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A new species of Gyrodactylus was described on the body surface of zebrafish (Danio rerio) in China. Basing on morphological characteristics and ITS sequence, we identified the parasite as a new member of the Gyrodactylus-wageneri group. Morphologically, Gyrodactylus sp. nov. is greatly similar to "G. zebrae", another species parasitic on zebrafish: both have moderately stout hamulus, marginal hook sickle with a prominent heel and toe, as well as a curved blade. However, distinct haptoral shape differences were detected between these two species. The dorsal bar is straight in Gyrodactylus sp. nov. but strongly curved in "G. zebrae", and the sickle shaft in Gyrodactylus sp. nov. is approximately perpendicular to the base, but in "G. zebrae" it is slanted downwards. The ITS1-5.8S-ITS2 sequence also indicate that Gyrodactylus sp. nov. exhibits the highest similarity to "G. zebrae": 95.7% sequence identity suggests interspecific differentiation. Phylogenetic analysis of the ITS1-ITS2 sequence showed that Gyrodactylus sp. nov. formed a sister clade with "G. zebrae", and exhibited a relatively close phylogenetic relationship with G. kobayashii, G. gurleyi, and G. longoacuminatus, all of which parasitise on goldfish, Carassius auratus. To test the susceptibility of zebrafish and goldfish to the Gyrodactylus sp. nov., ten gyrodactylids were inoculated to the caudal fin of zebrafish and goldfish. The gyrodactylids exhibited the ability to attach themselves to the goldfish, and some gyrodactylids reproduced a few days after the inoculation. On day 9, however, the mean abundance sharply decreased to zero on goldfish and increased to more than 30 on zebrafish. The result suggested that golfish is an unsuitable host for Gyrodactylus sp. nov. Therefore, on the basis of morphology, molecular sequence similarity, and host susceptibility, we conclude that the gyrodactylid found on the zebrafish is a new species, which we named Gyrodactylus banmae., (Copyright © 2021. Published by Elsevier B.V.)

Intestinal neoplasms and preneoplastic lesions are common in zebrafish research facilities. Previous studies have demonstrated that these neoplasms are caused by a transmissible agent, and two candidate agents have been implicated: a Mycoplasma sp. related to Mycoplasma penetrans and the intestinal parasitic nematode, Pseudocapillaria tomentosa , and both agents are common in zebrafish facilities. To elucidate the role of these two agents in the occurrence and severity of neoplasia and other intestinal lesions, we conducted two experimental inoculation studies. Exposed fish were examined at various time points over an 8-month period for intestinal histopathologic changes and the burden of Mycoplasma and nematodes. Fish exposed to Mycoplasma sp. isolated from zebrafish were associated with preneoplastic lesions. Fish exposed to the nematode alone or with the Mycoplasma isolate developed severe lesions and neoplasms. Both inflammation and neoplasm scores were associated with an increase in Mycoplasma burden. These results support the conclusions that P. tomentosa is a strong promoter of intestinal neoplasms in zebrafish and that Mycoplasma alone can also cause intestinal lesions and accelerate cancer development in the context of nematode infection.

Many scientific studies still use zebrafish from pet stores as animal models, even cutting-edge researches. However, these animals differ genotypically and phenotypically between them. The importance of the use of standardized models is widely recognized. Besides that, another consequence of using zebrafish from unknown origins is the acquisition of parasitized animals. This study aimed to relate the infection by Clinostomum sp. in zebrafish. Animals sold as "high standard" were acquired from a commercial company. Swimming alterations and superficial yellow dots were observed in five zebrafish with clinical signs, which were isolated, euthanized, and necropsied. Muscular yellow cysts with metacercaria associated with lesions were observed. The muscular cysts were responsible for the superficial yellow dots as well as the swimming alterations. The prevalence was 2.5%, and the mean infection intensity was 7 digeneans/host. The cysts measured a mean of 1251.43 μm long × 784.28 μm wide. Metacercariae measured a mean of 4847 μm long × 1353 μm wide. This first report about infection by Clinostomum sp. in zebrafish is globally relevant since the host and the parasite genus currently overlap worldwide. Furthermore, this study sheds light on the importance of the specific pathogen-free commercial creations or laboratory-reared zebrafish for research.

Infections by schistosomes result in granulomatous lesions around parasite eggs entrapped within the host tissues. The host and parasite determinants of the Schistosoma mansoni egg-induced granulomatous response are areas of active investigation. Some studies in mice implicate Tumor Necrosis Factor (TNF) produced in response to the infection whereas others fail to find a role for it. In addition, in the mouse model, the S. mansoni secreted egg antigen omega-1 is found to induce granulomas but the underlying mechanism remains unknown. We have recently developed the zebrafish larva as a model to study macrophage recruitment and granuloma formation in response to Schistosoma mansoni eggs. Here we use this model to investigate the mechanisms by which TNF and omega-1 shape the early granulomatous response. We find that TNF, specifically signaling through TNF receptor 1, is not required for macrophage recruitment to the egg and granuloma initiation but does mediate granuloma enlargement. In contrast, omega-1 mediates initial macrophage recruitment, with this chemotactic activity being dependent on its RNase activity. Our findings further the understanding of the role of these host- and parasite-derived factors and show that they impact distinct facets of the granulomatous response to the schistosome egg., Competing Interests: The authors have declared that no competing interests exist.

Schistosome eggs provoke the formation of granulomas, organized immune aggregates, around them. For the host, the granulomatous response can be both protective and pathological. Granulomas are also postulated to facilitate egg extrusion through the gut lumen, a necessary step for parasite transmission. We used zebrafish larvae to visualize the granulomatous response to Schistosomamansoni eggs and inert egg-sized beads. Mature eggs rapidly recruit macrophages, which form granulomas within days. Beads also induce granulomas rapidly, through a foreign body response. Strikingly, immature eggs do not recruit macrophages, revealing that the eggshell is immunologically inert. Our findings suggest that the eggshell inhibits foreign body granuloma formation long enough for the miracidium to mature. Then parasite antigens secreted through the eggshell trigger granulomas that facilitate egg extrusion into the environment. In support of this model, we find that only mature S. mansoni eggs are shed into the feces of mice and humans., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Toxoplasma gondii has long been considered a ubiquitous parasite possessing the capacity of infecting virtually all warm-blooded animals globally. Occasionally, this parasite can also infect cold-blooded animals such as fish if their body temperature reaches 37 °C. However, we are currently lacking an understanding of key details such as the minimum temperature required for T. gondii invasion and proliferation in these cold-blooded animals and their cells. Here, we performed in vitro T. gondii infection experiments with rat embryo fibroblasts (REF cells), grouper (Epinephelus coioides) splenocytes (GS cells) and zebra fish (Danio rerio) hepatocytes (ZFL cells), at 27 °C, 30 °C, 32 °C, 35 °C and 37 °C, respectively. We found that T. gondii tachyzoites could penetrate REF, GS nd ZFL cells at 27 °C but clear inhibition of multiplication was observed. Intriguingly, the intracellular tachyzoites retained the ability to infect mice after 12 days of incubation in GS cells cultured at 27 °C as demonstrated by bioassay. At 30 °C, 32 °C and 35 °C, we observed that the mammalian cells (REF cells) and fish cells (GS and ZFL cells) could support T. gondii invasion and replication, which showed a temperature-dependent relationship in infection and proliferation rates. Our data demonstrated that the minimum temperature for T. gondii invasion and replication was 27 °C and 30 °C respectively, which indicated that temperature should be a key factor for T. gondii invasion and proliferation in host cells. This suggests that temperature-dependent infection determines the differences in the capability of T. gondii to infect cold- and warm-blooded vertebrates., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Toxoplasma gondii is an obligate intracellular parasite capable of invading any nucleated cell. Three main clonal lineages (type I, II, III) exist and murine models have driven the understanding of general and strain-specific immune mechanisms underlying Toxoplasma infection. However, murine models are limited for studying parasite-leukocyte interactions in vivo , and discrepancies exist between cellular immune responses observed in mouse versus human cells. Here, we developed a zebrafish infection model to study the innate immune response to Toxoplasma in vivo By infecting the zebrafish hindbrain ventricle, and using high-resolution microscopy techniques coupled with computer vision-driven automated image analysis, we reveal that Toxoplasma invades brain cells and replicates inside a parasitophorous vacuole to which type I and III parasites recruit host cell mitochondria. We also show that type II and III strains maintain a higher infectious burden than type I strains. To understand how parasites are cleared in vivo , we further analyzed Toxoplasma- macrophage interactions using time-lapse microscopy and three-dimensional correlative light and electron microscopy (3D CLEM). Time-lapse microscopy revealed that macrophages are recruited to the infection site and play a key role in Toxoplasma control. High-resolution 3D CLEM revealed parasitophorous vacuole breakage in brain cells and macrophages in vivo , suggesting that cell-intrinsic mechanisms may be used to destroy the intracellular niche of tachyzoites . Together, our results demonstrate in vivo control of Toxoplasma by macrophages, and highlight the possibility that zebrafish may be further exploited as a novel model system for discoveries within the field of parasite immunity.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Research conducted on model organisms may be biased due to undetected pathogen infections. Recently, screening studies discovered high prevalence of the microsporidium Pseudoloma neurophilia in zebrafish (Danio rerio) facilities. This spore-forming unicellular parasite aggregates in brain regions associated with motor function and anxiety, and despite its high occurrence little is known about how sub-clinical infection affects behaviour. Here, we assessed how P. neurophilia infection alters the zebrafish´s response to four commonly used neurobehavioral tests, namely: mirror biting, open field, light/dark preference and social preference, used to quantify aggression, exploration, anxiety, and sociability. Although sociability and aggression remained unaltered, infected hosts exhibited reduced activity, elevated rates of freezing behaviour, and sex-specific effects on exploration. These results indicate that caution is warranted in the interpretation of zebrafish behaviour, particularly since in most cases infection status is unknown. This highlights the importance of comprehensive monitoring procedures to detect sub-clinical infections in laboratory animals.

Precise knowledge of the health status of experimental fish is crucial to obtain high scientific and ethical standards in biomedical research. In addition to the use of sentinel fish, the examination of diseased fish is a fundamental part of all health monitoring concepts. PCR assays offer excellent sensitivity and the ability to test a broad variety of pathogenic agents in different sample types. Recently, it was shown that analysis of environmental samples such as water, sludge or detritus from static tanks can complement PCR analysis of fish and is actually more reliable for certain pathogens. In our study, we investigated whether the analysis of filtered water mixed with detritus of tanks including fish showing clinical signs of illness is suitable to complement health monitoring programs in recirculating systems. The obtained data indicate that pathogens such as Pseudoloma neurophilia or Myxidium streisingeri were exclusively or mainly found in fish, while mycobacteria were predominantly present in environmental samples. A combination of both sample types seems to be required for the detection of a broad range of infectious agents in zebrafish colonies using real-time PCR technology., Competing Interests: The authors have declared that no competing interests exist.

Intensive fish farming relies on the use of feeds based on fish meal and oil as optimal ingredients; however, further development of the aquaculture sector needs new, nutritious and sustainable ingredients. According to the concept of circular economy, insects represent good candidates as aquafeed ingredients since they can be cultured through environmental-friendly, cost-effective farming processes, on by-products/wastes, and many studies have recently been published about their inclusion in fish feed. However, information about the physiological effects of insect-based diets over the whole life cycle of fish is presently missing. At this regard, the present study investigated, for the first time, the effects of Black Soldier Fly based diets (25 and 50% fish meal substitution) administration for a six months period in zebrafish (Danio rerio), from larvae to adults. A multidisciplinary approach, including biometric, biochemical, histological, spectroscopic and molecular analyses was applied. Aside a general reduction in fish growth and lipid steatosis, six-months feeding on Black Soldier Fly based diets did not show major negative effects on zebrafish. Gut histological analysis on intestine samples did not show signs of inflammation and both stress markers and immune response markers did not show significant differences among the experimental groups.

Background: Helminth parasites represent a significant threat to the health of human and animal populations, and there is a growing need for tools to treat, diagnose, and prevent these infections. Recent work has turned to the gut microbiome as a utilitarian agent in this regard; components of the microbiome may interact with parasites to influence their success in the gut, meaning that the microbiome may encode new anthelmintic drugs. Moreover, parasite infections may restructure the microbiome's composition in consistent ways, implying that the microbiome may be useful for diagnosing infection. The innovation of these utilities requires foundational knowledge about how parasitic infection, as well as its ultimate success in the gut and impact on the host, relates to the gut microbiome. In particular, we currently possess limited insight into how the microbiome, host pathology, and parasite burden covary during infection. Identifying interactions between these parameters may uncover novel putative methods of disrupting parasite success., Results: To identify interactions between parasite success and the microbiome, we quantified longitudinal associations between an intestinal helminth of zebrafish, Pseudocapillaria tomentosa, and the gut microbiome in 210 4-month-old 5D line zebrafish. Parasite burden and parasite-associated pathology varied in severity throughout the experiment in parasite-exposed fish, with intestinal pathologic changes becoming severe at late time points. Parasite exposure, burden, and intestinal lesions were correlated with gut microbial diversity. Robust generalized linear regression identified several individual taxa whose abundance predicted parasite burden, suggesting that gut microbiota may influence P. tomentosa success. Numerous associations between taxon abundance, burden, and gut pathologic changes were also observed, indicating that the magnitude of microbiome disruption during infection varies with infection severity. Finally, a random forest classifier accurately predicted a fish's exposure to the parasite based on the abundance of gut phylotypes, which underscores the potential for using the gut microbiome to diagnose intestinal parasite infection., Conclusions: These experiments demonstrate that P. tomentosa infection disrupts zebrafish gut microbiome composition and identifies potential interactions between the gut microbiota and parasite success. The microbiome may also provide a diagnostic that would enable non-destructive passive sampling for P. tomentosa and other intestinal pathogens in zebrafish facilities.

Due to their transparency, genetic tractability, and ease of maintenance, zebrafish (Danio rerio) have become a widely-used vertebrate model for infectious diseases. Larval zebrafish naturally prey on the unicellular protozoan Paramecium caudatum. This protocol describes the use of P. caudatum as a vehicle for food-borne infection in larval zebrafish. P. caudatum internalize a wide range of bacteria and bacterial cells remain viable for several hours. Zebrafish then prey on P. caudatum, the bacterial load is released in the foregut upon digestion of the paramecium vehicle, and the bacteria colonize the intestinal tract. The protocol includes a detailed description of paramecia maintenance, loading with bacteria, determination of bacterial degradation and dose, as well as infection of zebrafish by feeding with paramecia. The advantage of using this method of food-borne infection is that it closely mimics the mode of infection observed in human disease, leads to more robust colonization compared to immersion protocols, and allows the study of a wide range of pathogens. Food-borne infection in the zebrafish model can be used to investigate bacterial gene expression within the host, host-pathogen interactions, and hallmarks of pathogenicity including bacterial burden, localization, dissemination and morbidity.

The protective immune response in zebrafish (Danio rerio) against the parasitic ciliate Ichthyophthirius multifiliis, targeting host skin, fins and gills, comprises an accelerated and manifold elevated immunoglobulin gene expression as well as a significantly elevated number of neutrophils at infected sites. Experimental fish were subjected to a primary I. multifiliis infection followed by a series of secondary exposures before they were challenged by a high dosage of infective theronts. Immunized fish responded immediately with a protective response suggesting existence of immunological memory whereas fish exposed to the parasite for the first time obtained a marked infection. The primary response to infection was dominated by expression of genes encoding acute phase reactants and inflammatory cytokines as well as recruitment of neutrophils at infected locations. Immunized fish showed a significantly upregulated immunoglobulin gene expression following challenge, which indicates existence of a secondary response effected by antibodies. Both responses induced a significantly elevated expression of the Th2 signature cytokine Il13. The increased presence of neutrophils in immunized fish suggests that innate cell mediated immunity supplements or influence the protective response against the parasite., Competing Interests: The authors have declared that no competing interests exist.

Cryopreservation is a common method used to preserve the sperm of various animal species, and it is widely used with zebrafish (Danio rerio). As with other animals, there is a possibility of paternal pathogen transmission through sperm. We evaluated the ability of five common and important pathogens of zebrafish to survive cryopreservation as used with zebrafish sperm and freezing without cryopreservant. We evaluated Mycobacterium chelonae, Mycobacterium marinum, and Edwardsiella ictaluri, each originally isolated from zebrafish, eggs of Pseuodocapillaria tomentosa, and spores of Pseudoloma neurophilia. Each mycobacterial isolate showed relatively minimal reduction in survival after freezing and thawing, particularly when subjected to cryopreservation. E. ictaluri also showed survival after cryopreservation, but exhibited a several log reduction after freezing at -80°C without cryopreservant. With P. neurophilia, two separate experiments conducted 3 years apart yielded very similar results, showing some, but reduced, survival of spores by using three different viability assays: SYTOX stain, Fungi-Fluor stain, and presence of a spore vacuole. Eggs of P. tomentosa showed no survival based on larvation of eggs when subjected to either freezing method. Given that four of the five pathogens exhibited survival after cryopreservation, we recommend that sperm samples or donor male zebrafish fish be tested for pathogens when sperm are to be stored by using cryopreservation.

Excretory/secretory (ES) compounds isolated from third-stage larvae of the anisakid nematode Contracaecum osculatum parasitizing liver of Baltic cod were investigated for effects on immune gene expression in a zebrafish LPS-induced inflammation model. ES products containing a series of proteins, of which some had enzymatic activity, were injected solely or with LPS. ES proteins alone induced up-regulation of a number of immune-related genes, but generally to a lower degree compared to LPS. When co-injected with LPS, the worm products exacerbated merely expression of five genes affecting Th1, Th2, Th17 and innate responses compared to the LPS-injected group. However, the level of overexpression decreased in an inverse dose-dependent manner. The immune regulating action of C. osculatum ES products is interpreted as an important evolutionary ability of larval parasites in the transport host which makes it less susceptible to host immune responses whereby the probability of reaching the final host is increased.

Five amoeboid organisms of different origin (isolated from fish organs, soil and digestive tract of earthworm) that shared light microscopical and ultrastructural features including type and arrangement of mitochondrial cristae were subjected to phylogenetic analyses based on sequences of SSU rDNA and protein coding genes (actin, cytochrome oxidase I, and eukaryotic elongation factor 2). The reconstruction of multigene phylogeny of the strains studied (i) revealed that they belong to the same single-genus Copromyxa clade; (ii) strongly supported position of Copromyxa cantabrigiensis (syn. Hartmannella cantabrigiensis) within the genus; (iii) together with comparisons of light and electron microscopy data justified reclassification of Cashia limacoides (syn. Vexillifera expectata) to Copromyxa limacoides n. comb., and (iv) justified description of a new species, Copromyxa laresi n. sp., (© 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists.)

Specimens of Transversotrema patialense (sensu lato) ( Soparkar, 1924 ) Crusz and Sathananthan, 1960 (Digenea: Transversotrematidae) infected the skin (epidermal spaces beneath scales near pectoral fins) of 4 of 126 (prevalence 3%; mean intensity 1.8) zebrafish ( Danio rerio (Hamilton, 1822) [Cypriniformes: Cyprinidae]) purchased in 2009 and cultured by a California (USA) fish supplier. These fish were sold as "laboratory-reared" and "specific pathogen free," purportedly raised in a recirculating aquaculture system that included zebrafish only. We herein describe the morphological features of this transversotrematid using light and scanning electron microscopy, provide a comprehensive list of hosts (snails and fishes) and geographic locality records for specimens reported as T. patialense, which is perhaps a species complex, and provide a brief historical synopsis of the taxonomic and life history research that has been conducted on this fluke. No species of Transversotrema previously had been reported from the Americas; however, this discovery is not surprising given that: (1) a suitable intermediate host (red-rimmed melania, Melanoides tuberculata (Müller, 1774) [Cerithioidea: Thiaridae]) has been established in California and elsewhere in North America, (2) the zebrafish is a susceptible definitive host, and (3) T. patialense reportedly matures on a broad ecological and phylogenetic spectrum of freshwater fishes. To our knowledge, this is the northern-most geographic locality record for a species of this genus. We suspect this case study represents an example of a parasite that may now be established in North America by the fortuitous co-occurrence of a susceptible, exotic snail host (the red-rimmed melania) and a susceptible, widely distributed, exotic fish host (the zebrafish).

The zebrafish's potential as a model for human neurobehavioral research appears nearly limitless despite its relatively recent emergence as an experimental organism. Since the zebrafish has only been part of the research community for a handful of decades, pathogens from its commercial origins continue to plague laboratory stocks. One such pathogen is Pseudoloma neurophilia, a common microparasite in zebrafish laboratories world-wide that generally produces subclinical infections. Given its high prevalence, its predilection for the host's brain and spinal cord, and the delicate nature of neurobehavioral research, the behavioral consequences of subclinical P. neurophilia infection must be explored. Fish infected via cohabitation were tested for startle response habituation in parallel with controls in a device that administered ten taps over 10 min along with taps at 18 and 60 min to evaluate habituation extinction. After testing, fish were euthanized and evaluated for infection via histopathology. Infected fish had a significantly smaller reduction in startle velocity during habituation compared to uninfected tankmates and controls. Habituation was eliminated in infected and control fish at 18 min, whereas exposed negative fish retained partial habituation at 18 min. Infection was also associated with enhanced capture evasion: Despite the absence of external symptoms, infected fish tended to be caught later than uninfected fish netted from the same tank. The combination of decreased overall habituation, early extinction of habituation compared to uninfected cohorts, and enhanced netting evasion indicates that P. neurophilia infection is associated with a behavioral phenotype distinct from that of controls and uninfected cohorts. Because of its prevalence in zebrafish facilities, P. neurophilia has the potential to insidiously influence a wide range of neurobehavioral studies if these associations are causative. Rigorous health screening is therefore vital to the improvement of the zebrafish as a translational model for human behavior., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Pseudoloma neurophilia is a microsporidium of zebrafish (Danio rerio) that preferentially infects neural tissue. It is one of the most common pathogens of zebrafish in research laboratories based on diagnostic data from the Zebrafish International Resource Center diagnostic service (Eugene, OR). Five hundred fifty-nine zebrafish infected with P. neurophilia submitted to ZIRC from 86 laboratories between the years 2000 and 2013 were examined via histopathology to develop a retrospective study of the features of neural microsporidiosis. Parasite clusters (PCs) occurred in distinct axonal swellings, frequently with no associated inflammation. Inflammation was observed in viable cell bodies distant from PCs. Multiple PCs occasionally occurred within a single axon, suggesting axonal transport. PCs occurred most frequently in the spinal cord ventral white matter (40.3% of all PCs) and the spinal nerve roots (25.6%). Within the rhombencephalon, PCs were most common in the primary descending white matter tracts. Within the rhombencephalon gray matter, PCs occurred most frequently in the reticular formation and the griseum centrale (61% and 39%, respectively). High numbers of PCs within brain and spinal cord structures mediating startle responses and anxiety suggest that related behaviors could be altered by neural microsporidiosis. Infection could, therefore, introduce unacceptable variation in studies utilizing these behaviors.

Over several years of screening diagnostic cases, the Zebrafish International Resource Center Health Services has encountered a myxozoan parasite of the ducts associated with the kidney in zebrafish, Danio rerio , from an average of 21% of facilities submitting specimens over 5 yr. The parasite is coelozoic and is associated with no appreciable histological changes. Plasmodia bear ovoid spores with 3 sutural ridges. Spores are consistent with the genus Myxidium, but they are distinct from any known species and are thus described as Myxidium streisingeri n. sp. Phylogenetically, this parasite is a member of the polyphyletic urinary bladder clade, which is consistent with the site of infection. The common occurrence of a myxozoan in this closed husbandry system is unexpected because these parasites are known to have complex life cycles, alternating between a vertebrate and invertebrate host. It may be that biofilters provide habitat for suitable invertebrate hosts or perhaps M. streisingeri n. sp. can be transmitted directly. Future control of this parasite in zebrafish research laboratories depends on a better understanding of this life cycle.

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 d 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., (© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists.)

Like other vertebrate Toll-like receptors (TLRs), the TLRs of teleost fish can be subdivided into six major families, each of which recognize a general class of molecular patterns. However, there also are a number of Tlrs with unknown function, the presence of which seems unique to the bony fish, among which is Tlr20. We identified full-length complementary DNA (cDNA) sequences for tlr20 of zebrafish and common carp, two closely related fish species. Zebrafish have six copies of tlr20, whereas carp express only a single copy. Both zebrafish Tlr20 (at least Tlr20a-d) and carp Tlr20 have 26 leucine-rich repeats (LRRs). Three-dimensional modeling indicates a best fit to the crystal structure of TLR8. Phylogenetic analyses place Tlr20 in the TLR11 family closest to Tlr11 and Tlr12, which sense ligands from protozoan parasites in the mouse. Conservation of genes on zebrafish chromosome 9, which carries tlr20, with genes on mouse chromosome 14, which carries tlr11, indicates Tlr11 could be a possible ortholog of Tlr20. Confocal microscopy suggests a subcellular localization of Tlr20 at the endoplasmatic reticulum. Although in vitro reporter assays could not identify a ligand unique to Tlr20, in vivo infection experiments indicate a role for Tlr20 in the immune response of carp to protozoan parasites (Trypanoplasma borreli). Carp tlr20 is mainly expressed in peripheral blood leukocytes (PBL) with B lymphocytes, in particular, expressing relatively high levels of Tlr20. In vitro stimulation of PBL with T. borreli induces an upregulation of tlr20, supportive of a role for Tlr20 in the immune response to protozoan parasites.

Mycobacterial infections in laboratory zebrafish Danio rerio are common and widespread in research colonies. Mycobacteria within free-living amoebae have been shown to be transmission vectors for mycobacteriosis. Paramecium caudatum are commonly used as a first food for zebrafish, and we investigated this ciliate's potential to serve as a vector of Mycobacterium marinum and M. chelonae. The ability of live P. caudatum to transmit these mycobacteria to larval, juvenile and adult zebrafish was evaluated. Infections were defined by histologic observation of granulomas containing acid-fast bacteria in extraintestinal locations. In both experiments, fish fed paramecia containing mycobacteria became infected at a higher incidence than controls. Larvae (exposed at 4 d post hatch) fed paramecia with M. marinum exhibited an incidence of 30% (24/80) and juveniles (exposed at 21 d post hatch) showed 31% incidence (14/45). Adult fish fed a gelatin food matrix containing mycobacteria within paramecia or mycobacteria alone for 2 wk resulted in infections when examined 8 wk after exposure as follows: M. marinum OSU 214 47% (21/45), M. marinum CH 47% (9/19), and M. chelonae 38% (5/13). In contrast, fish feed mycobacteria alone in this diet did not become infected, except for 2 fish (5%) in the M. marinum OSU 214 low-dose group. These results demonstrate that P. caudatum can act as a vector for mycobacteria. This provides a useful animal model for evaluation of natural mycobacterial infections and demonstrates the possibility of mycobacterial transmission in zebrafish facilities via contaminated paramecia cultures.

Abstract Our zebrafish colony experienced a period of increased mortality rate of 6.5 times more deaths per month in a colony of over 13,000 zebrafish (Danio rerio), which developed over 3 months. We observed that before death, affected fish appeared emaciated, often with an abdominal bulge. We performed dissection on 18 fish that had this appearance and found in 15 that their gut was infected with a nematode that closely resembled Pseudocapillaria tomentosa. We devised a treatment protocol for this nematode infection, which involved addition of fenbendazole, a drug used to treat nematode infections in cattle and sheep, to the fish feed. Fenbendazole produced no severe side effects in the fish and several treatments have effectively eradicated the parasite from our colony. The mortality rate of our fish has decreased to a value of 0.7%/month (p<0.001, equal to that before the infection). We propose this protocol as an inexpensive alternative to having to rederive an entire colony from bleached eggs, and as a prophylactic measure used in quarantine facilities on a regular basis.

Background: Ichthyophthirius multifiliis, commonly known as Ich, is a highly pathogenic ciliate responsible for 'white spot', a disease causing significant economic losses to the global aquaculture industry. Options for disease control are extremely limited, and Ich's obligate parasitic lifestyle makes experimental studies challenging. Unlike most well-studied protozoan parasites, Ich belongs to a phylum composed primarily of free-living members. Indeed, it is closely related to the model organism Tetrahymena thermophila. Genomic studies represent a promising strategy to reduce the impact of this disease and to understand the evolutionary transition to parasitism., Results: We report the sequencing, assembly and annotation of the Ich macronuclear genome. Compared with its free-living relative T. thermophila, the Ich genome is reduced approximately two-fold in length and gene density and three-fold in gene content. We analyzed in detail several gene classes with diverse functions in behavior, cellular function and host immunogenicity, including protein kinases, membrane transporters, proteases, surface antigens and cytoskeletal components and regulators. We also mapped by orthology Ich's metabolic pathways in comparison with other ciliates and a potential host organism, the zebrafish Danio rerio., Conclusions: Knowledge of the complete protein-coding and metabolic potential of Ich opens avenues for rational testing of therapeutic drugs that target functions essential to this parasite but not to its fish hosts. Also, a catalog of surface protein-encoding genes will facilitate development of more effective vaccines. The potential to use T. thermophila as a surrogate model offers promise toward controlling 'white spot' disease and understanding the adaptation to a parasitic lifestyle.

Eosinophils are granulocytic leukocytes implicated in numerous aspects of immunity and disease. The precise functions of eosinophils, however, remain enigmatic. Alternative models to study eosinophil biology may thus yield novel insights into their function. Eosinophilic cells have been observed in zebrafish but have not been thoroughly characterized. We used a gata2:eGFP transgenic animal to enable prospective isolation and characterization of zebrafish eosinophils, and demonstrate that all gata2(hi) cells in adult hematopoietic tissues are eosinophils. Although eosinophils are rare in most organs, they are readily isolated from whole kidney marrow and abundant within the peritoneal cavity. Molecular analyses demonstrate that zebrafish eosinophils express genes important for the activities of mammalian eosinophils. In addition, gata2(hi) cells degranulate in response to helminth extract. Chronic exposure to helminth- related allergens resulted in profound eosinophilia, demonstrating that eosinophil responses to allergens have been conserved over evolution. Importantly, infection of adult zebrafish with Pseudocapillaria tomentosa, a natural nematode pathogen of teleosts, caused marked increases in eosinophil number within the intestine. Together, these observations support a conserved role for eosinophils in the response to helminth antigens or infection and provide a new model to better understand how parasitic worms activate, co-opt, or evade the vertebrate immune response.

Two Saprolegnia isolates, JY isolated from silver crucian carp (Carassius auratus gibelio Bloch) and BMY isolated from zebra fish (Brachydanio rerio Hamilton) came from infections occurring concurrently in different locations in China. To confirm whether the two isolates were from the same Saprolegnia clone, comparative studies have been carried out based on their morphological, physiological and molecular characteristics. Observations showed that morphologically (both asexual and sexual organs) the two isolates were broadly similar and both isolates underwent repeated zoospore emergence. Comparing 704 base pairs of internal transcribed spacer (ITS) region and the 5.8S rDNA, we found isolates JY and BMY shared an identical ITS sequence with a minor variation (99.6% similarity). Forty available sequences for representatives Saprolegnia spp. belonged to four phylogenetically separate clades. The two studied isolates fell within clade I that comprised a group of isolates which showed almost an identical ITS sequence but had been identified as a number of different morphological species. Our findings suggest that isolates JY and BMY appear to belong to the S. ferax clade and this clade (I) contains a number of closely related phylogenetic species. This is distinct from the more common fish pathogenic isolates, which belong to the S. parasitica clade (III) and are characterized by having cysts decorated by bundles of long hooked hairs and two further clades (II and IV) containing largely saprotrophic or soil born species.

Parasites represent a threat to endangered fish species, particularly when the parasite can host switch and the new host is vulnerable. If the parasite is highly host specific then successful host switching should be a rare occurrence; however, the host range of many parasites which are assumed to be specialists has never been tested. This includes the monogenean Gyrodactylus turnbulli, a well-studied ectoparasite found caudally on its known host, the guppy, Poecilia reticulata. In this study, we monitored parasite establishment and reproduction on a range of poeciliids and more distantly related fish. Individually maintained fish were experimentally infected with a single parasite and monitored daily to establish whether G. turnbulli could survive and reproduce on other fish species. Gyrodactylus turnbulli can infect a wider range of hosts than previously considered, highlighting the fact that host specificity can never be assumed unless experimentally tested. Our findings also have significant implications for parasite transmission to novel hosts and provide further insight into the evolutionary origins of this ubiquitous group of fish pathogens. Previous molecular evidence indicates that host switching is the key mechanism for speciation within the genus Gyrodactylus. Until recently, most Gyrodactylus spp. were assumed to be narrowly host specific. However, our findings suggest that even so-called specialist species, such as G. turnbulli, may represent a threat to vulnerable fish stocks. In view of the potential importance of host switching under artificial conditions, we propose to describe this as 'artificial ecological transfer' as opposed to 'natural ecological transfer', host switching under natural conditions.

Infections with capillarid nematodes were observed in zebrafish (Danio rerio) kept at several research facilities and in a large carcinogen exposure study previously conducted at Oregon State University. We report a morphologic description that identifies the worm as Pseudocapillaria tomentosa, a common nematode of cyprinid and other fishes. Pathologic lesions associated with the infection ranged from inflammatory changes to aggressive neoplasms of the intestine (i.e., intestinal carcinomas and mixed malignant neoplasms). Capillarid nematodes may have intermediate or paratenic hosts. Using a laboratory transmission study, we confirmed that the parasite has a direct life cycle.

A large colony of laboratory zebrafish (Brachydanio rerio) used in the study of early vertebrate embryogenesis began experiencing acute, unexplained mortality that approached 100% among approximately 30-day-old resident fry. The initial differential diagnosis included ammonia, nitrite, or chlorine toxicosis, as well as iatrogenically induced toxicosis associated with improper sanitation procedures of laboratory equipment. Necropsy of dead and moribund fry prior to fixation revealed swarms of ovoid-shaped, motile, ciliated protozoa with a "spiraling football" motion. Wet mount preparations of various water samples also contained high numbers of similar protozoa. Histologic examination of affected fry revealed numerous, periodic acid-Schiff-positive forms within the body coelom, and epithelial and muscle tissues. The protozoa were consistent morphologically with members of the genus Tetrahymena, which is usually a free-living, nonpathogenic ciliated protozoa in fresh and saltwater environments. Relevant disease associated with Tetrahymena spp. in viviparous fish has been reported as a result of concurrent disease, immunosuppression, or poor water quality conditions. To the authors' knowledge, this is the first report of an epizootic involving laboratory maintained zebrafish, and the diagnostic course and therapeutic interventions undertaken to alleviate Tetrahymena species-associated clinical disease.

An unusual xenoma-forming microsporidium was discovered in the central nervous system of moribund zebrafish from a laboratory colony in Eugene, Oregon. Infected fish were often emaciated and lethargic, and histological examination commonly revealed severe myelitis and myositis associated with the infection. Based on its structure, development, and small subunit ribosomal DNA sequence it is unique among fish microsporidia. Spores are uninucleate, ovoid to pyriform, with a prominent posterior vacuole. Spores average 5.4 x 2.7 microm with 13-16 coils of the polar filament. The microsporidium produces xenomas within the spinal cord and hindbrain of fish, and xenomas contained sporophorous vesicles with up to 16 spores. Sporoblasts and presporoblast stages (probably sporonts) are found occasionally in small aggregates dispersed randomly throughout xenomas. It clustered in the "Ichthyosporidium group" along with other fish microsporidian genera based on rDNA sequence analysis. The rDNA sequence of the zebrafish microsporidium was most similar to that of Ichthyosporidium, but showed only 12.1% similarity and therefore this microsporidium can be considered a distinct genus and species, which we have named Pseudoloma neurophilia n. g., n. sp.