Assessment of a quantitative 5′ nuclease real-time polymerase chain reaction using groEL gene for Ehrlichia and Anaplasma species in rodents in Brazil

Abstract

New genotypes of Anaplasmataceae agents have been detected in wild carnivores, birds and deer in Brazil. The present work aimed to investigate the presence of Ehrlichia and Anaplasma species in rodents sampled in Brazil. Additionally, a newly designed quantitative 5' nuclease real-time multiplex PCR for Ehrlichia and Anaplasma spp. detection based on groEL gene amplification was designed, showing high specificity and sensitivity (10 groEL fragment copy/μL). Between 2000 and 2011, different rodent species [n=60] were trapped in 5 Brazilian biomes. Among 458 rodent spleen samples, 0.4% (2/458) and 2.4% (11/458) were positive for Ehrlichia and Anaplasma spp., respectively. Of 458 samples, 2.0% (9/458) and 1.1% (5/458) were positive for Anaplasma sp. and Ehrlichia sp., respectively, using conventional 16S rRNA PCR assays. Maximum Likelihood phylogenetic analyse based on a small region of 16S rRNA genes positioned the Anaplasma genotypes in rodents near Anaplasma phagocytophilum or Anaplasma marginale and Anaplasma odocoilei isolates. Ehrlichia genotypes were closely related to E. canis. There was a low occurrence of Anaplasma and Ehrlichia in wild and synanthropic rodents in Brazil, suggesting the circulation of new genotypes of these agents in rodents in the studied areas.

Molecular detection of Anaplasmataceae agents in Dasyprocta azarae in northeastern Brazil

“…Additionally, prevalences of 0.44% and 2.40% for Ehrlichia sp. and Anaplasma sp., respectively, were reported in the same study, using a newly designed multiplex quantitative real-time PCR based on the groEL gene (BENEVENUTE et al, 2017). …”

“…in a specimen of Hylaeamys megacephalus that had been trapped in the Pantanal of the state of Mato Grosso. More recently, among 458 rodents sampled in different regions of Brazil, Benevenute et al (2017) reported prevalences of 1.09% and 1.96% for Ehrlichia sp. and Anaplasma sp., respectively, using previously described conventional PCR (cPCR) assays based on the 16S rRNA gene.…”

“…and Anaplasma spp. DNA was detected in free-living rodents in Brazil (WOLF et al, 2016;BENEVENUTE et al, 2017).…”

“…the phylogenetic positioning of this specimen was not accessed. More recently, Benevenute et al (2017) detected 16S rRNA DNA fragments of Anaplasma sp. in 21 (4.6%) out of 458 spleen samples from rodents that had been sampled in five distinct biomes in Brazil.…”

“…in 21 (4.6%) out of 458 spleen samples from rodents that had been sampled in five distinct biomes in Brazil. The Anaplasma genotypes were detected in rodents from the Caatinga, Cerrado and Atlantic Forest biomes (BENEVENUTE et al, 2017).…”