Proliferative enteropathy (PE) caused by Lawsonia intracellularis is a major diarrheal disease affecting swine worldwide. Routine laboratory diagnosis of PE is done by amplification of L. intracellularis-specific DNA sequences by PCR followed by agarose gel electrophoresis and staining of PCR products with ethidium bromide. We report the development of an enzyme-linked oligosorbent assay (ELOSA) for specific identification of chromosomal L. intracellularis 328-bp PCR amplified products. The ELOSA involved determination of optical density value at 450 nm (OD450) after hybridization of biotin-labelled PCR products with an amine-modified internal oligonucleotide capture probe immobilized in microwell plates, and avidin-biotin-peroxidase complex. A positive ELOSA cut-off value of ≥ 0.375 was established using the mean OD450 of negative control specimens plus three times the standard deviation. Using this value, the detection limit of PCR amplified L. intracellularis-specific products by ethidium bromide-stained agarose gel electrophoresis, Southern blot, and ELOSA were estimated to be 6.1 ng, between 0.8 and 3.0 ng, and 0.8 ng of DNA, respectively. Comparison of ethidium bromide-stained agarose gel analysis with ELOSA for detection of L. intracellularis-specific PCR products from 315 clinical specimens revealed 78% sensitivity, 100% specificity and 94% accuracy. The ELOSA produced a spectrophotometric signal that confirmed the authenticity of PCR products without subjective interpretation of ethidium bromide-stained PCR products after agarose gel electrophoresis. (C) 2000 Academic Press.
Bibliographical noteFunding Information:
The authors thank Michelle R. Mathiesen of the Department of Veterinary and Biomedical Sciences at the University of Nebraska-Lincoln for excellent technical assistance, veterinary practitioners and collaborators in the United States and Canada for providing clinical specimens for this study, and Jeffrey P. Knittel of Boehringer Ingelheim Vetmedica, Inc. for providing faecal specimens from swine farms free of proliferative enteropathy. The research was supported in part by funds provided by the National Pork Producers Council and the United States Department of Agriculture, Regional Research Project NC-62; Enteric diseases of swine and cattle: prevention, control and food safety. This paper is Paper No. 12292, Agricultural Research Division, Institute of Agriculture and Natural Resources, University of Nebraska-Lincoln, Lincoln, NE.
- Enzyme-linked oligosorbent assay
- Lawsonia intracellularis
- Proliferative enteropathy