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United States Department of Agriculture

Agricultural Research Service

Title: A Competitive Pcr Assay for Quantitative Detection of Neospora Caninum

Authors
item Liddell, Susan - 1265-40-00 & BRDC
item Jenkins, Mark
item Dubey, Jitender

Submitted to: International Journal for Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 6, 1999
Publication Date: N/A

Interpretive Summary: Neospora caninum is a parasite which was first identified as causing neuromuscular paralysis and death in dogs. Neospora-like organisms have also been found in naturally infected cattle, goats, horses, deer and sheep. Neosporosis has become a widely recognized disease of cattle and appears to be a significant cause of abortion and neonatal morbidity in dairy cattle in the United States and world-wide. Transfer of the parasite to the fetus may result in abortion, stillbirth, delivery of a diseased calf that dies a few days after birth, or an apparently normal, but infected calf. Infected dams and their congenitally infected offspring can transmit the parasite during subsequent pregnancies and vertical transmission appears to be a major route of infection within a herd. Another route of infection may be via foodstuffs contaminated by infective organisms found in fecal material of canids, the recently identified definitive host of the parasite. Prevention of parasite transmission is crucial to limiting economic losses in cattle incurred through neosporosis. Vaccination may be one approach to prevent the spread infection in bovines, canines and other hosts. The present study was conducted to improve upon a small animal model for neosporosis that can be used to conduct vaccine efficacy trials prior to testing in bovines. A sensitive molecular technique for detecting Neospora parasites was modified and expanded to permit the measurement of approximate numbers of Neospora parasites present in infected animal tissues. Furthermore, a component of the technique provides a useful control for the detection procedure and facilitates the identification of truly negative samples.

Technical Abstract: The purpose of the current study was to develop a quantitative-competitive polymerase chain reaction (QC-PCR) assay to detect and measure relative levels of Neospora caninum in animal tissues. A molecule was synthesized for use in PCR as a competitor to the target Neospora-specific Nc5 genomic sequence. Usefulness of the QC-PCR assay was evaluated the murine model of vertical infection of Neopora. Tissues (brain and lungs) from pups born to a dam infected with Neospora caniunum mid-gestation (day 11) were analyzed in the assay. The results revealed that similar levels of parasites were present in each littermate. Since the assay is capable of measuring parasite load in the tissues of infected animals in the murine model system, it should be useful in evaluation of vaccines against Neospora infection. An additional use for the competitor molecule is incorporation into the straighforward detection assay, providing a control for PCR failure and facilitating the identification of truly negative samples.

Last Modified: 4/21/2014