Partial alpha-tubulin gene amplification by nested PCR as a tool for Paracoccidioides ceti identification

Area of Science:

• Mycology
• Marine Mammal Pathology
• Molecular Biology

Background:

• Paracoccidioidomycosis ceti (PCMC), or lobomycosis, is a global cutaneous fungal disease in cetaceans.
• The causative agent, *Paracoccidioides ceti*, is an uncultivable fungus.
• The alpha-tubulin gene (TUB1) is a key marker for *Paracoccidioides* but remains unstudied in *P. ceti*.

Purpose of the Study:

• To investigate the utility of the alpha-tubulin gene (TUB1) for studying *Paracoccidioides ceti* (PCMC).
• To develop and apply a molecular method for identifying and analyzing *P. ceti* in clinical samples.

Main Methods:

• Nested polymerase chain reaction (PCR) targeting the TUB1 gene.
• Amplification of fungal DNA from skin lesions of two Brazilian cetaceans (Lahille's bottlenose dolphin and common bottlenose dolphin).
• Comparative sequence analysis and haplotype analysis with related *Paracoccidioides* species.

Main Results:

• The nested PCR assay successfully amplified TUB1 sequences from both PCMC cases.
• Sequences from infected dolphins were identical and closely related to the *P. brasiliensis* complex.
• Haplotype analysis placed *P. ceti* near *P. brasiliensis sensu stricto* and *P. americana*, with shared restriction sites with *P. americana*.

Conclusions:

• The nested PCR targeting TUB1 is effective for amplifying fungal DNA from the uncultivable *P. ceti*.
• This molecular tool facilitates phylogenetic studies of *P. ceti*.
• Findings enhance the understanding of PCMC pathogenesis and the evolutionary relationships within the *Paracoccidioides* genus.