In the Republic of Ireland slurry from herds infected with notifiable diseases, including tuberculosis and brucellosis, must be treated with Calcium Hydroxide suspension -20kg Ca(OH)2/m3 before spreading on pasture to minimise the risk of spreading disease. As the incidence of Johne's Disease is increasing it was desirable to see if a similar treatment was effective for inactivating Mycobacterium avium subsp. paratuberculosis (MAP) in slurry.

Method

300 ml quantities of sterile slurry (autoclaved at 130ºC for 60 mins) were spiked with a suspension of MAP type strain NCTC 8578 to give a concentration 1x10⁹cfu /ml. and incubated at 19ºC with shaking. Liquid Calcium Hydroxide was then added to the containers to achieve a pH of 12.7, and samples were subsequently taken from the treated containers and an untreated control at 6, 10, 24, 48 and 72 hrs, and 7 and 14 days post treatment and cultured. To prepare inoculum for culture 5ml of sample was suspended in 20ml of sterile phosphate buffered saline (PBS) and centrifuged briefly at 1000rpm for 60secs to precipitate large particles. The resulting supernatant was then centrifuged at 3000pm for 30mins to pellet the organisms, the pellet was washed 3 times with PBS, and finally resuspended in 2ml of PBS. 100µl of suspension was inoculated into Bactec B12B medium and HEY slants, incubated at 37ºC, for 12weeks and examined at weekly intervals for 12 weeks for growth.

Results

Colony counts on the solid medium showed a 10⁷ fold reduction in the number of viable organisms after 72hrs treatment compared to the untreated control, and in the liquid B12B medium time growth increased substantially up to 72hours. No organisms were recovered from either medium after 7days.

Conclusion

Survival of MAP after 24hrs and up to 72 hrs at pH 12.5 was unexpected and would render this method of treatment impractical. It is proposed to repeat the experiment using raw slurry and sample intervals of 12hrs for seven days, enumerating MAP colonies on solid media without decontamination with CPC.

Sponsorship

Attendance to this Congress was sponsored by the EU-funded project SSPE-CT-2004-501903