The use of singleplex and nested PCR to detect Batrachochytrium dendrobatidis in free-living frogs.
Many microorganisms are able to cause diseases in amphibians, and in the past few years one of the most reported has been Batrachochytrium dendrobatidis. This fungus was first reported in Brazil in 2005; following this, other reports were made in specimens deposited in museum collections, captive and free-living frogs. The aim of this study was to compare singleplex and nested-PCR techniques to detect B. dendrobatidis in free-living and apparently healthy adult frogs from the Brazilian Atlantic Forest. The sample collection area was a protected government park, with no general entrance permitted and no management of the animals there. Swabs were taken from the skin of 107 animals without macroscopic lesions and they were maintained in ethanol p.a. Fungal DNA was extracted and identification of B. dendrobatidis was performed using singleplex and nested-PCR techniques, employing specific primers sequences. B. dendrobatidis was detected in 61/107 (57%) and 18/107 (17%) animals, respectively by nested and singleplex-PCR. Nested-PCR was statistically more sensible than the conventional for the detection of B. dendrobatidis (Chi-square = 37.1; α = 1%) and the agreement between both techniques was considered just fair (Kappa = 0.27). The high prevalence obtained confirms that these fungi occur in free-living frogs from the Brazilian Atlantic Forest with no macroscopic lesions, characterizing the state of asymptomatic carrier. We concluded that the nested-PCR technique, due to its ease of execution and reproducibility, can be recommended as one of the alternatives in epidemiological surveys to detect B. dendrobatidis in healthy free-living frog populations.
Universidade Paulista, Laboratório de Biologia Molecular e Celular, Universidade Paulista, São Paulo, SP, Brasil, Laboratório de Biologia Molecular e Celular, Universidade Paulista, São Paulo, SP, Brazil.
Selene Dall'Acqua
SD
Coutinho
Universidade Paulista, Laboratório de Biologia Molecular e Celular, Universidade Paulista, São Paulo, SP, Brasil, Laboratório de Biologia Molecular e Celular, Universidade Paulista, São Paulo, SP, Brazil.
Julieta Catarina
JC
Burke
Universidade de São Paulo, Laboratório de Patologia Comparada de Animais Selvagens, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP, Brasil, Laboratório de Patologia Comparada de Animais Selvagens, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP, Brazil.
Catia Dejuste
CD
de Paula
Universidade de São Paulo, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP, Brasil, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP, Brazil.
Miguel Trefaut
MT
Rodrigues
Universidade de São Paulo, Laboratório de Patologia Comparada de Animais Selvagens, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP, Brasil, Laboratório de Patologia Comparada de Animais Selvagens, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP, Brazil.
José Luiz
JL
Catão-Dias
D000663:Amphibians / Q000382:microbiology*; D000818:Animals; D001938:Brazil; D002353:Carrier State / Q000382:microbiology / Q000662:veterinary*; D008411:Chytridiomycota / Q000235:genetics / Q000302:isolation & purification*; D008828:Microbiological Techniques / Q000379:methods; D025202:Molecular Diagnostic Techniques / Q000379:methods; D009181:Mycoses / Q000382:microbiology / Q000662:veterinary*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014730:Veterinary Medicine / Q000379:methods
Batrachochytrium dendrobatidis; Brazilian Atlantic Forest; PCR; chytridiomycosis; frogs