Description of Gyrodactylus banmae n. sp. (Monogenea, Gyrodactylidae) parasitic on zebrafish, Danio rerio.

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.

Pseudocapillaria tomentosa, Mycoplasma spp., and Intestinal Lesions in Experimentally Infected Zebrafish Danio rerio.

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.

First Record of Clinostomum sp. (Digenea: Clinostomidae) in Danio rerio (Actinopterygii: Cyprinidae) and the Implication of Using Zebrafish from Pet Stores on Research.

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.

Tumor Necrosis Factor and Schistosoma mansoni egg antigen omega-1 shape distinct aspects of the early egg-induced granulomatous response.

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.

Schistosoma mansoni Eggs Modulate the Timing of Granuloma Formation to Promote Transmission.

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.

Temperature is a key factor influencing the invasion and proliferation of Toxoplasma gondii in fish cells.

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.

Behavioural effects of the common brain-infecting parasite Pseudoloma neurophilia in laboratory zebrafish (Danio rerio).

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.

The zebrafish as a novel model for the in vivo study of Toxoplasma gondii replication and interaction with macrophages.

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.

Combining fish and environmental PCR for diagnostics of diseased laboratory zebrafish in recirculating systems.

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.

A six-months study on Black Soldier Fly (Hermetia illucens) based diets in zebrafish.

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.

Using the Protozoan Paramecium caudatum as a Vehicle for Food-borne Infections in Zebrafish Larvae.

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.

A longitudinal assessment of host-microbe-parasite interactions resolves the zebrafish gut microbiome's link to Pseudocapillaria tomentosa infection and pathology.

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.

Association between adaptive immunity and neutrophil dynamics in zebrafish (Danio rerio) infected by a parasitic ciliate.

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.

Survival of Bacterial and Parasitic Pathogens from Zebrafish (Danio rerio) After Cryopreservation and Thawing.

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.

Immunomodulatory effects of excretory/secretory compounds from Contracaecum osculatum larvae in a zebrafish inflammation model.

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.

Copromyxa laresi n. sp. (Amoebozoa: Tubulinea) and Transfer of Cashia limacoides (Page, 1967) to Copromyxa Zopf, 1885.

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.

First Record of Transversotrema Witenberg, 1944 (Digenea) from the Americas, with Comments on the Taxonomy of Transversotrema patialense (Soparkar, 1924) Crusz and Sathananthan, 1960, and an Updated List of Its Hosts and Geographic Distribution.

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 common neural parasite Pseudoloma neurophilia is associated with altered startle response habituation in adult zebrafish (Danio rerio): Implications for the zebrafish as a model organism.

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.