Tick-borne pathogens in small mammals from the Pantanal biome, Brazil

The order Piroplasmida (e.g., Babesia, Theileria, Cytauxzoon, and Rangelia) and order Eucoccidiorida, Suborder Adelorina (e.g., Hepatozoon) comprise protozoa of the phylum Apicomplexa. At the same time, the Anaplasmataceae family (Order Rickettsiales) encompasses important bacterial pathogens, notably the genera Anaplasma and Ehrlichia. All these genera of agents have pathogenic species transmitted to vertebrate hosts via ticks and pose significant economic, veterinary, and medical concerns. Biological samples of small mammals (Rodentia and Didelphiomorphia) collected between 2015 and 2018, as part of a study on the eco-epidemiological aspects of Brazilian spotted fever, were made available to investigate the molecular prevalence of Piroplasmida, Hepatozoon species, and Anaplasmataceae agents. The study included 28 marsupials of the species Gracilinanus agilis and 62 rodents, which comprised Cerradomys subflavus, Dasyprocta azarae, Hylaeamys megacephalus, Necromys lasiurus, and Oecomys mamorae. All individuals were captured in the municipality of Poconé in the Brazilian Pantanal, located in midwestern Brazil. Among 333 biological samples, including blood (n=85), spleen (n=85), liver (n=90), and lung (n=73), Hepatozoon were molecularly detected in cricetid rodents H. megacephalus (n=2) and Oecomys sp. (n=1), representing 3.3 % of the small mammals surveyed. Positive results for Anaplasma sp. were observed in 21 (23.3 %) animals, including H. megacephalus (n=15), Oecomys sp. (n=1), four rodents not identified at the species level, and the marsupial G. agilis (n=1). All biological samples were negative for Piroplasmida. Phylogenetic analysis of a partial 18S rRNA gene sequence from Hepatozoon sp. detected in H. megacephalus clustered into a clade with Hepatozoon sp. identified in Cuniculus paca from the Brazilian Amazon. In addition, a Bayesian phylogenetic inference based on the 16S ribosomal RNA gene (16S rRNA) from members of the Anaplasmataceae family obtained from H. megacephalus, Oecomys sp., and an unidentified rodent clustered together. This cluster was part of a sub-clade with two Anaplasma sp. sequences from Amblyomma coelebs nymphs collected from coatis (Nasau nasua) in southern Brazil and 'Candidatus Anaplasma sparouinense' detected in a human from French Guiana. In conclusion, we provide strong evidence of a high occurrence of Anaplasma genotypes infecting Cricetidae rodents in the Pantanal biome, which suggests that humans may be at risk of infection by an Anaplasma genotype closely related to 'Ca. A. sparouinense', responsible for an unusual case of human anaplasmosis in the Amazon rainforest. Given the potential involvement of rodents and marsupials in the natural cycle and transmission of these neotropical Anaplasma species, further investigations are needed to evaluate their role as sources of novel human infections.