Early Development and Tissue Distribution of Pseudoloma neurophilia in the Zebrafish, Danio rerio

Abstract

The early proliferative stages of the microsporidian parasite, Pseudoloma neurophilia were visualized in larval zebrafish, Danio rerio, using histological sections with a combination of an in situ hybridization probe specific to the P. neurophilia small-subunit ribosomal RNA gene, standard hematoxylin-eosin stain, and the Luna stain to visualize spores. Beginning at 5 days post fertilization, fish were exposed to P. neurophilia and examined at 12, 24, 36, 48, 72, 96, and 120 h post exposure (hpe). At 12 hpe, intact spores in the intestinal lumen and proliferative stages developing in the epithelial cells of the anterior intestine and the pharynx and within hepatocytes were observed. Proliferative stages were visualized in the pancreas and kidney at 36 – 48 hpe and in the spinal cord, eye, and skeletal muscle beginning at 72 hpe. The first spore stages of P. neurophilia were observed at 96 hpe in the pharyngeal epithelium, liver, spinal cord, and skeletal muscle. The parasite was only observed in the brain of larval fish at 120 hpe. The distribution of the early stages of P. neurophilia and the lack of mature spores until 96 hpe indicates that the parasite gains access to organs distant from the initial site of entry, likely by penetrating the intestinal wall with the polar tube.

Pseudoloma neurophilia: A Retrospective and Descriptive Study of Nervous System and Muscle Infections, with New Implications for Pathogenesis and Behavioral Phenotypes

“…Furthermore, Sanders et al 3 and Peterson et al 18 showed that light P. neurophilia infections, particularly those in the viscera, frequently require special staining to highlight small numbers of spores. Because this was a retrospective study ranging from 1999 to 2013 and because the Luna stain was not used for zebrafish diagnotics until *2008, we focused on neural and muscular infections observable by H&E staining alone.…”

“…This pattern of intravertebral infection could be explained if spores, consumed when the fish are larvae (or even as embryos in the case of vertical transmission), launch their polar tubules and hit the notochord. 3 This would allow for both the spread of the microsporidium throughout notochord cells and their retention within the notochord remnants of adjacent vertebrae in adult fish.…”

“…1,2 It has an overwhelming preference for neural tissue, particularly the nerve roots, spinal cord, and hindbrain. 3 First described in 2001, P. neurophilia has since been identified as one of the most common infectious organisms diagnosed in laboratory zebrafish. Clinical infection is most frequently characterized by emaciation and spinal deformities.…”

“…neurophilia has been extensively studied with regard to life cycle, transmission, microscopic and ultrastructural features, as well as the effects of stress on infection. [3][4][5][6]9,11 Zebrafish are now extensively used in behavior research, [12][13][14][15][16][17] and it is possible that these neural infections by P. neurophilia may introduce nonprotocol-induced variation to these experiments.…”

The common neural parasite Pseudoloma neurophilia is associated with altered startle response habituation in adult zebrafish (Danio rerio): Implications for the zebrafish as a model organism

“…P. neurophilia is spread primarily through the consumption of environmentally-resistant spores, either through scavenging of infected carcasses or through consumption of free spores released with eggs during spawning. Alternatively, spores can be transmitted vertically, as infections have been observed in both eggs and newly-hatched larvae [20–21]. P. neurophilia spores are a particular problem for zebrafish facilities because they can survive bleaching at a concentration of 25–50ppm, which is the standard concentration used in most facilities for embryo sterilization [22].…”