Transmission of a common intestinal neoplasm in zebrafish by cohabitation.

Intestinal neoplasms are common in zebrafish (Danio rerio) research facilities. These tumours are most often seen in older fish and are classified as small cell carcinomas or adenocarcinomas. Affected fish populations always contain subpopulations with preneoplastic lesions, characterized by epithelial hyperplasia or inflammation. Previous observations indicated that these tumours are unlikely caused by diet, water quality or genetic background, suggesting an infectious aetiology. We performed five transmission experiments by exposure of naïve fish to affected donor fish by cohabitation or exposure to tank effluent water. Intestinal lesions were observed in recipient fish in all exposure groups, including transmissions from previous recipient fish, and moribund fish exhibited a higher prevalence of neoplasms. We found a single 16S rRNA sequence, most similar to Mycoplasma penetrans, to be highly enriched in the donors and exposed recipients compared to unexposed control fish. We further tracked the presence of the Mycoplasma sp. using a targeted PCR test on individual dissected intestines or faeces or tank faeces. Original donor and exposed fish populations were positive for Mycoplasma, while corresponding unexposed control fish were negative. This study indicates an infectious aetiology for these transmissible tumours of zebrafish and suggests a possible candidate agent of a Mycoplasma species.

A method for collecting eggs of Pseudocapillaria tomentosa (Nematoda: Capillariidae) from zebrafish Danio rerio and efficacy of heat and chlorine for killing the nematode's eggs.

Pseudocapillaria tomentosa is a common pathogen of zebrafish (Danio rerio) in research facilities. We developed a method to collect and concentrate the nematode eggs using a modified sugar centrifugation method and documented their normal development. Embryonating stages with blastomere formation followed by elongation of the embryo prior to larva formation cumulated in developed larvae inside the eggs and hatching after 5-10 day. We then evaluated the efficacy of heat and chlorine to kill them based on a larva development assay. Eggs were exposed to 40, 50, 60 °C for 30 min and 1 h. Chlorine treatment was performed at 100, 250, 500, 1000, 3000 and 6000 ppm for 10 min. Samples exposed to 40 °C for 30 min or 1 h showed incidences of larvated eggs similar to controls. In contrast, no larvation occurred with eggs exposed to either 50 or 60 °C for 30 min or 1 h. Remarkably, in repeated assays, samples exposed to low doses of chlorine (100, 250, 500 and 1000 ppm for 10 min) showed significantly higher incidence of larvation than controls. Eggs treated with 3000 ppm for 10 min did not develop larvae, and no eggs were found after 6000 ppm treatment.

Husbandry stress exacerbates mycobacterial infections in adult zebrafish, Danio rerio (Hamilton).

Mycobacteria are significant pathogens of laboratory zebrafish, Danio rerio (Hamilton). Stress is often implicated in clinical disease and morbidity associated with mycobacterial infections but has yet to be examined with zebrafish. The aim of this study was to examine the effects of husbandry stressors on zebrafish infected with mycobacteria. Adult zebrafish were exposed to Mycobacterium marinum or Mycobacterium chelonae, two species that have been associated with disease in zebrafish. Infected fish and controls were then subjected to chronic crowding and handling stressors and examined over an 8-week period. Whole-body cortisol was significantly elevated in stressed fish compared to non-stressed fish. Fish infected with M. marinum ATCC 927 and subjected to husbandry stressors had 14% cumulative mortality while no mortality occurred among infected fish not subjected to husbandry stressors. Stressed fish, infected with M. chelonae H1E2 from zebrafish, were 15-fold more likely to be infected than non-stressed fish at week 8 post-injection. Sub-acute, diffuse infections were more common among stressed fish infected with M. marinum or M. chelonae than non-stressed fish. This is the first study to demonstrate an effect of stress and elevated cortisol on the morbidity, prevalence, clinical disease and histological presentation associated with mycobacterial infections in zebrafish. Minimizing husbandry stress may be effective at reducing the severity of outbreaks of clinical mycobacteriosis in zebrafish facilities.

Biochemical, molecular, and virulence characteristics of select Mycobacterium marinum isolates in hybrid striped bass Morone chrysops x M. saxatilis and zebrafish Danio rerio.

A panel of 15 Mycobacterium marinum isolates was characterized by biochemical tests, sequencing the ribosomal DNA intergenic spacer (ITS) region and the heat shock protein 65 gene (hsp65) and pulsed-field gel electrophoresis (PFGE). The biochemical characteristics of all isolates were similar, except for Tween 80 hydrolysis. DNA sequence of hsp65 for a subset of isolates were identical; however, at position 5 of the ITS rDNA, a single nucleotide polymorphism was identified. Isolates possessing a guanine residue at this position (G strains) were unable to hydrolyze Tween 80, while isolates that contained an adenine residue at this position (A strains) were positive for Tween 80 hydrolysis. PFGE successfully discriminated between the G and A strains; all G strains had identical AseI restriction enzyme-cutting patterns while the A strains exhibited a variety of cutting patterns. Eight isolates (4 G and 4 A strains) were further characterized for virulence by experimental infection of hybrid striped bass (HSB) Morone chrysops x M. saxatilis and zebrafish Danio rerio. Seven of the 8 strains produced cumulative mortality ranging from 13.3 to 83.3% in the HSB virulence trial. The M. marinum reference strain ATCC 927T did not produce mortality in HSB. HSB exposed to the G strains had significantly higher cumulative mortality than those exposed to the A strains. When these same isolates were tested in zebrafish, 6 of the 8 strains caused 100% cumulative mortality, with 2 of the A strains being the most pathogenic. In zebrafish, however, ATCC 927T was virulent and produced 28.5% mortality. Collectively, we conclude that the M. marinum G strains are unique and may represent a distinct virulence phenotype in HSB, but this trend was not consistent in zebrafish.

In vivo studies of gene expression via transient transgenesis using lipid-DNA delivery.

As the sequencing of the human genome proceeds, the need for a new screen for in vivo function is becoming apparent. Many investigators are turning to various transgenic models as a means of studying function. However, these approaches are very time consuming, with a transgene-expressing mouse model often taking months to establish. We have developed an efficient system for delivering genes in vivo, which allows the gene product to be studied as early as 24 h after introduction into the mouse model. The delivery system employs a novel cationic lipid, 1-[2-(9-(Z)-octadecenoyloxy)ethyl]-2-(8-(Z)-heptadecenyl)-3- (hydroxyethyl)imidazolinium chloride (DOTIM), and a neutral lipid, cholesterol, complexed with an expression vector containing the reporter gene chloramphenicol acetyl transferase (CAT). After a single intravenous injection of these complexes, several tissues were seen to express the transgene. High, persistent expression in the vascular endothelial cells in the mouse lung was obtained. Delivery of DNA in vivo has been evaluated by quantitative polymerase chain reaction and protein expression by CAT activity assays. In vivo studies showed reproducible expression in more than 500 mice injected via the tail vein. An early peak of expression was followed by lower, but sustained, expression for > 50 days. Transgene expression of CAT could also be identified by immunohistochemistry staining in mouse lung and appeared to be located within the capillaries. The pattern of in vivo expression could be modulated and targeted to specific organs by altering the lipid-DNA formulation. New expression vectors with altered introns and polyadenylation sites further improved expression. The expression reported here may be sufficient in magnitude, duration, and flexibility to be an attractive alternative, in some cases, to establishing transgenic animals by stable gene transfer.