Genotypic Discrimination of Chytrid Fungus Lineages in the Amphibian Trade.

Abstract

The international amphibian trade is a cause for concern due to its potential to introduce different lineages of the chytrid fungus (Batrachochytrium dendrobatidis, or Bd) into new environments, threatening native populations. Current diagnostic methods, particularly qPCR, lack the capability to differentiate between these lineages, necessitating more accurate genotyping approaches. Here we propose the application of TaqMan SNP genotyping assays to discriminate Bd genotypes (Bd-GPL, Bd-ASIA2/Bd-BRAZIL, and Hybrid) in skin swabs, tadpole mouthparts, and Bd cultures from Brazilian bullfrog farms. Employing two assays utilizing both qPCR and dPCR, we identified genotype presence and analyzed the relationship between Bd load and genotype determination. The genotyping assay achieved approximately 57% success overall, rising to nearly 82% in samples with over 1,000 genomic equivalents. The use of dPCR also enabled the recovery of genotypes in previously unresolved samples, enhancing lineage detection in challenging field contexts. Culture samples achieved a 100% success rate. We identified all Bd genotypes previously reported in Brazil-the targets of our assay-and detected coinfections and hybrids in high-density farms. We hereby present an efficient method for discriminating Bd genotypes, applicable to both pure cultures and field samples with low Bd loads. We emphasize the need for advanced discriminatory methods and comprehensive genetic studies, particularly regarding national regulations governing breeding sites and the global amphibian trade. Our findings highlight the feasibility and relevance of the method and support further research into infection dynamics by different Bd lineages to inform amphibian conservation efforts and trade regulatory policies.