Validation of a High-Throughput Microfluidic Real-Time PCR for the Detection of Vector-Borne Agents in Wild Birds from the Brazilian Pantanal

Abstract

Despite numerous studies on haemosporidians in wild birds from Brazil, the presence of other vector-borne agents (VBA) such as <i>Anaplasma</i> spp., <i>Bartonella</i> 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 <i>Anaplasma</i> spp., <i>Bartonella</i> spp., haemosporidians, and filariids were used. The molecular occurrence rates for the selected VBAs were 18.2% for <i>Anaplasma</i> spp., 0.36% for <i>Bartonella</i> spp., 6.2% for <i>Plasmodium</i> spp., 4.7% for <i>Haemoproteus</i> spp., and 6.5% for Onchocercidae filariids. The <i>Plasmodium</i> spp. <i>cyt</i>B sequence detected in a <i>Volatinia jacarina</i> clustered with <i>Plasmodium tejerai</i>, whereas the <i>Haemoproteus</i> spp. <i>cytB</i> sequence detected in a <i>Columbina squamata</i> clustered with <i>Haemoproteus columbae</i>. While Onchocercidae filariid <i>cox-</i>1 sequences were detected in specimens of <i>Ramphocelus carbo</i>, <i>Turdus amaurocalinus</i> and <i>Synallaxis albilora</i> grouped with <i>Aproctella</i> spp., one sequence detected in <i>R. carbo</i> was ancestral to the clade comprising <i>Splendidofilaria</i> spp. and <i>Eufilaria</i> 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.