Validation and standardization of a high-throughput microfluidic real-time PCR for the detection of vector-borne agents in wild birds from the Brazilian Pantanal

• Grant number: 23/08967-0
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: December 01, 2023
• End date: May 31, 2024
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Animal Pathology
• Principal Investigator: Marcos Rogério André
• Grantee: Amir Salvador Alabi Cordova
• Supervisor: Sara Moutailler
• Host Institution: Faculdade de Ciências Agrárias e Veterinárias (FCAV). Universidade Estadual Paulista (UNESP). Campus de Jaboticabal. Jaboticabal , SP, Brazil
• Institution abroad: Agence Nationale de Sécurité Sanitaire de l’Alimentation, de l’Environnement et du Travail (ANSES), France
• Associated to the scholarship: 20/14948-0 - Molecular detection and characterization of Anaplasmataceae, Bartonellaceae and Hepatozoidae agents in Passeriforms in the Brazilian Pantanal, BP.DR

Abstract

The composition of tropical bird communities results from a combination of permanent resident species, which live throughout the entire year in a specific area, and migratory species, which in turn are only seasonally present in a certain region. Even though several studies performed in Brazil investigated the occurrence and diversity of haemosporidians in wild birds in Brazil, the real occurrence of other vector-borne agents (Anaplasmataceae, Bartonellaceae, Trypanosoma and Hepatozoon) in birds in the country remains elusive. The low occurrence or lack of identification of such vector-borne agents might be associated with the low sensitivity of the molecular techniques used for the diagnosis in avian hosts, in which low bacteremia/parasitemia is expected. Microfluidic real-time PCR assays have emerged as promising molecular approaches to unravel the occurrence and diversity of vector-borne agents in both arthropod vectors and vertebrate hosts, due its high sensitivity. Considering the ecological importance of Pantanal wetlands for both resident and migratory bird species, the present work aims to validate and/or standardize a microfluidic real-time PCR to detect vector-borne agents (Anaplasma, Ehrlichia, Bartonella, Hepatozoon, Plasmodium, Haemoproteus and Leucocytozoon) in avian blood samples collected in the Pantanal wetland, state of Mato Grosso, central-western Brazil. For this purpose, selected positive avian blood DNA samples for Ehrlichia, Anaplasma, Bartonella (originated from the current project supported by FAPESP project number 2020/14948-0), haemosporidians and trypanosomes (data not shown and associated with Dr. Allan Fecchio' project) will be initially used to validate the microfluidic real-time PCR protocols used in Dr. Moutailler unit of research. In case of lack of amplification of these positive samples, whose molecular identity was already confirmed by sequencing and phylogenetic analyses, new primers and probes will be designed in order to standardize new protocols to catch the novel variants of vector-borne agents that circulate in wild birds from Brazil. After validation and/or standardization, microfluidic real-time PCR assays will be used to screen 300 wild birds' blood samples from the Pantanal of the state of Mato Grosso (sampling performed in 2022, July). The present work will validate and/or standardize a novel molecular technique that has never been applied to screen wild bird biological samples in Brazil to vector-borne agents. Additionally, the proposal will contribute to understanding the occurrence of co-infection by vector-borne pathogens in wild birds in the largest tropical wetland in the world. (AU)