Effects of Stocking Density on Stress Response and Susceptibility to Infectious Hematopoietic Necrosis Virus in Rainbow Trout.

The goals of this study were to examine the effect of stocking density on the stress response and disease susceptibility in juvenile rainbow trout (Oncorhynchus mykiss). Fish were sorted into one of 2 stocking densities (high density "HD", 20-40 kg/m³) or (low density, "LD", 4-8 kg/m³) and 3 stress indices (cortisol levels in serum and water, and neutrophil: lymphocyte (N:L) ratios from blood smears) were measured at multiple time points over 21 d. Serum cortisol was significantly increased at 1 h in LD samples and at 14 d in HD samples. Water cortisol concentrations were significantly higher in LD tanks as compared with HD tanks on day 14. N:L ratios were significantly higher in HD tanks on day 14 as compared with LD tanks and with baseline. The effect of stocking density on mortality after exposure to infectious hematopoietic necrosis virus (IHNV) was compared between fish held in HD or LD conditions, with or without prior acclimation to the different density conditions. No significant differences in survival were found between HD and LD treatments or between acclimated and nonacclimated treatments. Cumulative results indicate that 1) 1 to 4 gram rainbow trout did not generally demonstrate significant differences in stress indices at the density conditions tested over a 21-d period, 2) independent differences were found in 3 stress indices at day 14 after sorting into LD and HD holding conditions; and 3) LD and HD stocking densities did not have a significant effect on mortality due to IHNV.

Efficacy of cleaning and disinfection procedures in a zebrafish (Danio rerio) facility.

Appropriate cleaning and disinfection procedures in zebrafish (Danio rerio) laboratories are crucial in preventing the spread of aquatic animal pathogens and minimizing the build-up of waste products and biologic matter. The procedures selected should accomplish these goals and incorporate the individual needs of various laboratories. In this study of a single zebrafish facility, we assessed the efficacy of 2 different cleaning and disinfection procedures for nets, tanks, and lids. ATP levels were used as a surrogate biomarker for microbial burden. We measured the number of relative light units (RLU), as an expression of the amount of ATP present, on items before and after disinfection and calculated the percentage reduction. We compared daily replacement of a commercial net disinfection product in J lab with weekly replacement in H lab and found a 96.6% reduction in RLU in H lab and a 91.2% reduction in J lab. These results indicate that either replacement schedule is effective. Evaluation of tanks and lids soaked in a bleach disinfection bath for 30 or 60 min revealed a 99.7% reduction in RLU at 30 min compared with 97.1% at 60 min. Therefore a 30-min soak in a bleach bath achieved a similar level of disinfection as did a 60-min soak. The current results demonstrate that these cleaning and disinfection methods are efficacious.

Host immune response and acute disease in a zebrafish model of Francisella pathogenesis.

Members of the bacterial genus Francisella are highly virulent and infectious pathogens. New models to study Francisella pathogenesis in evolutionarily distinct species are needed to provide comparative insight, as the mechanisms of host resistance and pathogen virulence are not well understood. We took advantage of the recent discovery of a novel species of Francisella to establish a zebrafish/Francisella comparative model of pathogenesis and host immune response. Adult zebrafish were susceptible to acute Francisella-induced disease and suffered mortality in a dose-dependent manner. Using immunohistochemical analysis, we localized bacterial antigens primarily to lymphoid tissues and livers of zebrafish following infection by intraperitoneal injection, which corresponded to regions of local cellular necrosis. Francisella sp. bacteria replicated rapidly in these tissues beginning 12 h postinfection, and bacterial titers rose steadily, leveled off, and then decreased by 7 days postinfection. Zebrafish mounted a significant tissue-specific proinflammatory response to infection as measured by the upregulation of interleukin-1beta (IL-1beta), gamma interferon, and tumor necrosis factor alpha mRNA beginning by 6 h postinfection and persisting for up to 7 days postinfection. In addition, exposure of zebrafish to heat-killed bacteria demonstrated that the significant induction of IL-1beta was highly specific to live bacteria. Taken together, the pathology and immune response to acute Francisella infection in zebrafish share many features with those in mammals, highlighting the usefulness of this new model system for addressing both general and specific questions about Francisella host-pathogen interactions via an evolutionary approach.

Susceptibility of zebrafish (Danio rerio) to a model pathogen, spring viremia of carp virus.

To improve our understanding of the genetic basis of fish disease, we developed a pathogen model, using zebrafish (Danio rerio) and spring virema of carp virus (SVCV). Replicate groups of 10 fish were acclimated to 20 or 24 degrees C, then were exposed to SVCV concentrations of 10(3) to 10(5) plaque-forming units per milliliter (PFU/ml) of water and observed daily. In a second trial, fish were acclimated to 15 degrees C, and replicate groups of 10 fish were exposed to SVCV at a concentration of 10(5) PFU/ml; however, the temperature was raised 1 degrees C/wk. Moribund fish were collected for histologic examination, and dead fish were assayed for virus by use of cell culture and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Mortality exceeded 50% in fish exposed to 10(5) PFU of SVCV/ml at the lower temperatures. Clinical signs of disease became evident seven days after viral exposure and were observed most consistently in fish of the 10(5) PFU/ml groups. Affected zebrafish were anorectic and listless, with epidermal petechial hemorrhages followed by death. Use of plaque assays and RT-PCR analysis confirmed presence of SVCV at titers > or = 10(4) PFU/g of tissue. Histologic lesions included multifocal brachial necrosis and melanomacrophage proliferation in gills, liver, and kidneys. These results indicate that zebrafish are susceptible to infection by SVCV under conditions that mimic a natural route of exposure.