The zebrafish (Danio rerio) has become a very important animal model in biomedical research. In contrast with other models, such as mice, there has been relatively little documentation or control of subclinical disease in zebrafish research facilities. Several infectious and noninfectious conditions are consistently detected by histopathology in apparently healthy D. rerio. The most commonly observed infectious agent in zebrafish is Pseudoloma neurophilia, which is a microsporidian organism that targets the central nervous system, peripheral nerves, and occasionally other tissues. Mycobacteriosis, caused by Mycobacterium chelonae and other species, is also a frequent finding. Less commonly encountered agents include Pseudocapillaria tomentosa, which can cause extensive proliferative enteritis, and a myxozoan (Myxidium sp.) that inhabits the urinary tract but appears to cause few if any pathological changes. Noninfectious diseases that are often clinically unapparent in zebrafish include hepatic megalocytosis, bile and pancreatic ductal proliferation, and neoplasms of the ultimobranchial gland, gastrointestinal tract, and testis. To date, there is little information on the degree to which these conditions may impact research in subclinically affected fish, but there is reason to believe that they should be considered as potentially significant causes of nonprotocol variation in experiments. Therefore, it is imperative that research facilities monitor their stocks for the presence of these occult diseases and be aware of their existence when interpreting study results. Furthermore, for underlying disease conditions that cannot be readily eradicated, it is essential to determine the physiological and immunological changes that they elicit in zebrafish. Understanding the cause, modes of transmission, and distribution of the pathogens would provide useful information for the development of control and prevention strategies.

Pseudoloma neurophilia (Microsporidia) is a common disease of zebrafish Danio rerio, including those used as research models. We conducted a study comprised of 4 separate experiments to determine the effects of husbandry stress on preexisting and experimental P. neurophilia infections and the subsequent effects on survival, infection onset and intensity, fish growth, and reproduction. In fish (AB strain) with preexisting infections, stress or feeding cortisol significantly increased mortality over 7 wk compared to no stress or cortisol treatment. In contrast, no mortality was observed in fish (TL strain) experimentally exposed to P. neurophilia over 10 wk. A third experiment involved experimental exposure of AB fish to P. neurophilia and exposure to crowding and handling stressors. No mortality was associated with P. neurophilia regardless of stress treatment over a period of 20 wk. However, the onset of infection occurred sooner in stress-treated fish. Stress significantly increased the mean intensity of infection (described as xenoma area/spinal cord area in histological sections) at Week 20 post-exposure (PE). In fish with preexisting infections, myositis was significantly greater in stressed and cortisol-treated fish than those not stressed. With experimental exposure of AB fish, stressed and infected groups weighed significantly less than the control group at Week 20 PE. Regarding fecundity, the number of larvae hatched at 5 d post fertilization was negatively associated with mean infection intensity among P. neurophilia-infected and stressed AB fish. These experiments are the first to show empirically that P. neurophilia can be associated with reduced weight and fecundity, and that stress can exacerbate the severity of the infection.

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.