Abstract
Infections of avian haemosporidian parasites are regularly identified by molecular methods including multiplex PCR, which allows researchers to distinguish mixed infections of parasites from multiple genera. Here we extend the utility of a previously designed multiplex PCR by designing a primer set specific to parasites of the subgenus Haemoproteus (genus: Haemoproteus). The updated one-step multiplex PCR protocol we describe here allows for the detection of the genera Plasmodium and Leucocytozoon and the two subgenera (Haemoproteus and Parahaemoproteus) of the genus Haemoproteus. A sensitivity analysis showed that the multiplex PCR could amplify DNA of parasites in the subgenus Haemoproteus at very low levels of infection. We used this multiplex PCR to identify haemosporidian infections in 250 adult domestic pigeons (Columba livia) in Turkey. All samples were also screened by microscopy and a widely used nested PCR to compare with the results of multiplex PCR, to detect low levels of parasitemia, and to identify possible abortive infections. In total, 71 pigeons (28.4%) were found to be infected by all three methods. The multiplex PCR protocol successfully detected and discriminated both subgenera Haemoproteus and Parahaemoproteus infections. We compared our results with previous host species records to assess the host specificity of the parasite lineages we found. Our findings provide novel data on the prevalence of avian haemosporidians in domestic pigeons and demonstrate the utility of the new one-step multiplex PCR protocol for the determination of mixed avian haemosporidian infections. We expect that this protocol will contribute to a better understanding of the distribution, epizootiology, and ecology of avian haemosporidians.
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Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.
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Acknowledgements
Sumeyye Aygun is acknowledged for her assistance in the laboratory. We thank Zeynep Nurselin Colak and Cagatay Esin for their help with the fieldwork in Samsun. We thank Prof. Staffan Bensch for consultations on the amplification sensitivity analysis and Prof. Gediminas Valkiūnas for valuable comments on the microscopic images. Finally, we are grateful to all pigeon owners for their kind hospitality and help during the fieldwork.
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This work was supported by the Erciyes University Research Fund (grant number TSA-2020–10253).
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AC: Conceptualization, investigation, methodology, visualization, project administration, funding acquisition, writing—original draft, writing—review and editing; AY: Formal analysis, validation, supervision, writing—review and editing; DP: Investigation, writing—review and editing; GY: Investigation, formal analysis, writing—review and editing; NSS: Investigation, writing—review and editing; ES: Investigation, writing—review and editing; OD: Investigation, writing—review and editing; ZO: Investigation, writing—review and editing; NDK: Investigation, writing—review and editing; GZP: Investigation, writing—review and editing; VAE: Methodology, formal analysis, writing—review and editing; AI: Resources, validation, supervision, writing—review and editing.
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Permission for taking blood samples from the pigeons was approved by the Erciyes University Local Ethics Committee for Animal Experiments (05.02.2020–20/035) and the Republic of Turkey Ministry of Agriculture and Forestry, General Directorate of Nature Conservation and National Parks (23.03.2020/21264211–288.04-E.971590).
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Ciloglu, A., Yildirim, A., Pekmezci, D. et al. A novel one-step multiplex PCR protocol to detect avian haemosporidian parasites in the subgenus Haemoproteus (Kruse, 1890) used to quantify parasite prevalence in domestic pigeons (Columba livia) in Turkey. Vet Res Commun 47, 511–521 (2023). https://doi.org/10.1007/s11259-022-09962-z
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DOI: https://doi.org/10.1007/s11259-022-09962-z