Toyotome, T. | bioRxiv Science

bioRxiv2025

Adapting a commercial anti-Aspergillus IgG ELISA kit for penguin sera: A novel approach using anti-chicken IgY antibody for the detection of anti-Aspergillus IgY titer

Penguins are susceptible to aspergillosis and are affected by the causative fungal pathogens Aspergillus spp., under immunocompromised conditions. Currently, there are limited serological tests available for diagnosing Aspergillus infections in penguins, highlighting the need for new diagnostic methods. While an anti-Aspergillus IgG detection kit using ELISA is commercially available and widely used in human medicine, it is not applicable for penguins because it incorporates anti-human antibodies as the secondary antibody for detection. To address this issue, an anti-chicken IgY antibody was incorporated into a commercial anti-Aspergillus IgG detection ELISA kit. First, anti-chicken IgY antibody was examined for cross-reactivity to penguin IgY, and the antibody recognized IgY from King, Gentoo, and African penguins in sera. Subsequently, serum samples from healthy penguins and penguins with aspergillosis were examined using anti-chicken IgY antibody incorporated into the anti-Aspergillus IgG ELISA kit. The results suggest that the combination method is applicable for the detection of anti-Aspergillus penguin IgY antibodies. However, because background titers can vary among individuals, we propose that routine monitoring could aid in the early detection of aspergillosis, even before the onset of symptoms.

microbiology↗

bioRxiv2020

Identification of novel mutations contributing to azole tolerance of Aspergillus fumigatus through in vitro exposure to tebuconazole

Azole resistance of Aspergillus fumigatus is a global problem. The major resistant mechanism is a cyp51A alteration such as mutation(s) in the gene and the acquisition of a tandem repeat in the promoter. Although other azole tolerances and resistant mechanisms such as hmg1 mutation are known, few reports describe studies elucidating non-cyp51A resistance mechanisms. This study explored genes contributing to azole tolerance in A. fumigatus by in vitro mutant selection with tebuconazole, an azole fungicide. After three-round selection, we obtained four isolates with low susceptibility to tebuconazole. These isolates also showed low susceptibility to itraconazole and voriconazole. Comparison of the genome sequences of the obtained isolates and the parental strain revealed a non-synonymous mutation in MfsD (Afu1g11820, R337L mutation) in all isolates. Furthermore, non-synonymous mutations in AgcA (Afu7g05220, E535Stop mutation), UbcD (Afu3g06030, T98K mutation), AbcJ (Afu3g12220, G297E mutation), and RttA (Afu7g04740, A83T mutation), a protein responsible for tebuconazole tolerance, were found in at least one isolate. Clarification by constructing the MfsD R337L mutant suggests that the mutation contributes to azole tolerance. Disruption of the agcA gene and reconstruction of the A83T point mutation in RttA led to decreased susceptibility to azoles. The reversion of T98K mutation to wild type in UbcD led to the level of azole susceptibility comparable to the parental strain. These results suggest that these mutations contribute to lowered susceptibility to medical azoles and to agricultural azole fungicides.

microbiology↗