Batrachochytrium dendrobatidis no Ceará: detecção e prospecção por probióticos em ninhos de espuma de anuros

Abstract

Several amphibian species are undergoing population declines, making them the vertebrate group with the highest extinction rates. Multiple lines of evidence indicate chytridiomycosis as one of the main causes of these declines, a disease caused by the fungus Batrachochytrium dendrobatidis (Bd). In Brazil, infections caused by the chytrid fungus have been reported in nearly all biomes, particularly in the South and Southeast regions. In contrast, the Northeast region remains relatively understudied, although confirmed cases of Bd have already been recorded. In response, several conservation strategies have been proposed; among them, a promising approach is the use of probiotics. In this context, the present study aimed to (i) provide updates on the chytridiomycosis scenario in Ceará and (ii) prospect for probiotics in anuran foam nests. For Bd detection, 51 skin swab samples from anurans occurring in different regions of Ceará, collected in the field, had their DNA extracted and were evaluated by Polymerase Chain Reaction PCR. DNA extraction was performed using the PrepMan Ultra® reagent, and PCR was carried out using the Bd1a and Bd2a specific primers. The amplified 300 bp fragments indicating the presence of the chytrid fungus were visualized by electrophoresis on a 1% agarose gel. In parallel, 53 bacteria isolated from the foam nests of the anurans Leptodactylus vastus, Physalaemus cuvieri, and Adenomera hylaedactyla, belonging to the Microorganism Collection of the Laboratory of Genetic Resources (LarGen), were tested against the pathogen in vitro. To evaluate the inhibition of Bd growth by the isolates, semiquantitative assays were performed in 96-well plates with the following controls: positive control (1% tryptone + Bd zoospores), negative control (tryptone only), nutrient- depleted control (distilled water + Bd zoospores), and heat-killed Bd (heated at 70 °C for 1 hour + 1% tryptone). We used the panzootic lineage (Bd GPL 131) in our assays. The plate was monitored by absorbance readings (492 nm) on days 0, 4, 7, and 10, and the inhibition score was then obtained from the optical density data. Individuals of the species Rhinella dapsilis, Odontophrynus carvalhoi, and Adelophryne baturitensis in the municipality of Guaramiranga, Dendropsophus minusculus in Fortaleza, Proceratophrys cristiceps in Pacatuba, and Boana raniceps in Pacoti, tested positive for the presence of Bd, representing the first record for Fortaleza and for some of these species. The search for probiotics in foam nests yielded positive results, with more than 70% of the tested microorganisms strongly inhibiting the fungus. Among the genera identified, Pseudomonas, Stenotrophomonas, Chryseobacterium, Microbacterium, Streptomyces, Serratia, Bacillus, Enterobacter, Burkholderia, Brevundimonas, Comamonas, Dyella, Kurthia and Niabella stood out. This is one of the first studies to prospect probiotics isolated from foam nests with activity against the chytrid fungus and to detect Bd in the state of Ceará. Our results are of great importance for amphibian conservation, given the negative impact of chytridiomycosis on amphibian populations worldwide.