zebrafish to combinations of P. neurophilia infection and gamma irradiation to explore the interaction between this immunosuppressive experimental modality and a normally subclinical infection. Zebrafish infected with P. neurophilia and exposed to gamma irradiation exhibited higher mortality, increased parasite loads, and increased incidences of myositis and extraneural parasite infections than fish exposed either to P. neurophilia or gamma irradiation alone. This experiment highlights the devastating effects of opportunistic diseases on immunosuppressed individuals and should caution researchers utilizing immunosuppressive modalities to carefully monitor their stocks to ensure that their experimental animals are not infected.

Tolerance and efficacy of emamectin benzoate and ivermectin for the treatment of Pseudocapillaria tomentosa in laboratory zebrafish (Danio rerio).

Tolerance of adult zebrafish and efficacy of emamectin benzoate and ivermectin in eliminating Pseudocapillaria tomentosa infection were evaluated. In the tolerance study, behavioral changes, fecundity, histopathology, and mortality were evaluated for in-feed administration of emamectin (0.05, 0.10, and 0.25 mg/kg) and ivermectin (0.05 and 0.10 mg/kg). All doses of emamectin were well tolerated. Ivermectin 0.05 mg/kg administration resulted in mild behavioral changes and a transient decrease in fecundity. Ivermectin 0.10 mg/kg administration resulted in severe behavioral changes and some mortality. In the efficacy study, emamectin (0.05 and 0.25 mg/kg) and ivermectin (0.05 mg/kg) were evaluated for their efficacy in eliminating P. tomentosa infection. Emamectin reduced parasite burden in infected zebrafish, and ivermectin eliminated intestinal nematode infections. Despite a small margin of safety, ivermectin 0.05 mg/kg was effective at eliminating P. tomentosa infection in adult zebrafish. Higher doses or a longer course of treatment may be needed for complete elimination of P. tomentosa infection using emamectin. In this study, we propose two possible treatments for intestinal nematode infections in zebrafish.

Paramecium caudatum enhances transmission and infectivity of Mycobacterium marinum and M. chelonae in zebrafish Danio rerio.

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 d post hatch) fed paramecia with M. marinum exhibited an incidence of 30% (24/80) and juveniles (exposed at 21 d 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), and 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 P. 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.

Verification of intraovum transmission of a microsporidium of vertebrates: Pseudoloma neurophilia infecting the Zebrafish, Danio rerio.

Direct transmission from parents to offspring, referred to as vertical transmission, occurs within essentially all major groups of pathogens. Several microsporidia (Phylum Microsporidia) that infect arthropods employ this mode of transmission, and various lines of evidence have suggested this might occur with certain fish microsporidia. The microsporidium, Pseudoloma neurophilia, is a common pathogen of the laboratory zebrafish, Danio rerio. We previously verified that this parasite is easily transmitted horizontally, but previous studies also indicated that maternal transmission occurs. We report here direct observation of Pseudoloma neurophilia in the progeny of infected zebrafish that were reared in isolation, including microscopic visualization of the parasite in all major stages of development. Histological examination of larval fish reared in isolation from a group spawn showed microsporidian spores in the resorbing yolk sac of a fish. Infections were also observed in three of 36 juvenile fish. Eggs from a second group spawn of 30 infected fish were examined using a stereomicroscope and the infection was observed from 4 to 48 hours post-fertilization in two embryos. Intraovum infections were detected in embryos from 4 of 27 pairs of infected fish that were spawned based on qPCR detection of P. neurophilia DNA. The prevalence of intraovum infections from the four spawns containing infected embryos was low (∼1%) based on calculation of prevalence using a maximum likelihood analysis for pooled samples. Parasite DNA was detected in the water following spawning of 11 of the infected pairs, suggesting there was also potential for extraovum transmission in these spawning events. Our study represents the first direct observation of vertical transmission within a developing embryo of a microsporidian parasite in a vertebrate. The low prevalence of vertical transmission in embryos is consistent with observations of some other fish pathogens that are also readily transmitted by both vertical and horizontal routes.

Comparison of fixatives and fixation time for PCR detection of Mycobacterium in zebrafish Danio rerio .

Mycobacteriosis is a common disease of laboratory zebrafish Danio rerio. Different infection patterns occur in zebrafish depending on mycobacterial species. Mycobacterium marinum and M. haemophilum produce virulent infections associated with high mortality, whereas M. chelonae is more widespread and is not associated with high mortality. Identification of mycobacterial infections to the species level provides important information for making management decisions. Observation of acid-fast bacilli in histological sections or tissue imprints is the most common diagnostic method for mycobacteriosis in fish, but only allows for diagnosis to the genus level. Mycobacterial culture followed by molecular or biochemical identification is the traditional approach, but DNA of diagnostic value can also be retrieved from paraffin blocks. Here we investigated the type of fixative, time in fixative before processing, species of mycobacteria, and severity of infection as parameters to determine whether the hsp gene PCR assay (primer set HS5F/hsp667R) could detect and amplify mycobacterial DNA from paraffin-embedded zebrafish. Whole zebrafish were experimentally infected with either M. chelonae or M. marinum, and then preserved in 10% neutral buffered formalin or Dietrich's fixative for 3, 7, 21, and 45 d. Subsequently, fish were evaluated by hematoxylin and eosin and Fite's acid-fast stains to detect mycobacteria within granulomatous lesions. The PCR assay was quite effective and obtained PCR product from 75 and 88% of the M. chelonae- and M. marinum-infected fish, respectively. Fixative type, time in fixative, and mycobacterial species showed no statistical relationship with the efficacy of the PCR test.

Microsporidiosis in zebrafish research facilities.

Pseudoloma neurophilia (Microsporidia) is the most common pathogen detected in zebrafish (Danio rerio) from research facilities. The parasite infects the central nervous system and muscle and may be associated with emaciation and skeletal deformities. However, many fish exhibit subclinical infections. Another microsporidium, Pleistophora hyphessobryconis, has recently been detected in a few zebrafish facilities. Here, we review the methods for diagnosis and detection, modes of transmission, and approaches used to control microsporidia in zebrafish, focusing on P. neurophilia. The parasite can be readily transmitted by feeding spores or infected tissues, and we show that cohabitation with infected fish is also an effective means of transmission. Spores are released from live fish in various manners, including through the urine, feces, and sex products during spawning. Indeed, P. neurophilia infects both the eggs and ovarian tissues, where we found concentrations ranging from 12,000 to 88,000 spores per ovary. Hence, various lines of evidence support the conclusion that maternal transmission is a route of infection: spores are numerous in ovaries and developing follicles in infected females, spores are present in spawned eggs and water from spawning tanks based on polymerase chain reaction tests, and larvae are very susceptible to the infection. Furthermore, egg surface disinfectants presently used in zebrafish laboratories are ineffective against microsporidian spores. At this time, the most effective method for prevention of these parasites is avoidance.

Development and maintenance of a specific pathogen-free (SPF) zebrafish research facility for Pseudoloma neurophilia.

Pseudoloma neurophilia (Microsporidia) is very common in zebrafish Danio rerio research facilities. A new zebrafish facility has been established at the Sinnhuber Aquatic Resource Laboratory (SARL), Oregon State University, Corvallis, OR, U.S.A., and this was an opportunity to establish a specific pathogen-free (SPF) colony of zebrafish for this microsporidium. Progeny from 9 zebrafish lines (n=2203) were initially transferred to the SARL facility in 2007 following PCR screening of broodstock and a subpopulation of progeny (258 of 1000 fish from each family). Screening of fish for P. neurophilia within the facility was conducted as follows: (1) Moribund or dead fish were examined by histology. (2) Each line was regenerated on a 4 mo rotation, and a subsample of each of these major propagations (60 fry, in pools of 10) was PCR-screened at 10 d post hatch. (3) Adult fish (approximately 1 yr old) from each line were euthanized; 20 fish were examined by histology and the brains of another 60 fish (in pools of 5) were screened by PCR. (4) This screening was replicated on sentinel fish held in 4 tanks receiving effluent water from all tanks in the facility (20 fish per tank). (5) Four-month old fish (n=760) from a toxicology study conducted within the laboratory were examined by histology. To date, we have evaluated 2800 fish by PCR and 1222 fish by histology without detecting P. neurophilia. Thus, we have established 9 lines of zebrafish SPF for P. neurophilia. However, 26 fish exhibited mycobacteriosis, with acid-fast bacteria present in tissue sections, and 49 other fish had incidental lesions.

Animal cell cultures in microsporidial research: their general roles and their specific use for fish microsporidia.

The use of animal cell cultures as tools for studying the microsporidia of insects and mammals is briefly reviewed, along with an in depth review of the literature on using fish cell cultures to study the microsporidia of fish. Fish cell cultures have been used less often but have had some success. Very short-term primary cultures have been used to show how microsporidia spores can modulate the activities of phagocytes. The most successful microsporidia/fish cell culture system has been relatively long-term primary cultures of salmonid leukocytes for culturing Nucleospora salmonis. Surprisingly, this system can also support the development of Enterocytozoon bienusi, which is of mammalian origin. Some modest success has been achieved in growing Pseudoloma neurophilia on several different fish cell lines. The eel cell line, EP-1, appears to be the only published example of any fish cell line being permanently infected with microsporidia, in this case Heterosporis anguillarum. These cell culture approaches promise to be valuable in understanding and treating microsporidia infections in fish, which are increasingly of economic importance.

Pseudoloma neurophilia infections in zebrafish Danio rerio: effects of stress on survival, growth, and reproduction.

Pseudoloma neurophilia (Microsporidia) is a common disease of zebrafish Danio rerio, including those used as research models. We conducted a study comprised of 4 separate experiments to determine the effects of husbandry stress on preexisting and experimental P. neurophilia infections and the subsequent effects on survival, infection onset and intensity, fish growth, and reproduction. In fish (AB strain) with preexisting infections, stress or feeding cortisol significantly increased mortality over 7 wk compared to no stress or cortisol treatment. In contrast, no mortality was observed in fish (TL strain) experimentally exposed to P. neurophilia over 10 wk. A third experiment involved experimental exposure of AB fish to P. neurophilia and exposure to crowding and handling stressors. No mortality was associated with P. neurophilia regardless of stress treatment over a period of 20 wk. However, the onset of infection occurred sooner in stress-treated fish. Stress significantly increased the mean intensity of infection (described as xenoma area/spinal cord area in histological sections) at Week 20 post-exposure (PE). In fish with preexisting infections, myositis was significantly greater in stressed and cortisol-treated fish than those not stressed. With experimental exposure of AB fish, stressed and infected groups weighed significantly less than the control group at Week 20 PE. Regarding fecundity, the number of larvae hatched at 5 d post fertilization was negatively associated with mean infection intensity among P. neurophilia-infected and stressed AB fish. These experiments are the first to show empirically that P. neurophilia can be associated with reduced weight and fecundity, and that stress can exacerbate the severity of the infection.

Molecular systematics support the revival of Mycobacterium salmoniphilum (ex Ross 1960) sp. nov., nom. rev., a species closely related to Mycobacterium chelonae.

Mycobacterial infections in fish are usually attributed to strains of Mycobacterium marinum, Mycobacterium chelonae and Mycobacterium fortuitum. Bacteria identified as M. chelonae have been isolated numerous times from salmonid fishes. Recently, this bacterium has been associated with salmon mortalities in the aquaculture industry. An M. chelonae-like species from salmon, 'Mycobacterium salmoniphilum', was described in 1960. However, the species name lost standing in nomenclature when it was omitted from the 1980 Approved Lists of Bacterial Names because the species could not be distinguished with confidence from M. fortuitum. In the 1980s, mycobacteria isolated from salmon were characterized as a distinct subspecies, 'Mycobacterium chelonae subsp. piscarium'. Again, the uncertainty of the validity of the species resulted in the subsequent withdrawal of the name. Since then, most studies have considered isolates from salmon to be M. chelonae. Nucleotide sequence analysis of the small-subunit rRNA, hsp65 and rpoB genes was used to examine the taxonomic relatedness of type cultures and authentic isolates in our culture collection available from earlier studies. The M. chelonae-like strains from salmon were phylogenetically distinct from other Mycobacterium strains and members of the M. chelonae complex. Moreover, the cell-wall-bound mycolic acids were not representative of known mycolate patterns for M. chelonae-complex organisms. These results supported the status of the species as a separate taxon and effect the valid publication of the name 'M. salmoniphilum' as Mycobacterium salmoniphilum (ex Ross 1960) sp. nov., nom. rev., with the type strain SCT (=ATCC 13578T =DSM 43276T).

Spores of two fish microsporidia (Pseudoloma neurophilia and Glugea anomala) are highly resistant to chlorine.

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 post-exposure 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.