Abstract
Avian haemosporidian parasites have received considerable attention in ecology and evolution as a result of their wide distribution and ease of detection. However, conventional PCR-based detection methods may sometimes underestimate haemosporidian mixed infections, which are frequent in natural populations. This underestimation is due to differences in PCR sensitivity for detection of lineages within the mixed infections. Therefore, we designed new primers to amplify sequences that were not detected by the conventional primers and examined if our primers were useful for accurate detection of mixed infections. Blood samples were collected from 32 wild birds captured in Hokkaido, and 16 of these were positive for Leucocytozoon using the conventional primers, while 15 were positive using our primers. All positively amplified samples were sequenced, and we found that the conventional primers detected 16% (5/32) of multiple infections and none of them was a novel lineage, whereas our primers detected 44% (14/32) of multiple infections and ten of them were novel lineages. A phylogenetic analysis showed that the new primers can detect a wide range of Leucocytozoon lineages compared with that detected by the conventional primers. The results indicate that our primers are particularly suitable for revealing unique strains from multiple infections. Highly variable multiple infections in the same population of birds at the same location were found for the first time. We revealed a higher diversity of Leucocytozoon lineages in nature than expected, which would provide more information to better understand parasite diversity and host-vector interactions in wildlife.
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Funding
This work was supported by the Cooperative Research Grant (2020-joint-18) of National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, and the Japan Society for the Promotion of Science KAKENHI Grant Number 20K07461.
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Yui Honjo and Kenji Hikosaka designed the research; Yui Honjo, Shinya Fukumoto, and Kenji Hikosaka performed the research; Yui Honjo analyzed the data; and Yui Honjo, Hirokazu Sakamoto, and Kenji Hikosaka wrote the paper. All authors read and approved the final manuscript.
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All animal experiments in this study were conducted in accordance with the guidelines for the use of animals of Obihiro University of Agriculture and Veterinary Medicine, Japan (Permit number: 19–14). Consent to participate is not applicable.
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Honjo, Y., Fukumoto, S., Sakamoto, H. et al. New PCR primers targeting the cytochrome b gene reveal diversity of Leucocytozoon lineages in an individual host. Parasitol Res 121, 3313–3320 (2022). https://doi.org/10.1007/s00436-022-07667-5
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DOI: https://doi.org/10.1007/s00436-022-07667-5