BackgroundAnaplasma phagocytophilum is an emerging pathogen of humans, dogs and other animals, and it is transmitted by ixodid ticks. The objective of the current study was a) detect A. phagocytophilum in dogs and ixodid ticks using real-time Polymerase Chain Reaction (qPCR); and b) Determine important variables associated to host, environment and potential tick vectors that are related to the presence of A. phagocytophilum in dogs domiciled in Rio de Janeiro, Brazil.MethodsWe tested blood samples from 398 dogs and samples from 235 ticks, including 194 Rhipicephalus sanguineus sensu lato, 15 Amblyomma cajennense, 8 Amblyomma ovale and 18 pools of Amblyomma sp. nymphs. A semi-structured questionnaire was applied by interviewing each dog owner. Deoxyribonucleic acid obtained from ticks and dog buffy coat samples were amplified by qPCR (msp2 gene). The sequencing of 16S rRNA and groESL heat shock operon genes and a phylogenetic analysis was performed. The multiple logistic regression model was created as a function of testing positive dogs for A. phagocytophilum.ResultsAmong the 398 blood samples from dogs, 6.03% were positive for A. phagocytophilum. Anaplasma phagocytophilum was detected in one A. cajennense female tick and in five R. sanguineus sensu lato ticks (four males and one female). The partial sequences of the 16S rRNA, and groESL genes obtained were highly similar to strains of A. phagocytophilum isolated from wild birds from Brazil and human pathogenic strains. The tick species collected in positive dogs were R. sanguineus sensu lato and A. cajennense, with A.cajennense being predominant. Tick infestation history (OR = 2.86, CI = 1.98-14.87), dog size (OR = 2.41, IC: 1.51-12.67), the access to forest areas (OR = 3:51, CI: 1.52-16.32), hygiene conditions of the environment in which the dogs lived (OR = 4.35, CI: 1.86-18.63) and Amblyomma sp. infestation (OR = 6.12; CI: 2.11-28.15) were associated with A. phagocytophilum infection in dogs.ConclusionsThis is the first report of A. phagocytophilum in ixodid ticks from Brazil. The detection of A. phagocitophylum in A. cajennense, an aggressive feeder on a wide variety of hosts, including humans, is considered a public health concern.

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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.

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