Development of Quantitative Real-Time PCR Assays for Postmortem Detection ofMycobacteriumspp. Common in Zebrafish (Danio rerio) Research Colonies.
Mycobacterium spp. infections are common in zebrafish kept in research facilities. These comorbidities can substantially modulate the responses of these fish to external and internal stimuli. Therefore, diagnostic tests to detect Mycobacterium spp. infections in zebrafish colonies prove essential. Here, we outline the development of quantitative simplex real-time PCR assays to detect the 3 Mycobacterium species most commonly identified in laboratory zebrafish. The assays targeted the heatshock protein 65 gene of M. marinum, M. chelonae, and M. haemophilum. The assays are both highly specific and sensitive for fresh-frozen samples and highly specific and moderately sensitive for formalin-fixed paraffin-embedded (FFPE) samples. Two sampling techniques for FFPE samples of sagittally sectioned zebrafish were evaluated. Both paraffin cores targeting granulomas containing bacteria and scrolls from the entire fish yielded DNA of equivalent quantity and purity. The diagnostic sensitivity of cores was superior to that of scrolls for M. chelonae and M. haemophilum but not M. marinum. The assays are cost-effective and ideally suited to diagnosing common Mycobacterium spp. infections in zebrafish.
Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, Oregon.
Danielle M
DM
Meritet
Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Oregon State University, Corvallis, Oregon.
Donna M
DM
Mulrooney
Department of Biomedical Sciences, Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Oregon State University, Corvallis, Oregon, Department of Microbiology, College of Science, Oregon State University, Corvallis, Oregon.
Michael L
ML
Kent
Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, Oregon, Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Oregon State University, Corvallis, Oregon;, Email: christiane.loehr@oregonstate.edu.
Christiane V
CV
Löhr
D000818:Animals; D005393:Fish Diseases / Q000382:microbiology*; D007756:Laboratory Animal Science; D009161:Mycobacterium / Q000302:isolation & purification*; D009164:Mycobacterium Infections / Q000382:microbiology / Q000662:veterinary*; D060888:Real-Time Polymerase Chain Reaction; D015027:Zebrafish