Banca de DEFESA: ISAAC LEANDRO LIRA PINTO

Molecular detection, characterization and morphological analysis of bacteria from the Anaplasmataceae and Mycoplasmataceae family in populations of small mammals in areas of the Amazon Rainforest

Anaplasma, Ehrlichia, Mycoplasma, Bats, Rodents.

The objective of the present work was the molecular detection, characterization, and morphological analysis of bacteria of the families Anaplasmataceae and Mycoplasmataceae in populations of small mammals in areas of the Amazon forest, in the state of Acre, Brazil. The importance of this investigation is based on obligate intracellular bacteria that participate in wild cycles as zoonotic agents, and therefore of interest for human health, in addition to parasites of importance for domestic animals, and the animal's own ecology and life cycle. host. The study areas are concentrated in forested areas of the Amazon rainforest, in the city of Rio Branco, State of Acre, Brazil. For direct diagnosis, 186 samples of blood smear slides from previously captured rodents, marsupials and bats were used to detect, characterize, and measure inclusions indicative of bacteria from the families Anaplasmataceae and Mycoplasmataceae. Liver samples stored in RNA Later® with DNA extraction using DNeasy Blood & Tissue Kit (Qiagen®) were used for molecular diagnosis. Of the liver tissue samples analyzed, 14.52% (n=27/186) were considered positive for Mycoplasma sp. by PCR using primers Myco 322s and Myco 938as which amplify 620 base pairs of the 16S rDNA fragment. The diagnosis of Anaplasmataceae was hampered by the non-specificity of the PCR reactions with the tested primer pairs, and when the technique was specific, it resulted in a 16S rDNA fragment from Wolbachia sp. Therefore, these positive data were not considered for analysis. The positivity in blood smear slides for bacteria from the Anaplasmataceae family was 6.99% (n=13/186), while for hemotropic mycoplasmas it was 1.61% (n=3/186). Regarding the location, there was a significant statistical difference between the areas of São Raimundo and Floresta Piracema and the areas of Santa Cecília, EMBRAPA, APA do Amapá, and Colégio Agróciola, where São Raimundo and Floresta Piracema presented a higher percentage of positivity for Mycoplasma sp. compared to the others. Regarding the sequencing result of the 16S rDNA fragment of Mycoplasma sp., three samples from bats of the genera Glossophaga (22052), Artibeus (22108), Carollia (22152) had their sequences well read in the chromatogram, with approximately 600bp. The highest percentage of blast similarity for 22052 was 98.41% with a sample of Glossophaga commissarisi (LR699022), from Switzerland; for 22108 it was 99.82% with a sample of Platyrrhinus lineatus (MZ048291), from Mato Grosso do Sul, Brazil; and for 22152 it was 98.28% with a sample of Desmodus rotundus from Belize (KY932722). This study revealed different genotypes of Mycoplasma sp. in bats and demonstrated the presence of morulae in leukocytes and platelets, characteristic of bacteria of the Anaplasmataceae family in rodents, marsupials, and bats. These results suggest that hemotropic mycoplasmas are endemic in bats from Acre and that much remains to be discovered about the nature of obligate intracellular bacteria in these animals.