Despite numerous reports of Anaplasmataceae agents in mammals worldwide, few studies have investigated their occurrence in birds. The present study aimed to investigate the occurrence and molecular identity of Anaplasmataceae agents in birds from the Pantanal wetland, Brazil. Blood samples were collected from 93 different species. After DNA extraction, samples positive for the avian β-actin gene were subjected to both a multiplex quantitative real-time (q)PCR for Anaplasma and Ehrlichia targeting the groEL gene and to a conventional PCR for Anaplasmataceae agents targeting the 16S rRNA gene. As a result, 37 (7.4%) birds were positive for Anaplasma spp. and 4 (0.8%) for Ehrlichia spp. in the qPCR assay; additionally, 13 (2.6%) were positive for Anaplasmataceae agents in the PCR targeting the 16S rRNA gene. The Ehrlichia 16S rRNA sequences detected in Arundinicola leucocephala, Ramphocelus carbo, and Elaenia albiceps were positioned closely to Ehrlichia sp. Magellanica. Ehrlichia dsb sequences detected in Agelasticus cyanopus and Basileuterus flaveolus grouped with Ehrlichia minasensis. The 16S rRNA genotypes detected in Crax fasciolata, Pitangus sulphuratus and Furnarius leucopus grouped with Candidatus Allocryptoplasma. The 23S-5S genotypes detected in C. fasciolata, Basileuterus flaveolus, and Saltator coerulescens were related to Anaplasma phagocytophilum. In conclusion, novel genotypes of Anaplasma, Ehrlichia, and Candidatus Allocryptoplasma were detected in birds from the Pantanal wetland.

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.

Blood transfusions are indispensable in Veterinary Medicine, providing therapeutic support in cases of hematological disorders. Several pathogens can cause disease and/or exacerbate the condition of immunocompromised dogs or those requiring a transfusion. This study aimed to investigate the molecular occurrence of hemopathogens (Bartonella spp., Ehrlichia spp., Anaplasma spp., piroplasmids, and hemoplasmas) in blood donor and patient dogs using samples from a clinical veterinary laboratory in Brazil. One hundred blood samples were collected from each group. All dogs tested negative for Bartonella spp. in all performed assays. Among the 100 dogs from the clinical veterinary laboratory, 15% (95% CI: 9.3–23.3) tested positive for Ehrlichia spp., 6% (95% CI: 2.8–12.5) for Anaplasma spp., 3% (95% CI: 1.0–8.5) for Babesia spp., and 2% (95% CI: 0.6–7.0) for hemoplasmas. Blood donor dogs tested positive for hemoplasmas (5%) (95% CI: 2.2–11.2). Additional conventional and real-time PCR assays followed by sequencing confirmed the presence of Ehrlichia canis, Anaplasma platys, Babesia vogeli, ‘Candidatus Mycoplasma haematoparvum’, and Mycoplasma haemocanis. The molecular detection of E. canis, A. platys, ‘Ca. M. haematoparvum’, and M. haemocanis in dogs from midwestern Brazil reinforces the relevance of molecular tools in diagnosing hemopathogens. This is the first molecular detection of hemoplasmas in canine blood donors from Brazil. This finding indicates their silent circulation and highlights the importance of molecular screening to prevent the worsening of clinical conditions and the risk of turning recipients into new sources of infection.