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.

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.

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.

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.

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.

Assessing the accuracy of a polymerase chain reaction test for Ichthyophonus hoferi in Yukon River Chinook salmon Oncorhynchus tshawytscha.

Ichthyophonus hoferi Plehn & Mulsow, 1911, is a cosmopolitan, protistan pathogen of marine fishes. It is prevalent in mature returning Chinook salmon Oncorhynchus tshawytscha in the Yukon River watershed, and may be associated with prespawning mortality. We developed and evaluated a polymerase chain reaction (PCR) test for I. hoferi using primers specific to the parasite's small subunit rDNA. The test has a minimum detection limit of approximately 10(-5) parasite spores per reaction and does not cross-react with the closely related salmon parasites Dermocystidium salmonis or Sphaerothecum destruens. Sensitivity and specificity of the PCR test used on somatic muscle and heart tissue for detecting infected fish were determined using 334 Chinook salmon collected from the Yukon River at 2 locations (Tanana and Emmonak) in 2003 and 2004. The true infection status of the fish was determined by testing somatic muscle, heart and kidney tissue using histological evaluation, culture, and PCR. The severity of infection was grouped into 2 categories, light and heavy infection. The probability of detecting a heavily infected fish (sensitivity of the test) was generally much higher than the probability of detecting light infection, suggesting that more than one tissue and/or method should be used to accurately detect light or early infection by I. hoferi. The probability of correctly identifying a negative fish (specificity of the test) was always greater than 94% regardless of the tissue used, infection severity, sampling site or year of collection.

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.

Visceral nodular cartilaginous metaplasia in rockfishes (Sebastes spp.) in the eastern North Pacific Ocean.

Multiple, discrete, nodular foci of cartilaginous metaplasia were found in the spleens and kidneys of rockfishes taken from the northeastern Pacific Ocean during a survey to determine the incidence and the nature of diseases in these animals. These nodules sometimes occurred in association with granulomatous inflammation and distinct granulomas. Many of these fish were infected by Ichythophonus spp. or acid-fact bacteria (presumably Mycobacteria spp.). Some of the metaplastic foci contained encapsulated accumulations of eosinophilic vesicles and basophilic granular debris, described by other authors as "cysts of unknown etiology," which have been observed at different sites in a variety of temperate and tropical fish species.

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.

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).

Environmental stresses and skeletal deformities in fish from the Willamette River, Oregon.

The Willamette River, one of 14 American Heritage Rivers, flows through the most densely populated and agriculturally productive region of Oregon. Previous biological monitoring of the Willamette River detected elevated frequencies of skeletal deformities in fish from certain areas of the lower (Newberg pool [NP], rivermile [RM] 26 - 55) and middle (Wheatland Ferry [WF], RM 72 - 74) river, relative to those in the upper river (Corvallis [CV], RM 125-138). The objective of this study was to determine the likely cause of these skeletal deformities. In 2002 and 2003, deformity loads in Willamette River fishes were 2-3 times greater at the NP and WF locations than at the CV location. There were some differences in water quality parameters between the NP and CV sites, but they did not readily explain the difference in deformity loads. Concentrations of bioavailable metals were below detection limits (0.6 - 1 microg/ L). Concentrations of bioavailable polychlorinated biphenyls (PCBs) and chlorinated pesticides were generally below 0.25 ng/L. Concentrations of bioavailable polycyclic aromatic hydrocarbons were generally less than 5 ng/L. Concentrations of most persistent organic pollutants were below detection limits in ovary/oocyte tissue samples and sediments, and those that were detected were not significantly different among sites. Bioassay of Willamette River water extracts provided no evidence that unidentified compounds or the complex mixture of compounds present in the extracts could induce skeletal deformities in cyprinid fish. However, metacercariae of a digenean trematode were directly associated with a large percentage of deformities detected in two Willamette River fishes, and similar deformities were reproduced in laboratoryfathead minnows exposed to cercariae extracted from Willamette River snails. Thus, the weight of evidence suggests that parasitic infection, not chemical contaminants, was the primary cause of skeletal deformities observed in Willamette River fish.