Transmission of Mycobacterium chelonae and Mycobacterium marinum in laboratory zebrafish through live feeds.

The zebrafish (Danio rerio) is a popular vertebrate model organism used in a wide range of research fields. Mycobacteriosis, caused by Mycobacterium species, is particularly concerning because it is a common disease associated with chronic infections in these fish. Infections are also a source of uncontrolled experimental variance that may influence research results. Live feeds for zebrafish are common and include paramecia (Paramecium caudatum), brine shrimp (Artemia franciscana) and rotifers (Branchionus spp.). Although nutritionally beneficial, live feeds may pose a biosecurity risk. In this study, we investigate transmission of Mycobacterium chelonae and Mycobacterium marinum through these three live feeds. We show that all three live feeds ingest both M. marinum and M. chelonae and can transmit mycobacterial infections to zebrafish. This observation emphasizes the need for live feeds to be included in the consideration of potential biosecurity risks. This study is of importance to other beyond the zebrafish community, including those of additional aquatic models and those using live feeds for other types of aquaculture.

Perturbing the developing skull: using laser ablation to investigate the robustness of the infraorbital bones in zebrafish (Danio rerio).

BACKGROUND: The development of the craniofacial skeleton from embryonic mesenchyme is a complex process that is not yet completely understood, particularly for intramembranous bones. This study investigates the development of the neural crest derived infraorbital (IO) bones of the zebrafish (Danio rerio) skull. Located under the orbit, the IO bones ossify in a set sequence and are closely associated with the lateral line system. We conducted skeletogenic condensation and neuromast laser ablation experiments followed by shape analyses in order to investigate the relationship between a developing IO bone and the formation of the IO series as well as to investigate the highly debated inductive potential of neuromasts for IO ossification. RESULTS: We demonstrate that when skeletogenic condensations recover from laser ablation, the resulting bone differs in shape compared to controls. Interestingly, neighbouring IO bones in the bone series are unaffected. In addition, we show that the amount of canal wall mineralization is significantly decreased following neuromast laser ablation at juvenile and larval stages. CONCLUSIONS: These results highlight the developmental robustness of the IO bones and provide direct evidence that canal neuromasts play a role in canal wall development in the head. Furthermore, we provide evidence that the IO bones may be two distinct developmental modules. The mechanisms underlying developmental robustness are rarely investigated and are important to increase our understanding of evolutionary developmental biology of the vertebrate skull.

Assessment of Various Standard Fish Diets on Growth and Fecundity of Platyfish (Xiphophorus maculatus) and Medaka (Oryzias latipes).

Several small freshwater fish species are utilized as models for human conditions and disease in biomedical research. Research animal diets are generally tailored to optimize growth, fecundity, and produce healthy research animals. However, a lack of reference diets presents a barrier in comparative studies between aquatic animal models and even among laboratories using the same species. Therefore, the objective of this study was to determine feeding regime and dietary effects on growth and fecundity in two commonly used freshwater fish, platyfish and medaka. From 1 through 6 months of age, platyfish and medaka were fed one of three feeding regime/diets: (1) our custom feeding regime consists of commercial flake food, beef liver paste, and live brine shrimp (CON); (2) a commercially available zebrafish diet, Gemma (GEM); and (3) a laboratory defined reference feeding regime (WAT). Weight, size, brood numbers, and survival rates for both species were measured monthly. Numbers of platyfish fry and hatch rate of medaka embryos were also determined. We observed that custom feeding regime (CON) fed platyfish and medaka grew larger, exhibited a higher survival rate, and had higher fecundity than WAT or GEM fed fish. These observations suggest that diets and regimes designed for zebrafish are not optimal to maintain platyfish or medaka. Thus, base diets, with clearly defined components and regimes, need to be developed with compositions that can be adjusted in a species-specific manner.

Source or Sink: Examining the Role of Biofilms in Transmission of Mycobacterium spp. in Laboratory Zebrafish.

Zebrafish health is a primary research concern because diseases can have unintended impacts on experimental endpoints. Ideally, research would be conducted using disease-free fish or fish with known disease status. Mycobacteriosis is a common bacterial disease in wild and captive fishes, including zebrafish. Despite its prevalence, the dynamics of transmission and potential sources of mycobacterial infections in zebrafish are only partially understood. One suspected natural infection source is surface biofilms on tanks and other system components. This study investigates the role that tank biofilms play in mycobacteriosis in laboratory zebrafish by evaluating the establishment of biofilms from bacteria shed from fish, and conversely, the acquisition of infections in fish from surface biofilms. We found that zebrafish infected with Mycobacterium chelonae shed bacteria through feces, and bacteria are transmitted to tank biofilms from one to 16 weeks postinfection. We also found that zebrafish acquire M. chelonae infections as soon as 2 weeks when introduced to tanks with established M. chelonae biofilms. The results from this study highlight the role that tank biofilms play as both a reservoir and source of mycobacterial infections in zebrafish. Results support the inclusion of biofilm surveillance and prevention as part of a disease control program in zebrafish research facilities.

Inflammation-associated gene expression in RAW 264.7 macrophages induced by toxins from fungi common on damp building materials.

Most fungi that grow on damp building materials produce low molecular weight compounds, some of which are known to be toxic. In this study, we tested the hypothesis that exposure to some metabolites of fungi common on damp building materials would result in time-, dose-, and compound-specific responses in the production of various chemokines by RAW 264.7 cells. Cell cultures were exposed to a 10-7M or 10-8M metabolite dose for 2, 4, 8 or 24h. Metabolite concentrations used were based on those that might be expected in alveolar macrophages due to inhalation exposure from living or working in a damp building. Compared to controls, exposure provoked significant time-, dose- and compound-specific responses manifest as differentially elevated secretion of three of nine cytokines tested in culture supernatant of treated cells. The greatest number of cytokines produced in response to the metabolites tested were in andrastin A-treated cells (GM-CSF, TGFß1, Tnf-α) followed by koninginin A (TGFß1 and Tnf-α) and phomenone (GM-CSF, TGFß1). Chaetoglobosin A, chaetomugilin D and walleminone exposures each resulted in significant time-specific production of Tnf-α only. This investigation adds to a body of evidence supporting the role of low molecular weight compounds from damp building materials as pathogen associated molecular patterns (PAMPs). Along with fungal glucan and chitin, these compounds contribute to the non-allergy based respiratory outcomes for people living and working in damp buildings.

Zebrafish Embryo Disinfection with Povidone-Iodine: Evaluating an Alternative to Chlorine Bleach.

Mycobacteriosis is a common bacterial infection in laboratory zebrafish caused by several different species and strains of Mycobacterium, including both rapid and slow growers. One control measure used to prevent mycobacterial spread within and between facilities is surface disinfection of eggs. Recent studies have highlighted the effectiveness of povidone-iodine (PVPI) on preventing propagation of Mycobacterium spp. found in zebrafish colonies. We evaluated the effect of disinfection using 12.5-50 ppm PVPI (unbuffered and buffered) on zebrafish exposed at 6 or 24 h postfertilization (hpf) to determine if this treatment is suitable for use in research zebrafish. Our results show that 6 hpf embryos are less sensitive to treatment as fewer effects on mortality, developmental delay, and deformity were observed. We also found that buffered PVPI treatment results in a greater knockdown of Mycobacterium chelonae and Mycobacterium marinum, as well as results in decreased harmful effects on embryos. Treatments of shorter (2 min vs. 5 min) duration were also more effective at killing mycobacteria in addition to resulting in fewer effects on embryo health. In addition, we compared the efficacy of a rinsing regimen to rinsing and disinfecting. Based on the findings of this study, we recommend disinfecting embryos for 2 min with buffered PVPI at 12.5-25 ppm.

Activity of Antibiotics against Mycobacterium Species Commonly Found in Laboratory Zebrafish.

The Zebrafish Danio rerio is a popular vertebrate model organism used in a wide range of research fields. Importance is placed on Zebrafish health and the maintenance of disease-free laboratory fish so that experimental studies are not inadvertently affected. Mycobacteriosis, a common infection of laboratory Zebrafish, is caused by several Mycobacterium species. Little is known regarding the potential of antibiotic treatment for Zebrafish mycobacteriosis; however, treatment of infected Zebrafish may be appropriate to maintain valuable strains. Here, we investigated, in vitro, the antibiotic susceptibility of both rapid- and slow-growing isolates of Mycobacterium species from laboratory Zebrafish. Antibiotic testing was carried out using a commercially available 96-well microtiter plate format. Results indicated that some but not all antibiotics tested were effective at inhibiting mycobacterial growth and that susceptibility varied among species and strains. Tigecycline, tobramycin, clarithromycin, and amikacin were most effective at broad inhibition of rapid-growing mycobacteria; whereas, amikacin, clarithromycin, and rifampin were effective at inhibiting all slow-growing M. marinum strains tested. Results support the potential for targeted antibiotic treatment of Zebrafish infected with mycobacteria, but additional testing should be carried out in vivo.

Evaluating the effectiveness of common disinfectants at preventing the propagation of Mycobacterium spp. isolated from zebrafish.

Mycobacteriosis is a bacterial disease that is common in captive, wild and research fish. There is no one causative agent of mycobacteriosis, as several strains and species of Mycobacterium have been identified in zebrafish. With increased usage and investment in wild-type and mutant zebrafish strains, considerable value is placed on preserving zebrafish health. One control measure used to prevent mycobacterial spread within and between zebrafish facilities is egg disinfection. Here we investigate the effectiveness of three disinfectants [chlorine bleach, hydrogen peroxide, and povidone-iodine (PVPI)] commonly included in egg disinfection protocols for laboratory fish as well as aquaculture fish and compare the knockdown effect of these treatments on Mycobacterium spp. in vitro. Despite current usage, comparison of these disinfection regimes' abilities to prevent mycobacterial growth has not been tested. We found that the germicidal effect of different disinfectants varies by Mycobacterium spp. Hydrogen peroxide was the least effective disinfectant, followed by unbuffered chlorine bleach, which is commonly used to disinfect embryos in zebrafish facilities. Disinfection with 25ppm PVPI for 5min was very effective, and may be an improved alternative to chlorine bleach for embryo disinfection. Results from this study can be utilized by laboratory fish facilities in order to prevent the spread of mycobacteriosis in research fish.