Abstract
The aim of the present study was to develop a fast and reliable fluorescence staining technique in order to accurately determine the number of viable Mycobacterium avium ssp. paratuberculosis (MAP) cells in milk. Milk was artificially inoculated with known amounts of four different MAP strains. Different dilutions were tested by the combination of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and auramine orange (AO) staining. To validate this combined fluorescence staining technique, two additional methods were applied: culture and the FASTPlaqueTB™ assay. The detection limit of the combined CTC and AO staining was 102 colony-forming unit (CFU) mL−1 for spiked ultra-high-temperature (UHT) milk and 103 CFU mL−1for spiked raw milk (probability of detection >95 %). Combined CTC and AO staining provides the opportunity to determine the total cell count of acid fast bacteria as well as the number of respiratory active cells in milk within 8 h.
Similar content being viewed by others
References
Akineden Ö, Fernández-Silva JA, Weirich S, Abdulmawjood A, Bülte M (2011) Comparison of two decontamination procedures, three culture media, and real time-PCR assay for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) from artificially contaminated raw sausages. J Food Saf Food Qual 62:5–10
Altic LC, Rowe MT, Grant IR (2007) UV light inactivation of Mycobacterium avium subsp. paratuberculosis in milk as assessed by FASTPlaqueTB phage assay and culture. Appl Environ Microbiol 73:3728–3733
Anonymous (2010) Assessment of food as a source of exposure to Mycobacterium avium subspecies paratuberculosis (MAP). J Food Prot 73:1357–1397
Boulos L, Prévost M, Barbeau B, Coallier J, Desjardins R (1999) LIVE/DEAD BacLight: application of a new rapid staining method for direct enumeration of viable and total bacteria in drinking water. J Microbiol Methods 37:77–86
Cheng AG, Chang A, Farwell DG, Agoff SN (2005) Auramine orange stain with fluorescence microscopy is a rapid and sensitive technique for the detection of cervical lymphadenitis due to mycobacterial infection using fine needle aspiration cytology: a case series. Otolaryngol Head Neck Surg 133:381–385
Dixon WJ (1993) BMDP statistical software manual, volume 1 and 2. University of California Press, Berkeley, Los Angeles
Dundee L, Grant IR, Ball HJ, Rowe MT (2001) Comparative evaluation of four decontamination protocols for the isolation of Mycobacterium avium subsp. paratuberculosis from milk. Lett Appl Microbiol 33:173–177
Eltholth MM, Marsh VR, van Winden S, Guitian FJ (2009) Contamination of food products with Mycobacterium avium paratuberculosis: a systematic review. J Appl Microbiol 107:1061–1071
Foddai A, Elliott CT, Grant IR (2009) Optimization of a phage amplification assay to permit accurate enumeration of viable Mycobacterium avium subsp. paratuberculosis cells. Appl Environ Microbiol 75:3896–3902
Foddai A, Elliott CT, Grant IR (2010) Rapid assessment of the viability of Mycobacterium avium subsp. paratuberculosis cells after heat treatment, using an optimized phage amplification assay. Appl Environ Microbiol 76:1777–1782
Grant IR (2010) Mycobacterium avium subsp. paratuberculosis in animal-derived Foods and the Environment. In: Behr MA, Collins DM (eds) Paratuberculosis: organism, disease, control. CAB International, Oxfordshire, pp 29–35
Grant IR, Rowe MT (2004) Effect of chemical decontamination and refrigerated storage on the isolation of Mycobacterium avium subsp. paratuberculosis from heat-treated milk. Lett Appl Microbiol 38:283–288
Grant IR, Ball HJ, Rowe MT (1997) A novel staining technique for assessing clumping and viability of Mycobacterium paratuberculosis cells during pasteurization. Proceedings of the fifth international colloquium on paratuberculosis, pp. 374–376. http://www.paratuberculosis.info/proc5/page374.htm
Grant IR, Williams AG, Rowe MT, Muir DD (2005) Efficacy of various pasteurization time-temperature conditions in combination with homogenization on inactivation of Mycobacterium avium subsp. paratuberculosis in milk. Appl Environ Microbiol 71:2853–2861
Gunasekera TS, Sørensen A, Attfield PV, Sørensen SJ, Veal DA (2002) Inducible gene expression by nonculturable bacteria in milk after pasteurization. Appl Environ Microbiol 68:1988–1993
Kitaguchi A, Yamaguchi N, Nasu M (2005) Enumeration of respiring Pseudomonas spp. in milk within 6 hours by fluorescence in situ hybridization following formazan reduction. Appl Environ Microbiol 71:2748–2752
Kralik P, Nocker A, Pavlik I (2010) Mycobacterium avium subsp. paratuberculosis viability determination using F57 quantitative PCR in combination with propidium monoazide treatment. Int J Food Microbiol 141: 80–86
Larsen AB, Stalheim OH, Hughes DE, Appell LH, Richards WD, Himes EM (1981) Mycobacterium paratuberculosis in the semen and genital organs of a semen-donor bull. J Am Vet Med Assoc 179:169–171
Nielsen SS, Bjerre H, Toft N (2008) Colostrum and milk as risk factors for infection with Mycobacterium avium subspecies paratuberculosis in dairy cattle. J Dairy Sci 91:4610–4615
Pribylova R, Kubickova L, Babak V, Pavlik I, Kralik P (2012) Effect of short- and long-term antibiotic exposure on the viability of Mycobacterium avium subsp. paratuberculosis as measured by propidium monoazide F57 real time quantitative PCR and culture. Vet J 194:354–360
Rademaker JLW, Vissers MMM, Te Giffel MC (2007) Effective heat inactivation of Mycobacterium avium subsp. paratuberculosis in raw milk contaminated with naturally infected feces. Appl Environ Microbiol 73:4185–4190
Ricchi M, De Cicco C, Kralik P, Babak V, Boniotti MB, Savi R, Cerutti G, Cammi G, Garbarino C, Arrigoni N (2014) Evaluation of viable Mycobacterium avium subsp. paratuberculosis in milk using peptide-mediated separation and Propidium Monoazide qPCR. FEMS Microbiol Lett. doi:10.1111/1574-6968.12480
Richards OW, Kline EK, Leach RE (1941) Demonstration of tubercle bacilli by fluorescence microscopy. Am Rev Tuberc 44:255–266
Rodriguez GG, Phipps D, Ishiguro K, Ridgway HF (1992) Use of a fluorescent redox probe for direct visualization of actively respiring bacteria. Appl Environ Microbiol 58:1801–1808
Schaule G, Flemming HC, Ridgway HF (1993) Use of 5-cyano-2,3-ditolyl tetrazolium chloride for quantifying planktonic and sessile respiring bacteria in drinking water. Appl Environ Microbiol 59:3850–3857
Schönenbrücher H, Abdulmawjood A, Failing K, Bülte M (2008) New triplex real-time PCR assay for detection of Mycobacterium avium subsp. paratuberculosis in bovine feces. Appl Environ Microbiol 74:2751–2758
Slana I, Paolicchi F, Janstova B, Navratilova P, Pavlik I (2008) Detection methods for Mycobacterium avium subsp. paratuberculosis in milk and milk products: a review. Vet Med 53:283–306
Stabel JR (1997) An improved method for cultivation of Mycobacterium paratuberculosis from bovine fecal samples and comparison to three other methods. J Vet Diagn Invest 9:375–380
Stevenson K, Alvarez J, Bakker D, Biet F, de Juan L, Denham S, Dimareli Z, Dohmann K, Gerlach GF, Heron I, Kopecna M, May L, Pavlik I, Sharp JM, Thibault VC, Willemsen P, Zadoks RN, Greig A (2009) Occurrence of Mycobacterium avium subspecies paratuberculosis across host species and European countries with evidence for transmission between wildlife and domestic ruminants. BMC Microbiol 9:212
Swift BM, Denton EJ, Mahendran SA, Huxley JN, Rees CE (2013) Development of a rapid phage-based method for the detection of viable Mycobacterium avium subsp. paratuberculosis in blood within 48 h. J Microbiol Methods 94:175–179
Tasara T, Stephan R (2005) Development of an F57 sequence-based real-time PCR assay for detection of Mycobacterium avium subsp. paratuberculosis in milk. Appl Environ Microbiol 71:5957–5968
Whittington RJ (2010) Cultivation of Mycobacterium avium subsp. paratuberculosis. In: Behr MA, Collins DM (eds) Paratuberculosis: organism, disease, control. CAB International, Oxfordshire, pp 244–260
Whittington RJ, Marsh IB, Saunders V, Grant IR, Juste R, Sevilla IA, Manning EJB, Whitlock RH (2011) Culture phenotypes of genomically and geographically diverse Mycobacterium avium subsp. paratuberculosis isolates from different hosts. J Clin Microbiol 49:1822–1830
Yamaguchi N, Sasada M, Yamanaka M, Nasu M (2003) Rapid detection of respiring Escherichia coli O157:H7 in apple juice, milk, and ground beef by flow cytometry. Cytometry A 54:27–35
Acknowledgments
This study was supported by grants from the Federal Ministry of Education and Research (BMBF), support code 01KI1003E. Sandra Weirich was the recipient of a graduate scholarship from Justus Liebig University Giessen during this research.
Conflict of Interest
ÖA declares that he has no conflict of interest. SW declares that he has no conflict of interest. AA declares that he has no conflict of interest. KF declares that he has no conflict of interest. MB declares that he has no conflict of interest. This article does not contain any studies with human or animal subjects.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Akineden, Ö., Weirich, S., Abdulmawjood, A. et al. Application of a Fluorescence Microscopy Technique for Detecting Viable Mycobacterium avium ssp. paratuberculosis Cells in Milk. Food Anal. Methods 8, 499–506 (2015). https://doi.org/10.1007/s12161-014-9918-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12161-014-9918-3