Pleistophora hyphessobryconis (Microsporidia) infecting zebrafish Danio rerio in research facilities

Abstract

Zebrafish Danio rerio are important models for biomedical research, and thus, there is an increased concern about diseases afflicting them. Here we describe infections by Pleistophora hyphessobryconis (Microsporidia) in zebrafish from 3 laboratories. As reported in other aquarium fishes, affected zebrafish exhibited massive infections in the skeletal muscle, with no involvement of smooth or cardiac muscle. In addition, numerous spores within macrophages were observed in the visceral organs, including the ovaries. Transmission studies and ribosomal RNA (rRNA) gene sequence comparisons confirmed that the parasite from zebrafish was P. hyphessobryconis as described from neon tetra Paracheirodon innesi. Ten 15 d old zebrafish were exposed to P. hyphessobryconis collected from 1 infected neon tetra, and 7 of 10 fish became infected. Comparison of P. hyphessobryconis small subunit rRNA gene sequence from neon tetra with that obtained from zebrafish was nearly identical, with < 1% difference. Given the severity of infections, P. hyphessobryconis should be added to the list of pathogens that should be avoided in zebrafish research facilities, and it would be prudent to avoid mixing zebrafish used in research with other aquarium fishes.

Figure 1

Zebrafish infected with P. hyphessobryconis. Upper image shows mottled appearance with light areas (arrows) on flanks. Lower image is same fish with skin removed, exhibiting opaque regions in muscle (arrows) representing massive infection. Bar = 500 μm.

Figure 2

Wet mount of P. hyphessobryconis spores. Note prominent posterior vacuole (arrow). Bar = 10 μm.

Figure 3

Histological sections of zebrafish infected with P. hyphessobryconis. A. Numerous sporophorous vesicles with spores and developmental stages within myocytes. Hematoxylin and eosin (H&E). Bar = 20 μm B. Spores in phagocytes associated with chronic infections and myolysis (demarked by arrows). H&E. Bar = 20 μm. C. Mixed infection with P. hyphessobryconis in muscle and xenomas of Pseudoloma neurophilia in spinal cord (arrows). Accustain Gram. Bar = 20 μm. D. Meronts (m), and developing and mature spores (sp) in sporophorous vesicles. Some fully developed spores stain deep blue (arrows). Accustain Gram. Bar = 10 μm. E. Mature spores stain deep blue to purple (arrows), developmental stages are light orange (arrow heads). Lillie Twort Gram. Bar = 10 μm. F. Numerous spores (arrows) throughout all layers of the intestine and mesenteries. Accustain Gram. Bar = 20 μm. G. Masses of spores in phagocytes (arrows) in ovaries. Accustain Gram. Bar = 10 μm.

Figure 4

Phylogenetic tree of Pleistophora hyphessobryconis obtained from neon tetra and related microsporidia based on small subunit rRNA gene sequences. 1361 bp small subunit rRNA gene sequences from 20 microsporidia infecting fish were used to reconstruct phylogeny. Maximum likelihood tree is shown. Branch numbers are maximum likelihood bootstrap support based on 100 replicates/Bayesian posterior probabilities. Genus names shown are as recorded in GenBank with new genus designations in parentheses. The microsporidian parasite, Pseudoloma neurophilia was selected as an outgroup taxa.