Development and validation of a real-time PCR assay for <i>Cochlosoma anatis</i> in turkeys

<i>Cochlosoma anatis</i> is a flagellated protozoan parasite in the <i>Trichomonadidae</i> family that causes an enteric disease of turkeys and ducks known as cochlosomosis. Following the withdrawal of effective antiprotozoal medications from commercial poultry production, this organism has become of increased concern for the turkey industry. Historically, this organism has been detected by light microscopic evaluation of enteric mucosal scrapings. However, live trophozoites must be present in the sample for this method of detection because the parasite is differentiated from other protozoa by its characteristic movement. There is currently no real-time PCR (rtPCR) assay published for this organism; thus, we designed and validated a rtPCR assay. Ten-fold serial dilutions of gBlocks DNA representing the targeted region of the organism were evaluated to determine the limit of detection of the organism, which was ~50 copies per reaction. The assay was specific for <i>C. anatis</i>, with no cross-detection of 3 other poultry flagellates: <i>Histomonas meleagridis</i>, <i>Tetratrichomonas gallinarum</i>, and <i>Trichomonas gallinae</i>. Sensitivity and specificity compared to microscopic detection of live organisms were 100%. Coinfections of <i>C. anatis</i> with other pathogens have been reported. Thus, this assay can be added to syndromic panels for detection of enteric pathogens of turkeys.