Experimental Parasitology 109 (2005) 176–180 www.elsevier.com/locate/yexpr 0014-4894/$ - see front matter  2004 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2004.11.007 Anaplasma platys: an improved PCR for its detection in dogs Anthony R. Martin ¤ , Graeme K. Brown, R. Hugh Dunstan, Timothy K. Roberts The Molecular Structure and Detection Group, The University of Newcastle, Callaghan, NSW 2308, Australia Received 16 September 2004; received in revised form 23 November 2004; accepted 24 November 2004 Available online 6 January 2005 Abstract This study compares two PCR assays for the detection of Anaplasma platys in dog blood using primers based on the A. platys 16S rRNA gene. The Wrst approach utilized a “standard” PCR protocol composed of a “single-step” direct ampliWcation using an Ehr- lichia genus-speciWc primer set. The second assay being a “nested” PCR screen that Wrst involved a universal bacterial primer set that ampliWed the majority of the 16S rRNA gene, followed by the nested round of PCR using an A. platys-speciWc primer set. Of the 22 dogs sampled, 10 were found to contain A. platys DNA using both protocols, and an additional two dogs were found positive using the nested technique. An extract of A. platys positive genomic DNA was serially diluted and comparison of sensitivities determined between the nested PCR, and a direct assay using A. platys-speciWc primers. The nested protocol demonstrated an increased sensitiv- ity by at least 2 orders of magnitude when compared to the direct assay alone. Our results indicated that the nested PCR assay with its increased sensitivity would be useful for experimental research investigations as well as oVer the potential for use as a routine test in diagnostic pathology.  2004 Elsevier Inc. All rights reserved. Keywords: Anaplasma platys; PCR; Polymerase chain reaction; Nested PCR; Ehrlichia 1. Introduction Anaplasma platys, which causes canine infectious cyclic thrombocytopenia (CICT), was Wrst reported in the USA in 1978 (Harvey et al., 1978). A. platys is an obligate intracellular rickettsial organism that appears to only parasitize platelets of dogs. It is thought to be transmitted by the Brown Dog Tick Rhipicephalus sanguineus (Harrus et al., 1997; Woody and Hoskins, 1991). Diagnosis of A. platys infection has usually been made using microscopy, by demonstrating the organ- isms in platelets on blood Wlms or buVy-coat smears that have been stained using Romanowsky-type stains such as Giemsa or DiV-Quik. However, due to the cyclic parasitemia, this is not a reliable method and, additionally, in many regions of the world, the organisms are quite often absent or only present in very low numbers (Bradford et al., 1996; Harrus et al., 1997). A previous study examined free-roaming dogs in Cen- tral Australia to determine whether they harbored any blood-borne infectious agents of zoonotic signiWcance. Blood samples were initially tested by polymerase chain reaction (PCR) using primers to amplify a fragment of the 16S rDNA. DNA analysis of amplicons provided evidence of A. platys, and further analyses using a spe- ciWc primer set for A. platys led to the Wrst conWrmation and report of the carriage of A. platys by dogs in Austra- lia (Brown et al., 2001). The original PCR method employed a “single-step” direct ampliWcation using an Ehrlichia genus-speciWc primer set. To increase the sensitivity of the PCR, a nested PCR approach was developed in the present study. * Corresponding author. Fax: +61 2 4921 7281. E-mail address: Anthony.Martin@newcastle.edu.au (A.R. Martin).