Canine schistosomiasis is an infrequently diagnosed condition caused by Heterobilharzia americana. Infection with this trematode causes a variety of clinical signs, including weight loss, diarrhea, hematochezia, with or without systemic signs. This disease may be under-diagnosed due to the lack of clinical suspicion, vague clinical signs, and lack of sensitive diagnostic tests. For example, eggs of this parasite rarely float during routine fecal flotation and require sodium chloride sedimentation to be visualized. Therefore, the aim of this study was to develop a PCR based method for the detection of Heterobilharzia americana DNA in dog feces.
Specific primers were designed and optimized to amplify a section of the variable region of the 18S ribosomal DNA gene of Heterobilharzia americana. PCR products were purified and analyzed by automated cycle sequencing to confirm identification of the parasite. Heterobilharzia americana eggs were extracted from the feces of a dog that was positive for the parasite on sodium chloride sedimentation and were used as a positive control. The eggs were diluted to counts of 100, 50, 25, 10, 5, 2, and 1 egg per gram of fecal extract. Feces, from a healthy dog previously negative for the parasite, were spiked with these preparations to determine the sensitivity of the PCR assay. DNA was extracted from the fecal samples and the PCR assay was repeated 10 times for each sample with the 5 lowest egg counts to ensure reproducibility. Finally, DNA was extracted from fecal samples from two dogs that were positive for Heterobilharzia americana based on sodium chloride sedimentation. Extracted DNA was analyzed with the PCR assay. The PCR products were purified and sequenced directly to confirm the identity of parasite DNA.
Spiking experiments of Heterobilharzia americana eggs revealed that the average sensitivity of the PCR assay was 1.5 eggs per gram of feces. The reproducibility experiments demonstrated that the 5 lowest egg counts could be consistently assayed 100% of the time. The two clinical cases confirmed by sodium chloride sedimentation were also positive using the PCR assay and the identification of Heterobilharzia americana DNA was demonstrated by direct sequencing and comparison to the published sequence.
In conclusion, the PCR assay developed was sensitive and reproducible for the detection of Heterobilharzia americana DNA in dog feces. Further work is in progress to compare the sensitivity and reproducibility of this PCR assay with the current gold standard for the diagnosis of Heterobilharzia americana, sodium chloride sedimentation, and to determine the prevalence of Heterobilharzia americana infection in dogs in endemic areas.