Recipient Organization
UNIV OF PENNSYLVANIA
(N/A)
PHILADELPHIA,PA 19104
Performing Department
School Of Veterinary Medicine
Non Technical Summary
While botulism is fairly uncommon, it can strike swiftly and unexpectedly, taking the lives of many horses in outbreak situations. Equine botulism occurs across all of North America and numerous individual cases, as well as several outbreaks, occur each year. Last year, one particularly notable outbreak cost approximately 100 horses their lives within only a few days. Surviving botulism is largely dependent on prompt diagnosis followed by antitoxin administration as soon as possible and excellent nursing care throughout the course of the disease. Veterinary bills can be tens of thousands of dollars due to the cost of the antitoxin, extended hospital stays, and use of ventilatory support for foals. The significant economic impact only compounds the distress felt by owners of cases of botulism. The traditional mouse bioassay only identifies Clostridium botulinum in approximately 30% of fecal samples from adult horses highly suspected of having botulism, and results are often unavailable for 2-3 weeks after submission. Over the last 6 months, our laboratory has made steady progress in the development of a PCR test for type B botulism. This project will allow us to expand our PCR testing to look for types A and C botulism, the other two types of botulism that affect horses every year in this country. We will then be able to provide a much more rapid and comprehensive diagnostic test for botulism in horses than currently available in any laboratory in the United States, facilitating more timely treatment of exposed and sickened horses. The use of the PCR test will also permit the reduction in use of the mouse bioassay to confirm the diagnosis of botulism.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Our overall goal is to develop a multiplex quantitative real-time PCR (q-PCR) test for detection of Clostridium botulinum types A, B, and C in equine diagnostic samples that will be more rapid, economical, and sensitive than the traditional mouse bioassay, which has been the scientific gold standard for several decades. The availability of this new assay will be critical in more rapid diagnosis of affected horses, enabling prompt treatment and thereby decreasing the fatality rate of this disease.
Project Methods
The first experiments will be directed at PCR methods development. PCR primers, and detection probes for Botulinum type A, B, and C will be obtained from published sequences for those genes and amplicons respectively. Primer and probe concentrations and real-time PCR conditions will be optimized. An internal amplification control will be added, and the 3 primer/probe combinations will be added together in a multiplex PCR platform. Performance characteristics will first be verified using serial dilutions of purified genomic DNA. Test validation will be done, comparing the performance of the quantitative real-time PCR (qPCR) to the current laboratory standard, the mouse bioassay, using diagnostic material from previously confirmed clinical cases submitted to our laboratory. Samples consisting of feed material, intestinal contents, or feces from clinical cases will be set in anaerobic culture to allow sporulation of Clostridium botulinum organisms. From the culture supernatant, DNA will be extracted and subjected to the multiplex PCR. Supernatant will also be processed for testing in the mouse bioassay, and sensitivity and specificity of the two tests will be compared. Bacterial extracts from other bacterial species will also be tested by qrtPCR to verify specificity.