Modelling the destruction of Mycobacterium avium subsp. paratuberculosis In this section we are going to use simple mathematical techniques, associated with thermobacteriology, to investigate the survival of Mycobacterium avium subsp. paratuberculosis during high temperature short time, HTST, pasteurisation. This organism is often abbreviated M. paratuberculosis, M. avium sub. paratuberculosis or MAP. Two simple equations describing the death of microorganisms by thermal processing have been used to construct several calculators that make it fairly easy for students and others to predict MAP levels in milk following thermal processing.The methodology used could also be used to investigate the effects of pasteurisation on the destruction of salmonella or say Enterobacter sakazakii (E. sakazakii has recently been reclassified as a number of distinct species within a new Genus, Cronobacter).Why study MAP? MAP is interesting for several reasons e.g. there is public health concern about this organism since it has been linked to Crohn’s disease and there is also debate regarding whether it is killed by HTST treatments. At the end of the article there is an assignment that can be undertaken using the calculators. This exercise with appropriate adaption should be suitable for second and third year undergraduates in microbiology or food science and students undertaking taught Masters Degrees in Microbiology/Food Science/Food Technology. The exercise could also be used as part of an in- company staff development programme. Note prior to undertaking this exercise some prior knowledge of the effects of temperature on the death of microorganisms is required. The calculators can also be used to investigate the effects of thermal processing on the survival of pathogens and thermoduric/thermophilic organisms. PUBLIC HEALTH SIGNIFICANCE OF MAPBoth Crohn’s disease, a human illness of unknown cause and Johne’s disease,a disease of cattle and other ruminants caused by MAP, are chronic, incurable, inflammatory bowel diseases. Because the symptoms of these human and animal diseases are similar and that MAP has been isolated from gut-lesions combined with the the detection of an unique MAP-DNA insertion sequence (IS900) in intestinal tissue from some patients a link between Crohn’s disease and MAP has been suggested. This has still to be proven and MAP is not currently regarded as a human pathogen. MODELLING THE EFFECT OF HTST PASTEURISATION ON THE DESTRUCTION ON MAP IN MILK The number of microbial survivors, N2, following the heat treatment of a microbial population, N1, at a defined temperature for a particular time, t, can be calculated using equation 1 providing the Decimal Reduction Time of the organism, D, D is the time required to reduce the number of organisms by 1 log cycle, is known. While D is usually expressed in minutes or seconds it must be expressed in the same time units as t.Equation 1. t = D (log N1- log N2); Stumbo (1973). There is some scope, although limited, to vary t typically from 15 - 25 seconds. This can be done by changing the flow rate and/or the length of the holding tube in the pasteuriser. There is currently no scientifically validated reference value for D 72°C for MAP. D-values are difficult to obtain since MAP is is hard to culture and because of this, it is generally accepted that all or perhaps most methods of culture result in a significant underestimate of the number of viable organisms. Following a review by the UK Food Standards Agency, values of D 72°C for MAP of 12 to 14 seconds have been widely cited within the industry. However, even the 14-second value is low compared with data from Sung and Collins (1998) who described a strain, ATCC 19698, which had a D 71 of 16.5 seconds. However, there is older data suggesting that MAP is relatively heat sensitive. While this has been explained by differences in enumeration techniques and the difficulty in culturing MAP it is likely that there is also significant variation in the thermal resistance of strains this organism. For modelling purposes a D 71 value of 14 seconds will be used. However, if MAP were to be designated as a pathogen this value could be regarded as somewhat low. Establishing valid initial numbers of MAP for modelling purposes is also difficult. While MAP may be secreted directly into the milk in the udder, resulting in relatively low numbers, perhaps < 10 CFU/ml, the main source is thought to be faecal contamination. The faeces of infected animals can contain > 1 X 10 8 CFU/g. Grant et al. (1998) have indicated that the concentration of MAP in raw milk could be as high as 10 4 CFU/mL due to faecal contamination. Sung and Collins (1998) have suggested that a MAP concentration of 10 6 CFU/mL should be used when modelling MAP destruction for safety reasons. Predicting the number of MAP surviving heat treatment Equation 1, can be rewritten to give N2, the number of survivors, as shown in equation 2. Equation 2. log N2=logN1 - t/D N2 is obtained by finding the antilog of log N2. Equation 2 has been used to programme a calculator which can be accessed here. Using the calculator and the set values, which can be changed, we can investigate the effect of varying the initial number of MAP and holding time on the concentration of survivors. Calculating the holding time at 72°C to obtain a target number of MAP surviving heat treatment An alternative approach is to calculate the holding time at 72°C to reduce a designated population of MAP to a target number of viable organisms e.g. 0.1 CFU/mL. This can be done by using equation 1. Equation 1 has been used to programme a calculator for the determination of processing time. So far we have been able to investigate the effect of concentration of MAP and holding time on the number of survivors following a HTST treatment. It would also be useful to investigate the effect of higher temperatures on the survival of MAP. Predicting the effect of temperatures higher than 72° on the survival of MAP during HTST pasteurisation However we cannot investigate temperatures lower or higher than 72°C unless we have D-values for the desired temperatures. The D-value decreases as the temperature increases, since it takes less time to destroy microorganisms at a higher temperature. The Z-value is the temperature, typically in degrees Celsius in the UK , that is required for the thermal death curve, obtained by plotting log D-values versus temperature for an organism, to move (often called traverse) one log cycle. While the D-value gives us the time needed at a certain temperature to kill 90% of the population of a particular organism, the Z-value enables the death of an organism at different temperatures to be determined. Providing both the Z-value and the D-value of an organism are known the lethal effects of a range of temperatures can be calculated. The Z value for many vegative microorganisms is within the range 5-10°C; the higher the value, the more resistant the organism is to thermal processing. How to use a D-value at 72°C and the Z value for an organism to calculate a D value at a higher temperature. Given that the D value at 72°C for an organism is 14 seconds and the Z value is 7°C how can the D-value at 78°C be calculated? Since 78°C is 7°C more than 71°C and the Z-value is also 7°C the D-value is reduced by 1-log cycle i.e. 14 seconds (the D-value at 72°C) is divided by 10 to give a predicted D-value at 78°C of 1.4 seconds. Give a D-value at a particular temperature, D values at other temperatures can can also be calculated using a simple equation. Knowing the temperature, T1, at which DI was determined for an organism with a Z-value, z, the D2 value can be determined at temperature T2 using equation 4. Note the validity of this equation is dependent upon the curve describing the relationship between log D and time being linear. This is not always the case! Equation 3. LogD2=(T1-T2)/z +logD1; Stumbo (1973). Equation 3 has been used to programme a calculator for the determination of D2. Using the calculator and the set Z value of 8.246 (Sung and Collins, 1998)-other values can be used, the D-value of MAP at temperatures other than 72°C can be calculated. Using the calculated values for D, the number of survivors of MAP at particular temperatures can be obtained using equation 2. ASSIGNMENT This assignment is based on three exercises and answering a number of questions. Suggested input tables for recording the results of calculations are shown below. If there is sufficient interest I will expand this exercise and provide a facility to download a "Word" or PDF document at the end of this section. Part 1. Predict the effects of heat processing on the survival of MAP. Exercise 1, investigates the effect of the concentration of MAP and the holding time at 72°C, on the survival of MAP following HTST pasteurisation. Exercise 2, predicts the holding time at 72°C to reduce a specified concentration of MAP to a target number of survivors . Exercise 3, predicts the D-value for MAP at a range of temperatures. Exercise 4, investigates the effect of the concentration of MAP in raw milk, and the holding time at particular temperatures, on the survival of MAP following HTST pasteurisation. Using the calculators investigate the effect of HTST treatment on the survival of MAP using holding times of 15, 20 and 25 seconds and MAP concentrations of 10, 100, 10,000 and 1,000,000 CFU/ml. Use the input tables below to record your data. Using MAP concentrations of 10, 100 and 10,000 CFU/mL calculate how long the holding time at 72°C would have to be to reduce the number of survivors to 0.1 CFU/mL. Calculate the D 75, and D 80 values of MAP given that the Z value is 8.246 (Sung and Collins, 1998) and the D 71 value is 14 seconds. Using the D-values for MAP calculated at 75°C and 80°C predict the number of MAP that would survive 15 seconds at 75°C and 80°C assuming initial MAP populations of 100, 1000, 10,000 and 1,000,000 CFU/mL. Part 2.Test your understanding of the science, technology and societal issues concerned with MAP levels in milk. Discuss the survival of MAP during HTST pasteurisation of milk at 72°C. What HTST treatment of raw milk would you recommend to ensure that milk was free of viable cells of MAP? What do you understand by the statement "that milk does not contain viable cells of MAP?" Evaluate the case for specifying a minimal number of MAP in milk. If MAP was a pathogen what would be your advice for a standard specifying the number of MAP in milk? What actions would you recommend to lower the concentration of MAP in the farm and milking parlor environment in particular? Note wild animals including rabbits and foxes can host MAP. Explain the limitations of the models used to calculate the lethal effects of temperature on MAP. Input tables Table 1. Prediction of the number of viable cells of MAP surviving HTST treatment of milk at 72°C Initial number of MAP MAP (CFU/mL) surviving HTST treatment at 72°C Holding-time, seconds 15 20 25 10 100 1,000 10,000 1,000,000 Table 2. Prediction of the time at 72°C required to reduce the number of viable cells of MAP to 0.1/mL Initial numbers of MAP Time calculated to reduce the number of MAP surviving a HTST treatment at 72°C to 0.1/mL Holding time, seconds 10 100 1,000 10,000 1,000,000 Table 3 Prediction of the D-value of Mycobacterium avium subsp. paratuberculosis at 75°C and 80°C using the D 71 and associated Z values D-value, seconds Temperature, °C 75 80 Data set was taken from Sung and Collins (1998) and D-values determined using the Dairy Science and Food Technology D2 calculator and a D71 of 14 seconds and a Z value of 8.246. Table 4. Prediction of the number of viable cells of MAP surviving HTST treatment of milk using temperatures up to 80°C Initial numbers of MAP Predicted number of MAP survivors following HTST treatment at 72C, 75 and 80C 72 75 80 10 100 1,000 10,000 1,000,000 SELECTED LITERATURE Daniels, M.J., Hutchings, M.R., Beard, P.M., Henderson, D., Greig, A., Stevenson, K. and Sharp, J.M. (2003). Do non-ruminant wildlife pose a risk of paratuberculosis to domestic livestock and vice versa in Scotland? Journal of Wildlife Diseases. 39, 10-15. Food Standards Agency (2002). Strategy for the control of Mycobacterium avium subspecies paratuberculosis (MAP) in cows milk. [On-line] Available from: http://www.food.gov.uk/multimedia/pdfs/map_strategy.pdf. Accessed:10 December 2008. Grant, I.R., Ball, H.J., Neill, S. D. and Rowe, M. (1996). Inactivation of Mycobacterium paratuberculosis in cows' milk at pasteurisation temperatures. Appl. Environ. Microbiol. 62, 631-636. Grant, I. R., Ball, H. J. and Rowe, M. T. (1999). Effect of higher pasteurisation temperatures, and longer holding times at 72°C, on the inactivation of Mycobacterium paratuberculosis in milk. Letters in Applied Microbiology. 28, 461- 465. Grant, I.R., Hitchings, E.I., McCartney, A., Ferguson, F. and Rowe, M.T. (2002). Effect of commercial-scale high temperature, short time pasteurisation on the viability of Mycobacterium paratuberculosis in naturally infected cows’ milk. Appl. Environ. Microbiol. 68 (2): 602-607. Stumbo, C. R. (1973). Thermobacteriology in food processing, 2nd ed. Academic Press, New York.Sung, N. and Collins, M.T. (1998). Thermal tolerance of Mycobacterium paratuberculosis. Appl. Environ. Microbiol., 64, 999–1005. How to cite this article Mullan, W.M.A.(2008) . http://www.dairyscience.info/thermal-processing/161--mycobacterium-avium-subsp-paratuberculosis-.html . Accessed: 31 October, 2014. Text corrections made May 2013.