DISEASES OF AQUATIC ORGANISMS Vol. 48: 213–220, 2002 Published April 5 Dis Aquat Org Production of polyclonal antibodies to Batrachochytrium dendrobatidis and their use in an immunoperoxidase test for chytridiomycosis in amphibians Lee Berger1, Alex D. Hyatt1,*, Veronica Olsen1, Sandra G. Hengstberger1, Donna Boyle1, Gerry Marantelli2, Kaye Humphreys3, Joyce E. Longcore4 1Australian Animal Health Laboratory, CSIRO Livestock Industries, Private Bag 24, Geelong, Victoria 3220, Australia 2Amphibian Research Centre, PO Box 424, Brunswick, Victoria 3056, Australia 3Veterinary and Quarantine Centre, Taronga Zoo, PO Box 20, Mosman, New South Wales 2088, Australia 4Department of Biological Sciences, University of Maine, Orono, Maine 04469-5722, USA ABSTRACT: Polyclonal antibodies were produced for diagnosing chytridiomycosis in amphibians. Two sheep and 4 rabbits were inoculated with homogenized whole culture of Batrachochytrium den- drobatidis in Freund’s complete adjuvant or triple adjuvant. Antisera from all animals reacted strongly with all stages of B. dendrobatidis and stained the walls, cytoplasm, rhizoids and zoospores in an indirect immunoperoxidase test. Significant cross-reactivity occurred only with some fungi in the Chytridiomycota, and there are no members of this phylum besides B. dendrobatidis that infect frogs. The immunoperoxidase stain is a useful screening test when combined with recognition of the morphology and infection site of B. dendrobatidis. KEY WORDS: Batrachochytrium dendrobatidis · Chytridiomycosis · Fungus · Amphibians · Immuno- peroxidase · Polyclonal antibodies · Diagnosis Resale or republication not permitted without written consent of the publisher INTRODUCTION tat modification and multi-factorial causes (Alford & Richards 1999), the suspected introduction of chytrid- Chytridiomycosis is a fatal disease of amphibians iomycosis to wild amphibians is the most likely cause caused by the fungus Batrachochytrium dendrobatidis, of many declines in protected areas (Berger et al. 1999). the only member of the Chytridiomycota that causes Chytridiomycosis is a highly infectious disease that disease in vertebrates (Berger et al. 1999). This fungus appears to have spread nationally and internationally, has a broad amphibian host range and occurs world- possibly by movement of infected animals. Diagnosis wide. A total of 94 amphibian species from 15 families of infected amphibians will be an important aspect of have been found infected with B. dendrobatidis, from quarantine regulations aimed at preventing the intro- Australia, South America, Central America, North duction of disease to wild or captive populations. Cur- America, Europe, New Zealand and Africa (Speare et rent diagnostic tests rely on knowledge of the mor- al. 2001). Chytridiomycosis is the most common dis- phology of the fungus for identification by histology or ease of Australian frogs (Berger et al. 1999). Although examination of wet mounts of skin scrapings. Most sick amphibian population declines are often due to habi- frogs with chytridiomycosis have heavy infections with Batrachochytrium dendrobatidis that are easily recog- nized by standard histopathological techniques (Pessier *E-mail: [email protected] et al. 1999, Berger et al. 2000). However histological © Inter-Research 2002 · www.int-res.com 214 Dis Aquat Org 48: 213–220, 2002 diagnosis is insensitive when dealing with light infec- tralia, Ecuador, New Zealand and Germany were tions in healthy animals or autolysed samples, or when tested with the antisera. Ten other chytridiomycetes tests are performed by inexperienced workers. and 18 fungi from other phyla causing animal infec- Diagnostic methods with improved sensitivity and tions (including fungi from frogs, reptiles and fish) ease of testing are needed. The production of polyclonal were obtained from various collections (Table 1). Most antibodies and the introduction of an immunoperoxi- fungi were in agar cultures, but some identifiable fungi dase (IPX) stain are the first steps in the development within animal tissues were also used. Two protozoans of more sophisticated tests for the detection of antigen. in amphibian tissue were also tested: a myxozoan and a coccidian. Samples of agar or tissue were fixed in 10% neutral buffered formalin, processed into paraffin MATERIALS AND METHODS blocks and sectioned for IPX staining. IPX test. For indirect IPX staining, paraffin sections Antigen for immunization. A culture of Batrachochy- were dewaxed and incubated for 20 min with 0.1% trium dendrobatidis (isolate A98 1810/3) was obtained trypsin in 0.1% aqueous CaCl2 at 37°C for antigen from a sick, wild adult Australian lacelid (Nyctimystes unmasking. Slides were rinsed in distilled water and dayi). The culture was maintained on tryptone, gelatin PBSA, loaded into Sequenza cassettes (Shandon, UK) hydrolysate agar (Longcore et al. 1999) for 5 mo before and incubated with 200 µl of the anti-chytrid antisera use. Sporangia were harvested by lightly scraping a at various dilutions in 0.1% skim milk powder/PBSA 10 d old culture. Thirty milligrams of culture was mixed for 1 h at 37°C. After a 5 min rinse with PBSA, slides with 1 ml distilled water and left for 24 h at room tem- were incubated with biotinylated anti-rabbit, anti-goat perature, then manually homogenized in a sterile petri immunoglobulin (Dako large volume DAKO LSAB kit, dish and frozen at –80°C. After defrosting, the mixture DAKO Corp, Carpinteria, CA, USA) for 20 min at 37°C. was diluted with phosphate buffered saline (calcium- Slides were rinsed with PBSA then incubated with 3% and magnesium-free) (PBSA) to a final concentration H2O2 in distilled water for 20 min at room temperature of 3.25 mg ml–1. The protein concentration was deter- (22°C) to block endogenous peroxidase activity. After mined using a Pyr Unicam PU8800 UV/VIS spectro- rinsing in PBSA, slides were incubated with strepta- photometer at 280 nm, by interpolation of its absor- vidin peroxidase conjugated (DAKO LSAB kit) for bance from a standard curve calculated from known 20 min at 37°C, rinsed again, then removed from the concentrations of bovine serum albumin (0.25, 0.5, Sequenza cassettes. The antigenic complex was visu- 1.0 and 5.0 mg ml–1). Freund’s complete adjuvant and alized using a 3-amino-9-ethyl carbasole (AEC) chro- triple adjuvant (Quil A, DEAE-dextran, Montanide 888 mogen system (Sigma). The substrate and chromogen oil) (Prowse 2000) were prepared using a Sorvall Omn- (freshly made AEC solution [2 mg AEC powder in imixer for emulsification. The final protein concentra- 200 µl dimethyl formamide] added to 10 ml 0.05 M tion of antigen in both preparations was 0.5 mg ml–1 acetate buffer with 5 µl 30% hydrogen peroxide) were antigen. added and incubated at room temperature for 5 min. Immunization. Two rabbits (666 and 667) and 1 After washing, slides were counterstained in Lillie’s sheep (322) were inoculated with triple adjuvant intra- modified hemalum, blued in Scott’s tap water, rinsed dermally with boosters at 7 and 11 wk post inoculation in tap water and mounted in an aqueous mounting (pi). Two rabbits (668 and 669) and 1 sheep (386) were medium. inoculated with Freund’s complete adjuvant subcuta- Negative controls consisted of test slides incubated neously and boosted with Freund’s incomplete adju- with normal sera, pre-bleed sera or 1% skim milk vant at 7 and 11 wk pi. At each inoculation, the rabbits instead of primary antibody. Sections of Batrachochy- received 0.5 mg fungus in 1 ml adjuvant and the sheep trium dendrobatidis culture (A98 1810/3) and sec- received 1 mg fungus in 2 ml adjuvant. tions of infected skin from a green tree frog (Litoria At 13 wk pi, serum from all animals were strongly caerulea) (A99 1385/1) were used as positive control stained for Batrachochytrium dendrobatidis in the IPX slides. test (refer below), and at 15 wk pi animals were bled The IPX test was used to indicate the titer of anti- out under anesthesia (Australian Animal Health Labo- bodies from each animal (rabbits and sheep) and to ratory, animal ethics approval no. 97-797). Blood was characterize the cross-reactivity of antibodies with collected into SST gel and clot activator vacutainers. It other fungi. These fungi are listed in Table 1. was stored at 4°C for up to 2 d before centrifuging at Evaluation of the IPX test for diagnosis. Preliminary 3000 rpm (Beckman J-6 M centrifuge) for 3 min. Serum evaluation of the IPX as a diagnostic assay was con- was stored in 10 ml sterile Starstedt tubes at –80°C. ducted on 55 lightly infected and 15 control toe-clip Fungal isolates for cross reactivity testing. Speci- samples from an experimental infection using juve- mens of Batrachochytrium dendrobatidis from Aus- niles of Litoria caerulea. The toe-clips were collected Berger et al.: Production of polyclonal antibodies to Batrachochytrium dendrobatidis 215 Table 1. List of fungal species used, their strain identification numbers, the order and phylum they belong to, and their source. A: from Lee Berger, Australian Animal Health Laboratory, Australia; E: from Andrés Merino-Viteri, Museo de Zoologia, Quito, Ecuador; G: from Frank Mutschmann, Tierarztpraxis, Berlin, Germany; JEL: from the chytrid collection of Joyce Longcore, Uni- versity of Maine, Maine, USA; N: from Bruce Waldman and Richard Norman, Massey University, New Zealand; T: from Kaye Humphreys and Karrie Rose, Taronga Zoo, Australia Species Strain Order Phylum Source identification Batrachochytrium dendrobatidis A 98 1810/3 Chytridiales Chytridiomycota