Background

Mycobacterium avium subspecies paratuberculosis (Map) is an important pathogen causing ruminant paratuberculosis and human infection. paratuberculosis is characterized by chronic granulomatous enteritis, persistent diarrhea, progressivewasting, and finally death, and has resulted in significant economic losses to the dairy and cattle industries worldwide. Furthermore, the bacteria are speculated to be the cause of human Crohn's disease. One of the difficult issues in diagnosis and research of the infection is their significant slow growth. Even by using specially enriched Herrold's egg yolk medium (HEYM) with Mycobactin J, it takes 3 to 4 months to see minimal colonies. However, in a susceptibility test for anti-bacterial substance, evaluation of disinfectant or bactericidal activity of macrophages, for example, we need to know their viability for efficacy as soon as possible. Carboxyfluorescein diacetate succinimidyl (CFDA) and carboxyfluorescein diacetate succinimidyl ester (CFDA/SE) labelling has been used previously to study the adhesion of labelled bacteria to host cells and the uptake of labelled substrates by various cells using flowcytometry analysis and viability test of several bacteria, however no study was reported on Map. Therefore, we investigated the application of a viability test of Map by CFDA or CFDA/SE.

Results

The results revealed that CFDA is a useful reagent as a fluorescent probe to determine the quantitative viability of Map and as a tracer. CFDA/SE labelling heat-killed Map as well as live bacilli. Incubation of Map with CFDA at 100μM for 30min was practically the optimal condition for the viability test.

Conclusions

CFDA staining with fluorescent measurement is a useful tool in various required tests of viability, such as the evaluation of antibiotics, disinfectant, other sterilization conditions, and the bactericidal effect of activated phagocytes for Map.