Review of Pseudoloma neurophilia (Microsporidia): A common neural parasite of laboratory zebrafish (Danio rerio)

Abstract

Zebrafish (Danio rerio) is now the second most used animal model in biomedical research. As with other vertebrate models, underlying diseases and infections often impact research. Beyond mortality and morbidity, these conditions can compromise research end points by producing nonprotocol induced variation within experiments. Pseudoloma neurophilia, a microsporidium that targets the central nervous system, is the most frequently diagnosed pathogen in zebrafish facilities. The parasite undergoes direct, horizontal transmission within populations, and is also maternally transmitted with spores in ovarian fluid and occasionally within eggs. This transmission explains the wide distribution among research laboratories as new lines are generally introduced as embryos. The infection is chronic, and fish apparently never recover following the initial infection. However, most fish do not exhibit outward clinical signs. Histologically, the parasite occurs as aggregates of spores throughout the midbrain and spinal cord and extends to nerve roots. It often elicits meninxitis, myositis, and myodegeneration when it infects the muscle. There are currently no described therapies for the parasite, thus the infection is best avoided by screening with PCR-based tests and removal of infected fish from a facility. Examples of research impacts include reduced fecundity, behavioral changes, transcriptome alterations, and autofluorescent lesions.

Keywords: diagnostics; fish; infection; parasite; pathogenesis; transmission.

Figure 1.

Percentage of facilities and fish diagnosed with P. neurophilia by histopathology at the ZIRC diagnostic pathology service from 2006–2023. The number of submitting facilities or total number of fish is listed above each bar.

Figure 2.

Phylogenetic relationships among close relatives of P. neurophilia. The microsporidium clusters with other neurotropic microsporidia that do not form xenomas (swamp guppy microsporidium and M. cerebralis). Sequences were aligned using MAFFT v.7.520 (L-INS-i option) (Katoh et al. 2019) and trimmed using trimAl v1.4rev22 (Capella-Gutierrez et al. 2009) to remove all columns with gaps in more than 20% of the sequences. The maximum-likelihood tree was generated using the TIM3+F+I+G4 substitution model determined to be the best-fit using ModelFinder(Kalyaanamoorthy et al. 2017) in IQ-Tree v1.6.11 (Nguyen et al. 2015), and bootstrap values were generated using UFBoot with 1,000 replicates (Hoang et al. 2018). The tree was visualized in iTol (Letunic and Bork 2021).

Figure 3.

P. neurophilia spores in wet mount preparation of zebrafish (Danio rerio) spinal cord. Normarski phase contrast. Bar = 10 μm.

Figure 4.

P. neurophilia in histologic sections from zebrafish (Danio rerio). A, B, Several aggregates of spores (A) in ventral spinal cord. N = notochord. H&E. Bars = A, 200 μm, B = 20 μm. C. Chronic meninxitis (arrow) along dorsal aspect of spinal cord. H&E. Bar = 200 μm. D. Focal myositis and degeneration. H&E. Bar = 200 μm. E. Numerous spore aggregates, stained red with Luna stain, in spinal cord. Bar = 20 μm.um. F. Two fluorescening aggregates of spores using DAPI filter and Fungi-Fluor stain. Bar = 20 μm. G. Developing egg with spores. A = aggregates, Arrow = single spore. Gram stain. B = 25 μm.

Figure 5.

Ultrastructural morphology and development of P. neurophilia. Images adapted from Cali et al. (2012). A, B) Parasite cell starting the transition from proliferative to sporogonic development. Note the difference between it and the abutting proliferative cell (P) membrane which is still covered with its glycocalyx-like surface coat (G). The plasmalemma has numerous “blisters” starting to emerge (bold arrows) from it and the new plasmalemma is now “thickened” in the areas under the blisters. The blistering continues until it forms an isolation chamber, a sporophorous vesicle (SPOV). Nu = nucleus, Sp = sporont. ER = endoplasmic reticulum. Scale bar = 1.7 μm. C. Low-power image of a myelinated nerve with a long-term infection. The axoplasm filled with SPOVs containing spores; note the presence of a scant amount of axoplasm just inside the myelin (My) layers. Scale bar = 5 μm. D. A spore in the SPOV. The anterior structure of the spore (A) is well-illustrated. The lamellar polaroplast (LPp) surrounds the tubular portion (TPp) and clearly extends approximately half the length of the spore to the nucleus. The polar filament coil cross-sections are visible as 16 sections in a single row. PV = polar vacuole. Bar = 1 μm.