A rare cardiopulmonary parasite of the European badger, Meles meles: first description of the larvae, ultrastructure, pathological changes and molecular identification of Angiostrongylus daskalovi Janchev & Genov 1988

Background: Angiostrongylus daskalovi is a rare cardiopulmonary nematode infecting badgers. The parasite was described in 1988 and, since then, found only once in mustelids in Europe. The present study aims to report new cases of patent A. daskalovi infection in badgers from northern Romania and to provide new information on its ultrastructure, molecular diagnosis, and pathology. Methods: Eight road-killed or hunted badgers originating from Maramure. and Alba counties in Romania were collected and necropsied. Adults and larvae of cardio-pulmonary nematodes were collected and examined by light and scanning electron microscopy (SEM). Genomic DNA was extracted from adults and first-stage larvae (L1). PCR amplification of the internal transcribed spacer 2 (ITS2, similar to 500 bp) of the rRNA gene was performed. Amplicons were purified, sequenced, and compared to those available in the GenBank database. Histopathological examination of the lungs was performed and lesions described. Results: The necropsy revealed the presence of nematodes in the pulmonary arteries of three animals. All parasites were mature adults and the coproscopic examination showed the presence of eggs and L1 larvae in all three positive animals. Light microscopy examination confirmed the morphological and morphometric similarity of parasites to Angiostrongylus daskalovi. SEM highlighted the typical angiostrongylid structure of the rays of the copulatory bursa and the anterior extremity, with the presence of six sensory papillae surrounding the mouth opening in which a triangular tooth was visible. The first-stage larva (L1) of A. daskalovi is described here for the first time. Histopathological examination of the lungs showed chronic interstitial verminous pneumonia due to the presence of adult parasites. Molecular analysis showed 100 % nucleotide similarity to an Angiostrongylus sp. isolate originating from a badger from Spain, tentatively identified as A. daskalovi. Conclusions: Our study unequivocally demonstrates the presence of A. daskalovi in European badgers from Romania, provides the first description of the larvae and reveals new data about the ultrastructure of adult parasites and their pathological impact, contributing to the understanding of the phylogenetic relationships with other congeneric species.