Modification of a bovine ELISA to detect camelid antibodies to Mycobacterium paratuberculosis.

Mycobacterium avium subsp. paratuberculosis infection, or Johne's disease, reportedly has a low prevalence in South American camelid populations. Recently, however, single cases in the United States as well as an outbreak of the disease in Australian alpacas (Lama pacos) have been described. To provide a rapid and cost-effective method of diagnosing Johne's disease in this species, the bovine Parachek((R)) Johne's Absorbed EIA (CSL, Vic., Australia) was modified to create a camelid-specific serum antibody assay. An anti-llama IgG conjugated to horseradish peroxidase replaced the anti-bovine immunoglobulin. Checkerboard titration of principal reagents was performed using serum from nine tissue and/or fecal culture-positive camelids. Optimal dilutions of key components were determined in order to provide clear discrimination between positive and negative controls. Completion of a kinetic assay determined the optical density at which the enzyme-substrate reaction should be stopped. A herd of 100 camelids with no history of disease or exposure to M. a. paratuberculosis, a subset of which were tissue and/or fecal culture-negative, was tested to establish a cut-off value. Sample results were expressed as a percentage of the results for control sera by transforming optical density values to ELISA values (EV%). A preliminary EV% cut-off of 20 was established. Using this prototype assay, culture-positive animals showed significantly different antibody responses from culture-negative animals. These results indicate that this camelid-specific ELISA, once refined, may be a useful tool for screening camelid herds for M. a. paratuberculosis infection.

Specificity of four serologic assays for Mycobacterium avium ss paratuberculosis in llamas and alpacas: a single herd study.

An investigation was conducted for Mycobacterium avium ss paratuberculosis infections in a research herd of llamas and alpacas. Herd culture-negative status was established over a 23-month period by screening any individuals with any signs compatible with paratuberculosis (n = 1), high serology values (n = 8), or other health and research related reasons (n = 24). There were no M. avium ss paratuberculosis isolates from radiometric cultures of multiple tissue and fecal samples from these individuals and no known sources of exposure. Paratuberculosis is uncommon in North American llamas and alpacas: only 5 cases were identified after an extensive search of the Veterinary Medical Data Base, diagnostic laboratory records, publication databases, and personal communications. Therefore, serum samples from llamas (n = 84) and alpacas (n = 16) in the culture-negative herd were used to obtain preliminary estimates of test specificity for 3 enzyme-linked immunoassays (ELISAs) and an agar gel immunodiffusion (AGID) assay kit for detecting serum antibodies to M. avium ss paratuberculosis in South American camelids. The ELISAs were modifications of established bovine assays for antibody detection. With provisional cutoffs, ELISA-A had 52 false positives (specificity 48%), ELISA-B had 8 false positives (specificity 92%), ELISA-C had two false positives (specificity 98%), and the AGID had 0 false positives (specificity 100%). The range of ELISA values for culture-positive llamas and alpacas (n = 10) from other herds overlapped the range of values for culture-negative llamas and alpacas. The accuracy of the ELISAs may be improved by using age- and sex-specific cutoffs because uninfected male llamas and alpacas that were older than 1 year had higher values for some tests. These tests can be used for either llamas or alpacas; the protein-G conjugate ELISA (ELISA-B) may be useful for multispecies applications. These assays are best used for rapid presumptive diagnoses of llamas and alpacas with diarrhea and weight loss and as a screening tool for herds known to be exposed to infection. All seropositive results should be confirmed with culture.