Production of polyclonal antibodies to Batrachochytrium dendrobatidis and their use in an immunoperoxidase test for chytridiomycosis in amphibians

Abstract

Polyclonal antibodies were produced for diagnosing chytridiomycosis in amphibians. Two sheep and 4 rabbits were inoculated with homogenized whole culture of Batrachochytrium dendrobatidis 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 · Immunoperoxidase · Polyclonal antibodies · Diagnosis Resale or republication not permitted without written consent of the publisherDis Aquat Org 48: [213][214][215][216][217][218][219][220] 2002 diagnosis is insensitive when dealing with light infections in healthy animals or autolysed samples, or when tests are performed by inexperienced workers.Diagnostic methods with improved sensitivity and ease of testing are needed. The production of polyclonal antibodies and the introduction of an immunoperoxidase (IPX) stain are the first steps in the development of more sophisticated tests for the detection of antigen. MATERIALS AND METHODS Antigen for immunization.A culture of Batrachochytrium dendrobatidis (isolate A98 1810/3) was obtained from a sick, wild adult Australian lacelid (Nyctimystes dayi). The culture was maintained on tryptone, gelatin hydrolysate agar ) for 5 mo before use. Sporangia were harvested by lightly scraping a 10 d old culture. Thirty milligrams of culture was mixed with 1 ml distilled water and left for 24 h at room temperature, then manually homogenized in a sterile petri dish and frozen at -80°C. After defrosting, the mixture was diluted with phosphate buffered saline (calciumand magnesium-free) (PBSA) to a final concentration of 3.25 mg ml -1 . The protein concentration was determined using a Pyr Unicam PU8800 UV/VIS spectrophotometer at 280 nm, by interpolation of its absorbance from a standard curve calculated from known concentrations of bovine serum albumin (0.25, 0.5, 1.0 and 5.0 mg ml -1 ). Freund's complete adjuvant and triple adjuvant (Quil A, DEAE-dextran, Montanide 888 oil) (Prowse 2000) were prepared using a Sorvall Omnimixer for emulsification. The final protein concentration of antigen in both preparations was 0.5 mg ml -1 antigen.Immunization. Two rabbits (666 and 667) and 1 sheep (322) were inoculated with triple adjuvant intradermally with boosters at 7 and 11 wk post inoculation (pi). Two rabbits (668 and 669) and 1 sheep (386) were inoculated with Freund's complete adjuvant subcutaneously and boosted with Freund's incomplete adjuvant at 7 and 11 wk pi. At each inoculation, the rabbits received 0.5 mg fungus in 1 ml adjuvant and the sheep received 1 mg fungus in 2 ml...

Life cycle stages of the amphibian chytrid Batrachochytrium dendrobatidis

“…Rhizoids were rarely seen in skin sections examined by electron microscopy. They could not be seen in haematoxylin and eosin stained histology sections, but were discernible adjacent to sporangia when stained with the immunoperoxidase stain (Berger et al 2002). The fungi are often found in clusters on skin, 57…”

Chytridiomycosis in frogs of Mount Gede Pangrango, Indonesia

“…Histological and immunohistochemical analyses were conducted on the tadpole of L. hasseltii following the methods of Berger et al (2000) and Hyatt et al (2007), respectively. The immunoperoxidase stain employed rabbit 667 anti-Bd polyclonal antibody (Berger et al 2002). …”

Techniques for detecting chytridiomycosis in wild frogs: comparing histology with real-time Taqman PCR

“…However, disease surveys to date have been severely limited by the shortcomings of available diagnostic techniques, which until very recently have been: (1) insensitive, yielding many false-negatives (Berger et al 2002; (2) non-specific, leaving open the possibility for false-positives and observer bias; (3) invasive, requiring a skin sample such as webbing or toes (Berger et al 2002, Lips et al 2003; and (4) timeconsuming. While it has been suggested that chytridiomycosis in severely infected postmetamorphic individuals can be easily diagnosed by the presence of abnormal epidermal sloughing, reddening of the ventral surfaces, and behavioral changes such as lethargy and loss of righting reflex (Berger et al 1999a), animals at these later stages of infection are rarely found in the wild, due both to the short time span over which they are likely to survive, and the high rate at which they are scavenged (Green et al 2002).…”

“…Sensitivity and specificity of this technique has been improved slightly by the use of polyclonal antibodies and immunoperoxidase (IPX) staining (Berger et al 2002), but even this method may fail to detect a large proportion of infected animals ). More recently, a co-localisation technique ) has been designed to stain both Batrachochytrium dendrobatidis and the keratinized epidermis that the fungus infects, but no experiments have been undertaken to quantify the sensitivity of this technique.…”