A high number of swine males are colonized by A. suis in their preputial diverticula, and this colonization begins in the first weeks of life [1]. Due to its slow fastidious growth, A. suis has been difficult to isolate, a fact which may have impaired free overnight delivery estimates of its prevalence. Conventional culturing techniques for the identification of anaerobic bacteria can be time-consuming, are not always economically feasible and are beyond the capabilities of some smaller diagnostic laboratories [2]. As an alternative to direct bacterial isolation, indirect immunofluorescence (IF) has been used for A. suis detection [3�C7]. However, the disadvantages of the IF technique��such as the need for animals for antibody production, the paucity of antibody production laboratories for this agent, and the requirement of specialized equipment and personnel��make polymerase chain reaction (PCR) an affordable and promising alternative tool for the detection of A.

suis. Although PCR has not been used as a diagnostic method for this bacterium in pigs, this mechanism is already being applied to detect other species of the genus Actinobaculum sp. Bank et al. [8] described a PCR technique for the detection of Actinobaculum schaalii in human urine and found PCR to be a rapid and reliable method of detection, which contributed to more effective treatment and faster recovery of patients.The present study aims to develop a PCR strategy for the detection and identification of A. suis in pure cultures, urine samples, and preputial swabs; evaluate the PCR specificity and limits of detection; and compare the PCR results with those obtained using direct bacterial isolation techniques.

2. Material and Methods2.1. Sample CollectionOne hundred and ninety-two urine samples from sows and forty-five swabs of preputial diverticula from boars were collected from three swine herds in S?o Paulo State, Southeastern Brazil. Samples were kept at 4��C until processing. 2.2. Bacteriological ExaminationUrine samples (10mL) were centrifuged at 4,000��g for 10 minutes, and the obtained pellet or preputial swabs were spread in 5% sheep blood agar supplemented with colistin sulphate (10mg/L), nalidixic acid (15mg/L) and metronidazole (50mg/L). All antimicrobial powders were obtained from Sigma Chemical (St. Louis, MO, USA.). The plates were incubated in anaerobic conditions at 37��C for 72 hours.

The colonies presenting a characteristic dry, greyish-white, flattened, opaque surface, without hemolysis, were submitted for biochemical tests and the PCR described below. Morphology, catalase and urease production, hippurate Drug_discovery hydrolysis, nitrate reduction, and the fermentation of glucose, starch, lactose, maltose, and trehalose were all tested.2.3. DNA ExtractionPurified DNA was recovered according to the Boom et al.