Tracy S. Peterson

H. pylori virulence factor CagA increases intestinal cell proliferation by Wnt pathway activation in a transgenic zebrafish model

SUMMARY Infection with Helicobacter pylori is a major risk factor for the development of gastric cancer, and infection with strains carrying the virulence factor CagA significantly increases this risk. To investigate the mechanisms by which CagA promotes carcinogenesis, we generated transgenic zebrafish expressing CagA ubiquitously or in the anterior intestine. Transgenic zebrafish expressing either the wild-type or a phosphorylation-resistant form of CagA exhibited significantly increased rates of intestinal epithelial cell proliferation and showed significant upregulation of the Wnt target genes cyclinD1, axin2 and the zebrafish c-myc ortholog myca. Coexpression of CagA with a loss-of-function allele encoding the β-catenin destruction complex protein Axin1 resulted in a further increase in intestinal proliferation. Coexpression of CagA with a null allele of the key β-catenin transcriptional cofactor Tcf4 restored intestinal proliferation to wild-type levels. These results provide in vivo evidence of Wnt pathway activation by CagA downstream of or in parallel to the β-catenin destruction complex and upstream of Tcf4. Long-term transgenic expression of wild-type CagA, but not the phosphorylation-resistant form, resulted in significant hyperplasia of the adult intestinal epithelium. We further utilized this model to demonstrate that oncogenic cooperation between CagA and a loss-of-function allele of p53 is sufficient to induce high rates of intestinal small cell carcinoma and adenocarcinoma, establishing the utility of our transgenic zebrafish model in the study of CagA-associated gastrointestinal cancers.

Pleistophora hyphessobryconis (Microsporidia) infecting zebrafish Danio rerio in research facilities

Zebrafish Danio rerio are important models for biomedical research, and thus, there is an increased concern about diseases afflicting them. Here we describe infections by Pleistophora hyphessobryconis (Microsporidia) in zebrafish from 3 laboratories. As reported in other aquarium fishes, affected zebrafish exhibited massive infections in the skeletal muscle, with no involvement of smooth or cardiac muscle. In addition, numerous spores within macrophages were observed in the visceral organs, including the ovaries. Transmission studies and ribosomal RNA (rRNA) gene sequence comparisons confirmed that the parasite from zebrafish was P. hyphessobryconis as described from neon tetra Paracheirodon innesi. Ten 15 d old zebrafish were exposed to P. hyphessobryconis collected from 1 infected neon tetra, and 7 of 10 fish became infected. Comparison of P. hyphessobryconis small subunit rRNA gene sequence from neon tetra with that obtained from zebrafish was nearly identical, with < 1% difference. Given the severity of infections, P. hyphessobryconis should be added to the list of pathogens that should be avoided in zebrafish research facilities, and it would be prudent to avoid mixing zebrafish used in research with other aquarium fishes.

Edwardsiellosis Caused by Edwardsiella ictaluri in Laboratory Populations of Zebrafish Danio rerio

We report the first cases of Edwardsiella ictaluri causing epizootics in laboratory populations of Zebrafish Danio rerio. Edwardsiella ictaluri is primarily recognized as a disease of catfish species and is known to cause an economically important bacterial disease of farm-raised catfish in the USA and abroad; however, it has been isolated on occasion from 10 other genera of nonictalurid fishes. We isolated E. ictaluri from moribund Zebrafish held in quarantine at two different universities in two states and from a research facility in a third state between February 23 and December 6, 2011. Edwardsiellosis in Zebrafish can be described as a severe systemic disease characterized by tissue necrosis and the presence of large numbers of extracellular and intracellular bacteria, often within macrophages. The kidneys (pronephros and mesonephros), spleen, nares, and forebrain were the most commonly and severely affected tissues. In outbreaks, mortality was acute and numerous fish died over a 1-2 week period. Mortality continued until the majority of the population was lost, at which time the remaining fish were euthanized. In addition to these cases, four cultures of bacteria isolated from Zebrafish by another diagnostic laboratory were submitted to the Louisiana Aquatic Diagnostic Laboratory for identification and were confirmed as E. ictaluri. In total, eight cultures of E. ictaluri from Zebrafish from Louisiana, Massachusetts, Pennsylvania, and Florida were identified. The isolates were confirmed as E. ictaluri by biochemical phenotype, API 20E (bioMérieux), and amplification and sequencing of a portion of the 16S rRNA gene. Edwardsiella ictaluri isolates from Zebrafish are believed to comprise a unique group and were differentiated from catfish isolates by exhibiting weaker motility, autoaggregation in broth, a different plasmid profile (two plasmids of 4.0 and 3.5 kb), a different API 20E code (4204000), and lack of lipopolysaccharide recognition with Mab Ed9.

Luna stain, an improved selective stain for detection of microsporidian spores in histologic sections.

Microsporidia in histologic sections are most often diagnosed by observing spores in host tissues. Spores are easy to identify if they occur in large aggregates or xenomas when sections are stained with hematoxylin and eosin (H&E). However, individual spores are not frequently detected in host tissues with conventional H&E staining, particularly if spores are scattered within the tissues, areas of inflammation, or small spores in nuclei (i.e. Nucleospora salmonis). Hence, a variety of selective stains that enhance visualization of spores is recommended. We discovered that the Luna stain, used to highlight eosinophils, red blood cells, and chitin in arthropods and other invertebrates, also stains spores of Pseudoloma neurophilia. We compared this stain to the Gram, Fites acid fast, Giemsa, and H&E stains on 8 aquatic microsporidian organisms that were readily available in our 2 laboratories: Loma salmonae, Glugea anomala, Pseudoloma neurophilia, Pleistophora hyphessobryconis, Pleistophora vermiformis, Glugea sp., Steinhausia mytilovum, and an unidentified microsporidian from UK mitten crabs Eriocheir sinensis. Based on tinctorial properties and background staining, the Luna stain performed better for detection of 6 of the 8 microsporidia. Gram stain was superior for the 2 microsporidia from invertebrates: S. mytilovum and the unidentified microsporidian from E. sinensis.

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.

Neoplasia and Neoplasm Associated Lesions in Laboratory Colonies of Zebrafish Emphasizing Key Influences of Diet and Aquaculture System Design

During the past decade, the zebrafish has emerged as a leading model for mechanistic cancer research because of its sophisticated genetic and genomic resources, its tractability for tissue targeting of transgene expression, its efficiency for forward genetic approaches to cancer model development, and its cost effectiveness for enhancer and suppressor screens once a cancer model is established. However, in contrast with other laboratory animal species widely used as cancer models, much basic cancer biology information is lacking in zebrafish. As yet, data are not published regarding dietary influences on neoplasm incidences in zebrafish. Little information is available regarding spontaneous tumor incidences or histologic types in wild-type lines of zebrafish. So far, a comprehensive database documenting the full spectrum of neoplasia in various organ systems and tissues is not available for zebrafish as it is for other intensely studied laboratory animal species. This article confirms that, as in other species, diet and husbandry can profoundly influence tumor incidences and histologic spectra in zebrafish. We show that in many laboratory colonies wild-type lines of zebrafish exhibit elevated neoplasm incidences and neoplasm-associated lesions such as heptocyte megalocytosis. We present experimental evidence showing that certain diet and water management regimens can result in high incidences of neoplasia and neoplasm-associated lesions. We document the wide array of benign and malignant neoplasms affecting nearly every organ, tissue, and cell type in zebrafish, in some cases as a spontaneous aging change, and in other cases due to carcinogen treatment or genetic manipulation.

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 ciliates 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.

Investigating the impact of chronic atrazine exposure on sexual development in zebrafish.

Atrazine (ATZ) is a selective triazine herbicide used primarily for preemergent weed control in corn, sorghum, and sugar cane production. It is one of the most widely used herbicides in North America. Some research published over the last decade suggests that chronic exposure to environmentally relevant ATZ concentrations can adversely impact gonadal development and/or sexual differentiation in amphibians and fish, while other studies report no effect, or moderate effects. As a result, contrasting conclusions have been published regarding the potential effects of the herbicide ATZ on aquatic species. Two near-identical 4-month studies in 2009 (Study I) and 2010 (Study II) were performed investigating the potential for chronic ATZ exposure to affect zebrafish (Danio rerio) sexual development and differentiation. Zebrafish were chronically exposed to 0, 0.1, 1, 10 μM ATZ or 1 nM 17β-estradiol (E2). Fish were histologically examined to assign gender and to evaluate potential impacts of E2 or ATZ on gonadal development. Exposure to E2 consistently resulted in a significantly higher proportion of female fish to normal male fish when compared to unexposed fish (both studies). In both studies, ATZ exposure did not significantly influence the percentage of female or male fish when compared to unexposed fish. A greater percentage of abnormally developed male fish and fish lacking differentiated gonadal tissue was observed in Study II E2 exposures but not in ATZ exposures. Together, these studies indicate that long-term exposure to ATZ at or above environmentally relevant concentrations does not significantly impact zebrafish gonadal development or sexual differentiation.

Survey of Parasites In Threatened Stocks of Coho Salmon (Oncorhynchus kisutch) In Oregon By Examination of Wet Tissues and Histology

Abstract We are conducting studies on the impacts of parasites on Oregon coastal coho salmon (Oncorhynchus kistuch). An essential first step is documenting the geographic distribution of infections, which may be accomplished by using different methods for parasite detection. Thus, the objectives of the current study were to (1) identify parasite species infecting these stocks of coho salmon and document their prevalence, density, and geographic distribution; (2) assess the pathology of these infections; and (3) for the first time, determine the sensitivity and specificity of histology for detecting parasites compared with examining wet preparations for muscle and gill infections. We examined 576 fry, parr, and smolt coho salmon in total by histology. The muscle and gills of 219 of these fish also were examined by wet preparation. Fish were collected from 10 different locations in 2006–2007. We identified 21 different species of parasites in these fish. Some parasites, such as Nanophyetus salmincola and Myxobolus insidiosus, were common across all fish life stages from most basins. Other parasites, such as Apophallus sp., were more common in underyearling fish than smolts and had a more restricted geographic distribution. Additional parasites commonly observed were as follows: Sanguinicola sp., Trichodina truttae, Epistylis sp., Capriniana piscium, and unidentified metacercariae in gills; Myxobolus sp. in brain; Myxidium salvelini and Chloromyxum majori in kidney; Pseudocapillaria salvelini and adult digenean spp. in the intestine. Only a few parasites, such as the unidentified gill metacercariae, elicted overt pathologic changes. Histology had generally poor sensitivity for detecting parasites; however, it had relatively good specificity. We recommend using both methods for studies or monitoring programs requiring a comprehensive assessment of parasite identification, enumeration, and parasite-related pathology.

Early Development and Tissue Distribution of Pseudoloma neurophilia in the Zebrafish, Danio rerio

The early proliferative stages of the microsporidian parasite, Pseudoloma neurophilia were visualized in larval zebrafish, Danio rerio, using histological sections with a combination of an in situ hybridization probe specific to the P. neurophilia small‐subunit ribosomal RNA gene, standard hematoxylin‐eosin stain, and the Luna stain to visualize spores. Beginning at 5 d post fertilization, fish were exposed to P. neurophilia and examined at 12, 24, 36, 48, 72, 96, and 120 h post exposure (hpe). At 12 hpe, intact spores in the intestinal lumen and proliferative stages developing in the epithelial cells of the anterior intestine and the pharynx and within hepatocytes were observed. Proliferative stages were visualized in the pancreas and kidney at 36–48 hpe and in the spinal cord, eye, and skeletal muscle beginning at 72 hpe. The first spore stages of P. neurophilia were observed at 96 hpe in the pharyngeal epithelium, liver, spinal cord, and skeletal muscle. The parasite was only observed in the brain of larval fish at 120 hpe. The distribution of the early stages of P. neurophilia and the lack of mature spores until 96 hpe indicates that the parasite gains access to organs distant from the initial site of entry, likely by penetrating the intestinal wall with the polar tube.