Development of a validated quantitative polymerase chain reaction assay and fungal culture for the diagnosis of Macrorhabdus ornithogaster in budgerigars (Melopsittacus undulatus).

Abstract

OBJECTIVE: To develop and validate a quantitative PCR (qPCR) assay for detecting Macrorhabdus ornithogaster (MO) and reproducibly culture MO from infected budgerigars (Melopsittacus undulatus). METHODS: A TaqMan qPCR assay targeting a 94-bp segment of MO 18S rRNA was evaluated for limit of detection, dynamic range, intra-assay variability, interassay variability, and efficiency. Proventricular-ventricular samples and feces from deceased budgerigars positive for MO on cytology were plated with Basal Medium Eagle or chicken serum media, 20% fetal bovine serum, 5% sucrose, 100 U/mL penicillin, 100 &#xb5;g/mL streptomycin, and 25 &#xb5;g/mL chloramphenicol at pH 3 to 4 and 42 &#xb0;C under microaerophilic conditions. RESULTS: The qPCR was successfully developed and performed with high efficiency (slope = -3.355; R2 = 0.999; efficiency = 98.622) and low intra- and interassay variability (coefficient of variation < 2.63% at all dilutions). The dynamic range was 107 to 101 copies/reaction with a limit of detection of 10 target copies/reaction. Macrorhabdus ornithogaster was successfully cultured from 4 different infected budgerigars using this culture protocol; however, cultures did not maintain long enough for antifungal susceptibility testing. CONCLUSIONS: We developed and analytically validated a TaqMan qPCR assay for MO detection. Macrorhabdus ornithogaster culture is possible, but further research is needed for culture maintenance and susceptibility testing. CLINICAL RELEVANCE: This analytically validated qPCR MO assay will be a useful diagnostic tool for the detection and quantification of MO in infected budgerigar feces. Reliable culturing of MO will provide the basis for antifungal drug susceptibility testing to improve treatment methods for MO in birds.