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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., (© 2024 International Society of Protistologists.)

Here, we provide evidence that the freshwater parasitic copepod, Salmincola californiensis, acts as a vector for Aeromonas salmonicida. While investigating the effects of S. californiensis on Chinoook salmon (Oncorhynchus tshawytscha), we tangentially observed that fish infected with the copepod developed furunculosis, caused by A. salmonicida. This occurred despite being reared in pathogen-free well water in a research facility with no prior history of spontaneous infection. We further investigated the possibility of S. californiensis to serve as a vector for the bacterium via detection of fluorescently labelled A. salmonicida inside the egg sacs from copepods in which the fish hosts were experimentally infected with GFP-A449 A. salmonicida. We then evaluated copepod egg sacs that were collected from adult Chinook salmon from a freshwater hatchery with A. salmonicida infections confirmed by either culture or PCR. The bacterium was cultured on tryptic soy agar plates from 75% of the egg sacs, and 61% were positive by PCR. These three separate experiments indicate an alternative tactic of transmission in addition to direct transmission of A. salmonicida in captivity. The copepod may play an important role in transmission of the bacterium when fish are more dispersed, such as in the wild., (© 2023 John Wiley & Sons Ltd.)

Objective: We explore apparent infection of Salmincola californiensis arising during investigations involving this lernaeopodid copepod parasitic on Pacific salmon and trout Oncorhynchus spp., Methods: We noted occasional unusual coloration of adult female copepods collected from the wild. These females were bright blue and pink in contrast to the cream white coloration characteristic of the copepod. We also observed that similar color patterns developed under laboratory settings when copepod eggs were held for hatching. In paired egg cases, we found consistent hatching failure of blue and pink eggs and patterns in apparent disease development that would be consistent with both vertical and horizontal transmission., Result: Attempts to identify the cause of the apparent infection using genetic methods and transmission electron microscopy were inconclusive., Conclusion: Iridovirus infection was initially suspected, but bacterial infection is also plausible. This apparent reduced hatching success of S. californiensis warrants further exploration as it could reduce local abundances. Given the potential importance of a disease impacting this copepod, a parasite that itself affects endangered and commercially important Pacific salmon and trout, future research would benefit from clarification of the apparent infection through additional sequencing, primer development, visualization, and exploration into specificity and transmission., (© 2023 American Fisheries Society.)

Detecting the presence of important parasites within a host and its environment is critical to understanding the dynamics that influence a pathogen's ability to persist, while accurate detection is also essential for the implementation of effective control strategies. Pseudoloma neurophilia is the most common pathogen reported in zebrafish (Danio rerio) research facilities. The only assays currently available for P. neurophilia are through lethal sampling, often requiring euthanasia of the entire population for accurate estimates of prevalence in small populations. We present a non-lethal screening method to detect P. neurophilia in tank water based on the detection of environmental DNA (eDNA) from this microsporidium, using a previously developed qPCR assay that was adapted to the digital PCR (dPCR) platform to complement current surveillance protocols. Using the generated dPCR data, a multi-state occupancy model was also implemented to predict the probability of detecting the microsporidium in tank water under different flow regimes and pathogen prevalence. The occupancy model revealed that samples collected in static conditions were more informative than samples collected from flow-through conditions, with a probability of detection at 80% and 47%, respectively. There was also a positive correlation between the frequency of detection in water and prevalence in fish based on qPCR., (© American Society of Parasitologists 2022.)

Pseudoloma neurophilia is a critical threat to the zebrafish (Danio rerio) model, as it is the most common infectious agent found in research facilities. In this study, our objectives were two-fold: (1) compare the application of diagnostic tools for P. neurophilia and (2) track the progression of infection using PCR and histology. The first experiment showed that whole-body analysis by qPCR (WB-qPCR) can be a standardized process, providing a streamlined diagnostic protocol, without the need for extraction of specific tissues. Evaluating the course of infection in experimentally infected fish, we showed key dynamics in infection. Starting with a low dose exposure of 8000 spores/fish, the prevalence remained low until 92 days post-exposure (dpe), followed by a 30%-40% prevalence by histology or 40%-90% by PCR until the end of the experiment at 334 dpe. WB-qPCR positively detected infection in more fish than histology throughout the study, as WB-qPCR detected the parasite as early as 4 dpe, whereas it was undetected by histology until 92 dpe. We also added a second slide for histologic analyses, showing an increase in detection rate from 24% to 26% when we combined all data from our experiments, but this increase was not statistically significant., (© 2022 John Wiley & Sons Ltd.)

Although zebrafish continue to increase in popularity as a vertebrate animal model for biomedical research, chronic infectious diseases in laboratory populations remain prevalent. The presence of pathogens such as Pseudocapillaria tomentosa, a parasitic nematode found in the intestine of infected zebrafish, can significantly influence experimental endpoints and negatively impact reproducibility of research findings. Thus, there is a need for screening tests for zebrafish with the sensitivity to detect even low levels of pathogens present in tissues. Assays based on the detection of DNA are commonly used for such screening tests. Newer technologies such as digital PCR provide an opportunity to improve the sensitivity and precision of these assays, so they can be reliably used to detect pathogen DNA in water, reducing the need for lethal testing. We have designed a qPCR-based assay with the sensitivity to detect less than 5 copies of the P. tomentosa SSU-rDNA gene in tissues of infected zebrafish and environmental DNA from aquarium water housing infected fish. In addition, we adapted this test to a dPCR platform to provide a precise quantification of P. tomentosa DNA and demonstrate the resistance of this assay to inhibitors commonly found in freshwater aquaria., (© 2020 John Wiley & Sons Ltd.)

The use of zebrafish (Danio rerio) in biomedical research has expanded at a tremendous rate over the last two decades. Along with increases in laboratories using this model, we are discovering new and important diseases. We review here the important pathogens and diseases based on some 20 years of research and findings from our diagnostic service at the NIH-funded Zebrafish International Resource Center. Descriptions of the present status of biosecurity programmes and diagnostic and treatment approaches are included. The most common and important diseases and pathogens are two parasites, Pseudoloma neurophilia and Pseudocapillaria tomentosa, and mycobacteriosis caused by Mycobacterium chelonae, M. marinum and M. haemophilum. Less common but deadly diseases are caused by Edwardsiella ictaluri and infectious spleen and kidney necrosis virus (ISKNV). Hepatic megalocytosis and egg-associated inflammation and fibroplasia are common, apparently non-infectious, in zebrafish laboratories. Water quality diseases include supersaturation and nephrocalcinosis. Common neoplasms are spindle cell sarcomas, ultimobranchial tumours, spermatocytic seminomas and a small-cell carcinoma that is caused by a transmissible agent. Despite the clear biosecurity risk, researchers continue to use fish from pet stores, and here, we document two novel coccidia associated with significant lesions in zebrafish from one of these stores., (© 2020 John Wiley & Sons Ltd.)

The zebrafish is a widely used animal model in biomedical research. Despite this, pathogens continue to be common in laboratory zebrafish. It is important to determine and describe the pathophysiology of cryptic infections on zebrafish to elucidate the impacts on experimental endpoints. Body condition is a basic measurement used experimentally and in health monitoring of animals. We exposed three wild-type zebrafish strains: AB, WIK, and 5D to Pseudoloma neurophilia . After 8 weeks postexposure, we individually imaged and processed fish for histology. Morphometric analysis was performed on images and an index of body condition was calculated based on the ratio of length/width from the dorsal aspect. Histological sections from each fish were examined to establish sex, severity of infection, and tissue distribution. We observed a significant decrease in body condition in female fish that was not observed in males. In addition, we observed a negative correlation between the total gonadal area of P. neurophilia exposed females and unexposed controls. These results illustrate the sex-specific impacts of a common chronic pathogen on zebrafish health and a commonly used experimental endpoint, further supporting the establishment of rigorous health monitoring programs in laboratory zebrafish colonies that include screening for chronic infectious diseases.

Microsporidia are highly successful parasites that infect virtually all known animal lineages, including the model Danio rerio (zebrafish). The widespread use of this aquatic model for biomedical research has resulted in an unexpected increase in infections from the microsporidium Pseudoloma neurophilia, which can lead to significant physical, behavioral, and immunological modifications, resulting in nonprotocol variation during experimental procedures. Here, we seek to obtain insights into the biology of P. neurophilia by investigating its genome content, which was obtained from only 29 nanograms of DNA using the MiSeq technology and paired-end Illumina sequencing. We found that the genome of P. neurophilia is phylogenetically and genetically related to other fish-microsporidians, but features unique to this intracellular parasite are also found. The small 5.25-Mb genome assembly includes 1,139 unique open-reading frames and an unusually high number of transposable elements for such a small genome. Investigations of intragenomic diversity also provided strong indications that the mononucleate nucleus of this species is diploid. Overall, our study provides insights into the dynamics of microsporidian genomes and a solid sequence reference to be used in future studies of host-parasite interactions using the zebrafish D. rerio and P. neurophilia as a model., (© 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists.)

Gamma irradiation is commonly used as a bone marrow suppressant in studies of the immune system and hematopoiesis, most commonly in mammals. With the rising utility and popularity of the zebrafish (Danio rerio), gamma irradiation is being used for similar studies in this species. Pseudoloma neurophilia, a microparasite and common contaminant of zebrafish facilities, generally produces subclinical disease. However, like other microsporidia, P. neurophilia is a disease of opportunity and can produce florid infections with high morbidity and mortality, secondary to stress or immune suppression. In this study, we exposed zebrafish to combinations of P. neurophilia infection and gamma irradiation to explore the interaction between this immunosuppressive experimental modality and a normally subclinical infection. Zebrafish infected with P. neurophilia and exposed to gamma irradiation exhibited higher mortality, increased parasite loads, and increased incidences of myositis and extraneural parasite infections than fish exposed either to P. neurophilia or gamma irradiation alone. This experiment highlights the devastating effects of opportunistic diseases on immunosuppressed individuals and should caution researchers utilizing immunosuppressive modalities to carefully monitor their stocks to ensure that their experimental animals are not infected.

The microsporidium, Pseudoloma neurophilia, is the most common infectious organism found in laboratory zebrafish colonies. Many currently used zebrafish lines originally came from pet store fish, and the initial description of P. neurophilia came from zebrafish obtained from a retail pet store. However, as P. neurophilia has not been described from wild-caught zebrafish, whether P. neurophilia is a natural pathogen of zebrafish is an open question. The pooling of fish of different species in the aquarium fish trade is common and a generalist parasite could be transmitted to novel hosts in this scenario. We determined that P. neurophilia can infect seven species of fishes from five families by cohabitation with infected zebrafish: Betta splendens, Xiphophorus maculatus, Devario aequipinnatus, Pimephales promelas, Oryzias latipes, Carassius auratus and Paracheirodon innesi. Infections in these fishes were histologically similar to those of zebrafish. We include a case report of a laboratory population of fathead minnows with naturally acquired P. neurophilia infections. With such a broad host range, including several fish families, other laboratory fishes should be screened routinely for this and other microsporidian parasites.

Two myxozoan species were observed in the kidney of topsmelt, Atherinops affinis , during a survey of parasites of estuarine fishes in the Carpinteria Salt Marsh Reserve, California. Fish collected on 3 dates in 2012 and 2013 were sectioned and examined histologically. Large extrasporogonic stages occurred in the renal interstitium of several fish from the first 2 collections (5/8, 11/20, respectively) and, in some fish, these replaced over 80% of the kidney. In addition, presporogonic and polysporogonic stages occurred in the lumen of the renal tubules, collecting ducts, and mesonephric ducts. The latter contained subspherical spores with up to 4 polar capsules, consistent with the genus Chloromyxum. For the third collection (15 May 2013, n = 30), we portioned kidneys for examination by histology, wet mount, and DNA extraction for small subunit ribosomal (SSU rDNA) gene sequencing. Histology showed the large extrasporogonic forms in the kidney interstitium of 3 fish and showed 2 other fish with subspherical myxospores in the lumen of the renal tubules with smooth valves and 2 spherical polar capsules consistent with the genus Sphaerospora. Chloromyxum-type myxospores were observed in the renal tubules of 1 fish by wet mount. Sequencing of the kidney tissue from this fish yielded a partial SSU rDNA sequence of 1,769 base pairs (bp). Phylogenetic reconstruction suggested this organism to be a novel species of Chloromyxum, most similar to Chloromyxum careni (84% similarity). In addition, subspherical myxospores with smooth valves and 2 spherical polar capsules consistent with the genus Sphaerospora were observed in wet mounts of 2 fish. Sequencing of the kidney tissue from 1 fish yielded a partial SSU rDNA sequence of 1,937 bp. Phylogenetic reconstruction suggests this organism to be a novel species of Sphaerospora most closely related to Sphaerospora epinepheli (93%). We conclude that these organisms represent novel species of the genera Chloromyxum and Sphaerospora based on host, location, and SSU rDNA sequence. We further conclude that the formation of large, histozoic extrasporogonic stages in the renal interstitium represents developmental stages of Chloromyxum species for the following reasons: (1) Large extrasporogonic stages were only observed in fish with Chloromyxum-type spores developing within the renal tubules, (2) a DNA sequence consistent with the Chloromyxum sp. was only detected in fish with the large extrasporogonic stages, and (3) several Sphaerospora species have extrasporogonic forms, but they are considerably smaller and are composed of far fewer cells.

Direct transmission from parents to offspring, referred to as vertical transmission, occurs within essentially all major groups of pathogens. Several microsporidia (Phylum Microsporidia) that infect arthropods employ this mode of transmission, and various lines of evidence have suggested this might occur with certain fish microsporidia. The microsporidium, Pseudoloma neurophilia, is a common pathogen of the laboratory zebrafish, Danio rerio. We previously verified that this parasite is easily transmitted horizontally, but previous studies also indicated that maternal transmission occurs. We report here direct observation of Pseudoloma neurophilia in the progeny of infected zebrafish that were reared in isolation, including microscopic visualization of the parasite in all major stages of development. Histological examination of larval fish reared in isolation from a group spawn showed microsporidian spores in the resorbing yolk sac of a fish. Infections were also observed in three of 36 juvenile fish. Eggs from a second group spawn of 30 infected fish were examined using a stereomicroscope and the infection was observed from 4 to 48 hours post-fertilization in two embryos. Intraovum infections were detected in embryos from 4 of 27 pairs of infected fish that were spawned based on qPCR detection of P. neurophilia DNA. The prevalence of intraovum infections from the four spawns containing infected embryos was low (∼1%) based on calculation of prevalence using a maximum likelihood analysis for pooled samples. Parasite DNA was detected in the water following spawning of 11 of the infected pairs, suggesting there was also potential for extraovum transmission in these spawning events. Our study represents the first direct observation of vertical transmission within a developing embryo of a microsporidian parasite in a vertebrate. The low prevalence of vertical transmission in embryos is consistent with observations of some other fish pathogens that are also readily transmitted by both vertical and horizontal routes.

Neuroectodermal tumours in man, including medulloblastoma, medulloepithelioma, neuroblastoma, esthesioneuroblastoma, primitive neuroectodermal tumour and dysembryoplastic neuroepithelial tumour, typically occur in children and young adults. These tumour types are occasionally observed in juvenile and adult zebrafish (Danio rerio) either as induced tumours in carcinogen-exposed zebrafish or as an incidental finding in zebrafish≥2years of age. An adult zebrafish submitted for routine histological examination was sent for a second opinion consultation after an uncharacteristic brain mass was identified. Microscopically, the expansile and infiltrative extracortical mass arising from the cerebellum had a diffuse microcystic pattern with solid hypercellular regions occupying 80% of the extrameningeal space and effacing the endomeninx and significantly displacing the metencephalon. The mass was composed of dense sheets of oligodendrocyte-like cells, random neurons and pseudocysts containing 'floating neurons' within a scant mucinous matrix. Neoplastic cells demonstrated positive perinuclear and intracytoplasmic expression of S-100. Malignant dysembryoplastic neuroepithelial tumour was diagnosed based on the histological features of the brain mass, which were indistinguishable from the human tumour. To our knowledge, this is the first report of a dysembryoplastic neuroepithelial tumour in a zebrafish., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The zebrafish Danio rerio is an increasingly important biological model in many areas of research. Due to the potential for non-protocol-induced variation, diseases of zebrafish, especially those resulting in chronic, sub-lethal infections, are of great concern. The microsporidium Pseudoloma neurophilia is a common parasite of laboratory zebrafish. Current methods for detection of this parasite require lethal sampling of fish, which is often undesirable with poorly spawning mutant lines and small populations. We present here an improved molecular-based diagnostic assay using real-time polymerase chain reaction (PCR), and including sonication treatment prior to DNA extraction. Comparisons of several DNA extraction methods were performed to determine the method providing the maximum sensitivity. Sonication was found to be the most effective method for disrupting spores. Compared to previously published data on PCR-based assay using a dilution experiment, sensitivity is increased. This shows that our assay, which includes sonication, is capable of detecting parasite DNA at 1 log higher dilution than the conventional PCR-based assay, which does not include sonication. Furthermore, we demonstrate the application of this method to testing of water, eggs, and sperm, providing a potential non-lethal method for detection of this parasite in zebrafish colonies with a sensitivity of 10 spores 1(-1) of water, 2 spores per spiked egg sample, and 10 spores microl(-1) of spiked sperm sample.

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.

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.