title: A new quantitative PCR method for the detection of Anaplasma platys in dogs based on the citrate synthase gene.
abstract: Anaplasma platys is an obligate intracellular bacterium that primarily affects dogs, but it can also infect humans. Our study aimed to standardize a quantitative real-time (q)PCR method using the citrate synthase gene (gltA) as a specific target for A. platys detection in naturally infected dogs. Primers (gltA84F and gltA84R) and probe (PLATYSp) were designed to amplify an 84-bp fragment based on the gltA gene sequences of A. platys available in GenBank. A total of 186 dog blood samples originating from the Brazilian state of Rio de Janeiro were tested by qPCR. Additionally, the same samples were tested by cytology and a nested (n)PCR that targeted the 16S ribosomal DNA to determine the performance of our qPCR method compared to these existing techniques. Among the samples tested with qPCR, 17.2% were considered positive, significantly more than detected by nPCR (14.0%). Under optical microscopy, inclusions were observed in platelets of 25.3% of the samples, and among these samples, only 33.9% were identified as positive for A. platys using qPCR. The qPCR technique proved to be more specific than cytology and to have superior sensitivity to nPCR for detecting A. platys in dogs. The development of this new qPCR method contributes to the advancement of research involving A. platys Furthermore, it can be used to quantify the presence of this bacterium to evaluate the treatment of infected animals, or even as a more sensitive and specific tool for situations indicating possible clinical disease but with negative cytology.
authors.affiliation: Department of Animal Parasitology (Silva, Pires, Peckle, Costa, Vitari, Massard), Federal Rural University of Rio de Janeiro, Rio de Janeiro, BrazilDepartment of Epidemiology and Public Health, Veterinary Institute (HA Santos, Vilela), Federal Rural University of Rio de Janeiro, Rio de Janeiro, BrazilDepartment of Soils, Agronomy Institute (LA Santos), Federal Rural University of Rio de Janeiro, Rio de Janeiro, Brazil claudia_ufrrj@yahoo.com.br.
authors.forename: Claudia B
authors.initials: CB
authors.lastname: da Silva
authors.affiliation: Department of Animal Parasitology (Silva, Pires, Peckle, Costa, Vitari, Massard), Federal Rural University of Rio de Janeiro, Rio de Janeiro, BrazilDepartment of Epidemiology and Public Health, Veterinary Institute (HA Santos, Vilela), Federal Rural University of Rio de Janeiro, Rio de Janeiro, BrazilDepartment of Soils, Agronomy Institute (LA Santos), Federal Rural University of Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Marcus S
authors.initials: MS
authors.lastname: Pires
authors.affiliation: Department of Animal Parasitology (Silva, Pires, Peckle, Costa, Vitari, Massard), Federal Rural University of Rio de Janeiro, Rio de Janeiro, BrazilDepartment of Epidemiology and Public Health, Veterinary Institute (HA Santos, Vilela), Federal Rural University of Rio de Janeiro, Rio de Janeiro, BrazilDepartment of Soils, Agronomy Institute (LA Santos), Federal Rural University of Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Joice A R
authors.initials: JA
authors.lastname: Vilela
authors.affiliation: Department of Animal Parasitology (Silva, Pires, Peckle, Costa, Vitari, Massard), Federal Rural University of Rio de Janeiro, Rio de Janeiro, BrazilDepartment of Epidemiology and Public Health, Veterinary Institute (HA Santos, Vilela), Federal Rural University of Rio de Janeiro, Rio de Janeiro, BrazilDepartment of Soils, Agronomy Institute (LA Santos), Federal Rural University of Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Maristela
authors.initials: M
authors.lastname: Peckle
authors.affiliation: Department of Animal Parasitology (Silva, Pires, Peckle, Costa, Vitari, Massard), Federal Rural University of Rio de Janeiro, Rio de Janeiro, BrazilDepartment of Epidemiology and Public Health, Veterinary Institute (HA Santos, Vilela), Federal Rural University of Rio de Janeiro, Rio de Janeiro, BrazilDepartment of Soils, Agronomy Institute (LA Santos), Federal Rural University of Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Renata L
authors.initials: RL
authors.lastname: da Costa
authors.affiliation: Department of Animal Parasitology (Silva, Pires, Peckle, Costa, Vitari, Massard), Federal Rural University of Rio de Janeiro, Rio de Janeiro, BrazilDepartment of Epidemiology and Public Health, Veterinary Institute (HA Santos, Vilela), Federal Rural University of Rio de Janeiro, Rio de Janeiro, BrazilDepartment of Soils, Agronomy Institute (LA Santos), Federal Rural University of Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Gabriela L V
authors.initials: GL
authors.lastname: Vitari
authors.affiliation: Department of Animal Parasitology (Silva, Pires, Peckle, Costa, Vitari, Massard), Federal Rural University of Rio de Janeiro, Rio de Janeiro, BrazilDepartment of Epidemiology and Public Health, Veterinary Institute (HA Santos, Vilela), Federal Rural University of Rio de Janeiro, Rio de Janeiro, BrazilDepartment of Soils, Agronomy Institute (LA Santos), Federal Rural University of Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Leandro A
authors.initials: LA
authors.lastname: Santos
authors.affiliation: Department of Animal Parasitology (Silva, Pires, Peckle, Costa, Vitari, Massard), Federal Rural University of Rio de Janeiro, Rio de Janeiro, BrazilDepartment of Epidemiology and Public Health, Veterinary Institute (HA Santos, Vilela), Federal Rural University of Rio de Janeiro, Rio de Janeiro, BrazilDepartment of Soils, Agronomy Institute (LA Santos), Federal Rural University of Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Huarrisson A
authors.initials: HA
authors.lastname: Santos
authors.affiliation: Department of Animal Parasitology (Silva, Pires, Peckle, Costa, Vitari, Massard), Federal Rural University of Rio de Janeiro, Rio de Janeiro, BrazilDepartment of Epidemiology and Public Health, Veterinary Institute (HA Santos, Vilela), Federal Rural University of Rio de Janeiro, Rio de Janeiro, BrazilDepartment of Soils, Agronomy Institute (LA Santos), Federal Rural University of Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Carlos L
authors.initials: CL
authors.lastname: Massard
mesh_terms: D000709:Anaplasma / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000712:Anaplasmosis / Q000175:diagnosis* / Q000382:microbiology; D000818:Animals; D001938:Brazil; D002950:Citrate (si)-Synthase / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers; D004283:Dog Diseases / Q000175:diagnosis* / Q000382:microbiology; D004285:Dogs; D011237:Predictive Value of Tests; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary
chemical_list: D017931:DNA Primers; D002950:Citrate (si)-Synthase
keywords: Anaplasma platys; citrate synthase; dogs; real-time PCR