Vol. 91: 47–56, 2010 DISEASES OF AQUATIC ORGANISMS Published July 26 doi: 10.3354/dao02245 Dis Aquat Org Pleistophora hyphessobryconis (Microsporidia) infecting zebrafish Danio rerio in research facilities Justin L. Sanders1,*, Christian Lawrence2, Donald K. Nichols3, 4, Jeffrey F. Brubaker4, Tracy S. Peterson1, Katrina N. Murray5, Michael L. Kent1 1Department of Microbiology, Oregon State University, Corvallis, Oregon 97331, USA 2Children’s Hospital Boston, Aquatic Resources Program, Boston, Massachusetts 02115, USA 3U.S. Army Center for Environmental Health Research and 4U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland 21702, USA 5Zebrafish International Resource Center, University of Oregon, Eugene, Oregon 97403, USA ABSTRACT: Zebrafish Danio rerio are important models for biomedical research, and thus, there is an increased concern about diseases afflicting them. Here we describe infections by Pleistophora hyphessobryconis (Microsporidia) in zebrafish from 3 laboratories. As reported in other aquarium fishes, affected zebrafish exhibited massive infections in the skeletal muscle, with no involvement of smooth or cardiac muscle. In addition, numerous spores within macrophages were observed in the visceral organs, including the ovaries. Transmission studies and ribosomal RNA (rRNA) gene sequence comparisons confirmed that the parasite from zebrafish was P. hyphessobryconis as described from neon tetra Paracheirodon innesi. Ten 15 d old zebrafish were exposed to P. hyphesso- bryconis collected from 1 infected neon tetra, and 7 of 10 fish became infected. Comparison of P. hyphessobryconis small subunit rRNA gene sequence from neon tetra with that obtained from zebrafish was nearly identical, with <1% difference. Given the severity of infections, P. hyphesso- bryconis should be added to the list of pathogens that should be avoided in zebrafish research facili- ties, and it would be prudent to avoid mixing zebrafish used in research with other aquarium fishes. KEY WORDS: Zebrafish · Microsporidia · Pleistophora hyphessobryconis Resale or republication not permitted without written consent of the publisher INTRODUCTION Unfortunately, as is the case with any animal model, the zebrafish can be afflicted with a number of dis- The zebrafish Danio rerio is an important research eases, potentially confounding experimental results model for the study of infectious disease (Dooley & and causing growing concern among investigators. The Zon 2000), developmental and genetic biology most common infectious diseases found in laboratory (Grunwald & Eisen 2002, Ackermann & Paw 2003), zebrafish are mycobacteriosis (Kent et al. 2004) and mi- cancer (Amatruda et al. 2002), immunology (Yoder et crosporidiosis caused by the microsporidian parasite al. 2002, Trede et al. 2004), toxicology (Hill et al. Pseudoloma neurophilia. As the name implies, the mi- 2005), and drug discovery (Zon & Peterson 2005). crosporidium infects neural tissue in the brain and Their small size, relative ease of husbandry, large spinal cord of zebrafish (Matthews et al. 2001). research community, and ex vivo development of We recently detected another microsporidium in transparent embryos makes them an amenable zebrafish: Pleistophora hyphessobryconis. This is the model for such studies. As a result, numerous labora- causative agent of ‘neon tetra disease,’ which targets tory colonies have been established containing wild- the skeletal muscle of many aquarium fishes. The pri- type, mutant, and transgenic strains with a wide vari- mary and type host is the neon tetra Paracheirodon ety of genetic backgrounds. innesi (Characiformes: Characidae). However, this *Email: [email protected] © Inter-Research 2010 · www.int-res.com 48 Dis Aquat Org 91: 47–56, 2010 microsporidium shows broad host specificity, and has extraction from tissue with an initial proteinase K been reported from many species of aquarium fishes in digestion at 56°C for 3 h. Approximately 50 juvenile several families (Characidae, Cyprinidae, Cyprinodon- neon tetras with a suspected history of the infection tidae, Poecilidae, Cichlidae), including Danio rerio and were obtained from a private retail fish store in the D. nigrofasciatus (Steffens 1962). Some host range Corvallis, Oregon area, USA. Muscle tissue from 1 reports of this parasite were derived from cross trans- infected fish was frozen and processed for sequencing mission studies (Canning et al. 1986), but most reports as above. regarding the host range were from observations of PCR was performed using the general microsporid- naturally infected fishes. Therefore, it is conceivable ian small subunit rRNA gene primers V1F (5’-CAC that some of these infections may have been caused by CAG GTT GAT TCT GCC TGA C-3’) and 1492R (5’- other related, undescribed species that are morpholog- GTT ACC TTG TTA CGA CTT-3’). Amplifications ically indistinguishable. were performed on a Peltier 200 thermocycler (MJ We detected severe muscle infections of Pleistophora Research) with an initial denaturation at 94°C for hyphessobryconis (Lom & Dyková 1992, Shaw & Kent 2 min, 35 cycles at 94°C for 1 min, 55°C for 1 min, and 1999) in zebrafish from 3 separate research facilities. 68°C for 1 min with a final extension at 68°C for 7 min. We report here on the case histories, including macro- PCR products were cloned into TOPO TA Cloning vec- scopic and histological changes associated with the tors (Invitrogen) and sequenced in both directions infection in laboratory zebrafish. We demonstrate that using primers flanking the inserted sequence. To the parasite recently found in zebrafish from research exclude concomitant Pseudoloma neurophilia infec- facilities was P. hyphessobryconis by conducting cross tion, a reverse primer was designed using the Primer- transmission experiments and rRNA gene sequence BLAST program available online from the National comparisons with P. hyphessobryconis obtained from Center for Biotechnology Information (NCBI; http:// the type host, neon tetra. www3.ncbi.nlm.nih.gov/). The novel primer, PleistR (5’-TCT CGC TTG TTC GCG CCT GA-3’), was used with the forward primer, V1F, to perform the PCR on MATERIALS AND METHODS samples from Lab 3 using the same thermocycling con- ditions as described above. PCR products from these Case histories. Zebrafish from 3 research facilities samples were sequenced directly. All DNA analyzed in were examined by histology either as part of routine the study was sequenced on an ABI Prism®3730 health screening or because fish exhibited clinical dis- Genetic Analyzer with the BigDye® Terminator v. 3.1 ease. The index case (at Lab 1) was evaluated by 2 of Cycle Sequencing kit (Applied Biosystems). the authors (D.N. and J.B.), and the specimens from the Phylogenetic analysis. The 16S rRNA gene se- other 2 facilities were submitted to the Zebrafish Inter- quence of Pleistophora hyphessobryconis was aligned national Resource Center (ZIRC) diagnostic service with several Pleistophora sequences and other closely (http://zebrafish.org/zirc/health/index.php). Informa- related genera returned by BLAST query of the Gen- tion regarding fish husbandry and quarantine proce- Bank database (Altschul et al. 1990) using the dures was provided by the submitting client. ClustalW2 software (Larkin et al. 2007). Pseudoloma Histology. Fish were preserved in either 10% neurophilia and Glugea anomala (Microsporidia) were buffered formalin (Lab 1) or Dietrich’s fixative (Labs 2 selected as outgroup species. The jModelTest software and 3) and processed for routine histology. Transverse (Posada & Buckley 2004) was used to determine the sections were made of the fish from Lab 1; sagittal sec- most likely model of sequence evolution for the data- tions were prepared of the fish from Labs 2 and 3. All set, and phylogenetic analyses were performed using sections were stained with hematoxylin and eosin. Bayesian inference as implemented in MrBayes3.1.2 Additional tissue sections of the one infected fish from (Huelsenbeck et al. 2001) and maximum likelihood Lab 1 were stained with Lillie-Twort Gram stain algorithms (Guindon et al. 2009) as implemented in the (Culling 1974), and sections from several infected fish PHYML webserver (www.atgc-montpellier.fr/phyml/). from Lab 3 were also stained with Accustain™, MrBayes was run using the General Time Reversible (Sigma-Aldrich), a commercial Brown-Hopps stain. (GTR) model of nucleotide substitution with δ-distrib- rRNA gene sequencing. After diagnosis of the infec- uted rate variation across sites and a proportion of tion by histology in several fish from Lab 3, additional invariable sites (GTR I + G) for 1 000 000 generations. live fish were euthanized and skeletal muscle was PHYML was run using the GTR model of nucleotide examined by wet mount. Infected muscle tissues from substitution, and bootstrap support was based on 100 3 fish were processed for sequencing. Using the QIA- replicates. gen Blood and Tissue kit, ~25 mg of muscle tissue was The following sequences were obtained from Gen- extracted following the manufacturer’s protocol for Bank and used for alignment and subsequent phylo- Sanders et al.: Pleistophora hyphessobryconis infecting zebrafish in research facilities 49 genetic analysis: Pleistophora ovariae (AJ252955.1), in the established colony, consisting of dry flake food P. mirandellae (AJ252954.1), Ovipleistophora miran- (TetraMin tropical fish flake food) twice daily and live dellae (AF356223.1), Heterosporis anguillarum brine shrimp once daily.