In this study, products from all steps of anaerobic digestion at a farm-scale biogas plant supplied with manure from paratuberculosis-affected dairy cattle were examined and quantified for the presence of the causal agent of paratuberculosis, Mycobacterium avium subsp. paratuberculosis, using culture and quantitative real-time PCR (qPCR). Viable M. avium subsp. paratuberculosis cells were detected using culture in fermentors for up to 2 months; the presence of M. avium subsp. paratuberculosis DNA (10(1) cells/g) was demonstrated in all anaerobic fermentors and digestate 16 months after initiation of work at a biogas plant, using IS900 qPCR. F57 qPCR was able to detect M. avium subsp. paratuberculosis DNA (10(2) cells/g) at up to 12 months. According to these results, a fermentation process that extended beyond 2 months removed all viable M. avium subsp. paratuberculosis cells and therefore rendered its product M. avium subsp. paratuberculosis free. However, M. avium subsp. paratuberculosis DNA was found during all the examined periods (more than 1 year), which could be explained by either residual DNA being released from dead cells or by the presence of viable cells whose amount was under the limit of cultivability. As the latter hypothesis cannot be excluded, the safety of the final products of digestion used for fertilization or animal bedding cannot be defined, and further investigation is necessary to confirm or refute this risk.

Veterinary Research Institute Hudcova 70 621 00 Brno Czech Republic

Zobrazit více v PubMed

Baumgartner W., Damoser J., Khol J. L. 2005. Comparison of two studies concerning the prevalence of bovine paratuberculosis (Johne's disease) in Austrian cattle in the years 1995-1997 and 2002/03. Wien. Tierarztl. Monat. 92:274–277

Commission of the European Communities 2002. Animal by-products not intended for human consumption, regulation no. 1774 of the European Parliament and of the Council. Commission of the European Communities, Brussels, Belgium

Gantzer C., et al. 2001. Monitoring of bacterial and parasitological contamination during various treatment of sludge. Water Res. 35:3763–3770 PubMed

Gobec I., Ocepek M., Pogacnik M., Dobeic M. 2009. Inactivation of Mycobacterium avium paratuberculosis in sheep manure. Slov. Vet. Res. 46:105–113

Good M., et al. 2009. Prevalence and distribution of paratuberculosis (Johne's disease) in cattle herds in Ireland. Ir. Vet. J. 62:597–606 PubMed PMC

Grewal S. K., Rajeev S., Sreevatsan S., Michel F. C. 2006. Persistence of Mycobacterium avium subsp. paratuberculosis and other zoonotic pathogens during simulated composting, manure packing, and liquid storage of dairy manure. Appl. Environ. Microbiol. 72:565–574 PubMed PMC

Holm-Nielsen J. B., Al Seadi T., Oleskowicz-Popiel P. 2009. The future of anaerobic digestion and biogas utilization. Bioresour. Technol. 100:5478–5484 PubMed

Horan N. J., Fletcher L., Betmal S. M., Wilks S. A., Keevil C. W. 2004. Die-off of enteric bacterial pathogens during mesophilic anaerobic digestion. Water Res. 38:1113–1120 PubMed

Jorgensen J. B. 1977. Survival of Mycobacterium paratuberculosis in slurry. Nord. Med. 29:267–270 PubMed

Kearney T. E., Larkin M. J., Levett P. N. 1993. The effect of slurry storage and anaerobic digestion on survival of pathogenic bacteria. J. Appl. Bacteriol. 74:86–93 PubMed

Kralik P., et al. 2011. Development of a predictive model for detection of Mycobacterium avium subsp. paratuberculosis in faeces by quantitative real time PCR. Vet. Microbiol. 149:133–138 PubMed

Levy-Booth D. J., et al. 2007. Cycling of extracellular DNA in the soil environment. Soil Biol. Biochem. 39:2977–2991

Matlova L., et al. 2004. Impact of sawdust and wood shavings in bedding on pig tuberculous lesions in lymph nodes, and IS1245 RFLP analysis of Mycobacterium avium subsp. hominissuis of serotypes 6 and 8 isolated from pigs and environment. Vet. Microbiol. 102:227–236 PubMed

Moravkova M., et al. 2008. Strategy for the detection and differentiation of Mycobacterium avium species in isolates and heavily infected tissues. Res. Vet. Sci. 85:257–264 PubMed

Nielsen S. S., Toft N. 2009. A review of prevalences of paratuberculosis in farmed animals in Europe. Prev. Vet. Med. 88:1–14 PubMed

Olsen J. E., Larsen H. E. 1987. Bacterial decimation times in anaerobic digestions of animal slurries. Biol. Wastes 21:153–168

Olsen J. E., Jorgensen J. B., Nansen P. 1985. On the reduction of Mycobacterium-paratuberculosis in bovine slurry subjected to batch mesophilic or thermophilic anaerobic-digestion. Agric. Wastes 13:273–280

Pavlik I., et al. 2000. Parallel faecal and organ Mycobacterium avium subsp. paratuberculosis culture of different productivity types of cattle. Vet. Microbiol. 77:309–324 PubMed

Plymforshell L. 1995. Survival of salmonellas and Ascaris suum eggs in a thermophilic biogas plant. Acta Vet. Scand. 36:79–85 PubMed PMC

Reddacliff L. A., Vadali A., Whittington R. J. 2003. The effect of decontamination protocols on the numbers of sheep strain Mycobacterium avium subsp. paratuberculosis isolated from tissues and faeces. Vet. Microbiol. 95:271–282 PubMed

Sahlstrom L. 2003. A review of survival of pathogenic bacteria in organic waste used in biogas plants. Bioresour. Technol. 87:161–166 PubMed

Schnurer A., Schnurer J. 2006. Fungal survival during anaerobic digestion of organic household waste. Waste Manage. 26:1205–1211 PubMed

Slana I., Kralik P., Kralova A., Pavlik I. 2008. On-farm spread of Mycobacterium avium subsp. paratuberculosis in raw milk studied by IS900 and F57 competitive real time quantitative PCR and culture examination. Int. J. Food Microbiol. 128:250–257 PubMed

Watcharasukarn M., Kaparaju P., Steyer J. P., Krogfelt K. A., Angelidaki I. 2009. Screening Escherichia coli, Enterococcus faecalis, and Clostridium perfringens as indicator organisms in evaluating pathogen-reducing capacity in biogas plants. Microb. Ecol. 58:221–230 PubMed

Whittington R. J. 2009. Factors affecting isolation and identification of Mycobacterium avium subsp. paratuberculosis from fecal and tissue samples in a liquid culture system. J. Clin. Microbiol. 47:614–622 PubMed PMC

Whittington R. J., Marshall D. J., Nicholls P. J., Marsh I. B., Reddacliff L. A. 2004. Survival and dormancy of Mycobacterium avium subsp. paratuberculosis in the environment. Appl. Environ. Microbiol. 70:2989–3004 PubMed PMC

Whittington R. J., Marsh I. B., Reddacliff L. A. 2005. Survival of Mycobacterium avium subsp. paratuberculosis in dam water and sediment. Appl. Environ. Microbiol. 71:5304–5308 PubMed PMC

Culture- and quantitative IS900 real-time PCR-based analysis of the persistence of Mycobacterium avium subsp. paratuberculosis in a controlled dairy cow farm environment