DETECTION OF EHRLICHIA-RISTICII FROM FECES OF INFECTED HORSES BY IMMUNOMAGNETIC SEPARATION AND PCR

Potomac horse fever, caused by Ehrlichia risticii, is an important dis ease of equines. The major features of the disease are fever, leukopen ia, and diarrhea. The organism has been detected from the blood mononu clear cells of infected horses, but its presence in the feces has not been known. A method for immunomagnetic separation of E. risticii from the feces of infected horses was developed, and the separated organis ms were detected by PCR. Coating immunomagnetic beads (Dynabeads) with a 1:5 dilution of rabbit anti-E. risticii serum and incubating the Dy nabeads with fecal samples for 25 min at room temperature gave optimum results. E. risticii was detected from the feces during the course of diarrhea from two experimentally infected horses. In horse 1, watery diarrhea occurred from days 11 to 16 postinfection (p.i.), after which the feces became soft on day 17 p.i. and then returned to normal. The organisms were first detected from the feces on day 11 p.i., peaked o n day 13 p.i., and then gradually decreased until day 16 p.i., after w hich they became undetectable. In horse 2, first, on day 12 p.i., ther e was soft feces which continued and progressed to diarrhea on day 17 p.i. The feces became normal after day 18 p.i. The organisms in the fe ces of this horse were first detected on day 12 p.i. and peaked on day 14 p.i., after which they declined until day 16 p.i. and then became undetectable. In both horses, the number of organisms in the mononucle ar cells peaked on days 10 and 11 p.i., respectively, 3 days prior to the respective Peaks in the feces. E. risticii was not detected from t he plasma samples obtained from these horses. There was a drastic redu ction in PCR amplification of E. risticii DNA for fecal samples stored frozen at -20 degrees C in comparison with those stored at 4 degrees C. The presence of the organism in the feces only during the soft- or diarrheal-feces phase supports the previous hypothesis that the diarrh ea is caused by the organisms replicating in cells lining the intestin es. This rapid and simple method of detection of the organisms from th e feces will be helpful in diagnostic and epidemiologic studies of Pot omac horse fever.