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Optimisation of culture of Mycobacterium avium subspecies paratuberculosis from blood samples
Katrina Bower1, Douglas J Begg, Richard J Whittington
1Farm Animal and Veterinary Public Health, Faculty of Veterinary Science, University of Sydney, PMB 4003, Narellan, NSW 2567, Australia.
Abstract:
Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease or paratuberculosis, a chronic enteritis of ruminants, and has been suggested to play a role in Crohn's disease in humans. While disease expression is primarily in the gastrointestinal tract, isolation of MAP or MAP DNA at distant sites indicates that disseminated infections also occur. This is the first study to compare, optimise and critically evaluate different methods of concentrating MAP from spiked blood samples to enable its culture. Whole blood, erythrocytes, but not plasma, delayed the growth of MAP in BACTEC 12B medium. Culture of MAP from buffy coats (concentrated leukocytes) after lysis of erythrocytes and washing of the cells was an effective preparatory method. Several antibiotics were evaluated to reduce contamination of the slow growing MAP cultures with microbes which were derived from the skin during venipuncture, but were detrimental to the growth of MAP. However, decontamination of erythrocyte-lysed washed buffy coat samples in 0.75% hexadecylpyridinium chloride (HPC) for 72 h prior to inoculation of culture media did not inhibit the growth of MAP. The prepared samples can be stored at -80 degrees C prior to batch culture. MAP was isolated from the blood of 2 of 23 sheep 20 months after experimental inoculation. The optimised method has an analytical sensitivity of at least 10(1) MAP per ml of spiked whole blood and will enable trials to determine the incidence, duration and magnitude of mycobacteraemia in infected animals and humans.
Insights
This study optimized a method to culture Mycobacterium avium subspecies paratuberculosis (MAP) from blood, enabling detection of disseminated infections in animals and potentially humans with Johne's disease or Crohn's disease.
Area of Science:
- Veterinary Microbiology
- Infectious Diseases
- Diagnostic Bacteriology
Background:
- Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease in ruminants and is implicated in human Crohn's disease.
- MAP infections can disseminate beyond the gastrointestinal tract, necessitating blood-based detection methods.
- Current methods for culturing MAP from blood are not well-established or optimized.
Purpose of the Study:
- To compare, optimize, and evaluate methods for concentrating MAP from spiked blood samples for improved culture.
- To establish an effective preparatory method for MAP culture from blood components.
- To assess the impact of decontamination agents and storage conditions on MAP viability and culture.
Main Methods:
- Evaluation of MAP growth in BACTEC 12B medium using whole blood, erythrocytes, and plasma.
- Optimization of buffy coat preparation involving erythrocyte lysis and cell washing.
- Screening of antibiotics for contamination control and assessment of 0.75% hexadecylpyridinium chloride (HPC) for decontamination.
- Testing the storage stability of prepared samples at -80°C.
- Determining the analytical sensitivity of the optimized method.
Main Results:
- Erythrocytes, but not plasma, delayed MAP growth; buffy coat preparation was effective.
- Decontamination with 0.75% HPC for 72 hours did not inhibit MAP growth.
- Prepared samples could be stored at -80°C.
- The optimized method achieved an analytical sensitivity of at least 10(1) MAP per ml of spiked whole blood.
- MAP was successfully isolated from 2 of 23 experimentally inoculated sheep 20 months post-inoculation.
Conclusions:
- An optimized method for concentrating and culturing MAP from blood, specifically using washed buffy coats with HPC decontamination, has been developed.
- This method allows for sample storage and has high analytical sensitivity, facilitating further studies on MAP dissemination.
- The findings will enable investigations into the incidence, duration, and magnitude of mycobacteremia in infected animals and potentially humans.
