Partial Alpha-tubulin gene amplification by Nested PCR as a tool for Paracoccidioides ceti identification

Paracoccidioidomycosis ceti (PCMC), also called lobomycosis, is a cutaneous disease affecting cetaceans worldwide. It is caused by Paracoccidioides ceti, an uncultivable fungal species recently classified within the Paracoccidioides genus. Although several molecular markers have been used to investigate the PCMC pathogen, the alpha tubulin gene (TUB1), commonly utilized in genetic studies of cultivable Paracoccidioides, has remained unexplored in this taxon. In this study, we applied a nested PCR targeting TUB1 to amplify fungal sequences from two new cases of PCMC in Brazil: a Lahille’s bottlenose dolphin (Tursiops truncatus gephyreus), and a common bottlenose dolphin (Tursiops truncatus truncatus). Comparative analysis revealed that the sequences obtained from the infected dolphins were identical to each other and shared similarities with fungi belonging to the Paracoccidioides brasiliensis complex. In the haplotype analysis, P. ceti was found to be only a few mutational steps away from Paracoccidioides brasiliensis sensu stricto and Paracoccidioides americana. Notably, the latter shared the same cleavage sites as P. ceti in the in silico Restriction Fragment Length Polymorphism (RFLP) analysis. Our findings demonstrate that the nested PCR assay, originally developed for cultivable Paracoccidioides species, is also effective in amplifying TUB1 from P. ceti. Therefore, this method can be considered an additional marker for phylogenetic studies of this uncultivable species, contributing to a better understanding of this peculiar pathogen.