Here we review various aspects of the biology, pathogenesis, diagnostics, control, and impacts on research caused by Pseudoloma neurophilia (Microsporidia), the most frequently diagnosed pathogen in zebrafish research facilities. The benefit of aquatic models, notably zebrafish (Danio rerio), as part of an integrative approach to improving human health is being recognized by the scientific community (Phillips and Westerfield 2014). Zebrafish are now the second most commonly used animal model in biomedical research (Kinter et al., 2021). The NIH sponsored Zebrafish International Resource Center (ZIRC) listed 1,880 academic and 39 commercial users of zebrafish between 2016 and 2024 (Zoltan Varga, Director of ZIRC, pers. comm.). The use of zebrafish as a model for biomedical research began with investigations in developmental genetics, in which experimental endpoints involved primarily embryos or larval fish (Eisen 2020; Phillips and Westerfield 2020), and this line of research continues to advance with the application of CRISPR technologies (Hans et al., 2021). Adult zebrafish are now also used extensively as models throughout the biomedical research arena, for applications as varied as infectious disease susceptibility and immune system function (Torraca, and Mostowy 2018), toxicology (Tanguay 2018), behavior (Gerlai 2019), and oncology (Völkel et al., 2018; Yan et al., 2019). As with rodent models, underlying, often subclinical infections in zebrafish are a concern due to their potential and documented impacts on research. These underlying pathogens and diseases can cause significant non-protocol induced variation, yielding physiological impacts that confound study endpoints (Kent et al., 2012).

The typical zebrafish vivarium is comprised of multiple small aquaria on a recirculating system from commercial “racks” with about 30–50 tanks to large systems supporting hundreds of tanks. Regardless of size, the general design is recirculating effluent water through mechanical and biological filtration, followed by ultraviolet sterilization. The water source is usually provided by reverse osmosis in which salts are added to achieve a conductivity of 250 to 1,700 microsiemens (Hammer 2020). In their natural habitats in India, zebrafish generally thrive in much softer water (Lee et al. 2022), while the flow-through vivarium at Oregon State University holds them at less than 150 microsiemens. The initial supply water in the vivarium is presumed to be free of important zebrafish pathogens and fish are generally introduced to research vivaria in the form of chlorine or iodine disinfected embryos; however, infectious diseases are surprisingly common in research zebrafish (Kent et al., 2020a). Maternal transmission, including intraovum, transmission, is one way that pathogens circumvent biosecurity barriers, and pathogens may also be introduced when “eggs only” policies are ignored in research facilities. Moreover, pet stores also represent a potential source for the introduction of pathogens in research colonies because as some researchers introduce zebrafish from these stores directly into research facilities. Pet store zebrafish are exposed to a myriad of fish pathogens, including those from other fish species, with no pathogen screening.

P. neurophilia is readily transmitted vertically (Sanders et al., 2013), which likely explains why this microsporidium is the most prevalent pathogen diagnosed in zebrafish facilities (Kent et al., 2020a). We have reported two other microsporidia in zebrafish facilities, Pleistophora hyphessobriconis (Sanders et al., 2010) and a putative Nucleospora sp. (Kent et al., 2021), but these infections are rare. We have been investigating P. neurophilia in zebrafish since our first description (Matthews et al., 2001), and here we review studies on the parasite over the last two decades. This review covers primary papers, as well as reviews by our group (Kent et al., 2020c; Schuster et al., 2022c; Sanders et al., 2012; 2014) and others (Caballero-Huertes et al., 2021). We provide updated data on the prevalence and distribution of the parasite in zebrafish facilities, review of the pathogenesis and impacts on research, diagnostic methods, our recent findings on the phylogeny of P. neurophilia and recently discovered relatives, control and treatment, and potential uses of the parasite as a model in biomedical research.

Pseudoloma neurophilia and Mycobacterium spp. are the most frequently diagnosed pathogens in fish submitted to the Zebrafish International Resource Center (ZIRC) diagnostic pathology service (https://zebrafish.org/wiki/health/submission/report). Our report on occurrence of P. neurophilia infections was based on ZIRC data obtained between 2006–2023 (Figure 1). Submissions to the diagnostic service ranged from 20–68 facilities and 277–1,441 fish/year, with a total of 12,927 zebrafish examined. Over this time period, the parasite was detected in 19–74% of facilities, and 3–30% of the total fish submitted.

The parasite is transmitted both horizontally (fish to fish) and vertically (mother to progeny) (Sanders et al., 2013) and as with all microsporidia, P. neurophilia replicates intracellularly within the zebrafish host. The sequential development of P. neurophilia within larval zebrafish was described in Cali et al. (2012) and Sanders et al. (2014), where it was shown that infection initially begins with the ingestion of infectious spores in the environment.

With horizontal transmission, spores are ingested and then make their way through the intestine and into the coelomic organs and muscle, where they continue to undergo karyokinesis. In this case, this is the proliferative stage of the life cycle (Cali et al., 2012), which is in direct contact with the cytoplasm of host cells. In larval studies, proliferative stages were sequentially visualized in the pancreas and kidney, spinal cord, eye, and skeletal muscle (Sanders et al., 2014). The first spore stages of P. neurophilia were observed sequentially in the pharyngeal epithelium, liver, spinal cord, and skeletal muscle (Sanders et al., 2014). The parasite was visualized in the central nervous system (CNS) after many other organ systems were infiltrated. The distribution of the early stages of P. neurophilia and the lack of mature spores early in infection, indicates that the parasite gains access to organs distant from the initial site of entry. In adult zebrafish, the primary mode of transmission occurs horizontally, where zebrafish contract the infection by ingestion of spores shed from live fish in excretory material, spawning, and by ingestion of infected dead tissue or eggs.

Of note, the parasite is also commonly found in the ovaries, but has not been found in the testes. Vertical transmission can occur both inside the developing embryo (intraovum) and outside the egg in ovarian fluids (extraovum) further contributing the periodic release of resistant, infectious spores into the environment. Thus, spawning events can also represent a significant contributor to transmission (Sanders et al., 2013; Schuster et al., 2022a).

Pseudoloma neurophilia was first reported in zebrafish Danio rerio collected from a pet store over 40 years ago (de Kinkelin 1980), and two decades later it was assigned to a new genus and species (Matthews et al., 2001). At that time, phylogenetic analysis based on SSU rDNA placed it closest to Ichthyosporidium. Cali et al. (2012) redescribed P. neurophilia and assigned it to the family Ichthyosporidae. They conducted more detailed electron microscopy and showed that its early development occurs in direct contact with the host cell. They described a character that is unique to the genus; a surface layer reminiscent of the glycocalyx of gut epithelial cells, excluding the direct contact of host organelles with the parasite plasmalemma, sporogonic development proceeds in intracellular sporophorous vesicles (SPOVs), which are the outermost structures interfacing the host cytoplasm. Xenoma formation was absent.

SSU rDNA sequence has recently become available from three other microsporidia that appear to be related to P. neurophilia based on phylogenetic analysis. Brocklebank et al. (1995) described Microsporidium cerebralis from the CNS of Atlantic salmon (Salmo salar), and then Jones et al. (2017) provided sequence on the salmon parasite and Microsporidium lucoppercae from pike-perch (Sander lucioperca). We recently identified a genetically related parasite – a microsporidium infecting the brain of the swamp guppy (Micropoecilia picta) from Grenada (Schuster et al., 2024). These fish microsporidia form a clade of related species based on SSU rDNA (Fig. 2). We did not assign these all to the genus Pseudoloma as details of ultrastructure are lacking for some of these microsporidia, and only P. neurophilia has matching signature sequences, which are regions of the SSU gene that are genetically distinct from other Microsporidia, for the genus established by Cali et al. (2012). The observation of a microsporidium similar to P. neurophilia in a field survey suggests that there are many more species related to P. neurophilia. Whereas the swamp guppy microsporidium is clearly a distinct species from P. neurophilia, the former was detected by a conventional PCR test designed for P. neurophilia (Murray et al., 2016), but was not detected by a commonly used qPCR test targeting a 4 times smaller amplicon (Sanders et al., 2011). Larger amplicon size has been reported to decrease amplification efficiency, increasing the likelihood of non-specific amplification, as there may be more template available for off-target amplification (Dieffenbach et al. 1993, Suzuki & Giovannoni 1996). P. neurophilia infects a wide variety of fishes in different families, several of which are used as laboratory fish (Sanders et al., 2016). It is conceivable that there are other related microsporidia from other tropical freshwater fishes that would be capable of infecting zebrafish and other laboratory fishes and may cross react with diagnostic tests presently used for P. neurophilia in zebrafish facilities.

Based on various laboratory transmission studies (Table1), fish show histologically visible infections about 2–4 mo post-exposure, with increasing prevalence per sampling unit well over 50% as the disease progresses over many months to a year. Observations from natural exposures performed at the ZIRC show detection later (Murray et al., 2011; 2016), but the time of initial exposures in this case is unknown. Regardless, most infected fish show no clinical signs until much later in the course of the infection. As the infection progresses, fish within the population increasingly appear emaciated, and less frequently they may exhibit spinal deformations such as lordosis, kyphosis, and scoliosis. At the ZIRC, Matthews et al. (2001) noted the infection was more prevalent in fish with these clinical signs. Spagnoli et al. (2015) reviewed histologic slides of 550 infected fish submitted to the ZIRC diagnostic service from 86 laboratories between the years 2000 and 2013. Both apparently normal, sentinel fish and fish with clinical signs were submitted. For approximately 70% of these infected fish, no clinical signs were noted by the submitter, indicating that most infected fish do not show outward signs of disease. There was, however, no difference between clinical and normal fish in terms of severity of infection in the CNS, but the myositis associated with infection was linked to clinical disease. In another study, mortality was greatly increased in infected fish that were immunosuppressed by gamma irradiation (Spagnoli et al., 2016), while the groups of fish with the parasite or gamma irradiation exposure alone showed minimal mortality. Experimentally infected female fish, but not males, had reduced body condition scores due to ovarian mass being reduced by about one third (Sanders et al., 2019).

Various therapeutic drugs, particularly oral fumagillin, have been effectively used to treat several microsporidian infections in fishes (Kent et al., 2014). However, these treatments have not proved effective with P. neurophilia. For example, Lavin et al. (2023) evaluated oral treatments with fumagillin and albendazole for P. neurophilia infection, and neither were efficacious. Therefore, at this time, the infection is best controlled by avoidance.

Disinfection to prevent transmission of P. neurophilia is also challenging. Spores survive the typical concentrations of chlorine used for surface disinfection of embryos, i.e., 25–50 ppm unbuffered sodium hypochlorite (bleach) for 10 min (Ferguson et al., 2007). Higher doses are more effective, with about 80% of spores neutralized by 100 ppm unbuffered bleach. Complete neutralization is achieved by 200 ppm bleach and 100 ppm buffered to pH 7. However, these doses are also toxic to embryos and larvae (Kent et al., 2014). Crim et al. (2017) presented preliminary evidence that simply rinsing embryos with disinfected (UV-irradiated) fish water may reduce the likelihood of pathogen contamination of progeny. However, no amount of rinsing or surface disinfection will eliminate spores transmitted vertically within the egg (Sanders et al., 2013). High doses of bleach, i.e., >100 ppm, with sufficient rinsing or neutralization with sodium thiosulfate can be used for equipment disinfection. Peneyra et al (2018) tested different approaches with spawning chambers to reduce transmission using the Sanders and Kent (2011) PCR with sonication as a detection method. They found that 100 ppm bleach soak (pH 7.0), and a soak or spray with 75 ppm Wescodyne (an iodine product) (Steris Corp, Mentor, OH) was 100% effective at eliminating detection of the parasite. Disinfection of spawning chambers before spawning did not decrease P. neurophilia transmission when infected fish remained present in the breeding population, and hence they recommend not using infected brood fish to avoid infection. Mendoza et al. (2022) reviewed effects of various combinations of iodine or sodium hypochlorite on zebrafish egg and embryo viability, providing a useful guideline when developing disinfectants for killing P. neurophilia spores associated with eggs.

The spores are also resistant to freezing with cryoprotectant, a strategy commonly used to store zebrafish sperm (Norris et al., 2018). P. neurophilia spores have been detected by PCR in dissected testes and stripped sperm (Murray et al., 2011). What remains to be quantified is the risk of recovering infected progeny from in vitro fertilization using thawed sperm that was naturally contaminated with P. neurophilia prior to cryopreservation.

Ultraviolet sterilizers are essential for controlling the spread of infectious pathogens on recirculating aquaculture systems. Specific studies of the dose of UV irradiation required to neutralize P. neurophilia are not published. However, for several years ZIRC observed the pathogen in pre-filtration sentinel fish but not in sentinels receiving post-filtration water treated with 45,000 μWsec/cm² UV irradiance, evidence that the dose effectively prevented recirculation of infectious spores (Murray et al., 2011).

As the P. neurophilia life cycle and modes of transmission are well understood and sensitive diagnostic tests for the pathogen are available for in-house application and from diagnostic laboratories, P. neurophilia meets the criteria for exclusion Specific Pathogen Free (SPF) facilities (Murray et al., 2022). Indeed, the Sinnhuber Aquatic Research Laboratory (SARL) has successfully maintained a zebrafish colony that is SPF for P. neurophilia for over 10 years (Kent et al., 2011; Barton et al., 2016). As the parasite is maternally transmitted, the SARL strategy involves screening broodstock and progeny by PCR and a three-step quarantine to ensure that only Pseudoloma-free stocks are propagated. In addition, thorough testing of sentinel, moribund, and aged fish by histopathology and PCR is evidence that the colony remains SPF.

To avoid the potential negative impacts of P. neurophilia on research outcomes, from our conversations with lab animal veterinarians and facility managers it is apparent to us (K.M.) that several new zebrafish facilities have an intent to exclude the parasite. To achieve this objective, facilities may decline importation of fish from facilities with P. neurophilia or import lines into quarantine where progeny and broodstock are screened for the parasite before the line is established in the main facility. This strategy, while proven successful at SARL, requires time and resources that may be limiting factors when investigators need fish for grant-funded research.

Pseudoloma neurophilia has been found in 30% or more of zebrafish facilities submitting samples to the ZIRC diagnostic service (Figure 1). The persistence of the parasite likely reflects the difficulty of eliminating it from facilities once infections are established. While exclusion protocols are available, how to minimize and eliminate established endemic infections is a different and more complicated problem. One approach is to rederive individual contaminated fish lines by in vitro fertilization (IVF) using sperm from contaminated males and eggs from Pseudoloma-free females SPF AB wild-type fish are available for purchase from SARL (Ventura-Fernandes et al., 2021). Progeny must then lethally be tested by PCR for the infection, and the procedure may need to be repeated several times to obtain sufficient fish numbers to ensure statistical confidence that there is not occurrence in the rederived progeny, while ensuring that there is still a sufficient number of fish within the population that are not subjected to this lethal test screening. Additionally, sufficient numbers are needed to replace progeny that are infected from contaminated sperm samples. Progeny resulting from this cross will be heterozygous and must be backcrossed to establish a homozygous population. Furthermore, this strategy of relying on sperm to maintain a germline cannot be used for wild-type lines, where both males and females are required to preserve the line-specific genome, as opposed to preserving a genetic feature of a mutant or transgenic line.

The proportion of facilities with infected fish that have submitted samples to ZIRC appears to have decreased since a peak in 2010 (Figure 1), but conclusions as to the apparent cause should be accepted with caution as the submitters (facilities) are not selected at random from the overall research community. Nevertheless, it is possible that more researchers now appreciate the potential negative effects of the parasite beyond morbidity, and hence are taking measures to avoid the infection. These efforts include obtaining new fish from sources documented to be free of the infection and carrying out more rigorous quarantine procedures for these imports beyond simply introducing new stocks directly into the main facilities as surface disinfected eggs. In addition, more institutions are requiring their facilities to have health reports on zebrafish imports (Mocho et al., 2022).

The ZIRC’s strategy for minimizing P. neurophilia infections has evolved in tandem with our understanding of the parasite. To maintain genetic diversity, every third generation of mutant and transgenic lines is outcrossed to wild-type AB fish. Since 2006, the ZIRC outcrossing protocol has specified that AB fish cannot be used to outcross more than one stock (a specific generation of a genetic line), nor can they be returned to the original AB population after being used for outcross. This has prevented contaminated AB fish from spreading the parasite to other fish lines in the facility. In addition to robust biosecurity measures to prevent parasite spread between tanks, ZIRC has been identifying infected fish stocks by in-house PCR (Murray et al., 2011) and histopathology on moribund and randomly sampled colony fish since 2008. Infected stocks are euthanized, when possible, i.e., when a younger generation is sufficiently close to maturity, and genetically related stocks are monitored for the parasite. For a large facility, this is a slow process, but the ZIRC was able to decrease the parasite to below detectable levels by PCR and histopathology on moribund, pre-filtration sentinels, and randomly sampled colony fish from October 2010 to February 2013 (https://zebrafish.org/wiki/health/health_reports/start).

Prior to the establishment of new biosecurity measures, ZIRC imported new fish lines into quarantine and their surface-sanitized embryos were moved into the main fish colony. In addition, in 2011 ZIRC began importation of thousands of fish lines as cryopreserved sperm generated from large mutagenesis projects in the research community. These lines were regenerated at ZIRC by IVF to amplify sperm samples when they became depleted by customer orders. The combination of untested new fish lines in the facility and a 10-hour failure of UV sterilizers in September of 2013 had the unfortunate result of spreading P. neurophilia throughout the facility via untreated water in the recirculating system. In response, ZIRC intensified in-house testing and modified the importation strategy to include diagnostic testing and resulted in fewer new lines being moved into the main fish colony. As of 2016, all mutant and transgenic lines are imported to ZIRC via male fish. Sperm is collected in quarantine, cryopreserved, and stored in designated quarantine freezers. The males are then euthanized, fixed, and processed into paraffin blocks. Customer orders for these lines are filled by IVF using eggs from ZIRC AB females and shipped as surface-sanitized embryos. Before the line is regenerated and reared at ZIRC to reamplify sperm samples, histological sections are prepared from paraffin blocks of males that contributed the original sperm samples. Results of histological analysis for pathogens determines whether the line will be reared in quarantine or the main fish facility. By directing all imported lines through cryopreserved sperm, ZIRC avoids the risk of vertical and early horizontal transmission of P. neurophilia to progeny via infected females. The risk of contaminated sperm is mitigated by histological evaluation of males.

In 2018 ZIRC initiated a labor-intensive project to eliminate P. neurophilia from wild-type lines in the facility. For each line, at least 50 individual pair crosses were spawned. Clutches of 50 or embryos were surface-sanitized and held in isolation pending in-house PCR results for P. neurophilia on CNS dissected from both parents. Only embryos from Pseudoloma-free adults were pooled and reared to propagate the line. At least 25 clean clutches were required to maintain genetic diversity of each line. ZIRC continues to test pre- and post-filtration sentinel and moribund fish by histopathology. Every month, water from all 20-gallon tanks of 8-month fish is tested by dPCR for eDNA of P. neurophilia (Schuster et al., 2023). As of June 2022, P. neurophilia decreased below detection level. However, reintroduction of the parasite is a constant risk given the high rate of importation and IVF necessary to carry out the ZIRC mission as a stock center.

Sanders and Kent (2104) outlined potential uses of the Pseudoloma-infected zebrafish as a biomedical model for microsporidian infections. With the scalability (large sample sizes for low cost and space), optical clarity of the embryos, availability of SPF lines, and amenability for gene expression studies, both larvae and adult zebrafish provide excellent opportunities to study infectious diseases in this host. Other naturally occurring pathogens of zebrafish, such as Mycobacterium marinum (Myllymaki et al. 2016) and Pseudocapillaria tomentosa (Gaulke et al. 2109; Sharpton et al. 2020) have been leveraged into useful infectious disease models in zebrafish. Likewise, with its ease of transmission and readability of infection in all life stages, P. neurophilia has the potential to elucidate details of early development of microsporidia (Sanders et al. 2013) as well as facilitate investigations of downstream effects of specific pathogen-associated changes in the CNS. Moreover, with the publication of the genome of P. neurophilia (Ndikumana et al. 2016), there is potential to use the model to understand the evolution of the genome as it relates to the spliceosome, as it was shown that with only 14 predicted proteins, P. neurophilia may be the smallest spliceosome currently known (Whelan & Fast 2023).

P. neurophilia continues to be widespread in zebrafish vivaria, and it is clear now that this chronic neural infection of the CNS has many potential and demonstrated impacts on infected research animals. With recognition of the problem, and more recent implementation of control measures since a peak in detection in 2010 from facilities that report to ZIRC, the percentage of facilities where the parasite was detected has fallen below 50%. Sensitive and specific PCR-based tests are now available for screening, and we see even more utility of these tests as they transition into highly efficient eDNA tests that coupled with current surveillance efforts have the potential to help move facilities towards SPF status.