Microbe transmission from pet shop to lab-reared zebrafish reveals a pathogenic birnavirus.

Zebrafish are popular research organisms selected for laboratory use due in part to widespread availability from the pet trade. Many contemporary colonies of laboratory zebrafish are maintained in aquaculture facilities that monitor and aim to curb infections that can negatively affect colony health and confound experiments. The impact of laboratory control on the microbial constituents associated with zebrafish in research environments compared to the pet trade are unclear. Diseases of unknown causes are common in both environments. We conducted a metatranscriptomic survey to broadly compare the zebrafish-associated microbes in pet trade and laboratory environments. We detected many microbes in animals from the pet trade that were not found in laboratory animals. Cohousing experiments revealed several transmissible microbes including a newly described non-enveloped, double-stranded RNA virus in the Birnaviridae family we name Rocky Mountain birnavirus (RMBV). Infections were detected in asymptomatic animals from the pet trade, but when transmitted to laboratory animals RMBV was associated with pronounced antiviral responses and hemorrhagic disease. These experiments highlight the pet trade as a distinct source of diverse microbes that associate with zebrafish and establish a paradigm for the discovery of newly described pathogenic viruses and other infectious microbes that can be developed for study in the laboratory.

Microbe transmission from pet shop to lab-reared zebrafish reveals a pathogenic birnavirus.

Zebrafish are popular research organisms selected for laboratory use due in part to widespread availability from the pet trade. Many contemporary colonies of laboratory zebrafish are maintained in aquaculture facilities that monitor and aim to curb infections that can negatively affect colony health and confound experiments. The impact of laboratory control on the microbial constituents associated with zebrafish in research environments compared to the pet trade are unclear. Diseases of unknown causes are common in both environments. We conducted a metagenomic survey to broadly compare the zebrafish-associated microbes in pet trade and laboratory environments. We detected many microbes in animals from the pet trade that were not found in laboratory animals. Co-housing experiments revealed several transmissible microbes including a newly described non-enveloped, double-stranded RNA virus in the Birnaviridae family we name Rocky Mountain birnavirus (RMBV). Infections were detected in asymptomatic animals from the pet trade, but when transmitted to laboratory animals RMBV was associated with pronounced antiviral responses and hemorrhagic disease. These experiments highlight the pet trade as a distinct source of diverse microbes that associate with zebrafish and establish a paradigm for the discovery of newly described pathogenic viruses and other infectious microbes that can be developed for study in the laboratory.

Natural rodent model of viral transmission reveals biological features of virus population dynamics.

Emerging viruses threaten global health, but few experimental models can characterize the virus and host factors necessary for within- and cross-species transmission. Here, we leverage a model whereby pet store mice or rats-which harbor natural rodent pathogens-are cohoused with laboratory mice. This "dirty" mouse model offers a platform for studying acute transmission of viruses between and within hosts via natural mechanisms. We identified numerous viruses and other microbial species that transmit to cohoused mice, including prospective new members of the Coronaviridae, Astroviridae, Picornaviridae, and Narnaviridae families, and uncovered pathogen interactions that promote or prevent virus transmission. We also evaluated transmission dynamics of murine astroviruses during transmission and spread within a new host. Finally, by cohousing our laboratory mice with the bedding of pet store rats, we identified cross-species transmission of a rat astrovirus. Overall, this model system allows for the analysis of transmission of natural rodent viruses and is a platform to further characterize barriers to zoonosis.

Linking Virus Discovery to Immune Responses Visualized during Zebrafish Infections.

The discovery of new viruses currently outpaces our capacity for experimental examination of infection biology. To better couple virus discovery with immunology, we genetically modified zebrafish to visually report on virus infections. After generating a strain that expresses green fluorescent protein (GFP) under an interferon-stimulated gene promoter, we repeatedly observed transgenic larvae spontaneously expressing GFP days after hatching. RNA sequencing comparisons of co-housed GFP-positive and GFP-negative zebrafish revealed a naturally occurring picornavirus that induced a canonical interferon-mediated response and hundreds of antiviral defense genes not observed following immunostimulatory treatments or experimental infections with other viruses. Among the many genes induced by picornavirus infection was a large set encoding guanosine triphosphatase (GTPase) of immunity-associated proteins (GIMAPs). The GIMAP gene family is massively expanded in fish genomes and may also play a crucial role in antiviral responses in mammals, including humans. We subsequently detected zebrafish picornavirus in publicly available sequencing data from seemingly asymptomatic zebrafish in many research institutes and found that it altered gene expression in a previous study of zebrafish development. Experiments revealed a horizontal mode of virus transmission, highlighting a system for studying the spread of picornavirus infections within and between individuals. Our study describes a naturally occurring picornavirus that elicits strong antiviral responses in zebrafish and provides new strategies for simultaneously discovering viruses and their impact on vertebrate hosts.

Natural variation in the roles of C. elegans autophagy components during microsporidia infection.

Natural genetic variation can determine the outcome of an infection, and often reflects the co-evolutionary battle between hosts and pathogens. We previously found that a natural variant of the nematode Caenorhabditis elegans from Hawaii (HW) has increased resistance against natural microsporidian pathogens in the Nematocida genus, when compared to the standard laboratory strain of N2. In particular, HW animals can clear infection, while N2 animals cannot. In addition, HW animals have lower levels of initial colonization of Nematocida inside intestinal cells, compared to N2. Here we investigate how this natural variation in resistance relates to autophagy. We found that there is much better targeting of autophagy-related machinery to parasites under conditions where they are cleared. In particular, ubiquitin targeting to Nematocida cells correlates very well with their subsequent clearance in terms of timing, host strain and age, as well as species of Nematocida. Furthermore, clearance correlates with targeting of the LGG-2/LC3 autophagy protein to parasite cells, with HW animals having much more efficient targeting of LGG-2 to parasite cells than N2 animals. Surprisingly, however, we found that LGG-2 is not required to clear infection. Instead, we found that LGG-2/LC3 regulates Nematocida colonization inside intestinal cells. Interestingly, LGG-2/LC3 regulates intracellular colonization only in the HW strain, and not in N2. Altogether these results demonstrate that there is natural genetic variation in an LGG-2-dependent process that regulates microsporidia colonization inside intestinal cells, although not microsporidia clearance.

Identification of microsporidia host-exposed proteins reveals a repertoire of rapidly evolving proteins.

Pathogens use a variety of secreted and surface proteins to interact with and manipulate their hosts, but a systematic approach for identifying such proteins has been lacking. To identify these 'host-exposed' proteins, we used spatially restricted enzymatic tagging followed by mass spectrometry analysis of Caenorhabditis elegans infected with two species of Nematocida microsporidia. We identified 82 microsporidia proteins inside of intestinal cells, including several pathogen proteins in the nucleus. These microsporidia proteins are enriched in targeting signals, are rapidly evolving and belong to large Nematocida-specific gene families. We also find that large, species-specific families are common throughout microsporidia species. Our data suggest that the use of a large number of rapidly evolving species-specific proteins represents a common strategy for microsporidia to interact with their hosts. The unbiased method described here for identifying potential pathogen effectors represents a powerful approach to study a broad range of pathogens.

Cell-to-cell spread of microsporidia causes Caenorhabditis elegans organs to form syncytia.

The growth of pathogens is dictated by their interactions with the host environment1. Obligate intracellular pathogens undergo several cellular decisions as they progress through their life cycles inside host cells2. We have studied this process for microsporidian species in the genus Nematocida as they grew and developed inside their co-evolved animal host, Caenorhabditis elegans3-5. We found that microsporidia can restructure multicellular host tissues into a single contiguous multinucleate cell. In particular, we found that all three Nematocida species we studied were able to spread across the cells of C. elegans tissues before forming spores, with two species causing syncytial formation in the intestine and one species causing syncytial formation in the muscle. We also found that the decision to switch from replication to differentiation in Nematocida parisii was altered by the density of infection, suggesting that environmental cues influence the dynamics of the pathogen life cycle. These findings show how microsporidia can maximize the use of host space for growth and that environmental cues in the host can regulate a developmental switch in the pathogen.

A wild C. elegans strain has enhanced epithelial immunity to a natural microsporidian parasite.

Microbial pathogens impose selective pressures on their hosts, and combatting these pathogens is fundamental to the propagation of a species. Innate immunity is an ancient system that provides the foundation for pathogen resistance, with epithelial cells in humans increasingly appreciated to play key roles in innate defense. Here, we show that the nematode C. elegans displays genetic variation in epithelial immunity against intestinal infection by its natural pathogen, Nematocida parisii. This pathogen belongs to the microsporidia phylum, which comprises a large phylum of over 1400 species of fungal-related parasites that can infect all animals, including humans, but are poorly understood. Strikingly, we find that a wild C. elegans strain from Hawaii is able to clear intracellular infection by N. parisii, with this ability restricted to young larval animals. Notably, infection of older larvae does not impair progeny production, while infection of younger larvae does. The early-life immunity of Hawaiian larvae enables them to produce more progeny later in life, providing a selective advantage in a laboratory setting--in the presence of parasite it is able to out-compete a susceptible strain in just a few generations. We show that enhanced immunity is dominant to susceptibility, and we use quantitative trait locus mapping to identify four genomic loci associated with resistance. Furthermore, we generate near-isogenic strains to directly demonstrate that two of these loci influence resistance. Thus, our findings show that early-life immunity of C. elegans against microsporidia is a complex trait that enables the host to produce more progeny later in life, likely improving its evolutionary success.

Caenorhabditis elegans as a model for intracellular pathogen infection.

The genetically tractable nematode Caenorhabditis elegans is a convenient host for studies of pathogen infection. With the recent identification of two types of natural intracellular pathogens of C. elegans, this host now provides the opportunity to examine interactions and defence against intracellular pathogens in a whole-animal model for infection. C. elegans is the natural host for a genus of microsporidia, which comprise a phylum of fungal-related pathogens of widespread importance for agriculture and medicine. More recently, C. elegans has been shown to be a natural host for viruses related to the Nodaviridae family. Both microsporidian and viral pathogens infect the C. elegans intestine, which is composed of cells that share striking similarities to human intestinal epithelial cells. Because C. elegans nematodes are transparent, these infections provide a unique opportunity to visualize differentiated intestinal cells in vivo during the course of intracellular infection. Together, these two natural pathogens of C. elegans provide powerful systems in which to study microbial pathogenesis and host responses to intracellular infection.

C. elegans detects pathogen-induced translational inhibition to activate immune signaling.

Pathogens commonly disrupt host cell processes or cause damage, but the surveillance mechanisms used by animals to monitor these attacks are poorly understood. Upon infection with pathogenic Pseudomonas aeruginosa, the nematode C. elegans upregulates infection response gene irg-1 using the zip-2 bZIP transcription factor. Here we show that P. aeruginosa infection inhibits mRNA translation in the intestine via the endocytosed translation inhibitor Exotoxin A, which leads to an increase in ZIP-2 protein levels. In the absence of infection we find that the zip-2/irg-1 pathway is upregulated following disruption of several core host processes, including inhibition of mRNA translation. ZIP-2 induction is conferred by a conserved upstream open reading frame in zip-2 that could derepress ZIP-2 translation upon infection. Thus, translational inhibition, a common pathogenic strategy, can trigger activation of an immune surveillance pathway to provide host defense.

Small molecule-mediated activation of the integrin CD11b/CD18 reduces inflammatory disease.

The integrin CD11b/CD18 (also known as Mac-1), which is a heterodimer of the α(M) (CD11b) and ß(2) (CD18) subunits, is critical for leukocyte adhesion and migration and for immune functions. Blocking integrin-mediated leukocyte adhesion, although beneficial in experimental models, has had limited success in treating inflammatory diseases in humans. Here, we used an alternative strategy of inhibiting leukocyte recruitment by activating CD11b/CD18 with small-molecule agonists, which we term leukadherins. These compounds increased the extent of CD11b/CD18-dependent cell adhesion of transfected cells and of primary human and mouse neutrophils, which resulted in decreased chemotaxis and transendothelial migration. Leukadherins also decreased leukocyte recruitment and reduced arterial narrowing after injury in rats. Moreover, compared to a known integrin antagonist, leukadherins better preserved kidney function in a mouse model of experimental nephritis. Leukadherins inhibited leukocyte recruitment by increasing leukocyte adhesion to the inflamed endothelium, which was reversed with a blocking antibody. Thus, we propose that pharmacological activation of CD11b/CD18 offers an alternative therapeutic approach for inflammatory diseases.

Clonal analysis of hematopoietic progenitor cells in the zebrafish.

Identification of hematopoietic progenitor cells in the zebrafish (Danio rerio) has been hindered by a lack of functional assays to gauge proliferative potential and differentiation capacity. To investigate the nature of myeloerythroid progenitor cells, we developed clonal methylcellulose assays by using recombinant zebrafish erythropoietin and granulocyte colony-stimulating factor. From adult whole kidney marrow, erythropoietin was required to support erythroid colony formation, and granulocyte colony-stimulating factor was required to support the formation of colonies containing neutrophils, monocytes, and macrophages. Myeloid and erythroid colonies showed distinct morphologies and were easily visualized and scored by their expression of lineage-specific fluorescent transgenes. Analysis of the gene-expression profiles after isolation of colonies marked by gata1:DsRed or mpx:eGFP transgenes confirmed our morphological erythroid and myeloid lineage designations, respectively. The majority of progenitor activity was contained within the precursor light scatter fraction, and more immature precursors were present within the lymphoid fraction. Finally, we performed kinetic analyses of progenitor activity after sublethal irradiation and demonstrated that recovery to preirradiation levels occurred by 14 days after irradiation. Together, these experiments provide the first report of clonal hematopoietic progenitor assays in the zebrafish and establish the number, characteristics, and kinetics of myeloerythroid progenitors during both steady-state and stress hematopoiesis.

Identification of dendritic antigen-presenting cells in the zebrafish.

In mammals, dendritic cells (DCs) form the key link between the innate and adaptive immune systems. DCs act as immune sentries in various tissues and, upon encountering pathogen, engulf and traffic foreign antigen to secondary lymphoid tissues, stimulating antigen-specific T lymphocytes. Although DCs are of fundamental importance in orchestrating the mammalian immune response, their presence and function in nonmammalian vertebrates is largely unknown. Because teleosts possess one of the earliest recognizable adaptive immune systems, we sought to identify antigen-presenting cells (APCs) in the zebrafish to better understand the potential origins of DCs and their evolutionary relationship to lymphocytes. Here we present the identification and characterization of a zebrafish APC subset strongly resembling mammalian DCs. Rare DCs are present in various adult tissues, and can be enriched by their affinity for the lectin peanut agglutinin (PNA). We show that PNA(hi) myeloid cells possess the classical morphological features of mammalian DCs as revealed by histochemical and ultrastructural analyses, phagocytose-labeled bacterial preparations in vivo, and exhibit expression of genes associated with DC function and antigen presentation, including il12, MHC class II invariant chain iclp1, and csf1r. Importantly, we show that PNA(hi) cells can activate T lymphocytes in an antigen-dependent manner. Together, these studies suggest that the cellular constituents responsible for antigen presentation are remarkably conserved from teleosts to mammals, and indicate that the zebrafish may serve as a unique model to study the origin of APC subsets and their evolutionary role as the link between the innate and adaptive immune systems.

Eosinophils in the zebrafish: prospective isolation, characterization, and eosinophilia induction by helminth determinants.

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.