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Title: SPECIFIC DETECTION OF NEOSPORA CANINUM OOCYSTS IN FECAL SAMPLES FROM EXPERIMENTALLY INFECTED DOGS USING THE POLYMERASE CHAIN REATION

Author
item Hill, Dolores
item Dubey, Jitender
item LIDDELL, SUSAN - 1265-40-00
item Jenkins, Mark

Submitted to: Journal of Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/21/2000
Publication Date: N/A
Citation: N/A

Interpretive Summary: Neospora caninum is a protozoan parasite which has been shown to cause abortion in dairy and beef cattle, as well as neurological disorders in dogs and horses. Recently, dogs have been shown to be a definitive host of N. caninum. Though vertical transmission has been documented in cattle, little is known about how definitive hosts, such as dogs, become infected or how they contribute to transmission of N. caninum to cattle. Epidemiological studies which could lead to risk reduction strategies on the farm have been hampered by an inability to morphologically distinguish N. caninum oocysts in dog feces from oocysts of other canine coccidian parasites such as H. heydorni, or oocysts contaminating dog feces such as H. hammondia or T. gondii. The inability to follow oocyst shedding patterns in infected dogs, the inability to identify contamination routes of cattle feed and facilities, and the inability to determine to what extent wild canids, such as foxes, contribute to the transmission cycle are a direct result of the lack of sensitive methods to specifically detect N. caninum oocysts. In this study, we have developed a method using the pNc-5 genomic DNA sequence to specifically detect N. caninum oocysts isolated from dog feces by PCR, and to quantify the number of N. caninum oocysts isolated from dog fecal samples.

Technical Abstract: Neospora caninum oocysts, passed in the feces of a definitive host (dog), were isolated and genomic DNA extracted. PCR, targeting the N. caninum- specific pNc-5 genomic sequence was performed using the isolated DNA. A synthesized competitor molecule containing part of the pNc-5 sequence was included in the assay as a check against false-negative PCR results and to quantify N. caninum oocyst DNA in fecal samples. A standard curve of the ratio of fluorescence intensity of competitor:oocyst DNA was constructed to compare oocyst equivalents from fecal samples containing unknown numbers of N. caninum oocysts, and to assess the sensitivity of the assay. The specificity of the assay was determined using the pNc-5 specific primers in PCR assays against other parasites likely to be found in canine feces. Genomic DNA sequences from the canine coccidian parasites H. heydorni, Cryptosporidium parvum, Sarcocystis cruzi, S. tenella, and Isospora ohioensis, and the canine helminth parasites Strongyloides stercoralis, Toxocara canis, Dipylidium caninum, and Ancylostoma caninum were not amplified. In addition, genomic DNA sequences from oocysts of coccidian parasites that might contaminate dog feces such as Hammondia hammondia, Toxoplasma gondii, or Eimeria tenella were not amplified in the PCR assay. The assay should be useful in epidemiological surveys of both domestic and wild canine hosts and in investigations of oocyst biology in experimental infections.