Specific detection of Neospora caninum oocysts in fecal samples from experimentally-infected dogs using the polymerase chain reaction

Neospora caninum oocysts, passed in the feces of a definitive host (dog), w ere isolated, and genomic DNA was extracted. A polymerase cahin reaction (P CR) targeting the N. caninum-specific Nc 5 genomic sequence was performed u sing the isolated DNA. A synthesized competitor molecule containing part of the Nc 5 sequence was included in the assay as a check against false-negat ive PCR results and to quantify N. caninum oocyst DNA in fecal samples. A s tandard curve of the ratio of fluorescence intensity of PCR-amplified compe titor to that of oocyst DNA was constructed to compare oocyst equivalents f rom fecal samples containing unknown numbers of N. caninum oocysts and to a ssess the sensitivity of the assay. The specificity of the assay was determ ined using the Nc 5-specific primers in PCR assays against other parasites likely to be found in canine feces. Genomic DNA sequences from the canine c occidians Hammondia heydorni. Cryptosporidium parvum. Sarcoystis cruzi, S. tenella, and Isospora ohioensis and the canine helminth parasites Strongloi des stercoralis. Toxocura canis. Dipylidium caninum, and Ancylostoma caninu m were not amplified. In addition, genomic DNA sequences from oocysts of co ccidian parasites that might contaminate dog feces, such as Hammondia hammo ndi, Taxoplasma gondii, or Eimeria tenella, were not amplified in the PCR a ssay. The assay should be useful in epidemiological surveys of both domesti c and wild canine hosts and in investigations of oocyst biology in experime ntal infections.