CASE NOTES
Bovine Johne's Disease ELISA Testing — Answers for Frequent Questions
Steven Hum, Veterinary Research Officer, NSW Department of Primary Industries, Elizabeth Macarthur Agricultural Institute
Posted Flock & Herd August 2010
Introduction
Bovine Johnes Elisa testing is most often conducted for the Johnes Disease Market Assurance Program, export testing and for the diagnosis of the disease. For regulatory requirements an initial positive test is followed up by a repeat Elisa and a definitive cultural technique to establish if the animal is actually infected (culture positive - true Elisa positive) or not infected (culture negative - false Elisa positive).
Problems arise during this repeat tests where a previously positive Elisa reaction is not confirmed or previous positive test result is confirmed with a different numerical value. In addition questions often asked about the reasons of false positive reactions. The following paragraphs briefly summarise some of this issues.
The ELISA test kit used at the SVDL
We use the Institute Pourquier commercial Elisa kit (IP-Elisa kit), which was introduced after extensive in-house evaluation in early 2006. One advantage of this kit is that it gives quantitative results comparing to other kits with report only positive or negative test results.
This quantitation can be quite useful analysing background reactions when titres cluster around the cut of point,e.g. previously marginally positive/inconclusive test result turns out to be negative on retest, but still close to the negative cut of point, indicating some acceptable numerical variations in Elisa test results.
Reasons for the numerical variations in ELISA test results
Reactions measured in optical densities (OD) and results expressed as percentages of the sample/positive control ratio according to the formula;
OD sample - negative control
SP Ratio % = ------------------------------------------------------ X 100
OD positive control - negative control
As there are multiple steps and dilutions, additions, incubations and washes, room temperature variations effecting enzyme activities, minor changes in optical densities are to be expected, therefore repeat testing will not yield identical results. The time interval in follow up testing is an other variable since the concentration of circulating IgG antibody is dynamic.
As a protein it is rapidly catabolised by normal processes: it has a finite half life of approximately 30 days in humans and so probably similar in cattle and is replenished in an ongoing manner as fresh secretion by active plasma cells. Other minor factors responsible for the variations of the numerical expression of the ELISA values includes - variations in antigen binding to plates, sample collection, transport storage, haemolysis and so on.
The cause of false positive reations
The IP Elisa is designed to detect circulating IgG antibodies by capturing them with antigen prepared from Mycobacterium avium paratuberculosis (MAP). Unfortunately, there are other environmental organisms which share similar or overlapping antigenic make up to MAP including soil saprophytic mycobacteria or other ‘related bacteria’ in the environment like Coryne/Nocardia/Actinomyces spp. these organisms could sensitize certain animals and sera collected from those will react in the IP Elisa.
The saprophytic or ‘related organisms’ tend to be more prevalent in certain geographic areas (usually more hot and humid) and may explain the fact that specificity of the IP Elisa declines when heading from the southern part of Australia to the north. For example the reported specificity of the IP ELISA in South West Australia is 99.8% while the northern part of the state drops to 97.4%.
Since S (sheep) or C (cattle) MAP strains are antigenically identical the IP-Elisa cannot differentiate between the two strains.