MS #16491 Revised
The effect of pasteurization on the viability of Mycobacterium paratuberculosis
R. J. Chiodini1 and J. Hermon-Taylor2
1Mycobacteriology Unit, Division of Gastrointestinal Diseases, Department of Medicine, Rhode Island Hospital and Brown University, Division of Biological and Medical Sciences, 593 Eddy Street, Providence, Rhode Island 02903; 2Department of Surgery, St. George's Hospital Medical School, University of London, Cranmer Terrace, London SW17 ORE, U.K.
Mycobacterium paratuberculosis is the etiologic agent of ruminant paratuberculosis, commonly referred to as Johne's disease. The disease is characterized by a chronic granulomatous ileocolitis which ultimately terminates in diarrhea, weight loss, debilitation, and death.6 The disease is transmitted between ruminants predominately through feces when animals are at a young age. In the United States, the national disease prevalence has been suggested to be 2.6% of the dairy and 0.8% of the beef cattle,15 although regional surveys suggest local prevalences ranging from 11-17%.1,5
Cattle infected with M. paratuberculosis shed organisms in feces often at concentrations approaching 108 colony forming units (cfu) per gram.6 The environment of an infected herd is therefore considered to be heavily contaminated.6,17 In addition to feces, other body fluids may also shed M. paratuberculosis, including milk. Some studies have suggested that as many as 35% of clinically infected cattle21 and 11.6% of asymptomatic carriers20 have detectable quantities of M. paratuberculosis in their milk.
In recent years, there has been an interest in the possible association of paratuberculosis and human Crohn's disease, a chronic granulomatous ileocolitis of unknown etiology. The possible etiological association between these two diseases has largely been prompted by the isolation of M. paratuberculosis from human patients with Crohn's disease in the United States, Australia, the Netherlands, and France.2 Of the 3 primary groups currently engaged in the culture of human tissues, M. paratuberculosis has been isolated from 20, 33, and 38% of patients with Crohn's disease, but from only 0.8% (1 in 121) of controls.4 These human isolates have been shown to be of the bovine-type rather than of ovine-caprine origin,9,22 and indistinguishable from strains isolated from cattle.3,4,9,11,14
Recently, a species-specific insertion sequence in M. paratuberculosis, IS900,11 was used in the polymerase chain reaction to examine tissues from human patients for the presence of this organism. IS900 was detected in 65% of tissues from patients with Crohn's disease, but in only 12.5% of controls and 4.3% of cases of ulcerative colitis.19 The finding of IS900 in 12.5% of controls indicates a previously unrecognized environmental distribution of this organism, suggesting that human contact with M. paratuberculosis may be common.18 The low presence of M. paratuberculosis in ulcerative colitis patients suggest that colonization of the disrupted or abnormal intestinal mucosa by M. paratuberculosis does not occur. Therefore, the high prevalence (65%) in Crohn's disease patients would suggest some etiologic role for this known chronic enteric pathogen in humans as in many other animal species.18,19
Since these findings suggest human contact with M. paratuberculosis other than direct exposure to infected cattle, we sought to examine indirect avenues of human contact with the dairy industry. As part of that examination, the effects of pasteurization methods on the viability of M. paratuberculosis were determined.
Two strains of M. paratuberculosis of bovine origin (strains 3737 and ATCC 19698) and 2 strains isolated from diseased human tissues (Linda and Ben) were grown in 7H9 broth containing DOAC and 0.05% Tween 80 as previously described.7,8 Cultures were lightly sonicated to disrupt large clumps and initial cfu per ml estimated spectrophotometrically as previously described.7 A wild-type strain of Mycobacterium bovis (hawaiian strain) and M. bovis BCG (TMC 1011) were used as controls.
Fresh milk was obtained asceptically from a Holstein cow known to be free of paratuberculosis, chilled on ice, and maintained at 4oC until use. In order to insure accurate cfu determinations, organisms were suspended in whole milk at approximately 1 x 103, 1 x 104, and 1 x 105 per ml, thereby insuring a minimum of 100 colonies per undiluted inoculated tube at a 0% kill rate. M. paratuberculosis was suspended in a total volume of 10 ml raw milk for each pasteurization cycle. Prior to and after pasteurization, milk containing M. paratuberculosis were vigorously mixed, subjected to serial 10 fold dilutions in phosphate buffered saline containing 0.05% Tween 80, and 100 ul samples inoculated onto a minimum of 5 slants of Herrold's egg yolk medium containing 2 ug/ml mycobactin. Cfu's were determined by colony counts before and after pasteurization. Only dilution tubes containing between 5-50 colonies were counted to determine cfu per ml milk since greater than 50 colonies could not be reliably counted and there was too great a variability in dilution tubes containing less than 5 colonies. All strains were exposed to 3 separate pasteurization cycles on the same day. Results are reported as the average of the 3 pasteurizations, thereby representing the average colony count of at least 15 tubes or 1.5 ml of the total milk sample.
Pasteurizations were performed according to methods as defined by the Public Health Service, Food and Drug Administration.16 The Standard Holder method was performed by heating milk in a water bath to 63oC (145oF) and maintaining that temperature for 30 minutes with continuous mixing. For the High Temperature Short Time (HTST) method, milk was heated to 72oC (161oF) and maintained at that temperature for 15 seconds with continuous mixing. An electronic temperature probe and a mercury filled thermometer were used to monitor milk temperature. After pasteurization, milk samples were allowed to gradually cool at room temperature, at 4oC, and in an ice bath. Three routes of cooling were employed since, although no standards apply, most processing plants cool the milk after pasteurization and the rate of such cooling is unknown.
Results of the various methods of pasteurization on the viability of M. paratuberculosis are summarized in Tables 1 and 2. In all cases, pasteurization effectively killed both M. bovis strains (data not shown). By the standard holder method of pasteurization, the viability of over 90% of the bovine M. paratuberculosis isolates was effectively destroyed; however, 5-9% of the organisms per ml were still viable after pasteurization. The faster the cooling rate, the larger the number of organisms surviving. This observation was likely due to the reduced exposure to increased heat (after the 30 minute cycle) in the more rapidly cooled samples. In contrast to the bovine isolates, human isolates had a higher resistance to the standard holder method of pasteurization with only 60-80% of these organisms being killed by pasteurization. As with the bovine isolates, faster cooling increased the number of surviving organisms.
The HTST pasteurization method appeared more effective against bovine isolates, as compared with the standard holder method, with over 95% of the organisms destroyed; however, 3-5% still survived pasteurization. In contrast, the HTST method was generally less effective against the human isolates, particularly with rapid cooling. Cooling at 4oC caused an increased survival from 24.8 to 31.4% as compared to gradual cooling at room temperature. Rapid cooling on ice resulted in an unexplained increased number of colonies (18-21%) of the human strains after pasteurization. Although this increase in colony counts after pasteurization could not be explained, it may be the result of breaking up of the bacillary clumps characteristic of M. paratuberculosis4 by the high heat shock and rapid cooling, thereby causing an increased number of individual colonies rather than an actual increase in the number of organisms. This was not apparent in the bovine isolates presumably because of their increased sensitivity to heat, i.e., breaking up of clumps of dead bacilli would not increase cfu's.
These results suggest that current pasteurization methods are not completely effective against strains of M. paratuberculosis, particularly those strains originating from humans. Although pasteurization is generally considered to be effective in destroying human pathogens, recent studies have suggested that some strains of other organisms, such as Listeria monocytogenes,10 have the ability to survive normal pasteurization procedures.
In addition to the resistance of M. paratuberculosis to complete killing by both the standard holder and HTST pasteurization methods, pasteurized milk may also become contaminated with M. paratuberculosis or other pathogens by contamination of pasteurized milk with raw milk12 or with excrement from the environment. The presence of coliforms in pasteurized bottled milk suggests that post-pasteurization contamination occurs frequently.13
In our efforts to identify human sources of this pathogen, studies are currently in progress to determine if M. paratuberculosis exists within pasteurized retail dairy products. The finding that M. paratuberculosis survives pasteurization suggests that the introduction of new public health procedures and other measures for the prevention of human disease may be appropriate.
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2. Chiodini, R. J. 1989. Crohn's disease and the Mycobacterioses: a review and comparison of two disease entities. Clin. Microbiol. Rev. 2:90-117.
3. Chiodini, R. J. 1990. Characterization of Mycobacterium paratuberculosis and organisms of the Mycobacterium avium complex by restriction polymorphism of the rRNA gene region. J. Clin. Microbiol. 28:489-494.
4. Chiodini, R. J. 1992. Historical overview and current approaches in determining a mycobacterial aetiology of Crohn's disease. In: Mulder C. J. J., and Tytgat G. N. J., eds. Is Crohn's disease a mycobacterial disease. Dordrecht: Kluwer Academic Publishers. pp 1-15.
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14. McFadden, J.J., Butcher, P.D., Chiodini, R.J., and Hermon-Taylor J. 1987. Crohn's disease-isolated mycobacteria are identical to Mycobacterium paratuberculosis, as determined by DNA probes that distinguish between mycobacterial species. J. Clin. Microbiol. 25:796-801.
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16. Public Health Service, Food and Drug Administration. 1985. Grade A pasteurized milk ordinance. US PHS Publication No. 229.
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20. Sweeney R. W., R. H. Whitlock, and A. E. Rosenberger. 1992. Mycobacterium paratuberculosis cultured from milk and supramammary lymph nodes of infected asymptomatic cows. J Clin Microbiol 30:166-171.
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22. Whipple, D., P. Kapke, and C. Vary. 1990. Identification of restriction fragment length polymorphisms in DNA from Mycobacterium paratuberculosis. J. Clin. Microbiol. 28:2561-2564.