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 10³ to 10⁴ organisms/ml were processed with combinations of three temperatures of 72, 75, and 78°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°C for 15 s, 75°C for 25 s, and 78°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.     

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.     

Thermal Tolerance of Mycobacterium paratuberculosis

D values (decimal reduction time; the time required to kill 1 log concentration of bacteria) were determined for both human and bovine strains (Dominic, Ben, BO45, and ATCC 19698) of Mycobacterium paratuberculosis in 50 mM lactate solution (pH 6.8) and in milk at four temperatures (62, 65, 68, and 71°C). Viable M. paratuberculosis organisms were quantified by a radiometric culture method (BACTEC). Thermal death curves for the M. paratuberculosis strains tested were generally linear, with R² of ≥0.90, but a few curves (R², 0.80 to 0.90) were better described by a quadratic equation. The human strains (Dominic and Ben) had similar D values in milk and in lactate solution. However, D values for the bovine strains (BO45 and ATCC 19698) were significantly different depending on the menstruum. D values for low-passage clinical strains (Dominic, Ben, and BO45) were lower than those of the high-passage laboratory strain (ATCC 19698). The D value based on pooled data for clinical strains of M. paratuberculosis in milk at 71°C (D_71°C) was 11.67 s. Pooled D_62°C, D_65°C, and D_68°C of clinical M. paratuberculosis strains in milk were 228.8, 47.8, and 21.8 s, respectively. The Z value (the temperature required for the decimal reduction time to traverse 1 log cycle) of clinical strains in milk was 7.11°C. The D values of clumped and single M. paratuberculosis cells were not significantly different. The D values of all M. paratuberculosis strains tested were considerably higher than those published for Listeria, Salmonella, and Coxiella spp. and estimated for Mycobacterium bovis, indicating that M. paratuberculosis is more thermally tolerant. This study supports the premise that M. paratuberculosis may survive high-temperature, short-time pasteurization when the initial organism concentration is greater than 10¹ cells/ml.     

Heat inactivation data for Mycobacterium avium subsp. paratuberculosis: implications for interpretation

AIMS: We discuss several factors that are critical for heat inactivation experiments and which should be taken into account for future research. METHODS AND RESULTS: On the basis of examples from the literature we discuss critical factors influencing the calculated heat inactivation of Mycobacterium avium subsp. paratuberculosis (MAP). Furthermore, using a modelling approach, we show that tailing of the inactivation curve of MAP is caused by the presence of cell clumps and not by a more heat-resistant cell fraction. CONCLUSIONS: The experimental conditions of the MAP heat inactivation studies of different research groups vary significantly and lead to considerable differences in results and conclusions. Therefore, a more consensual approach should be employed in future studies. In addition, our model on clumping of MAP can be used to predict the decimal reduction of MAP during heat treatment and to study the effect of clumping on other lethal effects. SIGNIFICANCE AND IMPACT OF THE STUDY: We discuss several factors that should be carefully considered in heat resistance experiments. This is essential for a thorough interpretation of results from experiments and should be given proper attention in future experiments and publications on this topic.     

Possible Association of GroES and Antigen 85 Proteins with Heat Resistance of Mycobacterium paratuberculosis

Conflicting reports on the heat resistance of Mycobacterium paratuberculosis prompted an examination of the effect of culture medium on this property of the organism. M. paratuberculosis was cultured in three types of media (fatty acid-containing medium 7H9-OADC (oleic acid-albumin-dextrose-catalase supplement) and glycerol-containing media WR-GD and 7H9-GD [glycerol-dextrose supplement]) at pH 6.0. M. paratuberculosis grown under these three culture conditions was then tested for heat resistance in distilled water at 65°C. Soluble proteins and mycolic acids of M. paratuberculosis were evaluated by two-dimensional electrophoresis (2-DE) and thin-layer chromatography (TLC), respectively. The type of culture medium used significantly affected the heat resistance of M. paratuberculosis. The decimal reduction times at 65°C (D_65°C values; times required to reduce the concentration of bacteria by a factor of 10 at 65°C) for M. paratuberculosis strains grown in 7H9-OADC were significantly higher than those for the organisms grown in WR-GD medium (P < 0.01). When the glycerol-dextrose supplement of WR was substituted for the fatty acid supplement (OADC) in 7H9 medium (resulting in 7H9-GD), the D_65°C value was significantly lower than that for the organism grown in 7H9-OADC medium (P = 0.022) but higher than that when it was cultured in WR-GD medium (P = 0.005). Proteomic analysis by 2-DE of soluble proteins extracted from M. paratuberculosis grown without heat stress in the three media (7H9-OADC, 7H9-GD, and WR-GD) revealed that seven proteins were more highly expressed in 7H9-OADC medium than in the other two media. When the seven proteins were subjected to matrix-assisted laser desorption ionization-mass spectrometric analysis, four of the seven protein spots were unidentifiable. The other three proteins were identified as GroES heat shock protein, alpha antigen, and antigen 85 complex B (Ag85B; fibronectin-binding protein). These proteins may be associated with the heat resistance of M. paratuberculosis. Alpha antigen and Ag85B are both trehalose mycolyltransferases involved in mycobacterial cell wall assembly. TLC revealed that 7H9-OADC medium supported production of more trehalose dimycolates and cell wall-bound mycolic acids than did WR-GD medium. The present study shows that in vitro culture conditions significantly affect heat resistance, cell wall synthesis, and protein expression of M. paratuberculosis and emphasize the importance of culture conditions on in vitro and ex vivo studies to estimate heat resistance.     

Possible association of GroES and antigen 85 proteins with heat resistance of Mycobacterium paratuberculosis

Conflicting reports on the heat resistance of Mycobacterium paratuberculosis prompted an examination of the effect of culture medium on this property of the organism. M. paratuberculosis was cultured in three types of media (fatty acid-containing medium 7H9-OADC (oleic acid-albumin-dextrose-catalase supplement) and glycerol-containing media WR-GD and 7H9-GD [glycerol-dextrose supplement]) at pH 6.0. M. paratuberculosis grown under these three culture conditions was then tested for heat resistance in distilled water at 65 degrees C. Soluble proteins and mycolic acids of M. paratuberculosis were evaluated by two-dimensional electrophoresis (2-DE) and thin-layer chromatography (TLC), respectively. The type of culture medium used significantly affected the heat resistance of M. paratuberculosis. The decimal reduction times at 65 degrees C (D(65 degrees C) values; times required to reduce the concentration of bacteria by a factor of 10 at 65 degrees C) for M. paratuberculosis strains grown in 7H9-OADC were significantly higher than those for the organisms grown in WR-GD medium (P < 0.01). When the glycerol-dextrose supplement of WR was substituted for the fatty acid supplement (OADC) in 7H9 medium (resulting in 7H9-GD), the D(65 degrees C) value was significantly lower than that for the organism grown in 7H9-OADC medium (P = 0.022) but higher than that when it was cultured in WR-GD medium (P = 0.005). Proteomic analysis by 2-DE of soluble proteins extracted from M. paratuberculosis grown without heat stress in the three media (7H9-OADC, 7H9-GD, and WR-GD) revealed that seven proteins were more highly expressed in 7H9-OADC medium than in the other two media. When the seven proteins were subjected to matrix-assisted laser desorption ionization-mass spectrometric analysis, four of the seven protein spots were unidentifiable. The other three proteins were identified as GroES heat shock protein, alpha antigen, and antigen 85 complex B (Ag85B; fibronectin-binding protein). These proteins may be associated with the heat resistance of M. paratuberculosis. Alpha antigen and Ag85B are both trehalose mycolyltransferases involved in mycobacterial cell wall assembly. TLC revealed that 7H9-OADC medium supported production of more trehalose dimycolates and cell wall-bound mycolic acids than did WR-GD medium. The present study shows that in vitro culture conditions significantly affect heat resistance, cell wall synthesis, and protein expression of M. paratuberculosis and emphasize the importance of culture conditions on in vitro and ex vivo studies to estimate heat resistance.     

Variation in resistance of Mycobacterium paratuberculosis to acid environments as a function of culture medium

Acid resistance of Mycobacterium paratuberculosis was examined as a function of growth conditions (i.e., in vitro growth medium and pH). M. paratuberculosis was cultured in either fatty acid-containing medium (7H9-OADC) or glycerol-containing medium (WR-GD or 7H9-GD) at two culture pHs (pHs 6.0 and 6.8). Organisms produced in these six medium and pH conditions were then tested for resistance to acetate buffer at pHs 3, 4, 5, and 6 at 20 degrees C. A radiometric culture method (BACTEC) was used to quantify viable M. paratuberculosis cell data at various acid exposure times, and D values (decimal reduction times, or the times required to kill a 1-log(10) concentration of bacteria) were determined. Soluble proteins of M. paratuberculosis grown under all six conditions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to identify proteins that may be associated with acid resistance or susceptibility. The culture medium affected growth rate and morphology: thin floating sheets of cells were observed in 7H9-OADC versus confluent, thick, waxy, and wrinkled pellicles in WR-GD. Culture medium pH affected growth rate (which was highest at pH 6.0), but it had little or no effect on D values for M. paratuberculosis at any test pH. When grown in 7H9-OADC, M. paratuberculosis was more acid resistant at all test pHs (higher D values) than when grown in WR-GD. Glycerol appeared to be the culture medium component most responsible for lower levels of M. paratuberculosis acid resistance. When glycerol was substituted for OADC in the 7H9 medium, D values were significantly lower than those of 7H9-OADC-grown M. paratuberculosis and were approximately the same as those for M. paratuberculosis grown in WR-GD medium. Comparison of the SDS-PAGE protein profiles for M. paratuberculosis cultures grown in 7H9-OADC, WR-GD, or 7H9-GD medium revealed that increased expression of 34.2- and 14.0-kDa proteins was associated with higher levels of acid resistance of M. paratuberculosis grown in 7H9-OADC medium and that 56.6- and 41.3-kDa proteins were associated with lower levels of acid resistance. This is the first report showing that in vitro culture conditions significantly affect growth characteristics, acid resistance, and protein expression of M. paratuberculosis, and the results emphasize the importance of culture conditions for in vitro susceptibility studies.     

Effect of IS900 Gene of Mycobacterium paratuberculosis on Mycobacterium smegmatis

Mycobacterium paratuberculosis is mycobactin dependent and contains multiple copies of the IS900 gene that encodes for p43 (46.5K protein). The correlation between the two characteristics has been investigated. A 3.2-kb BamHI fragment from M. paratuberculosis containing the 1.451 kb IS900 gene was cloned in Escherichia coli and Mycobacterium smegmatis with pcDNA II and pNEZ6.3 plasmids, respectively. Surprisingly, the recombinant M. smegmatis grew poorly and slower in 7H9 broth supplemented with OADC (12 day) compared with M. smegmatis wild type or to M. smegmatis transformed with pNEZ6.3 (2 day). The growth rate of the recombinant M. smegmatis was restored by the addition of 2.4 μM ferric mycobactin J to the media. There was no effect on the growth rate of E. coli recombinants. Western blot analysis with p43-specific anti-peptide antibodies resulted in the expression of 46.5K and a cleaved form of 33.5K protein bands in the recombinant E. coli. There was no expression in the recombinant M. smegmatis. A lower expression of 33.5K protein band was detected in the native M. paratuberculosis protein. The nucleotide sequence of the 3.2-kb fragment confirmed the presence of p43-encoded ORF. There was no additional encoding sequence in the fragment. This suggests that the IS900 gene and/or its encoding products are involved in mycobactin dependency and possibly the slow growth rate of M. paratuberculosis. Received: 12 May 1998 / Accepted: 6 July 1998     

Growth and Metabolic Characteristics of Mycobacterium paratuberculosis

The cultural characteristics of newly isolated strains of Mycobacterium paratuberculosis on a variety of media were studied. The mycobactin dependence ascribed to this species of Mycobacterium was found to be easily circumvented by incorporation of 1% ferric ammonium citrate in serum or egg yolk media, but the earliest and most abundant growth occurred on serum medium that contained mycobactin and sodium pyruvate. In the presence of ferric ammonium citrate, respiration did not decrease during 5 days; but in the presence of all other agents studied, respiration decreased after the first day.     

Bovine PGLYRP1 polymorphisms and their association with resistance to Mycobacterium avium ssp. paratuberculosis

Mycobacterium avium ssp. paratuberculosis (MAP) causes a chronic, granulomatous inflammatory condition of the intestines in ruminants and wild-type species. It causes significant economic losses to the dairy and beef industries owing to reduced productivity, premature culling and mortality. Bovine peptidoglycan recognition protein 1 is an important pattern recognition molecule that is capable of directly killing microorganisms. The goal of this study was to identify single nucleotide polymorphisms (SNPs) in the gene encoding bovine peptidoglycan recognition protein 1 and to assess their association with susceptibility to MAP infection in dairy cattle. Blood and milk samples were collected from Holsteins in Southwestern and Eastern Ontario and tested for MAP infection using blood and milk ELISAs. A resource population consisting of 197 infected (S/P > 0.25) and 242 healthy (S/P < 0.10) cattle was constructed. Sequencing of pooled DNA was used to identify three SNPs (c.102G>C, c.480G>A and c.625C>A) that were genotyped in the resource population. Statistical analysis was performed using a logistic regression model fitting the additive and dominance effects of each SNP in the model. SNP c.480G>A (P = 0.054) was found to be associated with susceptibility to MAP infection. Cows with a copy of the major allele 'G' at this locus had an odds ratio of 1.51 (95% CI: 0.99-2.31) for being infected with MAP.     

Killing of Mycobacterium avium subspecies paratuberculosis within macrophages

Background  

Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is a facultative intracellular pathogen that resides within host macrophages during infection of ruminant animals. We examined survival of M. paratuberculosis infections within cultured macrophages to better understand the interplay between bacterium and host.     

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.     

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 10² to 3.5 × 10⁵ 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°C at holding (mean residence) times of 6 to 15 s. Following 72°C and a holding time of 6 s, 70°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 lnk₀ of 107.2, corresponding to a D value of 1.2 s at 72°C and a Z value of 7.7°C. Homogenization did not significantly affect the inactivation. The conclusion can be drawn that HTST pasteurization conditions equal to 15 s at ≥72°C result in a more-than-sevenfold reduction of M. avium subsp. paratuberculosis.     

Antigenicity of Mycobacterium paratuberculosis superoxide dismutase in mice

Mycobacterium paratuberculosis (MPT) is the etiologic agent of paratuberculosis. The disease is prevalent in cattle worldwide, and exacts a heavy financial toll. Effective control requires the development of acellular vaccines offering a better protection than the current available vaccines without side effects and allowing the discrimination between infected and vaccinated animals. We studied the immune response of mice to the MPT superoxide dismutase (SOD) alone or adjuvanted by Ribi. We cloned, overexpressed and purified this antigen in Escherichia coli. Spleen cells from immunized mice, after exposure to recombinant MPT SOD (MPT rSOD), produced significant levels of IFNgamma, TNFalpha and IL-6. IFNgamma and TNFalpha production was increased by the addition of Ribi. In contrast, low levels of NO, IL-4 and IL-10 were secreted by spleen cells culture from immunized mice. The immunoglobulin isotype distribution analysis showed that Ribi adjuvant clearly induced a significantly higher anti-MPT rSOD antibody production of all classes tested and decreased the IgG1/IgG2a ratio thus improving the Th1 response. Delayed-type hypersensitivity responses in mice footpads were observed only in mice immunized with MPT rSOD emulsified in Ribi. Vaccination of MPT rSOD emulsified with Ribi induced both a Th2 and Th1 type of immune response with the later slightly more pronounced. The results presented here on the immunogenicity of MPT SOD suggest that this antigen should be further tested as a candidate antigen for a future acellular vaccine against paratuberculosis.     

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°C (2,500 pulses at 30 kV/cm) reduced the level of viable M. paratuberculosis cells by approximately 5.3 and 5.9 log₁₀ 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°C for 25 min or at 72°C for 25 s (extended high-temperature, short-time pasteurization) resulted in reductions of M. paratuberculosis of approximately 0.01 and 2.4 log₁₀ CFU/ml, respectively. TEM studies revealed that exposure to PEF treatment resulted in substantial damage at the cellular level to M. paratuberculosis.     

Mycobacterium paratuberculosis zoonosis is a One Health emergency

EcoHealth - A singular pathogen has been killing animals, contaminating food and causing an array of human diseases. Mycobacterium avium subspecies paratuberculosis (MAP) is the cause of a fatal...