Efficacy of Various Pasteurization Time-Temperature Conditions in Combination with Homogenization on Inactivation of Mycobacterium avium subsp. paratuberculosis in Milk

The effect of various pasteurization time-temperature conditions with and without homogenization on the viability of Mycobacterium avium subsp. paratuberculosis was investigated using a pilot-scale commercial high-temperature, short-time (HTST) pasteurizer and raw milk spiked with 10¹ to 10⁵ M. avium subsp. paratuberculosis cells/ml. Viable M. avium subsp. paratuberculosis was cultured from 27 (3.3%) of 816 pasteurized milk samples overall, 5 on Herrold's egg yolk medium and 22 by BACTEC culture. Therefore, in 96.7% of samples, M. avium subsp. paratuberculosis had been completely inactivated by HTST pasteurization, alone or in combination with homogenization. Heat treatments incorporating homogenization at 2,500 lb/in², applied upstream (as a separate process) or in hold (at the start of a holding section), resulted in significantly fewer culture-positive samples than pasteurization treatments without homogenization (P < 0.001 for those in hold and P < 0.05 for those upstream). Where colony counts were obtained, the number of surviving M. avium subsp. paratuberculosis cells was estimated to be 10 to 20 CFU/150 ml, and the reduction in numbers achieved by HTST pasteurization with or without homogenization was estimated to be 4.0 to 5.2 log₁₀. The impact of homogenization on clump size distribution in M. avium subsp. paratuberculosis broth suspensions was subsequently assessed using a Mastersizer X spectrometer. These experiments demonstrated that large clumps of M. avium subsp. paratuberculosis cells were reduced to single-cell or “miniclump” status by homogenization at 2,500 lb/in². Consequently, when HTST pasteurization was being applied to homogenized milk, the M. avium subsp. paratuberculosis cells would have been present as predominantly declumped cells, which may possibly explain the greater inactivation achieved by the combination of pasteurization and homogenization.     

Effective heat inactivation of Mycobacterium avium subsp. paratuberculosis in raw milk contaminated with naturally infected feces

The effectiveness of high-temperature, short holding time (HTST) pasteurization and homogenization with respect to inactivation of Mycobacterium avium subsp. paratuberculosis was evaluated quantitatively. This allowed a detailed determination of inactivation kinetics. High concentrations of feces from cows with clinical symptoms of Johne's disease were used to contaminate raw milk in order to realistically mimic possible incidents most closely. Final M. avium subsp. paratuberculosis concentrations varying from 10(2) to 3.5 x 10(5) cells per ml raw milk were used. Heat treatments including industrial HTST were simulated on a pilot scale with 22 different time-temperature combinations, including 60 to 90 degrees C at holding (mean residence) times of 6 to 15 s. Following 72 degrees C and a holding time of 6 s, 70 degrees C for 10 and 15 s, or under more stringent conditions, no viable M. avium subsp. paratuberculosis cells were recovered, resulting in >4.2- to >7.1-fold reductions, depending on the original inoculum concentrations. Inactivation kinetic modeling of 69 quantitative data points yielded an E(a) of 305,635 J/mol and an lnk(0) of 107.2, corresponding to a D value of 1.2 s at 72 degrees C and a Z value of 7.7 degrees C. Homogenization did not significantly affect the inactivation. The conclusion can be drawn that HTST pasteurization conditions equal to 15 s at > or =72 degrees C result in a more-than-sevenfold reduction of M. avium subsp. paratuberculosis.     

Inactivation of Mycobacterium paratuberculosis in cows' milk at pasteurization temperatures.

The thermal inactivation of 11 strains of Mycobacterium paratuberculosis at pasteurization temperatures was investigated. Cows' milk inoculated with M. paratuberculosis at two levels (10(7) and 10(4) CFU/ml) was pasteurized in the laboratory by (i) a standard holder method (63.5 degrees C for 30 min) and (ii) a high-temperature, short-time (HTST) method (71.7 degrees C for 15 s). Additional heating times of 5, 10, 15, 20, and 40 min at 63.5 degrees C were included to enable the construction of a thermal death curve for the organism. Viability after pasteurization was assessed by culture on Herrold's egg yolk medium containing mycobactin J (HEYM) and in BACTEC Middlebrook 12B radiometric medium supplemented with mycobactin J and sterile egg yolk emulsion. Confirmation of acid-fast survivors of pasteurization as viable M. paratuberculosis cells was achieved by subculture on HEYM to indicate viability coupled with PCR using M. paratuberculosis-specific 1S900 primers. When milk was initially inoculated with 10(6) to 10(7) CFU of M. paratuberculosis per ml, M. paratuberculosis cells were isolated from 27 of 28 (96%) and 29 of 34 (85%) pasteurized milk samples heat treated by the holder and HTST methods, respectively. Correspondingly, when 10(3) to 10(4) CFU of M. paratuberculosis per ml of milk were present before heat treatment, M. paratuberculosis cells were isolated from 14 of 28 (50%) and 19 of 33 (58%) pasteurized milk samples heat treated by the holder and HTST methods, respectively. The thermal death curve for M. paratuberculosis was concave in shape, exhibiting a rapid initial death rate followed by significant "tailing." Results indicate that when large numbers of M. paratuberculosis cells are present in milk, the organism may not be completely inactivated by heat treatments simulating holder and HTST pasteurization under laboratory conditions.     

Effect of commercial-scale high-temperature, short-time pasteurization on the viability of Mycobacterium paratuberculosis in naturally infected cows' milk

Raw cows' milk naturally infected with Mycobacterium paratuberculosis was pasteurized with an APV HXP commercial-scale pasteurizer (capacity 2,000 liters/h) on 12 separate occasions. On each processing occasion, milk was subjected to four different pasteurization treatments, viz., 73 degrees C for 15 s or 25 s with and without prior homogenization (2,500 lb/in(2) in two stages), in an APV Manton Gaulin KF6 homogenizer. Raw and pasteurized milk samples were tested for M. paratuberculosis by immunomagnetic separation (IMS)-PCR (to detect the presence of bacteria) and culture after decontamination with 0.75% (wt/vol) cetylpyridinium chloride for 5 h (to confirm bacterial viability). On 10 of the 12 processing occasions, M. paratuberculosis was detectable by IMS-PCR, culture, or both in either raw or pasteurized milk. Overall, viable M. paratuberculosis was cultured from 4 (6.7%) of 60 raw and 10 (6.9%) of 144 pasteurized milk samples. On one processing day, in particular, M. paratuberculosis appeared to have been present in greater abundance in the source raw milk (evidenced by more culture positives and stronger PCR signals), and on this occasion, surviving M. paratuberculosis bacteria were isolated from milk processed by all four heat treatments, i.e., 73 degrees C for 15 and 25 s with and without prior homogenization. On one other occasion, surviving M. paratuberculosis bacteria were isolated from an unhomogenized milk sample that had been heat treated at 73 degrees C for 25 s. Results suggested that homogenization increases the lethality of subsequent heat treatment to some extent with respect to M. paratuberculosis, but the extended 25-s holding time at 73 degrees C was found to be no more effective at killing M. paratuberculosis than the standard 15-s holding time. This study provides clear evidence that M. paratuberculosis bacteria in naturally infected milk are capable of surviving commercial high-temperature, short-time pasteurization if they are present in raw milk in sufficient numbers.     

Development of an F57 sequence-based real-time PCR assay for detection of Mycobacterium avium subsp. paratuberculosis in milk

A light cycler-based real-time PCR (LC-PCR) assay that amplifies the F57 sequence of Mycobacterium avium subsp. paratuberculosis was developed. This assay also includes an internal amplification control template to monitor the amplification conditions in each reaction. The targeted F57 sequence element is unique for M.avium subsp. paratuberculosis and is not known to exist in any other bacterial species. The assay specificity was demonstrated by evaluation of 10 known M. avium subsp. paratuberculosis isolates and 33 other bacterial strains. The LC-PCR assay has a broad linear range (2 x 10(1) to 2 x10(6) copies) for quantitative estimation of the number of M. avium subsp. paratuberculosis F57 target copies in positive samples. To maximize the assay's detection sensitivity, an efficient strategy for isolation of M. avium subsp. paratuberculosis DNA from spiked milk samples was also developed. The integrated procedure combining optimal M. avium subsp. paratuberculosis DNA isolation and real-time PCR detection had a reproducible detection limit of about 10 M. avium subsp. paratuberculosis cells per ml when a starting sample volume of 10 ml of M. avium subsp. paratuberculosis-spiked milk was analyzed. The entire process can be completed within a single working day and is suitable for routine monitoring of milk samples for M. avium subsp. paratuberculosis contamination. The applicability of this protocol for naturally contaminated milk was also demonstrated using milk samples from symptomatic M. avium subsp. paratuberculosis-infected cows, as well as pooled samples from a dairy herd with a confirmed history of paratuberculosis.     

Mycobacterium avium subspecies paratuberculosis cultured from locally and commercially pasteurized cow's milk in the Czech Republic

Between November 2002 and April 2003, 244 bottles and cartons of commercially pasteurized cow's milk were obtained at random from retail outlets throughout the Czech Republic. During the same period, samples of raw milk and of milk that was subsequently subjected to a minimum of 71.7 degrees C for 15 s in a local pasteurization unit were also obtained from two dairy herds, designated herds A and B, with low and high levels, respectively, of subclinical Mycobacterium avium subsp. paratuberculosis infection, and from one herd, herd C, without infection. Infection in individual cows in each herd was tested by fecal culturing. Milk samples were brought to the Veterinary Research Institute in Brno, Czech Republic, processed, inoculated onto Herrold's egg yolk slants, and incubated for 32 weeks. Colonies were characterized by morphology, Ziehl-Neelsen staining, mycobactin J dependency, and IS900 PCR results. M. avium subsp. paratuberculosis was cultured from 4 of 244 units (1.6%) of commercially pasteurized retail milk. M. avium subsp. paratuberculosis was also cultured from 2 of 100 (2%) cartons of locally pasteurized milk derived from infected herds A and B and from 0 of 100 cartons of milk from uninfected herd C. Raw milk from 1 of 10 (10%) fecal culture-positive cows in herd A and from 13 of 66 (19.7%) fecal culture-positive cows in herd B was culture positive for M. avium subsp. paratuberculosis. These findings confirm that M. avium subsp. paratuberculosis is present in raw milk from subclinically infected dairy cows. The culture of M. avium subsp. paratuberculosis in the Czech Republic from retail milk that had been pasteurized locally or commercially to the required national and European Union standards is in agreement with similar research on milk destined for consumers in the United Kingdom and the United States and shows that humans are being exposed to this chronic enteric pathogen by this route.     

Fate of Mycobacterium avium subsp. paratuberculosis in Swiss hard and semihard cheese manufactured from raw milk

Raw milk was artificially contaminated with declumped cells of Mycobacterium avium subsp. paratuberculosis at a concentration of 10(4) to 10(5) CFU/ml and was used to manufacture model hard (Swiss Emmentaler) and semihard (Swiss Tisliter) cheese. Two different strains of M. avium subsp. paratuberculosis were tested, and for each strain, two model hard and semihard cheeses were produced. The survival of M. avium subsp. paratuberculosis cells was monitored over a ripening period of 120 days by plating out homogenized cheese samples onto 7H10-PANTA agar. In both the hard and the semihard cheeses, counts decreased steadily but slowly during cheese ripening. Nevertheless, viable cells could still be detected in 120-day cheese. D values were calculated at 27.8 days for hard and 45.5 days for semihard cheese. The most important factors responsible for the death of M. avium subsp. paratuberculosis in cheese were the temperatures applied during cheese manufacture and the low pH at the early stages of cheese ripening. Since the ripening period for these raw milk cheeses lasts at least 90 to 120 days, the D values found indicate that 10(3) to 10(4) cells of M. avium subsp. paratuberculosis per g will be inactivated.     

Peptide aMptD-mediated capture PCR for detection of Mycobacterium avium subsp. paratuberculosis in bulk milk samples

A peptide-mediated capture PCR for the detection of Mycobacterium avium subsp. paratuberculosis in bulk milk samples was developed and characterized. Capture of the organism was performed using peptide aMptD, which had been shown to bind to the M. avium subsp. paratuberculosis MptD protein (J. Stratmann, B. Strommenger, R. Goethe, K. Dohmann, G. F. Gerlach, K. Stevenson, L. L. Li, Q. Zhang, V. Kapur, and T. J. Bull, Infect. Immun. 72:1265-1274, 2004). Consistent expression of the MptD receptor protein and binding of the aMptD ligand were demonstrated by capturing different Mycobacterium avium subsp. paratuberculosis type I and type II strains and subsequent PCR analysis using ISMav2-based primers. The analytical sensitivity of the method was determined to be 5 x 10(2) CFU ml(-1) for artificially contaminated milk. The specificity of aMptD binding was confirmed by culture and competitive capture assays, showing selective enrichment of M. avium subsp. paratuberculosis (at a concentration of 5 x 10(2) CFU ml(-1)) from samples containing 100- and 1,000-fold excesses of other mycobacterial species, including M. avium subsp. avium and M. avium subsp. hominissuis. The aMptD-mediated capture of M. avium subsp. paratuberculosis using paramagnetic beads, followed by culture, demonstrated the ability of this approach to capture viable target cells present in artificially contaminated milk. Surface plasmon resonance experiments revealed that the aMptD peptide is a high-affinity ligand with a calculated association rate constant of 9.28 x 10(3) and an association constant of 1.33 x 10(9). The potential use of the method on untreated raw milk in the field was investigated by testing 423 bulk milk samples obtained from different dairy farms in Germany, 23 of which tested positive. Taken together, the results imply that the peptide-mediated capture PCR might present a suitable test for paratuberculosis screening of dairy herds, as it has an analytical sensitivity sufficient for detection of M. avium subsp. paratuberculosis in bulk milk samples under field conditions, relies on a defined and validated ligand-receptor interaction, and is adaptable to routine diagnostic laboratory automation.     

Heat inactivation of Mycobacterium avium subsp. paratuberculosis in milk

The effectiveness of pasteurization and the concentration of Mycobacterium avium subsp. paratuberculosis in raw milk have been identified in quantitative risk analysis as the most critical factors influencing the potential presence of viable Mycobacterium paratuberculosis in dairy products. A quantitative assessment of the lethality of pasteurization was undertaken using an industrial pasteurizer designed for research purposes with a validated Reynolds number of 62,112 and flow rates of 3,000 liters/h. M. paratuberculosis was artificially added to raw whole milk, which was then homogenized, pasteurized, and cultured, using a sensitive technique capable of detecting one organism per 10 ml of milk. Twenty batches of milk containing 10(3) to 10(4) organisms/ml were processed with combinations of three temperatures of 72, 75, and 78 degrees C and three time intervals of 15, 20, and 25 s. Thirty 50-ml milk samples from each processed batch were cultured, and the logarithmic reduction in M. paratuberculosis organisms was determined. In 17 of the 20 runs, no viable M. paratuberculosis organisms were detected, which represented > 6-log10 reductions during pasteurization. These experiments were conducted with very heavily artificially contaminated milk to facilitate the measurement of the logarithmic reduction. In three of the 20 runs of milk, pasteurized at 72 degrees C for 15 s, 75 degrees C for 25 s, and 78 degrees C for 15 s, a few viable organisms (0.002 to 0.004 CFU/ml) were detected. Pasteurization at all temperatures and holding times was found to be very effective in killing M. paratuberculosis, resulting in a reduction of > 6 log10 in 85% of runs and > 4 log10 in 14% of runs.     

Development of a new, combined rapid method using phage and PCR for detection and identification of viable Mycobacterium paratuberculosis bacteria within 48 hours

The FASTPlaqueTB assay is an established diagnostic aid for the rapid detection of Mycobacterium tuberculosis from human sputum samples. Using the FASTPlaqueTB assay reagents, viable Mycobacterium avium subsp. paratuberculosis cells were detected as phage plaques in just 24 h. The bacteriophage used does not infect M. avium subsp. paratuberculosis alone, so to add specificity to this assay, a PCR-based identification method was introduced to amplify M. avium subsp. paratuberculosis-specific sequences from the DNA of the mycobacterial cell detected by the phage. To give further diagnostic information, a multiplex PCR method was developed to allow simultaneous amplification of either M. avium subsp. paratuberculosis or M. tuberculosis complex-specific sequences from plaque samples. Combining the plaque PCR technique with the phage-based detection assay allowed the rapid and specific detection of viable M. avium subsp. paratuberculosis in milk samples in just 48 h.     

Effect of three factors in cheese production (pH, salt, and heat) on Mycobacterium avium subsp. paratuberculosis viability

Low pH and salt are two factors contributing to the inactivation of bacterial pathogens during a 60-day curing period for cheese. The kinetics of inactivation for Mycobacterium avium subsp. paratuberculosis strains ATCC 19698 and Dominic were measured at 20 degrees C under different pH and NaCl conditions commonly used in processing cheese. The corresponding D values (decimal reduction times; the time required to kill 1 log(10) concentration of bacteria) were measured. Also measured were the D values for heat-treated and nonheated M. avium subsp. paratuberculosis in 50 mM acetate buffer (pH 5.0, 2% [wt/vol] NaCl) and a soft white Hispanic-style cheese (pH 6.0, 2% [wt/vol] NaCl). Samples were removed at various intervals until no viable cells were detected using the radiometric culture method (BACTEC) for enumeration of M. avium subsp. paratuberculosis. NaCl had little or no effect on the inactivation of M. avium subsp. paratuberculosis, and increasing NaCl concentrations were not associated with decreasing D values (faster killing) in the acetate buffer. Lower pHs, however, were significantly correlated with decreasing D values of M. avium subsp. paratuberculosis in the acetate buffer. The D values for heat-treated M. avium subsp. paratuberculosis ATCC 19698 in the cheese were higher than those predicted by studies done in acetate buffer. The heat-treated M. avium subsp. paratuberculosis strains had lower D values than the nonheated cells (faster killing) both in the acetate buffer (pH 5, 2% [wt/vol] NaCl) and in the soft white cheese. The D value for heat-treated M. avium subsp. paratuberculosis ATCC 19698 in the cheese (36.5 days) suggests that heat treatment of raw milk coupled with a 60-day curing period will inactivate about 10(3) cells of M. avium subsp. paratuberculosis per ml.     

Culture- and Quantitative IS900 Real-Time PCR-Based Analysis of the Persistence of Mycobacterium avium subsp. paratuberculosis in a Controlled Dairy Cow Farm Environment

The aim of this study was to monitor the persistence of Mycobacterium avium subsp. paratuberculosis in environmental samples taken from a Holstein farm with a long history of clinical paratuberculosis. A herd of 606 head was eradicated, and mechanical cleaning and disinfection with chloramine B with ammonium (4%) was carried out on the farm; in the surrounding areas (on the field and field midden) lime was applied. Environmental samples were collected before and over a period of 24 months after destocking. Only one sample out of 48 (2%) examined on the farm (originating from a waste pit and collected before destocking) was positive for M. avium subsp. paratuberculosis by cultivation on solid medium (Herrold's egg yolk medium). The results using real-time quantitative PCR (qPCR) showed that a total of 81% of environmental samples with an average mean M. avium subsp. paratuberculosis cell number of 3.09 × 10(3) were positive for M. avium subsp. paratuberculosis before destocking compared to 43% with an average mean M. avium subsp. paratuberculosis cell number of 5.86 × 10(2) after 24 months. M. avium subsp. paratuberculosis-positive samples were detected in the cattle barn as well as in the calf barn and surrounding areas. M. avium subsp. paratuberculosis was detected from different matrices: floor and instrument scrapings, sediment, or scraping from watering troughs, waste pits, and cobwebs. M. avium subsp. paratuberculosis DNA was also detected in soil and plants collected on the field midden and the field 24 months after destocking. Although the proportion of positive samples decreased from 64% to 23% over time, the numbers of M. avium subsp. paratuberculosis cells were comparable.     

UV light inactivation of Mycobacterium avium subsp. paratuberculosis in milk as assessed by FASTPlaqueTB phage assay and culture

UV light inactivation of Mycobacterium avium subsp. paratuberculosis in Middlebrook 7H9 broth and whole and semiskim milk was investigated using a laboratory-scale UV machine that incorporated static mixers within UV-penetrable pipes. UV treatment proved to be less effective in killing M. avium subsp. paratuberculosis suspended in milk (0.5- to 1.0-log(10) reduction per 1,000 mJ/ml) than that suspended in Middlebrook 7H9 broth (2.5- to 3.3-log(10) reduction per 1,000 mJ/ml). The FASTPlaqueTB phage assay provided more rapid enumeration of surviving M. avium subsp. paratuberculosis (within 24 h) than culture on Herrold's egg yolk medium (6 to 8 weeks). Despite the fact that plaque counts were consistently 1 to 2 log(10) lower than colony counts throughout the study, UV inactivation rates for M. avium subsp. paratuberculosis derived using the phage assay and culture results were not significantly different (P = 0.077).     

A molecular beacon-based real-time NASBA assay for detection of Mycobacterium avium subsp. paratuberculosis in water and milk

A molecular beacon-based real-time NASBA assay for detection and identification of Mycobacterium avium subsp. paratuberculosis has been developed. It targets and amplifies sequences from the dnaA gene which are specific for this bacterium. The assay includes an internal amplification control, to allow identification of inhibited reactions. The assay was tested against 18 isolates of M. avium subsp. paratuberculosis, 17 other mycobacterial strains and 25 non-mycobacterial strains, and was fully selective in that it detected all the targets but none of the non-targets. The lowest number of cells which the assay can detect with 99% probability is 150-200 cells per reaction (as determined using pure culture suspensions). Using centrifugation and nucleic acid extraction as sample treatment, the assay was able to consistently detect 10(3) M. avium subsp. paratuberculosis cells in 20 ml artificially contaminated drinking water. With a simple detergent and enzymatic sample pretreatment before centrifugation and nucleic acid extraction, the assay was able to consistently detect 10(4) M. avium subsp. paratuberculosis cells in 20 ml artificially contaminated semi-skimmed milk. The assay will be a useful addition to the range of diagnostic tools available for the study of M. avium subsp. paratuberculosis.     

Comparative evaluation of the MGIT and BACTEC culture systems for the recovery of Mycobacterium avium subsp. paratuberculosis from milk

AIMS: To compare the detection capabilities of the non-radiometric MGIT (Mycobacteria Growth Indicator Tubes) and radiometric BACTEC 460TB culture systems (Becton Dickinson, Cowley, Oxford, UK) for recovering Mycobacterium avium subsp. paratuberculosis from milk. METHODS AND RESULTS: Ultra heat treated (UHT) milk samples spiked with different levels of M. paratuberculosis (10-107 cells ml-1) were inoculated into MGIT and BACTEC media (containing recommended supplements) with and without prior chemical decontamination of the milk samples with 0.75% (w/v) cetylpyridinium chloride for 5 h. Time for the detection of growth in days was recorded for each culture system, and a M. paratuberculosis count for each milk sample was calculated from BACTEC readings using a published formula. Correlation between MGIT and BACTEC detection times was 0.6983. Both culture systems were capable of detecting 10-100 M. paratuberculosis cells ml-1 in milk within 30-40 days when no decontamination treatment was applied, but only 102-103 cells ml-1 or greater when chemical decontamination was applied before culture. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF STUDY: The non-radiometric MGIT system could be substituted for the radiometric BACTEC system for the culture of M. paratuberculosis from milk without loss of detection sensitivity. Chemical decontamination before culture caused a significant reduction in numbers of viable M. paratuberculosis in all spiked milk samples resulting in decreased detection capability for both culture systems.     

Persistence of Mycobacterium avium subsp. paratuberculosis at a farm-scale biogas plant supplied with manure from paratuberculosis-affected dairy cattle

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.     

Effect of high-temperature, short-time (HTST) pasteurization on milk containing low numbers of Mycobacterium paratuberculosis

The efficacy of high-temperature, short-time (HTST) pasteurization (72 °C/15 s) when low numbers (≤ 10³ cfu ml ⁻¹ ) of Mycobacterium paratuberculosis are present in milk was investigated. Raw cows' milk spiked with Myco. paratuberculosis (10³ cfu ml⁻¹, 10² cfu ml⁻¹, 10 cfu ml⁻¹, and 10 cfu 50 ml⁻¹) was subjected to HTST pasteurization using laboratory pasteurizing units. Ten bovine strains of Myco. paratuberculosis were tested in triplicate. Culture in BACTEC Middlebrook 12B radiometric medium detected acid-fast survivors in 14·8% and 10% of HTST-pasteurized milk samples at the 10³ and 10² cfu ml⁻¹ inoculum levels, respectively, whereas conventional culture on Herrold's egg yolk medium containing mycobactin J detected acid-fast survivors in only 3·7% and 6·7% of the same milk samples. IS900-based PCR confirmed that these acid-fast survivors were Myco. paratuberculosis . No viable Myco. paratuberculosis were isolated from HTST-pasteurized milk initially containing either 10 cfu ml⁻¹ or 10 cfu 50 ml⁻¹.     

Inactivation of Mycobacterium avium subsp. paratuberculosis in cow's milk by means of high hydrostatic pressure at mild temperatures

Two strains of Mycobacterium avium subsp. paratuberculosis (3644/02 and ATCC 19698) were inoculated (approximately 6 log CFU/ml) into sterilized milk to evaluate inactivation by high hydrostatic pressure. Reductions of M. avium subsp. paratuberculosis increased with pressure level. Significant differences were also found between M. avium subsp. paratuberculosis strains and between the media used. Average reductions of 4 log CFU/ml after treatment with 500 MPa are comparable to those caused by thermal treatments.     

Inactivation of Mycobacterium paratuberculosis by pulsed electric fields

The influence of treatment temperature and pulsed electric fields (PEF) on the viability of Mycobacterium paratuberculosis cells suspended in 0.1% (wt/vol) peptone water and in sterilized cow's milk was assessed by direct viable counts and by transmission electron microscopy (TEM). PEF treatment at 50 degrees C (2,500 pulses at 30 kV/cm) reduced the level of viable M. paratuberculosis cells by approximately 5.3 and 5.9 log(10) CFU/ml in 0.1% peptone water and in cow's milk, respectively, while PEF treatment of M. paratuberculosis at lower temperatures resulted in less lethality. Heating alone at 50 degrees C for 25 min or at 72 degrees C for 25 s (extended high-temperature, short-time pasteurization) resulted in reductions of M. paratuberculosis of approximately 0.01 and 2.4 log(10) CFU/ml, respectively. TEM studies revealed that exposure to PEF treatment resulted in substantial damage at the cellular level to M. paratuberculosis.     

New triplex real-time PCR assay for detection of Mycobacterium avium subsp. paratuberculosis in bovine feces

In the present study, a robust TaqMan real-time PCR amplifying the F57 and the ISMav2 sequences of Mycobacterium avium subsp. paratuberculosis from bovine fecal samples was developed and validated. The validation was based on the recommendations of International Organization for Standardization protocols for PCR and real-time PCR methods. For specificity testing, 205 bacterial strains were selected, including 105 M. avium subsp. paratuberculosis strains of bovine, ovine, and human origin and 100 non-M. avium subsp. paratuberculosis strains. Diagnostic quality assurance was obtained by use of an internal amplification control. By investigating six TaqMan reagents from different suppliers, the 100% detection probability was assessed to be 0.1 picogram M. avium subsp. paratuberculosis DNA per PCR. The amplification efficiency was 98.2% for the single-copy gene F57 and 97.8% for the three-copy insertion sequence ISMav2. The analytical method was not limited due to instrument specificity. The triplex real-time PCR allowed the reliable detection of M. avium subsp. paratuberculosis DNA using the ABI Prism 7000 sequence detection system, and the LightCycler 1.0. TaqMan(mgb) and locked nucleic acid fluorogenic probes were suitable for fluorescent signal detection. To improve the detection of M. avium subsp. paratuberculosis from bovine fecal samples, a more efficient DNA extraction method was developed, which offers the potential for automated sample processing. The 70% limit of detection was assessed to be 10(2) CFU per gram of spiked bovine feces. Comparative analysis of 108 naturally contaminated samples of unknown M. avium subsp. paratuberculosis status resulted in a relative accuracy of 98.9% and a sensitivity of 94.4% for fecal samples containing <10 CFU/g feces compared to the traditional culture method.