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.     

Effect of chemical decontamination and refrigerated storage on the isolation of Mycobacterium avium subsp. paratuberculosis from heat-treated milk

AIMS: To assess the impact of chemical decontamination and refrigerated storage before culture on the recovery of Mycobacterium avium subsp. paratuberculosis from heat-treated milk. METHODS AND RESULTS: Five-millilitre samples of ultra heat-treated (UHT) milk spiked with Myco. paratuberculosis NCTC 8578, B4 or 806R (ca 10(6) CFU ml(-1)) were heated at 63 degrees C for 20 or 30 min by submersion in a water bath. Heat-treated milk (0.5 ml) was cultured immediately into BACTEC 12B medium or refrigerated at 4 degrees C for 48 h before culture. Milk samples that received a 20-min heat treatment were also subjected to decontamination with 0.75% cetylpyridinium chloride (CPC) for 5 h at room temperature before inoculation into BACTEC 12B medium when tested immediately and after 48 h at 4 degrees C. BACTEC vials were monitored for evidence of growth over an 18-week incubation period at 37 degrees C. CPC decontamination resulted in a significant reduction in the number of culture-positive milk samples recovered immediately after heating (P < 0.05) and after refrigerated storage for 48 h (P < 0.01). Refrigerated storage for 48 h before testing did not have any significant effect, beneficial or detrimental, on Myco. paratuberculosis recovery rates. CONCLUSIONS: CPC decontamination applied to milk immediately or 48 h after heating will adversely affect the recovery of viable Myco. paratuberculosis, possibly leading to nonrecovery of the organism although viable cells are present in the original milk sample. SIGNIFICANCE AND IMPACT OF THE STUDY: Published pasteurization studies in which milk samples were decontaminated before culture will have underestimated the survival capability of Myco. paratuberculosis after high-temperature, short-time pasteurization. CPC decontamination should not be applied to pasteurized milk in future studies.     

Evaluation of culture media for the recovery of Mycobacterium avium subsp. paratuberculosis from Cheddar cheese

AIMS: The study evaluated the efficacy of four Mycobacterium avium subsp. paratuberculosis (MAP) culture media in suppressing commonly used starter cultures and typical nonstarter microflora present during the manufacture and ripening of Cheddar cheese, with a view to identify a suitable medium for the enumeration of MAP during laboratory-scale Cheddar production. METHODS AND RESULTS: Four Cheddar starter cultures and Cheddar cheese manufactured with these starters were inoculated onto Herrold's egg yolk medium (HEYM); HEYM supplemented with vancomycin, amphotericin B and nalidixic acid (HEYM/VAN); Middlebrook 7H10 agar containing polymyxin, amphotericin B, nalidixic acid, trimethoprim and azlocillin (PANTA) antibiotic supplement; and BACTEC 12B radiometric medium with and without a preliminary decontamination step (0.75% w/v hexadecylpyridinium chloride (HPC), 5 h). The inclusion of a decontamination step inhibited all Cheddar cheese starter and nonstarter micro-organisms. The medium 7H10/PANTA and to a lesser extent HEYM/VAN were effective inhibitors of cheese microflora when no decontamination step was employed. CONCLUSIONS: Middlebrook 7H10 medium, supplemented with PANTA antibiotics, suppressed all micro-organisms associated with ripening Cheddar cheese manufactured with pasteurized milk. SIGNIFICANCE AND IMPACT OF THE STUDY: A MAP culture medium has been identified, which may be used to enumerate this bacterium during the laboratory manufacture and ripening of Cheddar cheese and hence facilitate further research into the persistence of this pathogen in the product.     

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.     

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.     

Persistence of Mycobacterium paratuberculosis during manufacture and ripening of cheddar cheese

Model Cheddar cheeses were prepared from pasteurized milk artificially contaminated with high 10(4) to 10(5) CFU/ml) and low (10(1) to 10(2) CFU/ml) inocula of three different Mycobacterium paratuberculosis strains. A reference strain, NCTC 8578, and two strains (806PSS and 796PSS) previously isolated from pasteurized milk for retail sale were investigated in this study. The manufactured Cheddar cheeses were similar in pH, salt, moisture, and fat composition to commercial Cheddar. The survival of M. paratuberculosis cells was monitored over a 27-week ripening period by plating homogenized cheese samples onto HEYM agar medium supplemented with the antibiotics vancomycin, amphotericin B, and nalidixic acid without a decontamination step. A concentration effect was observed in M. paratuberculosis numbers between the inoculated milk and the 1-day old cheeses for each strain. For all manufactured cheeses, a slow gradual decrease in M. paratuberculosis CFU in cheese was observed over the ripening period. In all cases where high levels (>3.6 log(10)) of M. paratuberculosis were present in 1-day cheeses, the organism was culturable after the 27-week ripening period. The D values calculated for strains 806PSS, 796PSS, and NCTC 8578 were 107, 96, and 90 days, respectively. At low levels of contamination, M. paratuberculosis was only culturable from 27-week-old cheese spiked with strain 806PSS. M. paratuberculosis was recovered from the whey fraction in 10 of the 12 manufactured cheeses. Up to 4% of the initial M. paratuberculosis load was recovered in the culture-positive whey fractions at either the high or low initial inoculum.     

Improved template DNA preparation procedure for detection of Mycobacterium avium subsp. paratuberculosis in milk by PCR

Factors affecting the detection of Mycobacterium avium subsp. paratuberculosis (MAP) by PCR in raw milk and their interactions were investigated. Three day old bulk tank raw milk (50 ml) samples were seeded with MAP at a level of an estimated 30 CFU/ml. Heat-treatment of raw milk before centrifugation significantly affected the partitioning of MAP in the cream, whey and pellet fractions. Based on the IS900 PCR results, MAP preferentially partitioned into the cream fraction in unheated raw milk, and into the pellet fraction in the heat-treated milk. Treatment with 0.75% hexadecylpyridinium chloride (HPC) helped collect MAP in cream fraction. Heat treatment, use of pooled cream and pellet fractions and treatment with HPC improved the detection by PCR significantly, while washing of pellets prior to DNA extraction did not. The limit of detection using our optimized procedure was an estimated 15-50 CFU in 50 ml, or 相似文献   

Bactericidal effect of chlorine on Mycobacterium paratuberculosis in drinking water

AIMS: One possible route of transmission of Mycobacterium paratuberculosis from cattle to humans is via contaminated water supplies. The aim of this work was to determine whether this organism can survive standard water treatment processes. METHODS AND RESULTS: Two strains of M. paratuberculosis (bovine strain, NCTC 8578 and human strain Linda, ATCC 43015) were subjected to various chlorine concentrations (0.5, 1.0 and 2.0 microg ml(-1)) for 15 and 30 min. Chlorine test solutions were made up in two types of water, sterile water that had been deionized and subjected to reverse osmosis (DRO) and DRO water containing MgCl(2), CaCl(2), NaHCO(3) and bovine serum albumin (0.3% w/v), the latter to mimic conditions the organism would experience in commercial water treatment operations. CONCLUSION: The data showed that when initial inoculum levels were high (10(6) cfu ml(-1)) neither M. paratuberculosis strain was completely killed at the free chlorine concentrations and contact times applied. Log10 reductions in the range 1.32-2.82 were observed. The greatest log(10) reduction in cell numbers (2.82 and 2.35 for the bovine and human strains, respectively) was observed at the highest chlorine concentration (2 microg ml(-1)) and longest contact time (30 min). SIGNIFICANCE AND IMPACT OF THE STUDY: This work highlights the need for further research into the survival of M. paratuberculosis during water treatment.     

Surveillance of bulk raw and commercially pasteurized cows' milk from approved Irish liquid-milk pasteurization plants to determine the incidence of Mycobacterium paratuberculosis

Over the 13-month period from October 2000 to November 2001 (inclusive), the Food Safety Authority of Ireland (FSAI) carried out surveillance of Irish bulk raw (n = 389) and commercially pasteurized (n = 357) liquid-milk supplies to determine the incidence of Mycobacterium paratuberculosis. The pasteurization time-temperature conditions were recorded for all pasteurized samples. Overall, 56% of whole-milk pasteurized samples had been heat treated at or above a time-temperature combination of 75 degrees C for 25 s. All analyses were undertaken at the Department of Food Science (Food Microbiology) laboratory at Queen's University Belfast. Each milk sample was subjected to two tests for M. paratuberculosis: immunomagnetic separation-PCR (IMS-PCR; to detect the presence of M. paratuberculosis cells, live or dead) and chemical decontamination and culture (to confirm the presence of viable M. paratuberculosis). Overall, M. paratuberculosis DNA was detected by IMS-PCR in 50 (12.9%; 95% confidence interval, 9.9 to 16.5%) raw-milk samples and 35 (9.8%; 95% confidence interval, 7.1 to 13.3%) pasteurized-milk samples. Confirmed M. paratuberculosis was cultured from one raw-milk sample and no pasteurized-milk samples. It is concluded that M. paratuberculosis DNA is occasionally present at low levels in both raw and commercially pasteurized cows' milk. However, since no viable M. paratuberculosis was isolated from commercially pasteurized cows' milk on retail sale in the Republic of Ireland, current pasteurization procedures are considered to be effective.     

Incidence of Mycobacterium paratuberculosis in bulk raw and commercially pasteurized cows' milk from approved dairy processing establishments in the United Kingdom

Over a 17-month period (March 1999 to July 2000), a total of 814 cows' milk samples, 244 bulk raw and 567 commercially pasteurized (228 whole, 179 semi-skim, and 160 skim), from 241 approved dairy processing establishments throughout the United Kingdom were tested for the presence of Mycobacterium paratuberculosis by immunomagnetic PCR (to detect all cells living and dead) and culture (to detect viable cells). Overall, M. paratuberculosis DNA was detected by immunomagnetic PCR in 19 (7.8%; 95% confidence interval, 4.3 to 10.8%) and 67 (11.8%; 95% confidence interval, 9.0 to 14.2%) of the raw and pasteurized milk samples, respectively. Confirmed M. paratuberculosis isolates were cultured from 4 (1.6%; 95% confidence interval, 0.04 to 3.1%) and 10 (1.8%; 95% confidence interval, 0.7 to 2.8%) of the raw and pasteurized milk samples, respectively, following chemical decontamination with 0.75% (wt/vol) cetylpyridinium chloride for 5 h. The 10 culture-positive pasteurized milk samples were from just 8 (3.3%) of the 241 dairy processing establishments that participated in the survey. Seven of the culture-positive pasteurized milk samples had been heat treated at 72 to 74 degrees C for 15 s; the remainder had been treated at 72 to 75 degrees C for the extended holding time of 25 s. When typed by restriction fragment length polymorphism and pulsed-field gel electrophoresis methods, some of the milk isolates were shown to be types distinct from those of laboratory strains in regular use within the testing laboratory. From information gathered at the time of milk sample collection, all indications were that pasteurization had been carried out effectively at all of the culture-positive dairies. That is, pasteurization time and temperature conditions complied with the legal minimum high-temperature, short-time process; all pasteurized milk samples tested phosphatase negative; and post-process contamination was considered unlikely to have occurred. It was concluded that viable M. paratuberculosis is occasionally present at low levels in commercially pasteurized cows' milk in the United Kingdom.     

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 10(1) to 10(5) 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.     

IS900 PCR to detect Mycobacterium paratuberculosis in retail supplies of whole pasteurized cows' milk in England and Wales.

IS900 PCR for Mycobacterium paratuberculosis was applied to cream, whey, and pellet fractions of centrifuged whole cows' milk. The test and simultaneous control reactions gave correct results for spiked milk and for native milk samples obtained directly from M. paratuberculosis-free, subclinically infected, and clinically infected cows. The test was then applied to units of whole pasteurized cows' milk widely obtained from retail outlets throughout central and southern England from September 1991 to March 1993. With peak periods in January to March and in September to November, when up to 25% of units were affected, an overall 22 of 312 samples (7%) tested positive for M. paratuberculosis. In 18 of the 22 positive samples (81%), the PCR signal segregated to the cream or pellet fractions or both, consistent with the presence of intact mycobacteria. Nine of 18 PCR-positive milk samples (50%) and 6 of 36 PCR-negative milk samples (16%) yielded long-term liquid cultures which tested positive for M. paratuberculosis after 13 to 40 months of incubation, despite overgrowth by other organisms. Taken together with data on the prevalence of M. paratuberculosis infection in herds in the United Kingdom, the known secretion of M. paratuberculosis in milk from subclinically infected animals, and the inability of laboratory conditions simulating pasteurization to ensure the killing of all these slowly growing or unculturable organisms, there is a high risk, particularly at peak times, that residual M. paratuberculosis will be present in retail pasteurized cows' milk in England.     

Effect of higher pasteurization temperatures, and longer holding times at 72°C, on the inactivation of Mycobacterium paratuberculosis in milk

Raw cow's milk spiked with 10(6) cfu ml-1 of Mycobacterium paratuberculosis was subjected to heat treatments of 72, 75, 78, 80, 85 or 90 degrees C for 15 s, and 72 degrees C for 20 and 25 s, using laboratory pasteurizing units. Three bovine strains of Myco. paratuberculosis were studied (NCTC 8578, B2 and DVL 943). Each strain was subjected to all the heat treatments indicated on three separate occasions. Although each of the heat treatments achieved a substantial (5-6 log10) reduction in numbers of viable Myco. paratuberculosis, small numbers of the organism (4-16 cfu 10 ml-1) survived in a proportion of the milk samples at each of the higher temperatures investigated, right up to 90 degrees C for 15 s. A longer holding time of 25 s at 72 degrees C was found to be more effective at inactivating Myco. paratuberculosis. Only one of the three strains studied, B2, yielded small numbers of survivors after heating at 72 degrees C for 20 s, but it was completely inactivated by extending the holding time at 72 degrees C by a further 5 s to 25 s. It was concluded that a longer holding time is more likely to achieve the complete inactivation of Myco. paratuberculosis in milk than a higher pasteurization temperature.     

Development of an IMS-PCR assay for the detection of Mycobacterium avium ssp. paratuberculosis in water

AIMS: To develop a sensitive detection method for Mycobacterium avium ssp. paratuberculosis (Map) in water by modifying and optimizing an existing immunomagnetic separation polymerase chain reaction (IMS-PCR) technique. METHODS AND RESULTS: Sterile distilled water (50 ml) spiked with 10(6) Map ml(-1) was subjected to either filtration (0.45 microm pore size) followed directly by IS900 PCR (method 1) or centrifugation (2500 g for 20 min) followed by IMS and IS900 PCR (method 2). Method 2 permitted the detection of Map, whereas method 1 did not. Method 2 was then optimized by adding different concentrations of Tween 80 (0.05, 0.1, 0.2, 0.4 and 0.6% v/v) to water samples spiked with Map (10(6)-1 CFU ml(-1)) prior to centrifugation, and assessing the impact of this action on the detection sensitivity of subsequent IMS-PCR. The optimum Tween 80 concentration was found to be 0.4%, which permitted the detection of 10 Map CFU ml(-1) in spiked water samples by IMS-PCR. CONCLUSIONS: This method will be used to determine the incidence of Map in water destined for domestic use in future studies. SIGNIFICANCE AND IMPACT OF THE STUDY: A sensitive method for the detection of Map in water involving addition of 0.4% Tween 80, centrifugation and IMS-PCR was developed.     

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.     

Rapid real-time PCR assay for detection and quantitation of Mycobacterium avium subsp. paratuberculosis DNA in artificially contaminated milk

Using fluorescence resonance energy transfer technology and Lightcycler analysis, we developed a real-time PCR assay with primers and probes designed by using IS900 which allowed rapid detection of Mycobacterium avium subsp. paratuberculosis DNA in artificially contaminated milk. Initially, the PCR parameters (including primer and probe levels, assay volume, Mg(2+) concentration, and annealing temperature) were optimized. Subsequently, the quantitative ability of the assay was tested and was found to be accurate over a broad linear range (3 x 10(6) to 3 x 10(1) copies). The assay sensitivity when purified DNA was used was determined to be as low as five copies, with excellent reproducibility. A range of DNA isolation strategies was developed for isolating M. avium subsp. paratuberculosis DNA from spiked milk, the most effective of which involved the use of 50 mM Tris HCl, 10 mM EDTA, 2% Triton X-100, 4 M guanidinium isothiocyante, and 0.3 M sodium acetate combined with boiling, physical grinding, and nucleic acid spin columns. When this technique was used in conjunction with the real-time PCR assay, it was possible to consistently detect <100 organisms per ml of milk (equivalent to 2,000 organisms per 25 ml). Furthermore, the entire procedure (extraction and PCR) was performed in less than 3 h and was successfully adapted to quantify M. avium subsp. paratuberculosis in spiked milk from heavily and mildly contaminated samples.     

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.     

Comparison of the sensitivity of manual and automated immunomagnetic separation methods for detection of Shiga toxin-producing Escherichia coli O157:H7 in milk

AIM: To determine the sensitivity of methods for detection of injured and uninjured Escherichia coli O157:H7 (E. coli O157) in raw and pasteurized milk. METHODS AND RESULTS: Raw milk, pasteurized milk with 1.5% fat content and pasteurized milk with 3.5% fat content were spiked with E. coli O157 at low levels. The samples were enriched in modified tryptone soya broth with novobiocin (mTSBn) at 37 degrees C. Aliquots of the enriched culture were analysed either by manual immunomagnetic separation (MIMS) and culturing on sorbitol MacConkey agar with or without cefixime and potassium tellurite (SMACct or SMAC), or by automated immunomagnetic separation and integrated ELISA (EiaFosstrade mark). Uninjured E. coli O157 organisms were detected in milk by both methods at 1 cfu 10 ml-1 sample). Injured organisms were detected at levels of about 4 cfu 10 ml-1 sample. Direct enrichment in mTSBn (22 h incubation) showed better sensitivity for injured cells than enrichment in buffered peptone water (BPW, 22 h incubation), or in a two-step enrichment consisting of BPW (6 h, 37 degrees C) and mTSBn (16 h, 37 degrees C), successively. CONCLUSIONS: The methods showed equal sensitivity in that they were both able to detect 1 cfu 10 ml-1 milk sample. Injured organisms can be detected and isolated at a level almost as low as this. A resuscitation step is not recommended for the detection and isolation of injured and non-injured E. coli O157 from milk. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to the dilution of contamination in the bulk tank, analysis of milk for the presence of E. coli O157 requires a very sensitive method. Both methods described here are useful for such analysis.     

The use of spiral plating and microscopic colony counting for the rapid quantitation of Mycobacterium paratuberculosis

AIMS: To evaluate a spiral plating and microscopic colony counting technique to hasten the quantitation of Mycobacterium paratuberculosis. METHODS AND RESULTS: Broth and milk cultures of M. paratuberculosis were spirally plated onto Middlebrook agar plates and microscopically counted at 8 and 14 days of incubation. The same plates were recounted at 27-28 days of incubation when grossly visible colonies were present. The results were statistically compared with no difference in CFU ml-1 derived from the shorter vs longer incubation times. Other mycobacteria isolates were also plated and microscopically examined and found to be easily distinguishable from M. paratuberculosis. CONCLUSIONS: Microscopic quantitation of spirally plated M. paratuberculosis cultures can be achieved within 8-14 days of plate incubation and compare favourably to counts derived after prolonged incubations. SIGNIFICANCE AND IMPACT OF THE STUDY: The technique could greatly hasten the quantitation of viable M. paratuberculosis.     

An intra-laboratory cultural and real-time PCR method comparison and evaluation for the detection of subclinical paratuberculosis in dairy herds

Mycobacterium avium subsp. paratuberculosis (MAP) is a vigorous microorganism which causes incurable chronic enteritis, Johne’s disease (JD) in cattle. A target of control programmes for JD is to accurately detect MAP-infected cattle early to reduce disease transmission. The present study evaluated the efficacy of two different cultural procedures and a TaqMan real-time PCR assay for detection of subclinical paratuberculosis in dairy herds. Therefore, sixty-one faecal samples were collected from two Dutch dairy herds (n = 40 and n = 21, respectively) which were known to be MAP-ELISA positive. All individual samples were assessed using two different cultural protocols in two different laboratories. The first cultural protocol (first laboratory) included a decontamination step with 0.75% hexadecylpyridinium chloride (HPC) followed by inoculation on Herrold’s egg yolk media (HEYM). The second protocol (second laboratory) comprised of a decontamination step using 4% NaOH and malachite green-oxalic acid followed by inoculation on two media, HEYM and in parallel on modified Löwenstein-Jensen media (mLJ). For the TaqMan real-time PCR assay, all faecal samples were tested in two different laboratories using TaqMan® MAP (Johne’s) reagents (Life Technologies). The cultural procedures revealed positive reactions in 1.64% of the samples for cultivation protocol 1 and 6.56 and 8.20% of the samples for cultivation protocol 2, respectively. The results of the TaqMan real-time PCR performed in two different laboratories yielded 13.11 and 19.76% positive reaction. The kappa test showed proportional agreement 0.54 between the mLJ media (second laboratory) and TaqMan® real-time PCR method (second laboratory). In conclusion, the TaqMan real-time PCR could be a strongly useful and efficient assay for the detection of subclinical paratuberculosis in dairy cattle leading to an improvement in the efficiency of MAP control strategies.