Geographic disparities in IDEXX ELISA

Accurate identification of cattle infected with Mycobacterium bovis is essential for the prevention and control of bovine tuberculosis. One method of identifying infected cattle is an ELISA developed by IDEXX Laboratories, which detects antibodies against two M. bovis proteins, MPB70 and MPB83. The sensitivity of the test varies by geographic region, with sensitivities of 77%, 45% and 9% in bovine serum samples from the UK (n=126), USA (n=146) and Mexico (n=128), respectively .

We hypothesized that geographically biased sequence variation in mpb70 and mpb83, or in the genes that regulate their expression (sigK and rskA), may account for these different sensitivities. This hypothesis was tested by comparing the sequences of these four genes in 455 M. bovis strains isolated from cattle in the aforementioned countries.

For each gene, a single common sequence was identified in most of the genomes of the M. bovis strains collected in the three countries. Twelve of the 455 strains were isolated from infected cattle for which the IDEXX ELISA was also performed. Five of the seven ELISA positive genomes and three of the five ELISA negative genomes contained the most common sequence of the four genes. Thus, sequence variation in mpb70, mpb83, sigK, and rskA does not explain geographic disparities in IDEXX ELISA sensitivity.

The bacterium Mycobacterium bovis is the causative agent of bovine tuberculosis, a persistent infection that primarily affects the lungs but can also spread to other body systems. It is a member of the Mycobacterium tuberculosis complex, which is a group of bacterial species that cause tuberculosis. As it has a significant impact on both animal health and international trade, substantial economic resources are devoted to the prevention, management and control of outbreaks of bovine tuberculosis1,2.

In addition to cattle, M. bovis can infect many other hosts, including humans1,3, although Mycobacterium tuberculosis, another member of the Mycobacterium tuberculosis complex, is responsible for the majority of human tuberculosis cases. Human-to-human transmission of M. bovis is rare, with most human infections resulting from consumption of unpasteurized dairy products or inhalation of respiratory droplets from infected cattle4.

Due to the economic consequences associated with M. bovis infections in cattle, as well as its zoonotic potential, methods for rapid and accurate identification of infected cattle are of considerable importance. One such method is an enzyme-linked immunosorbent assay (ELISA) developed by IDEXX Laboratories (Westbrook, ME), which allows rapid detection of antibodies specific to M. bovis in bovine serum or milk samples5,6.

This test detects antibodies against the antigenic proteins MPB70 (where "MPB" is the acronym for M. bovis protein7 and "70" refers to the relative electrophoretic mobility of the protein on a polyacrylamide gel8) and MPB83, which are usually produced in large quantities by M. bovis7.

The IDEXX ELISA has been tested on serum and milk samples from cattle not infected with M. bovis and found to have a specificity of approximately 98%, i.e. the test gives a result positive for only 2% of uninfected cattle5,6.

The sensitivity of the ELISA was assessed using cattle verified for M. bovis infection using independent tests. Its sensitivity varied according to the geographical origin of the infected cattle, oscillating between 30% and 90% and was globally around 62%5,6. The reasons for these marked geographic differences in test sensitivity are currently unknown.

In this study, we present additional data regarding the sensitivity of the IDEXX ELISA in different geographical regions and show that the test has good sensitivity for serum samples from cattle from the UK, moderate sensitivity for samples serum from cattle from the United States and low sensitivity for serum samples from cattle from Mexico.

We then test the hypothesis of a sequence variation in the mpb70 and mpb83 genes (including both their coding sequences and their upstream and downstream regions), or in the genes whose corresponding proteins are involved in the regulation of their expression. (sigK and rskA)9,10,11,12, is responsible for these geographical disparities. (See Supplemental Discussion S1 for more information on MPB70 and MPB83, the genes that encode them, and the proteins involved in regulating their expression). This hypothesis has been studied