Metabarcoding characterization of gastrointestinal strongyle nematodes in captive Asian elephants (Elephas maximus) and white rhinoceroses (Ceratotherium simum) in a private zoo, Thailand.

Gastrointestinal strongyle nematodes pose significant health risks to captive megaherbivores, including Asian elephants (Elephas maximus) and white rhinoceroses (Ceratotherium simum). Traditional diagnostic methods often fail to accurately identify species due to morphological similarities, limiting understanding of parasite diversity and host-specificity. This study is among the first in Southeast Asia to apply high-throughput internal transcribed spacer-2 (ITS-2) rDNA metabarcoding to characterize strongyle nematode communities in these endangered hosts. Fecal samples from six rhinoceroses and four elephants housed in a private zoo in Thailand were processed using flotation, larval culture, and DNA extraction protocols. Amplicon sequencing was conducted on the Illumina MiSeq platform, and taxonomic assignments were performed using the DADA2 pipeline and NCBI/GenBank databases. Our results revealed the presence of strongyle infections. Murshidia spp. were detected in both host species, while Kiluluma ceratotherii was found exclusively in rhinoceroses. Phylogenetic analysis based on ITS-2 rDNA sequences demonstrated clear host-associated clades and suggested potential cryptic species within Kiluluma and Murshidia lineages. These findings provide new genetic evidence of host specificity and evolutionary divergence among strongylid nematodes in captive wildlife. The study underscores the utility of DNA metabarcoding for non-invasive parasite surveillance and highlights the urgent need to expand molecular databases for better taxonomic resolution in wildlife parasitology.