Use of Recombinant Proteins to Identify Antibody Responses Associated with Equine Proliferative Enteropathy.

Subject: Lawsonia intracellularis is the causative bacterium of equine proliferative enteropathy (EPE), a disease of young horses characterized by a protein losing enteropathy with occasional fatalities. Currently, diagnosis of clinical EPE cases involves the presence of non-specific clinical signs in addition to fecal PCR and serologic detection of bacterial exposure. While the latter clinical tests are specific for the bacterium, they are not capable of differentiating exposed from clinically affected horses. Significance: L. intracellularis and the disease it causes in horses, EPE, is an emerging pathogen of increasing importance to the horse industry from both an economic and welfare standpoint. The hallmark of EPE is a protein-losing enteropathy where affected horses suffer weight loss and some ultimately succumb to the disease despite aggressive treatment. While a majority of young horses are exposed to L. intracellularis, EPE is a sporadic disease, though some farms experience clinical cases year after year. This project will use both an expression library and 2-dimensional gel immunoscreening to identify immunoreactive proteins of L. intracellularis that could be used to identify individuals at risk for developing clinical EPE. Current diagnosis of EPE relies on the presence of clinical signs concurrent with suggestive, but non-confirmatory tests of infection. Serological detection of infection would be the most specific and cost-effective means of diagnosis, but current serological tests for L. intracellularis detect only exposure to the bacterium and fail to provide confirmation of a clinical infection. Hypothesis/Objectives: Our hypothesis is that the antibody response to specific L. intracellularis antigens can be used to identify EPE affected horses. This will be accomplished by creating a DNA expression library (Objective #1), which we will use to obtain L. intracellularis recombinant proteins for immunoblot screening (Objective #3). We will also use 2D-SDS-PAGE gel electrophoresis of whole L. intracellularis (Objective #2) blotted with serum from L. intracellularis-exposed horses to identify immunoreactive proteins (Objective #3). The immunoreactive recombinant proteins will then be used to screen sera from horses representing clinically EPE affected, exposed but not clinically affected, and non-exposed horses (Objective #4). This will allow us to identify antibody responses to specific proteins that may be diagnostic for either clinical EPE or protective immunity. Proteins that do demonstrate variable immunoreactivity will be explored for possible use in a new ELISA.