Smear diagnosis has low sensitivity, as there are few bacteria present in the samples, morulae can be visualized Ganetespib molecular weight only during the acute phase, and the percentage of infected cells is usually less than 1% [6]. Additionally, the presence of A. platys is cyclical, and the bacteria are easily mistaken as nonspecific inclusion bodies and staining artifacts [1, 7]. Serology is hampered by cross-reactions and cannot discriminate between a current infection and previous exposure to the pathogen. Moreover, antibody titers tend to persist for several months to years after treatment, making serology an unreliable tool for posttreatment diagnosis [8].The first PCR-based diagnostic method for ehrlichiosis amplified the 16S rRNA gene and was reported by Iqbal et al. in 1994 [9].

Further improvements and the use of other target genes increased the sensitivity of the tests. The p30-based nested PCR (nPCR) assay has been shown to be more sensitive than the 16S rRNA-based nPCR assay [10], possibly because E. canis contains multiple copies of the p30 gene but only one copy of the 16S rRNA gene [11]. As opposed to single-step PCR, nPCR amplification of the 16S rRNA gene has been used more often to detect E. canis and A. platys. In both single step PCR and nPCR, the peripheral blood is frequently used as a DNA source [1, 5, 12]. Only a single report has described the use of mononuclear cells as a DNA source [9].There is a high prevalence of canine ehrlichiosis, but there are few reports on the identification of the infectious agents; therefore, a practical diagnostic technique that can be routinely used in veterinary medicine must be established.

The nPCR assay may fulfill this requirement, but the blood fraction that serves as the best DNA source must be determined beforehand. The aim of the present study was to compare the effectiveness of whole blood (WB) and blood fractions��buffy coat (BC), granulocytes (G), mononuclear fraction (M) and blood clot (C)��by nPCR to diagnose canine ehrlichiosis and anaplasmosis.2. Batimastat Methods2.1. Samples and Cell FractionationBlood was collected from 21 dogs bearing suggestive clinical signs of either ehrlichiosis or anaplasmosis (petechia, ecchymosis, fever, and anorexia) and harboring ticks. Some animals also had intracytoplasmic morulae, as indicated by direct examination of blood smears and/or hematological parameters suggestive of ehrlichiosis and anaplasmosis. The dogs were selected from the veterinary hospital Universidade Federal de Campina Grande (UFCG), the Veterinary Medical Center Dr. Leonardo Torres at Patos, State of Paraiba, and at the Veterinary Hospital at Universidade Federal Rural de Pernambuco (UFRPE), at Recife, State of Pernambuco.2.2.