Validation of a High-Throughput Microfluidic Real-Time PCR for the Detection of Vector-Borne Agents in Wild Birds from the Brazilian Pantanal
2025
Alabí Córdova, Amir Salvador | Pinho, João Batista | Pereira, Amanda Garcia | Galon, Clémence | Ferreira, Tiago Valadares | das Neves, Lorena Freitas | de Oliveira Lopes, Gabrielly | Machado, Rosangela Zacarias | Moutailler, Sara | André, Marcos Rogério | Universidade Estadual Paulista Júlio de Mesquita Filho = São Paulo State University (UNESP) | Universidade Federal de Mato Grosso (UFMT) | Biologie moléculaire et immunologie parasitaires et fongiques (BIPAR) ; École nationale vétérinaire d'Alfort (ENVA)-Laboratoire de santé animale, sites de Maisons-Alfort et de Normandie ; Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE) | This research was supported by “Fundação de Amparo à Pesquisa do Estado de São Paulo” (FAPESP) (BEPE-FAPESP Process Number #2023/08967-0;) e CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico; Productivity Fellowship conceived to MRA [Process CNPq #303701/2021-8]). Amir Alabi received Ph.D. Scholarship from FAPESP (Process #2020/14948-0 and BEPE-FAPESP Process Number #2023/08967-0, respectively), and CAPES-PROEX (code 001).
The sequences generated and analyzed during the present study were submitted in the NCBI Genbank (https://www.ncbi.nlm.nih.gov/genbank/. Sequences can be accessed by the following accession numbers: PQ450485, PQ450488, PQ450487, PQ452771, PQ452776, PQ452777, PQ452778, PQ452774, PQ452775, PQ452772, FR823331, PQ452773 and PV083549.
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Mostrar más [+] Menos [-]Inglés. Despite numerous studies on haemosporidians in wild birds from Brazil, the presence of other vector-borne agents (VBA) such as Anaplasma spp., Bartonella spp., and Onchocercidae filariids in avian hosts remains largely unknown. The low occurrence of these VBAs might be due to the low sensitivity of traditional molecular techniques. The microfluidic real-time PCR assay, known for its high sensitivity, has emerged as a promising method to detect and study the occurrence and diversity of VBAs in both arthropod vectors and vertebrate hosts. To validate previously and standardize newly designed microfluidic real-time PCR protocols, selected positive avian blood DNA samples for Anaplasma spp., Bartonella spp., haemosporidians, and filariids were used. The molecular occurrence rates for the selected VBAs were 18.2% for Anaplasma spp., 0.36% for Bartonella spp., 6.2% for Plasmodium spp., 4.7% for Haemoproteus spp., and 6.5% for Onchocercidae filariids. The Plasmodium spp. cytB sequence detected in a Volatinia jacarina clustered with Plasmodium tejerai, whereas the Haemoproteus spp. cytB sequence detected in a Columbina squamata clustered with Haemoproteus columbae. While Onchocercidae filariid cox-1 sequences were detected in specimens of Ramphocelus carbo, Turdus amaurocalinus and Synallaxis albilora grouped with Aproctella spp., one sequence detected in R. carbo was ancestral to the clade comprising Splendidofilaria spp. and Eufilaria spp. High-throughput microfluidic real-time PCR assay can be used for screening VBAs in avian hosts from South America, but new primers/probe sets should be designed for VBA genotypes present in Brazil.
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