MAP in the Environment

Overview

The Paratuberculosis Problem

MAP in Water

MAP in Food

Overview

MAP in Dairy Products

MAP in Beef Products

Marshfield Clinic Study

Photo Gallery

MAP IN FOOD:
MAP in Dairy Products

Introduction

The purpose of this document is to review scientific research which has shown that live bacteria of the pathogenic bacterial species Mycobacterium avium subspecies paratuberculosis (MAP) may be present in the retail supplies of dairy products in the United States. This document contains a summary and detailed review of this scientific evidence, and a discussion of the human health implications of this evidence. 

Since MAP is not classified as a human pathogen, meat, milk and other products from animals infected with MAP may be continually entering the human food chain. There is a wealth of evidence which appears to indicate that MAP is capable of surviving the food processing methods that we employ to protect us from disease, such as cooking and pasteurization. This scientific evidence is thoroughly reviewed and discussed in this section as well as the  MAP in Beef Products, and Marshfield Clinic Study sections of our website.  

Summary

Scientific research has been conducted in the USA and Europe which demonstrates the following

• Mycobacterium avium subspecies paratuberculosis (MAP) causes a chronic and fatal intestinal disease in a wide range of animal species ¹.
  
  
• Mycobacterium avium subspecies paratuberculosis (MAP) has, on at least one occasion, been documented as crossing the species barrier to cause disease in a human ².

• MAP has been implicated in causing the human disease Crohn's disease ³. Crohn's disease is a chronic, debilitating and potentially fatal disease. The number of Americans who suffer from Crohn's disease has never been satisfactorily recorded, but is somewhere between 400,000 and 1,000,000 ³. Each year, at least 20,000 Americans are clinically diagnosed as having Crohn's disease ³.
  
  
• MAP infection in animals (Johne's Disease) bears extensive clinical, pathologic, and systemic similarity to Crohn's disease in humans ⁴.
  
  
• MAP bacteria isolated from the intestines of sufferers of Crohn's disease is of the bovine subtype, i.e. the bacteria originated in cattle. ⁵.
  
  
• MAP infection in cattle is known as Bovine Johne's Disease (BJD).
  
  
• Cattle with Bovine Johne's Disease shed live MAP in their milk and in their feces ^1,6.
  
  
• Milk in the USA is pasteurized by heating it to 71.7 °C (161 °F ) for 15 seconds. This process is known as "High Temperature Short Time" (HTST) pasteurization.
  
  
• MAP has been shown to be far more resistant to heat than Mycobacterium bovis, the bacterium which current milk pasteurization techniques are designed to kill ¹⁰. Strains of MAP cultured from the intestines of patients with Crohn's disease are the most heat resistant of all strains of MAP ¹⁰.
  
  
• Five independent research groups in three countries (USA ^7,9,10, Northern Ireland ^8,13, Australia ¹¹) have reported that MAP may be capable of surviving commercial milk pasteurization, and thus may be present in the retail milk supply.
  
  
• Milk taken from cattle with Bovine Johne's Disease and treated by the conditions of commercial pasteurization is considered unsafe to feed to newborn calves ⁹.
  
  
• Live MAP has been shown capable of surviving HTST pasteurization, as conducted by a commercial-scale HTST milk pasteurization unit, in research conducted in Australia ¹¹. The Australian researchers were able to culture MAP from milk which had been heated to 82 °C (179.6 °F ) for 15 seconds, demonstrating that MAP can survive temperatures which are 10.3 °C (18.5 °F ) higher than commercial milk pasteurization temperatures used in the United States.
  
  
• Live MAP has been cultured from the retail milk supply in England and Wales ¹². Both England and Wales use the same commercial milk pasteurization process as the United States.
  
  
• Between 20% and 40% of US dairy herds are infected with Bovine Johne's Disease ¹⁵.
  
  
• An estimated one-third of cheese in the United States is made from unpasteurized milk. ¹⁶
  
  

MAP in Milk from Cattle with Bovine Johne's Disease(BJD)

Cattle infected with Bovine Johne's Disease contaminate their milk with MAP in two main ways

1. Intracellular bacteria.  MAP is an intracellular pathogen that colonizes and multiplies in white blood cells of cows¹. Such white blood cells are filled with MAP. Since the milk of cattle, like that of all mammals, contains white blood cells, milk from cattle infected with BJD is certain to contain white blood cells which are infected with MAP.
    
2. Fecal contamination. Cattle heavily infected with BJD can shed up to 5 trillion (5,000,000,000,000) MAP bacteria per day, or over 100 million (100,000,000) MAP bacteria per gram (milliliter) of feces ¹. Cattle infected with BJD have uncontrollable diarrhea, which "sprays" from the cow's behind in a liquid form. Due to the close proximity of the cow's behind to its udders, it is nearly unavoidable that the infected cow's udders will be smeared with contaminated feces, thus leading to contamination of their milk with high numbers of MAP.
   

Cow with diarrhea, caused by BJD

MAP is More Heat Resistant Than Other
Disease-Causing Bacteria

Sung and Collins ¹⁰ used standard food microbiology techniques to determine whether or not MAP is capable of surviving commercial milk pasteurization. They measured the D-values and Z-values of MAP, which are the standard measurements needed to calculate the parameters of pasteurization. The higher the D-value of a bacterium, the more resistant is that bacterium to heat.

The parameters of HTST pasteurization were chosen to provide a wide safety margin for the destruction of disease causing bacteria which exist in milk, such as Listeria monocytogenes(Listeriosis), Salmonella typhimurium(Salmonellosis), Coxiella burnetti(Q Fever) and Mycobacterium bovis(Tuberculosis). Sung and Collins ¹⁰ demonstrated that MAP is more heat resistant than all of these bacteria, and thus may be able to survive the conditions of commercial milk pasteurization.

Sung and Collins ¹⁰ concluded that MAP is only killed by pasteurization if there are less than 10 MAP bacteria per milliliter of milk. Fecal contamination of cow's milk does occur, and a severely infected cow can shed up to 100 million MAP bacteria per gram (< 1 milliliter) of feces ¹, leading to the conclusion that more than 10 MAP bacteria are likely to be present in raw milk, and thus viable MAP bacteria may be present in retail milk that has been pasteurized. Sung and Collins presented a table of the times necessary to kill MAP when the bacterium is present in various concentrations.

Their results indicate that when 100 MAP bacteria per milliliter are present in milk, that milk must be treated at 71 °C for 23 seconds before all the bacteria are killed, i.e. 8 seconds longer than commercial milk pasteurization techniques used in the United States. When 1,000 MAP bacteria per milliliter are present, the milk must be treated at 71 °C for 35 seconds before all the bacteria are killed, i.e. 20 seconds longer than commercial milk pasteurization techniques used in the United States, etc.

Laboratory Simulations of HTST Pasteurization

Although laboratory simulations are, by definition, insufficient to definitively answer the real question: "Are the MAP bacteria present in retail dairy products alive or dead?", we must include a discussion of these simulations, because the U.S. Food and Drug Administration has chosen to base national dairy safety policy in relation to MAP on a single simulation of commercial milk pasteurization.

Several groups of researchers have conducted laboratory simulations of pasteurization. Laboratory simulations are, by definition, only an approximation of reality, and thus cannot give definitive answers as to whether or not MAP bacteria that are present in retail supplies of dairy products are alive. (To provide an analogy, relying on lab simulations to ensure that the retail dairy supply is free of contamination with live MAP is equivalent to asking airline passengers to fly in an aircraft that has only ever flown as a scale model in a wind-tunnel, or as a computer simulation.)

These laboratory simulations give rise to much argument, because of the parameters that researchers choose to implement in their experiment. Among the parameters which can vary between experiments are

• Number of MAP bacteria per milliliter.
  
  
• Pasteurization temperature.
  
  
• Number of different strains of MAP bacteria tested.
  
  
• Culture media used to detect viable (capable of multiplying) bacteria after pasteurization. Liquid culture media have been reported by Condron et al ¹¹, Grant et al ¹³ and Sung et al ¹⁰ to be more effective than solid culture media for culturing "sublethally injured bacteria".
  
  
• Length of culture time. In the veterinary world it is widely accepted that MAP cultures must be left for a minimum of 16 weeks before they can be declared negative. Bacteria that have been "sublethally injured" by pasteurization most likely require much more time than this. The shortest culture time used was by Stabel et al (USDA-ARS) ¹⁴, who cultured for a maximum of 12 weeks, whereas Sung et al ¹⁰ cultured for 8 months.
  
  
• Number of samples taken. It is widely accepted that a large number of samples must be cultured, in order to obtain results that have statistical significance.
  
  
• Agitation of milk. Commercial milk pasteurization machines stir milk to ensure that all particles of the milk are exposed to the pasteurizing heat.
  

Chiodini and Hermon-Taylor were the first researchers to find that MAP is capable of surviving pasteurization ⁷. They conducted laboratory simulations of the HTST pasteurization process, and were able to culture MAP from the pasteurized milk samples. Although they applied to the U. S. Department of Agriculture in 1993 and 1994 for funding to conduct more detailed studies, their grant applications were refused on three separate occasions (see "Prevalence of Mycobacterium Paratuberculosis in Retail Dairy Products, 3rd Revision" for details).

In February 1996, Grant et al described the results of their first laboratory simulation of HTST pasteurization ⁸. They added between 1,000 and 10 million MPTB bacteria per milliliter to milk and submitted that milk to pasteurization in a test tube. They cultured their samples for up to 18 weeks, in liquid and solid culture media. In milk where they had added between 1 million and 10 million bacteria per milliliter to milk, they were able to culture MAP from 29 out of 34 (85%) of the samples. In milk where they had added between 1,000 and 10,000 bacteria per milliliter to milk, they were able to culture MAP from 19 out of 33 (58%) of the samples.

In 1998, Grant et al described the results of their second laboratory simulation of HTST pasteurization ¹³. Their first study had been criticized for adding excessively high numbers of MAP bacteria to milk. Their second paper was entitled "Effect of high-temperature, short-time (HTST) pasteurization on milk containing low numbers of MAP". In this study, Grant et al added between 0.2 and 1,000 MPTB bacteria per milliliter of milk. They cultured their samples for 18 weeks, in liquid and solid culture media. In milk where they had added 1,000 bacteria per milliliter to milk, they were able to culture MAP from 4 out of 27 (14.8%) of the samples. In milk where they had added 100 bacteria per milliliter to milk, they were able to culture MAP from 3 out of 30 (10%) of the samples. They concluded "HTST pasteurisation was only completely effective when < 10 CFU of Myco. paratuberculosis/ml were present in the milk."

Meylan et al tested the survival of MAP in pasteurized colostrum from cattle infected with BJD ⁹. Colostrum is the name given to the first milk produced by cattle after they have given birth. They found that MAP survived their simulation of the conditions of pasteurization. The researchers concluded :- "The pasteurization procedure did significantly reduce, but did not completely eliminate M. paratuberculosis from the pasteurized colostrum samples". They go on to state that "[Pasteurization is] not sufficient by itself to prevent potential infection of newborn calves". It is for this reason that dairy farmers are advised, in order to combat Bovine Johne's Disease, not to feed milk from BJD-infected cattle to newborn calves, whether it has been pasteurized or not.

Condron et al conducted a study of HTST pasteurization in Australia ¹¹. In contrast to other researchers who used laboratory scale equipment to simulate pasteurization, Condron et al used a commercial-scale pasteurizer unit for their simulation of pasteurization. They were able to culture live MAP from 8 of 33 contaminated samples heated to 72 °C (161.6 °F ) for 15 seconds, from 1 of 10 samples which had been contaminated with a human strain of MAP (strain Ben, which was originally cultured from a patient with Crohn's disease named Ben) and heated to 73 °C (163.4 °F ) for 15 seconds, and also from 1 of 18 samples which had been heated to 82 °C (179.6 °F ) for 15 seconds. This last result indicates that MAP may be capable of surviving temperatures that are a full 10.3 °C (18.5 °F ) above HTST pasteurization temperatures used in the United States.

Although the validity and accuracy of laboratory simulations of commercial HTST pasteurization is open to interpretation, this experiment provides solid evidence, through the use of an actual, commercial-scale HTST pasteurization unit, that MAP can survive HTST pasteurization and thus may be present in the retail milk supply.
 

USDA Research

One study that found that MAP did not survive a simulation of HTST pasteurization was conducted on behalf of the USDA-ARS (US Department of Agriculture - Agricultural Research Service) by Stabel et al ¹⁴. The USDA researchers used a "laboratory-scale pasteurizer" to conduct simulations of commercial pasteurization. They found that MAP did not survive their simulation of HTST pasteurization. However, their choice of parameters for their simulation are highly controversial. In particular, the treatment to which the USDA researchers the bacteria were subjected is widely questioned:-

• Starved: The USDA researchers grew their supply of MAP bacteria "until they reached a concentration of 10⁸ to 10⁹ cells per ml (A540=1.15)", meaning that the bacteria had left "log-phase growth" and were in decline through lack of available nutrients, i.e. they were weakened by starvation before they were submitted to pasteurization.
  
  
• Frozen: The USDA researchers stored their bacteria at -80 °C (-112 °F ). Storage of MAP at this temperature has been demonstrated to kill a percentage of the bacteria ¹⁷, obviously leaving whatever bacteria were left alive "sublethally injured". When ice crystals form inside bacteria, the bacteria's DNA chains are severed and their cell walls are sliced open.
  
  
• Blasted: Before submitting them to pasteurization, the USDA researchers "sonicated" the MAP bacteria, at 35 watts for 15 seconds. Sonication is a process used in vaccine preparation to kill and rip apart bacteria, using resonant sound waves.
  
  
• Heated: This is the only way that the USDA researchers should have treated the MAP bacteria, i.e. the only adverse conditions that the bacteria would be submitted to under real-world pasteurization. Note, however, that the USDA researchers used heat treatment of 72 °C (161.6 °F ), which is 0.3 °C higher than the 71.7 °C (161.0 °F ) used in commercial HTST pasteurization in the United States.
  
  
• Blasted again: After pasteurizing the MAP bacteria, The USDA researchers "sonicated" the bacteria again at 35 watts for 15 seconds.
  
  
• Inadequate culture time: The USDA researchers cultured their samples for between 4 and 12 weeks. In the veterinary world, MAP cultures are held for a minimum of 16 weeks before they are declared negative. This is particularly important for bacteria that have been "sublethally injured" by pasteurization (and "starvation", and "freezing" and "blasting" and re-"blasting"). In contrast, Sung and Collins ¹⁰ cultured their samples for 8 months (32 weeks).
  
  
• Inadequate culture media: The USDA researchers used solid culture media (HEYM), as opposed to the liquid culture media used by all other researchers in pasteurization studies. All of the other researchers that have studied pasteurization have found the liquid culture media to be more sensitive at recovering MAP that had been "sublethally injured" by pasteurization. To quote Grant et al ¹³:- "If only conventional culture on HEYM had been used to detect survivors after HTST pasteurisation in this study, the number of milk samples containing viable Myco. paratuberculosis would have been considerably underestimated".
  
  

The scientific methods used in the USDA-ARS study can be criticized for the following reasons:

• The USDA researchers omitted necessary details in describing their experiment, making it impossible to reproduce the research. For example, the numbers of bacteria that the USDA researchers added to milk are not definitively specified. They state:- "In these experiments, raw milk (1 to 2 liters) was inoculated with two concentrations of M. paratuberculosis (10⁴ and 10⁶ cells per ml) and mixed thoroughly prior to introduction into the holding vessel." However, they do not specify if "10⁴ and 10⁶ cells per ml" is the concentration of the inoculum or the final concentration in milk.
  
  
• The USDA researchers studied only 2 strains of MAP. In contrast, Grant et al studied 10 and 11 strains respectively in their two experiments ^8,13.
  
  
• The USDA-ARS researchers did not conduct enough control experiments to ensure that their methodology was correct, e.g. they should have run their simulation without heating the milk at all, to assure them that the MAP bacteria were not sticking to the inside of the holding tube, for example.
  
  
• The USDA researchers based their conclusions about HTST pasteurization on the results of 4 culture samples, making it impossible to draw any statistically significant conclusions from their work. In contrast Condron et al ¹¹ cultured 296 samples, and Grant et al cultured 123 and 114 samples in their two respective experiments ^8,13.
  
  
• The USDA researchers did not use standard food microbiology techniques to conduct their experiments, i.e. they did not measure D-values or Z-values.
  

It should be noted that the U.S. Food and Drug Administration has chosen to base U.S. national dairy safety policy in relation to MAP on this USDA research, a single simulation of commercial milk pasteurization, and one which disagrees with at least ten other documented studies.

Live MAP Has Been Cultured from Retail Milk Supplies in England and Wales

In 1996, Hermon-Taylor et al described the results of their search for MAP in milk purchased from retail outlets across England and Wales ¹². They found that on average 7% of retail milk samples contained DNA from MAP, with up 25% of retail milk samples testing positive during Fall and Winter. The finding of DNA does not prove that the bacteria were alive, since DNA is present in both dead and live bacteria. However, the finding does prove that a substantial proportion of milk submitted for pasteurization and eventual retail distribution in those countries is from cattle infected with BJD.

The researchers attempted to culture samples of milk that had tested positive for the presence MAP DNA and samples that had tested negative for the presence of paratuberculosis DNA (i.e. samples where the level of paratuberculosis DNA present was below the detection sensitivity of the experiment).

After 13 to 40 months of culture, the positive samples developed small clumps of mycobacteria which tested positive for paratuberculosis DNA, but which could not be definitively identified as MAP because of extreme overgrowth by other organisms present in the milk, such as fungi and species of bacteria which grow faster than MAP. (This problem of overgrowth is only encountered when testing actual retail milk. Simulation studies all have used sterile milk which is guaranteed to be free of these contaminating organisms).

However, the culture results from the negative samples were conclusive. Samples which had tested negative for the presence of MAP DNA were cultured for between 13 and 40 months. 16% of these samples changed from testing negative to testing positive for MAP DNA after culture. This increase in the amount of MAP DNA from below the detection limit of the experiment to above the detection limit of the experiment can only be explained by the presence of live and multiplying MAP bacteria. DNA from dead bacteria cannot multiply.

In May of 2002, a study was published, entitled "Incidence of Mycobacterium paratuberculosis in Bulk Raw and Commercially Pasteurized Cows' Milk from Approved Dairy Processing Establishments in the United Kingdom"  (Grant, I.R.; Ball, H J.; Rowe, M.T.  Applied and Environmental Biology, May 2002, p. 2428-2435, Vol. 68. No. 5).¹⁴   Results from this study confirmed that MAP survives pasteurization standards used in the UK, pasteurization standards which equal or exceed those used in the U.S.
 

MAP in Cheese

As noted by McDowell and McElvaine ¹⁶, an estimated one-third of cheese on sale in the United States is made from unpasteurized milk. The presence of MAP in retail cheese has not been studied anywhere in the world.

Discussion

Whether or not live MAP are present in retail supplies of milk and derived dairy products is a question that many researchers have tried to answer. The overwhelming majority of laboratory simulation studies found that the bacterium was able to survive pasteurization. Evidence from the study which used a commercial-scale pasteurizer demonstrated most forcefully that the bacterium can indeed survive commercial milk pasteurization.

The study published by Grant, et al (referenced above) entitled "Incidence of Mycobacterium paratuberculosis in Bulk Raw and Commercially Pasteurized Cows' Milk from Approved Dairy Processing Establishments in the United Kingdom"  confirmed that MAP survives pasteurization standards used in the UK, pasteurization standards which equal or exceed those used in the U.S.

Therefore, the debate should be over!  MAP does survive pasteurization.  Regardless, despite MANY pleas from PARA since mid 1997,  the USDA and FDA have not taken measures to ensure that the food we give our children is free from contamination with MAP.
 

Instead, the federal government is funding more "simulation" studies.  

What must be remembered is that the question "Can MAP survive commercial milk pasteurization?" is a very different question from "Are the MAP bacteria present in the retail milk supply alive or not ?". Research which attempts to answer the first question is necessarily based upon assumptions and approximations of reality.

In the words of Chiodini:- "Funding laboratory studies is misguided simply because they are laboratory studies, and do not reflect the true conditions of a commercial pasteurization study, even with 'mini-pasteurizers'". He also says :- "The definitive evidence needed is to determine not if MAP can survive pasteurization, but whether or not the MAP found in retail milk are viable".

It is vital to answer this question urgently. Hermon-Taylor et al, on Feb 7 ^th 1998, published the first known case of MAP being found in relation to disease in a human ². In the author's own words:

Meylan et al ⁹ noted that:- "[Pasteurization is] not sufficient by itself to prevent potential [MAP] infection of neonate calves". If pasteurized milk from cattle with BJD is not safe for consumption by newborn calves, is it safe for consumption by humans, and particularly, is it safe for consumption by human infants?

Since an estimated one-third of cheese on sale in the United States is made from unpasteurized milk ¹⁶, it is also imperative to determine whether or not retail supplies of cheese are a vehicle for human infection with MAP. The same questions apply to butter, yogurt, cream and other dairy-derived products.

If, as extensive research strongly suggests, MAP is a cause of Crohn's disease, then any such potential contamination of retail dairy supplies may be responsible, in part or in whole, for the 20,000 new cases of Crohn's disease diagnosed each year in the United States.

In 1993 and 1994, Chiodini and Hermon-Taylor submitted a proposal to test the retail milk supply for the presence of viable MAP to the USDA National Research Initiative Cooperative Grants Program. That proposal was rejected three times. To quote that proposal:-

PARA deeply regrets that these important questions were not answered in 1993, and have still not been answered in 2003. Since 1993, at least 200,000 Americans have been diagnosed with Crohn's disease, many of them children. As long as these questions remain unanswered, hundreds of thousands of Americans will continue to suffer, perhaps unnecessarily because the cause of their illness could have been prevented.

Marshfield Clinic is Testing Retail Milk in the U.S.

The Marshfield Clinic in Marshfield, Wisconsin, is conducting a study involving the testing of retail milk for the presence of MAP.  This is milk taken from supermarket shelves.  Results of that study will be published in the summer of 2003.   (For more information about this, please visit the Marshfield Clinic section of our website.)

The FDA was contacted by PARA for a comment on the Marshfield Study and clarification of their position about the safety of retail milk supplies in the United States, in light of the publication of the Study by Grant, et al in May of 2002.  Not surprisingly, no one at FDA's CFSAN would speak with PARA, instead referred PARA to the Risk Assessment Department, to their attorneys.   Sadly, it appears that FDA considers PARA, the organization, more of a risk than PARA, the bacterium.  FDA's position remains the same: 

They will take no action! 
They will not exert the precautionary principle.

Because of the mounting evidence, it is now long past the time for food safety regulators in the United States (FDA, FSIS, USDA) to take immediate measures to protect the public by eliminating MAP from the food supply.

The Dairy Industry

The dairy industry is aware of the research which shows evidence of an association between Bovine Johne's Disease and Crohn's disease. However, the industry apparently prefers to do nothing about the presence of MAP in its products until it is proven beyond all possible doubt that MAP does in fact cause Crohn's disease. Essentially, the dairy industry is gambling that MAP is not the cause of Crohn's disease, a strategy which allows them to continue to ignore Bovine Johne's Disease, as they have done for fifty years.

Of course, the dairy industry is gambling with the health of consumers of dairy foods, as well as their own financial health. If conclusive evidence ultimately appears that MAP is actually a cause of Crohn's disease, consumers will lose faith in dairy products as healthy and nutritious foods. The market for dairy foods may collapse, just as the market for British beef collapsed when British consumers discovered that they could contract Creutzfeld-Jakob Disease (CJD) from eating beef from cattle with Mad Cow Disease (BSE).

Instead of tackling the difficult problem of Mad Cow Disease, the British beef industry spent all of their money and energy on PR campaigns reassuring the consumer that British beef was completely safe. When the proof finally did arrive that Mad Cow Disease did cause Creutzfeld-Jakob Disease in humans, beef consumers realized the hollowness and false basis of these PR campaigns, and both the home and export markets for British beef collapsed. The British beef industry suffered a devastating blow, from which it has not yet recovered and may never recover. British beef, previously perceived as healthy food, necessary for good nutrition, is now perceived in Britain and elsewhere as tainted and potentially health-destroying.

The British beef industry gambled that Mad Cow Disease could not cause disease in humans. They lost their bet. PARA wishes to send the following message to the U.S. dairy industry:-

References

Hyperlinked references will appear in a new browser window

Mycobacterium avium subspecies paratuberculosis is a pathogenic (disease-causing) bacterium, which causes chronic intestinal disease in a wide range of animals, including sub-human primates and cattle. Mycobacterium avium subspecies paratuberculosis has been associated with disease in humans. Mycobacterium avium subspecies paratuberculosis cultured from the intestines of sufferers of Crohn's disease is of the bovine subtype.

1	1984		Ruminant Paratuberculosis (Johne's disease):- The current status and future prospects Chiodini RJ, Van Kruiningen HJ, Merkal RS Cornell Veterinarian 74(3):218-262
2	1998		Mycobacterium paratuberculosis Cervical Lymphadenitis followed five years later by terminal ileitis similar to Crohn's Disease Barnes N, Clarke C and Finlayson C, Hermon-Taylor J British Medical Journal 7 th Feb 1998.
3	1998		Scientific facts about Mycobacterium paratuberculosis and Crohn's disease Source: http://members.aol.com/ParaTBweb/crohn.htm
4	1989		Crohn's disease and the mycobacterioses: a review and comparison of two disease entities Chiodini RJ Clin Microbiol Rev 2:90-117
5	1995		Characterization by restriction endonuclease analysis and DNA hybridization using IS900 of bovine, ovine, caprine and human dependent strains of Mycobacterium paratuberculosis isolated in various localities. Pavlík I; Bejcková L; Pavlas M; Rozsypalová A; Kosková S Vet Microbiol:1995:45:311-318.

Mycobacterium avium subspecies paratuberculosis is shed in the milk of cattle infected with BJD. In four out of six pasteurization simulation studies it was shown that Mycobacterium avium subspecies paratuberculosis may be capable of surviving pasteurization. Live Mycobacterium avium subspecies paratuberculosis has been detected in the retail milk supply of the United Kingdom.

6	1992	USA	Mycobacterium paratuberculosis cultured from milk and supramammary lymph nodes of infected asymptomatic cows Sweeney RW; Whitlock RH; Rosenberger AE J Clin Microbiol, 30:166-171.
7	1993	USA	The thermal resistance of Mycobacterium paratuberculosis in raw milk under conditions simulating pasteurization Chiodini RJ; Hermon-Taylor J J Vet Diagn Invest, 5:629-631.
8	1996	N. Ireland	Inactivation of Mycobacterium paratuberculosis in cows' milk at pasteurization temperatures Grant IR; Ball HJ; Neill SD; Rowe MT Appl Environ Microbiol 62:631-6
9	1996	USA	Survival of Mycobacterium paratuberculosis and preservation of immunoglobulin G in bovine colostrum under experimental conditions simulating pasteurization. Meylan M, Rings DM, Shulaw WP, Kowalski JJ, Bech-Nielsen S, Hoffsis GF Am. J. Vet Res. 57:1580-1585
10	1998	USA	Thermal tolerance of Mycobacterium paratuberculosis. Sung N, and MT Collins Applied and Environmental Microbiology 64(3),999-1005.
11	1996	Australia	Pasteurization of Mycobacterium paratuberculosis in whole milk AF Hope, PA Tulk, and RJ Condron Proceedings of the Fifth International Colloquium on Paratuberculosis:p377-382.
12	1996	UK	IS900 PCR to detect Mycobacterium paratuberculosis in retail supplies of whole pasteurized cows milk in England and Wales Millar D, et al Applied and Environmental Microbiology:1996 Sept:pages 3446-3452.
13	1998	N. Ireland	Effect of high-temperature, short-time (HTST) pasteurization on milk containing low numbers of Mycobacterium paratuberculosis. Grant, I.R., Ball, H.J. and M.T. Rowe Letters in Applied Microbiology:1998: 26 (2), 166-170.
14	2002	N. Ireland	Incidence of Mycobacterium paratuberculosis in Bulk Raw and Commercially Pasteurized Cows' Milk from Approved Dairy Processing Establishments in the United Kingdom   Grant, I.R., Ball, H.J. and M.T. Rowe.  Applied and Environmental Biology, May 2002, P. 2428-2435, Vol 68, No. 5

Between 20% and 40% of US dairy herds are infected with Bovine Johne's Disease. An estimated one-third of cheese on sale in the United States is made from unpasteurized milk.

15	1997		Johne's Disease on U.S. Dairy Operations US Department of Agriculture report, October 1997.
16	1997		Long-term Sequelae to foodborne disease McDowell RM and McElvaine MD 1997, Rev. sci. tech. Off. int. Epiz: 16(2), 337-341
17	1981		Effects of physical and chemical factors on the viability of Mycobacterium paratuberculosis. Richards WD 1981 Nov, J Clin Microbiol, 14(5):587-8
18	1973		Thermobacteriology in food processing, 2nd ed. Stumbo, C. R. (ed.) 1973. Academic Press, New York, N.Y.
19	1981		Food processing technology. In W. T. Hubbert and H. V. Hagstad (ed.), Food safety and quality assurance. William, T. H., H. V. Hagstad, and E. Spangler 1991. Iowa State University Press, Ames, Iowa.