A modified method for molecular identification of Baylisascaris transfuga in European brown bears (Ursus arctos)

Abstract

Baylisascaris transfuga is a roundworm that has been reported worldwide in most bear species. In mammals and possibly humans, the larvae of B. transfuga can migrate in the tissues of aberrant hosts with larva migrans syndrome. The current study was performed to identify B. transfuga in faecal samples from free-ranging brown bears in the Tatra Mountains National Park in southern Poland. A commercial kit was used to extract genomic DNA directly from faecal samples. Additionally, a Chelex resin-based technique was successfully implemented to prepare a PCR template from eggs retrieved by flotation. Based on the flotation results of 32 collected faecal samples, the prevalence of B. transfuga was 15.6%. The parasite was confirmed in samples found to be positive during the initial flotation by a molecular assay using DNA isolated directly from faeces. The retrieved eggs were confirmed as B. transfuga after their DNA was extracted using the Chelex protocol. Based on PCR amplification and sequencing of a 413-bp segment of cytochrome c oxidase 1 (COI), the obtained sequence was 100% identical to the COI segment of B. transfuga after a BLAST comparison to the GenBank™ database. The current study includes the first molecular confirmation of B. transfuga in brown bears in the western part of the Carpathians. We show that direct extraction of parasite DNA from bear faeces is efficient for molecular assays. As an alternative, we present the effectiveness of a Chelex-based technique for fast and convenient DNA isolation from the difficult-to-disrupt eggs of B. transfuga for PCR. Molecular tests of parasite DNA extracted directly from faecal material have limits of detection related to the amount of eggs in the samples. Thus, using classical flotation to obtain eggs for PCR may increase the credibility of the results, particularly in cases with a low number of excreted eggs. The Chelex resin protocol has potential for application in studies of intestinal parasites in wildlife for which conventional flotation is routinely used for microscopy.