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Pasteurization of Mycobacterium paratuberculosis in whole milk

AF Hope, PA Tulk, and RJ Condron

Victorian Institute of Animal Science,
475 Mickleham Road,
Attwood 3049,
Australia

Table of contents.

Abstract

It is preferable to use commercial pasteurizing units to assess bacterial thermosusceptibility and establish the efficacy of pasteurization because the heating and cooling differentials generated in laboratory heat treatment may not simulate commercial conditions. Seventeen batches of raw milk were loaded with 102-105 CFU/mL of Mycobacterium paratuberculosis and pasteurized in a small scale commercial pasteurizing unit at temperatures ranging from 72-90oC for 15-35 seconds. Up to 20 samples were tested from each batch and M. paratuberculosis was not isolated from 96% (275/286) of pasteurized milk samples, representing 104 reductions in mycobacterial concentrations as radiometric culture could detect about one colony forming unit per milliliter. Viable mycobacteria were not recovered when raw whole milk was loaded with less than 104 mycobacteria per milliliter. They were not detected in any of five batches of milk pasteurized at 72-73oC for 25-35 seconds, which are the minimum conditions applied when this machine is used commercially to correct for laminar flow in the holding tube. At shorter times than recommended no viable bacteria were isolated from seven batches, however small numbers of viable bacteria were cultured from four of eight batches heat treated at 72-73oC for 15 seconds and one of four batches treated at 82-92oC for 15 seconds. In the five batches where M. paratuberculosis was recovered the raw whole milk was loaded with more than 104 mycobacteria per milliliter. Survival of M. paratuberculosis in experimentally inoculated batches of milk in the small-scale commercial unit cannot be directly extrapolated to commercial pasteurization of naturally infected milk in dairy factories because of artificially high mycobacterial loads used in these experiments, possible differences in the thermosusceptibility of laboratory cultured mycobacteria, and features of the small-scale unit. Pasteurization in a small-scale commercial unit used in these experiments appears to be more efficient at killing mycobacteria than laboratory heat treatment systems.

Introduction

Mycobacterium paratuberculosis is not a recognized agent of food-borne disease, however there have been recent investigations of its thermosusceptiblity following its detection in patients with Crohn's disease3.

M. paratuberculosis has been isolated from raw milk. Organisms excreted in feces are the most likely source of milk contamination, although endogenous infection has also been demonstrated by culture of the mycobacteria from asceptically collected milk17,20,23, supramammary lymph nodes17, and deep udder tissue6. M. paratuberculosis has been isolated from the milk of up to 12% of subclinically infected cows16,17,19, from the milk of between 5% and 82% of clinically infected cattle4,17,19,23, and from colostrum16. Using a highly sensitive method of detection, DNA segments specific to M. paratuberculosis were found in 6.25% of 336 retail milk samples in the United Kingdom12.

Pasteurization, heating to 72.4oC for at least 15 seconds and immediately cooling to 3.5oC, destroys milk-borne pathogens and delays development of spoilage bacteria. Viable M. paratuberculosis were recovered after heating at 72oC for 15 seconds in two recent laboratory experiments3,8. Estimates of bacterial thermal death time are influenced by the heating and cooling lags generated within the heat treatment systems13,18. It is preferable to establish bacterial thermosusceptibility using commercial pasteurization units, although in practice their use is limited by their availability and the large volumes of milk required for each experiment. The aims of this study were to establish the effect of pasteurization under commercial conditions on the viability of M. paratuberculosis, using a small-scale continuous flow pasteurizing machine.

Materials and Methods

Seventeen batches of 5 L to 15 L of raw milk, artificially seeded with M. paratuberculosis, were pasteurized by the high-temperature short-time (HTST) method in a small-scale commercial pasteurizing unit

The pasteurizing unit featured a positive displacement pumpa, Alfa-Laval P20-HB heat exchange unit with a bank of 30 plates, an Alfa-Laval GM2 hot water pump with thermostat, a 117 cm stainless steel holding line with thermometer at the terminal end, and glycol chilling. The small-scale pasteurizing unit used in this study differed from commercial units in the following features; it had a linear holding tube, it did not have filters to remove large particulate debris, an homogenizer, or flow diversion valve. Holding times were altered by changing the rate of flow of milk through the pasteurizing unit. Milk particles flowing through straight sections of the holding tube were subject to laminar flow rather than turbulent flow generated in the holding tubes of larger units.

M. paratuberculosis was obtained from field cases of bovine and caprine Johne's disease in Australia, an isolate from an Australian Crohn's patient, and a reference culture ATCC 19698. Large quantities of mycobacteria used to inoculate batches of milk were cultured in Watson Reid synthetic medium, adjusted to pH 5.8, and supplemented with 2mg/L mycobactin Jb. The bovine field isolates were passed no more than five times. M. paratuberculosis was recovered from the grossly thickened and corrugated ileum of two dairy cattle in advanced stages of Johne's disease (in vivo inoculum). A total of 1,200 g of macerated mucosa was digested in 0.5% trypsin at pH 7.8, centrifuged, and the pellet decontaminated in 0.75% hexadecylpridiniumchloride (HPC).

Initial mycobacterial concentrations in raw milk were estimated from five 50 mL samples. Following centrifugation the pellets were decontaminated in 0.75% HPC overnight and an additional incubation at 37oC for four hours with 0.2 mL of a commercial antibiotic mix (PANTA Plusc)in 5 mL water. Five 50 mL samples from ten-fold dilutions were cultured onto Herrold Egg Yolk slopes containing mycobactin (HEYJ).

Pasteurized milk samples (50 mL) were collected and immediately processed for radiometric culture. 0.1 mL of suspension was injected into modified 12B Bactecd bottles supplemented with 1.0 mL of egg yolk/PBS suspension, 4 mg of mycobactin J, and 0.2 mL of PANTA Plus. The bottles were incubated at 37oC and monitored weekly for evidence of bacterial growth. Results from samples containing viable M. paratuberculosis were considered to be binomially distributed in each batch and their 95% confidence intervals were estimated using two-tailed tests in batches yielding M. paratuberculosis and one-tailed tests in batches that did not yield growth15.

The sensitivity of the radiometric culture system was determined for a bovine field strain, in vivo mycobacteria pooled from two clinical cases, an isolate from a Crohn's patient, and Strain 316V. These mycobacteria were placed in 10 mL of pasteurized whole milk, and ten-fold dilutions were cultured by radiometric and conventional methods. Mean mycobacterial concentrations (CFU/mL) in raw milk were estimated using arithmetic means from ten-fold sample dilutions which yielded 10-80 colonies per slope. A small random selection of pasteurized milk samples (11) which were negative on radiometric culture were examined for the presence of the DNA insertion sequence (IS900) by polymerase chain reaction (PCR)22 and all gave positive reactions.

Results

M. paratuberculosis was added to batches of raw milk resulting in concentrations of 102 to 105 CFU/mL (Table 1). M. paratuberculosis was not recovered by radiometric culture in 96% (275 of 286) of pasteurized milk samples. Twelve of 17 batches of pasteurized milk tested negative, including ten batches loaded with 102-105 CFU/mL bovine field strains, and single batches containing 102 CFU/mL ATCC 19698, and an isolate from a Crohn's patient 104 CFU/mL. Viable mycobacteria were not recovered from any batch containing initial loads of less than 104 CFU/mL. M. paratuberculosis DNA was detected by PCR in seven pasteurized milk samples which were negative by radiometric culture.

Eleven samples yielding viable M. paratuberculosis were from Batches A, G, K, M, and O (Table 1) comprising 4 batches pasteurized at 72-73oC for 15 seconds, and one batch pasteurized at 82-90oC for 15 seconds. The samples were collected from the unit's outlet during early (4), mid (3) and late (4) processing of the batches. The highest rate of recovery was from a batch of skim milk.

Radiometric culture was more sensitive than conventional culture and was able to detect M. paratuberculosis at concentrations of about 1 CFU/mL (Table 2). Conventional culture was not suitable for enumeration of organisms in pasteurized samples.

Discussion

The commercial pasteurizing unit efficiently killed mycobacteria, with no M. paratuberculosis recovered by radiometric culture from 96% of pasteurized milk samples, representing in the order of 104 reductions in mycobacterial concentrations. Commercial pasteurization appeared to be more effective than heat treatment in laboratory systems, as mycobacteria were recovered from a higher percentage of milk samples treated in laboratory models of pasteurization3,8.

Small numbers of M. paratuberculosis survived pasteurization in half of the batches of milk at 72-73oC for 15 seconds in the small-scale continuous flow pasteurizing machine used in these experiments. Important functional differences between the small-scale commercial pasteurizing unit and larger commercial units were the lack of a homogenizer and a straight holding tube which results in laminar flow of milk particles rather than turbulent flow. To correct for laminar flow effects, the minimum holding time of 25 seconds is applied when this machine is used commercially. M. paratuberculosis was not recovered from any of five batches of milk held at 72-73oC for 25 seconds. In the small number of batches treated adequate holding time appeared to be more effective in killing M. paratuberculosis than higher temperatures.

Survival of M. paratuberculosis in some batches of milk cannot be directly extrapolated to commercial pasteurization of naturally infected milk in dairy factories because of the artificially high mycobacterial loads used in the experiments, possible differences in the thermosusceptibility of laboratory cultured mycobacteria, and physical limitations of the small-scale unit. The thermosusceptibility of laboratory cultured mycobacteria varies with their culture media and age and growth phase of cultures through mechanisms such as changes in cell membrane viscosity and the production of heat shock proteins2,6,9,24. Laboratory cultured mycobacteria may have greater thermotolerance compared with in vivo mycobacteria11. Mycobacterial concentrations used in this experiment were one million times higher than reported concentrations of M. paratuberculosis in naturally infected milk19.

The proportion of pasteurized milk samples containing viable mycobacteria was greater in a skim milk compared to whole milk batches. This may reflect a larger inoculum of mycobacteria in the skim milk batch, improved repair of sublethally affected mycobacteria in skim milk, or increased efficiency of recovery from skim milk. Pasteurization efficacy in whole milk may differ from skim milk or homogenized milk if killing of lipophilic mycobacteria is affected by the distribution of milk fat globules in these liquids.

Pasteurization was effective in reducing concentrations of M. paratuberculosis to below the detectable limit (about 1 CFU/mL) in twelve batches of raw whole milk. The inability to recover bacteria post-pasteurization does not ensure sterility. Criticisms of pasteurization trials yielding negative results are; suboptimal cultural conditions for bacterial recovery, employment of sampling methods unlikely to detect viable bacteria, and extrapolation of heat treatment of bacterial suspensions to bacteria that are usually found within cells. Although it has been postulated that bacteria located within bovine phagocytes will have a higher heat tolerance5, many studies have found pasteurization is still effective against intracellular pathogens1,7,10. There was no evidence from these experiments that M. paratuberculosis in tissues (in vivo inoculum) was resistant to heat treatment. It has not been established whether radiometric culture is optimum for the recovery of heat damaged mycobacteria. Injured organisms are sensitive to their immediate chemical environment, and it is possible the radiometric liquid media was more conducive to recovery of mycobacteria than conventional culture media despite its antibiotic supplementation. Sample volumes of 50 mL were selected to increase the likelihood of detecting bacteria14.

Pasteurization studies investigating the control of potentially hazardous milk-borne bacteria often give conflicting results. Kill rates in pasteurization studies depend on heat differentials generated in the heat treatment systems, and varying come-up times, and cooling lags explain contrasting results between laboratory and commercial systems18. Further studies using commercial pasteurization of milk from naturally infected cows and herds will provide confidence in the assessment of pasteurization efficacy against M. paratuberculosis.

Acknowledgments

This work was financially supported by the Dairy Research and Development Corporation. The authors would like to thank the Victorian College of Agriculture and Horticulture for access to their pasteurizing unit, with special thanks to Selwyn Stokes for his assistance and guidance.

References

1.Bunning VK, Donnelly CW, Peeler JT, Briggs EH, Bradshaw JG, Crawford RG, Beliveau CM, and Tierney JT (1988) Thermal inactivation of Listeria monocytogenes within bovine milk phagocytes Appl Environ Microbiol 54(2):364-370
2.Bunning VK, Crawford RG, Tierney JT, and Peeler JT (1992) Thermotolerance of heat-shocked Listeria monocytogenes in milk exposed to high-temperature, short-time pasteurization Appl Environ Microbiol 58(6):2096-2098
3.Chiodini RJ, and Hermon-Taylor (1993) The thermal resistance of Mycobacterium paratuberculosis in raw milk under conditions simulating pasteruization J Vet Diagn Invest 5:629-631
4.Doyle TM (1954) Isolation of Johne's bacilli from the udders of clinically affected cow Brit Vet J 110:215-218
5.Doyle MP, Glass KA, Beery JT, Garcia GA, Pollard DJ, and Schultz RD (1987) Survival of Listeria monocytogenes in milk during High-temperature, Short-time pasteurization Appl Environ Microbiol 53(7):1433-1438
6.Farber JM, Daley E, Coates F, Emmons DB, and McKellar R (1992) Factors influencing survial of Listeria monocytogenes in milk in a high-temperature short-time pasteurizer J Food Prot 55(12)946-951
7.Farber JM, Sanders GW, Speirs JI, D'Aoust JY, Emmons DB, and McKellar R (1988) Thermal resistance of Listeria monocytogenes in inoculated and naturally contaminated raw milk Int J Food Microbiol 7:277-286
8.Grant IR, Ball HJ, Neill SD and Rowe MT (1996) Inactivation of Mycobacterium paratuberculosis in cow's milk at pasteurization temperatures Appl Environ Microbiol 62(2):631-636
9.Knabel SJ, Walker HW, Hartman PA, and Mendonca AF (1990) Effects of growth temperature and strictly anaerobic recovery on the survival of Listeria monocytogenes during pasteurization Appl Environ Microbiol 56(2):370-376
10.Lovett J, Wesley IV, Vandermaaten MJ, Bradshaw JG, Francis DW, Crawford RG, Donnelly CW, and Messer JW (1990) High-temperature short-time pasteurization inactivates Listeria monocytogenes J Food Prot 53(9):734-738
11.Merkal RS, Sneed Lyle P, and Whipple DL (1981) Heat inactivation of in vivo- and in vitro- grown mycobacteria in meat products Appl Environ Microbiol 41(6):1484-1485
12.Millar DS, For J, Sanderson JD, Tizard MLV, Kempsell K, Lake RJ and Hermon-Taylor J (1994) IS900 PCR testing for Mycobacterium paratuberculosis in units of whole pasteurized cows milk widely obtained from retail outlets in England and Wales [Abstract] In Proc 4th Inter Coll Paratuberculosis Eds Chiodini RJ, Collins MT, and Bassey EOE p320
13.North CE, and Park WH (1927) Standards for milk pasteurization Am J Hygiene 7:147-173
14.Siebert KJ (1993) Computer simulation of microbiological sampling of liquid foods J Food Protection 56(6):518-524
15.Sokal RR and Rohlf FJ (1969) in Biometry The Principles and practice of statistics in biological research Second Edition, WH Freeman and Company, New York
16.Streeter RN, Hoffsis GF, Bech-Nielsen S, Shulaw WP, and Rings M (1995) Isolation of Mycobacterium paratuberculosis from colostrum and milk of subclinically infected cows Am J Vet Res 56(10):1322-1324
17.Stuart P (1965) Vaccination against Johne's disease in cattle exposed to experimental infection Brit Vet J 121:289-318
18.Stumbo CR (1973) Thermobacteriology in food processing Second Edition, In Food Science and Technology, Academic Press, London pp152-189
19.Sweeney RW, Whitlock RH, and Rosenberger AE (1992) Mycobacterium paratuberculosis cultured from milk and supramammary lymph nodes of infected asymptomatic cows J Clin Microbiol 30(1):166-171
20.Sweeney RW, Whitlock RH, Buckley CL, Spencer P, Rosenberger AE, and Hutchinson LJ (1994) Diagnosis of paratuberculosis in dairy cattle, using enzyme-linked immunosorbent assay for detection of antibodies against Mycobacterium paratuberculosis in milk Am J Vet Res 55(7):905-909
21.Taylor TK, Wilks CR, and McQueen DS (1981) Isolation of Mycobacterium paratuberculosis from the milk of a cow with Johne's disease Vet Rec pp532-533
22.Vary PH, Andersen PR, Green E, Hermon-Taylor J, and McFadden JJ (1990) Use of highly specific DNA probes and the polymerase chain reaction to detect Mycobacterium paratuberculosis in Johne's disease J Clin Microbiol 28(5):933-937
23.Williams Smith H (1960) The examination of milk for the presence of Mycobacterium johnei J Path Bacteriol 80:440-442
24.Yamamori T, and Yura T (1982) Genetic control of heat-shock protein synthesis and its bearing on growth and thermal resistance in Escherichia coli K-12 Proc Natl Acad Sci 79:860-864


Table One: Pasteurisation of raw whole milk artificially seeded with Mycobacterium paratuberculosis using a small-scale commercial unit

M. paratuberculosis strainBatchPasteurisation conditionsVolume treated (L)Mycobacterial concentration in raw milk, determined by conventional culture
(CFU/mL, ± standard deviation)
Proportion of radiometric culture positive samples post-pasteurisation
Bovine field strain "27/10"A72.0oC, 15s103.5 x 104 (±1.1 x 104)3/20
Bovine field strain "27/10"B71.0oC, 34s51.3 x 102 (±0.6 x 102)a0/14c
Bovine field strain "27/10"C73.0oC, 35s101.1 x 105 (±0.2 x 105)0/27
Bovine field strain "27/10"D72.0oC, 25s15Contaminated0/20
Bovine field strain "27/10"E73.0oC, 25s104.6 x 102 (±1.4 x 102)0/20
Bovine field strain "27/10"F75.0oC, 25s150.6 x 102 (±0.3 x 102)0/20
Bovine field strain "27/10"G82.0oC, 15s103.2 x 104 (±0.8 x 104)1/18c
Bovine field strain "27/10"H82.5oC, 15s105.2 x 103 (±1.2 x 103)0/15
Bovine field strain "27/10"I92.0oC, 15s104.4 x 103 (±1.4 x 103)0/19
Bovine mixed field strainsJ90.0oC, 15s109.3 x 103 (±2.5 x 103)0/20
Bovine mixed field strainsKr72.0oC, 1510Not done5/13c
Bovine mixed field strainsL73.0oC, 15s106.8 x 105 (±1.7 x 105)0/16
Caprine field strainsM72.5oC, 15s103.5 x 104 (±1.0 x 104)1/16c
Crohn's isolateN73.0oC, 15s55.2 x 104 (±2.5 x 104)0/10
Crohn's isolateO73.0oC, 15s 53.2 x 104 (±0.7 x 104)1/10
ATCC 19698P73.0oC, 15s57.0 x 102 (±2.1 x 102)0/19
Bovine in vivo isolateR73.0oC, 15s55.2 x 103 (±1.6 x 103)0/9c

a Estimate based on counts <10
c Contaminated samples excluded from report
r Raw skim used in this batch


Table Two: Level of detection of culture for Mycobacterium paratuberculosis in milk

Level of detection replicatesM. paratuberculosis strainaMycobacterial concentration in original sample determined by conventional culture
(CFU/mL, ±standard deviation)
Greatest dilution in which growth was detected by conventional cultureaGreatest dilution in which growth was detected by radiometric culturebLevel of detection of radiometric culture
3Bovine field strain "27/10"2.8 x 104 (±1.1 x 104)10-410-63 CFU/100mL
5Bovine field strain "27/10"1.2 x 105 (±0.4 x 105)10-510-51 CFU/mL
7Bovine field strain "27/10"1.5 x 105 (±0.4 x 105)10-510-5, 10-7Not estimated
10Bovine field strain "27/10"2.3 x 106 (±0.4 x 106)10-510-62 CFU/mL
11316V1.6 x 104 (±0.3 x 104)10-410-320 CFU/mL
21(1)316V3.0 x 104 (±0.8 x 104)10-310-43 CFU/mL
12Bovine in vivo isolate3.8 x 106 (±0.4 x 106)10-610-74 CFU/10mL
14Bovine in vivo isolate5.7 x 105 (± 0.0 x 103)10-510-56 CFU/mL
22Crohn's isolate1.7 x 106 (±0.3 x 106)10-410-62 CFU/mL

a On any of five HEYJ slopes
b In a single modified Bactec 12B bottle