Toxicity of chlorine to zebrafish embryos.

Surface disinfection of fertilized fish eggs is widely used in aquaculture to reduce extraovum pathogens that may be released from brood fish during spawning, and this is routinely used in zebrafish Danio rerio research laboratories. Most laboratories use approximately 25 to 50 ppm unbuffered chlorine solution for 5 to 10 min. Treatment of embryos with chlorine has significant germicidal effects for many Gram-negative bacteria, viruses, and trophozoite stages of protozoa, but is less effective against cyst or spore stages of protozoa and certain Mycobacterium spp. Therefore, we evaluated the toxicity of unbuffered and buffered chlorine solutions to embryos exposed at 6 or 24 h post-fertilization (hpf) to determine whether higher concentrations can be used for treating zebrafish embryos. Most of our experiments entailed using an outbred line (5D), with both mortality and malformations as endpoints. We found that 6 hpf embryos consistently were more resistant than 24 hpf embryos to the toxic effects of chlorine. Chlorine is more toxic and germicidal at lower pH, and chlorine causes elevated pH. Consistent with this, we found that unbuffered chlorine solutions (pH ca. 8-9) were less toxic at corresponding concentrations than solutions buffered to pH 7. Based on our findings here, we recommend treating 6 hpf embryos for 10 min and 24 hpf embryos for 5 min with unbuffered chlorine solution at 100 ppm.

A retrospective study of the prevalence and classification of intestinal neoplasia in zebrafish (Danio rerio).

For over a decade, spontaneous intestinal neoplasia has been observed in zebrafish (Danio rerio) submitted to the ZIRC (Zebrafish International Resource Center) diagnostic service. In addition, zebrafish displayed preneoplastic intestinal changes including hyperplasia, dysplasia, and enteritis. A total of 195 zebrafish, representing 2% of the total fish submitted to the service, were diagnosed with these lesions. Neoplastic changes were classified either as adenocarcinoma or small cell carcinoma, with a few exceptions (carcinoma not otherwise specified, tubular adenoma, and tubulovillous adenoma). Tumor prevalence appeared similarly distributed between sexes and generally occurred in zebrafish greater than 1 year of age, although neoplastic changes were observed in fish 6 months of age. Eleven lines displayed these preneoplastic and neoplastic changes, including wild-types and mutants. Affected zebrafish originated from 18 facilities, but the majority of fish were from a single zebrafish research facility (hereafter referred to as the primary facility) that has submitted numerous samples to the ZIRC diagnostic service. Zebrafish from the primary facility submitted as normal sentinel fish demonstrate that these lesions are most often subclinical. Fish fed the diet from the primary facility and held at another location did not develop intestinal lesions, indicating that diet is not the etiologic agent.

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.