Abstract
The international amphibian trade raises concerns regarding its impact
on native populations due to the potential introduction of different
lineages of the chytrid fungus (Batrachochytrium dendrobatidis or
Bd) into new environments. 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 [hereafter Bd-BRAZIL], and Hybrid) in
samples from Brazilian bullfrog farms. We collected samples, including
skin swabs, tadpole mouthparts, and pure Bd cultures, from bullfrog
farms across Brazil. 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 a 56.6 %
success rate, notably improving with higher Bd loads, reaching 81.8 %
for loads over 1,000 genomic equivalents. Culture samples achieved a 100
% success rate. We identified all Bd genotypes in the bullfrog farms,
highlighting the issue of coinfections and hybrids in densely populated
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
research underscores the feasibility and significance of the proposed
method and advocates for further investigations into infection dynamics
by different Bd lineages to inform amphibian conservation efforts and
trade regulatory policies.