This report is based on a colloquium, sponsored by the American Academy of Microbiology, convened June 15-17, 2007, in Salem, Massachusetts.
The American Academy of Microbiology is the honorific leadership group of theAmerican Society for Microbiology. The mission of the American Academy ofMicrobiology is to recognize scientific excellence and foster knowledge andunderstanding in the microbiological sciences. The Academy strives to includeunderrepresented scientists in all its activities.
The American Academy of Microbiology is grateful for the generosity of thefollowing organizations for support of this project: U.S. Department of Agriculture, Animal and Plant Health Inspection Service (APHIS) U.S. Department of Agriculture, Cooperative State Research, Education, and Extension Service (CSREES) U.S. Department of Agriculture, CSREES, Johne's Disease Integrated Program(USDA-CSREES-NRI Award No. 2007-01019) Canadian Foundation for Infectious Diseases Crohn's & Colitis Foundation of Canada Kauver Foundation Sequella, Inc.
TREK Diagnostic Systems, Inc.
The opinions expressed in this report are those solely of the colloquiumparticipants and do not necessarily reflect the official positions of our sponsors or the American Society for Microbiology.
R. John Collier, Ph.D. (Chair) Carol A. Nacy, Ph.D. (Chair) Harvard University Medical School Sequella, Inc., Rockville, Maryland Kenneth I. Berns, M.D., Ph.D.
Marcel A. Behr, M.D.
University of Florida Genetics Institute Montreal General Hospital, Montreal,Québec, Canada Edward DeLong, Ph.D.
Massachusetts Institute of Technology Charles N. Bernstein, M.D.
University of Manitoba, Winnipeg, E. Peter Greenberg, Ph.D.
University of Washington Judith Lipton, M.D.
Carol A. Gross, Ph.D.
Redmond, Washington University of California, San Francisco Mary E. Torrence, D.V.M., Ph.D.
Lonnie O. Ingram, Ph.D.
U.S. Department of Agriculture (ARS), University of Florida Greenbelt, Maryland J. Michael Miller, Ph.D.
Carol Colgan, Director Centers for Disease Control American Academy of Microbiology Stephen A. Morse, Ph.D.
Centers for Disease Control and Prevention Merry Buckley, Ph.D.
Ithaca, New York Edward G. Ruby, Ph.D.
University of Wisconsin-Madison Patricia Spear, Ph.D.
Northwestern University George F. Sprague, Jr., Ph.D.
MYCOBACTERIUM AVIUM PARATUBERCULOSIS: Infrequent Human Pathogen or Public Health Threat? Yrjo T. Grohn, D.V.M., Ph.D.
Cornell University Douwe Bakker, Ph.D.
Michael A. Carter, D.V.M., M.P.H.
Central Institute for Animal John Hermon-Taylor, FRCS, MChir National Johne's Disease Program, Disease Control, CIDC-Lelystad, St. George's Hospital Medical USDA-APHIS Veterinary Services, School, London, England Riverdale, Maryland Marcel A. Behr, M.D.
Vivek Kapur, Ph.D.
Ian Durrant, Ph.D.
Montreal General Hospital, Montreal, University of Minnesota Oxford Immunotec Ltd., Abington, Oxfordshire, England (grant support from the Broad Preston Linn, M.S.
Foundation and the Crohn's & Colitis Durham, North Carolina Foundation of Canada) Giaconda Limited, Sydney, New Judith Lipton, M.D.
South Wales, Australia Charles N. Bernstein, M.D.
Redmond, Washington University of Manitoba, Winnipeg, Biljana Mihajlovic Elizabeth J.B. Manning, (grant support from the Crohn's & D.V.M., M.P.H.
Colitis Foundation of Canada) School of Veterinary Medicine, Morris E. Potter, D.V.M.
University of Wisconsin-Madison U.S. Food and Drug Administration, Edgar C. Boedeker, M.D.
New Mexico VA Health Science Carol A. Nacy, Ph.D.
Center, Albuquerque, New Mexico Sequella, Inc., Rockville, Maryland Nadine M. Sullivan, Ph.D.
(founder, chief executive officer, and TREK Diagnostic Systems, Inc., Thomas J. Borody, M.D., Ph.D.
chair of the board, Sequella, Inc.) Sun Prairie, Wisconsin Center for Digestive Diseases, FiveDock, New South Wales, Australia Saleh A. Naser, Ph.D.
Ronald E. Weinberg, M.B.A.
(grant support from the Broad University of Central Florida, Hawk Corporation, Cleveland, Ohio Foundation and shareholder and chief medical officer, Giaconda) (grant support from the BroadFoundation) Sheldon T. Brown, M.D.
Veterans Affairs Medical Center, Mihai G. Netea, M.D.
Radboud University Nijmegen MedicalCenter, The Netherlands Michael T. Collins, D.V.M., Ph.D.
School of Veterinary Medicine, Warwick Selby, MBBS, M.D., FRACP University of Wisconsin-Madison University of Sydney, New South (consultant: BD Diagnostic Systems) Daniel N. Frank, Ph.D.
Mary E. Torrence, D.V.M., Ph.D.
EXECUTIVE SUMMARY Crohn's Disease (CD) is a devastating illness in search of a cause and a cure.
More than 800,000 people in North America suffer from CD, a gastrointestinal disorder characterized by severe abdominal pain, diarrhea, bleeding, bowel obstruction, and a variety of systemic symptoms that can impede the ability to lead a normal life during chronic episodes that span months to years.
Researchers and clinicians agree that onset of CD requires a series of events; implicated are certain inherited genetic traits, an environmental stimulus, and an overzealous immune and inflammatory response. The combination of these factors contributes to a disease whose course is variable among patients and whose symptoms range from mild to devastating on any given day. The economic and social impact of this disease is substantial for the patient, the family, the community, and the healthcare system.
Long considered an autoimmune and chronic inflammatory disorder, current CD therapies are designed to treat symptoms of overactive inflammation in the gut. Chronic inflammation, however, does not generally induce itself. Inflammation is normally caused by a "foreign body," an inanimate object (i.e., splinter) or animate objects like rogue cells (i.e., cancer) or microorganisms (i.e., bacterium, virus, or fungus). Until the cause of inflammation is eliminated, the body continues to send in its clean-up crew, the white blood cells of inflammation whose job it is to expel the tissue invader. Inflammation only subsides when the causative agent is finally banished.
There is suspicion, supported by reports of genetic inability to interact appropriately with certain bacteria or bacterial products in some patients, that CD may have a currently unrecognized infectious origin, perhaps environmentally derived. That CD is a set of wide-ranging symptoms, more like a syndrome than a specific disease, suggests that if its origin is microbial, more than one etiologic agent may ultimately be identified. Bacterial suspects at the moment include a Mycobacterium and a variant of the normal bacterial flora of the gut, Escherichia coli. The possibility of more than one infectious cause that leads to a similar set of symptoms confounds the research agenda to find both a cause and a cure for CD.
One acknowledged potential microbial agent of CD is Mycobacterium avium sub- species paratuberculosis (MAP), a microorganism that causes a gastrointestinal disease similar to CD in ruminants, including dairy cattle, called Johne's disease (or paratuberculosis). People with CD have 7:1 odds of having a documented pres- ence of MAP in blood or gut tissues than those who do not have CD, thus the association of MAP and CD is no longer in question (see Figure 1, page 11). The critical issue today is not whether MAP is associated with CD, but whether MAP causes CD or is only incidentally present, not an inciter or participant in the disease process.
If MAP is involved in the disease process of CD or other gastrointestinal disor- ders, then we need to determine how people are exposed to this microorganism, how to prevent that exposure, and how to treat the infection.
Despite its prevalence in the U.S. population in numbers that exceed most cancers, CD is not a focus of research attention in the same way as these other feared diseases. The American Academy of Microbiology convened a colloquium with experts in medicine, microbiology, veterinary pathology, epidemiology, infectious diseases, and food safety to describe the state of knowledge about the relationship between MAP and CD and to make recommendations for effective research that will move the field forward.
The general consensus of the assembled experts was that there are certainly reasons to suspect a role for MAP in CD: MAP persists in contaminated soil and water, which links the environmental factor of CD to the disease.
MAP has a cell wall that contains muramyl dipeptide (MDP). One genetic trait that is affiliated in certain patients with CD is the NOD2 gene, which regulates ability to respond appropriately to MDP, thus the link between the genetic trait and MAP, or other bacteria.
MAP causes Johne's disease, an illness of cattle and other ruminants that has many similarities with CD. The similarities of MAP disease in animals, for which the etiologic agent is known, and CD, for which the etiologic agent is unknown, provide a symptomatic link between agent and disease.
MAP can survive standard milk pasteurization processes and has been identified in off-the-shelf milk in retail grocery stores in the U.S. and the Euro- pean Union (E.U.). There is increasing concern that MAP can also be found in cheese made from the milk of MAP-infected cattle and meat from Johne's diseased animals. These observations could provide the exposure route of CD patients to MAP.
Treatment of some CD patients with antibiotics that have activity on certain other Mycobacteria, although not specifically selected for their activity against MAP, provides short-term or long-term relief or remission of symptoms.
Circumstantially, these observations appear to make a compelling case for MAP as involved in CD. On the other hand, the ability to definitively identify MAP as the cause of CD, or the cause of a significant number of CD cases, has been stymied by the elusive characteristics the organism itself, the lack of broadly available and validated clinical tools to easily and definitively identify MAP in accessible tissues, and the late symptomatic stage at which CD is finally diagnosed, where the origin of the destructive inflammation could have MYCOBACTERIUM AVIUM PARATUBERCULOSIS: INFREQUENT HUMAN PATHOGEN OR PUBLIC HEALTH THREAT?
been years before the patient sought medical care. Most important, however, is the lack of resources, financial and scientific, to generate the tools that clinicians and patients need to determine whether MAP is involved in the disease process or not.
Several important clinical trials of antibiotics have been attempted in CD patients, with variable results. Treating CD patients with existing antibiotics with activity against other Mycobacteria (M. tuberculosis, which causes TB, and M. avium complex, or MAC, which is pathogenic in immune compromised persons) have either failed to provide relief (TB drugs) or produced promising outcomes for some patients, but not all (MAC drugs). Confounding these clinical results is the lack of information about which patients in the clinical trial population were actually infected with MAP, and whether any MAP organisms in infected patients were susceptible to the antibiotics used in the trials. Without sensitive and specific diagnostics that can detect early MAP infection, knowledge of how and where to isolate MAP for antibiotic susceptibility studies, and drugs that are known to be active against MAP itself, alone or in combination, the role of MAP in CD will remain circumstantial and the controversy over CD etiology will continue.
There is little known about where exactly viable MAP can be found in human tissues or, since most pathogenic Mycobacteria are intracellular, in which cells MAP can live and grow in humans. While the site of infection and tissue pathologies of MAP in animals can be assessed at necropsy, there is enough dissimilarity between digestive processes of ruminants and humans that this information may not necessarily inform studies in humans.
Of concern from a public health perspective is the ongoing presence of MAP disease in commercial livestock that supply the U.S. with dairy and meat products.
If, in fact, CD is a zoonotic infection (one that is passed from animals to humans) and MAP is the (or one) cause of CD, then early identification of MAP disease in veterinary practice and appropriate management of these animals to safeguard the food supply will be critical to guard the public health.
Even in animals, it is nearly impossible to diagnose Johne's disease in the early stages of disease. Diagnosis is by a combination of clinical observation (wasting and reductions in milk production in dairy cattle, for instance) and microbiological, histopathological, and immunological testing of Johne's disease suspects. Although efforts to eliminate Johne's disease and MAP from livestock herds are ongoing, the lack of an accurate and easily-administered diagnostic for early disease onset is hampering these efforts. The results are mixed, and food products containing MAP or MAP DNA can be found on supermarket shelves. Veterinary diagnostics that are sensitive (detect MAP at early stages of infection) and specific (identify MAP and not other microorganisms) will be necessary to eliminate Johne's disease from the commercial food supply. Research to discover and validate these techniques may also shed light on diagnosis of human disease.
Colloquium participants agreed that research to elucidate the role of MAP in CD must address two major unknowns: (1) whether MAP from livestock and other ani- mals is transmissible to humans and how it is transmitted and (2) whether humans are susceptible to infection and disease after exposure to MAP. No single study will fill all the gaps in our understanding of the possible relationship between MAP and CD. Furthermore, participants agreed that validated, reproducible biological markers confirming human MAP infection are desperately needed. If MAP can be causally associated with CD using reproducible analytical techniques, appropriate patient populations can be treated with antibiotics that are selected for their MAP activity.
Then, at least MAP-infected CD patients will have both a cause and a cure.
CD was named after Burrill B. Crohn, an American physician who published a paper in 1932 clearly distinguishing CD from intestinal TB (1). CD is a syndrome characterized by chronic and debilitating inflammation of the gastrointestinal tract that can be accompanied by mild to excruciating pain, frequent bouts of diarrhea, and malnutrition due to rapid passage of food through the inflamed intestinal tract. Some patients must be fed intravenously during the most difficult of their episodes. These devastating and episodic symptoms can force patients to maintain a limited work schedule or to refrain from working altogether, and the psychological effects of the disease are profound. The uncertainty of their condi- tion and the ever-present possibility of symptomatic flare-ups often drive patients into anxiety, depression, and isolation. As of 2001, the Centers for Disease Control and Prevention estimated that over 500,000 people in the U.S. are living with CD (2), and more recent statistics show that 800,000 people in North America and 1 in 1000 persons in westernized countries have diagnosed CD. In the U.S., roughly 50% of CD patients are children. There is currently no cure for CD.
With the right anti-inflammatory drug or immunomodulatory biologic regimen, CD patients can experience temporary remission of symptoms, but long-term flare-ups inevitably follow, coming on suddenly or developing gradually over time.
Surgery to remove inflamed sections of the bowel is the only option to alleviate the symptoms of many people living with CD. Many other patients endure endless regimens of drugs to alleviate some of their symptoms, but these drugs, too, have side effects, some that can even be life threatening.
Table 1. Drugs for Therapy of Crohn's Disease, 20081
acute, then taper acute use only,taper acute increasethen taper acute, then taperif possible cost offset bymedicare n/a in U.S. Free inrest of world BORODY COCKTAIL (CLARITHROMYCIN, RIFABUTIN, CLOFAZIMINE)
Table 1. Drugs for Therapy of Crohn's Disease, 20081
Cost of infusion same as AWP Initialcost = $7,626.00 Cost of infusion infusion total cost $6,000/2 mo Immune suppressant,now considered TNF inhibitor STEM CELLS
Risk: sepsis/death Narcotic, not for IMMUNE STIMULANTS - PARASITES
1 Table compiled by J. Lipton, M.D. with assistance from F. Cunningham (UC Berkeley, CA) and I. Barash, Ph.D., M.D. (UCSD). 2 Average Wholesale Prices (AWP), provided B. Faulkner, Woodinville Medical Center Pharmacy, Woodinville, WA 98072 USA on February 29, 2008. NOTE: AWP change from day to day based on information from the drug manufacturers and/ or by calculations made by drug wholesalers. The AWP does not always accurately reflect the retail cost of a drug, especially in the case of many generic drugs which canbe purchased and sold below the AWP in many markets. AWP is the price used in comparing prices of drugs, although for some drugs it is not an accurate reflection of cost to the patient.
For decades, CD was considered an autoimmune disease, where the immune system erroneously identifies tissues of the gastrointestinal tract, specifically the large intestine, as "altered" or "foreign" and begins a relentless attack. In recent years, however, the pathologies associated with CD have been observed in other conditions that affect the regulation of immune and inflammatory cells to cope with bacterial pathogens, such as Chronic Granulomatous Disease (CGD) and Chronic Variable Immunodeficiency (CVI). Moreover, studies of white blood cells present in tissue biopsies from CD patients demonstrate a dysfunction in the abil- ity of these cells to ingest particles, a preliminary event for clearance of bacteria that invade tissues (3). These more recent studies call into question the autoim- mune etiology of CD. This disease does not appear to be a result of immune activities directed specifically against human tissue.
Studies strongly suggest that CD has a genetic component (4, 5), one that controls an immune defect. Whole genome studies of CD patients identified an association of the disease with genes of the NOD2 family that influence the immune system (3, 5). Mutations in the NOD2 gene found in CD patients disrupt the ability of immune cells to recognize certain signals that are present in pathogenic bacteria. But it is also clear that heredity is not the full story, nor is an overactive immune system. The inflammation of CD may be due to a malfunction of the immune system that is infectious in origin, genetically controlled, and environmentally exacerbated.
The incidence of CD has been rising over the last several decades, as observed in multiple countries by independent investigators. There is an association between rising economic conditions and increasing reports of CD in a population. This has been interpreted as evidence that an environmental trigger is as, if not more, important in the etiology of CD as human genetics.
That NOD2 gene mutations are found in 25-35% of CD patients suggests a microbial factor in disease onset for at least a subset of patients. CD tissue pathology and symptoms could be due either to an inappropriate response to otherwise harmless bacteria, or to an appropriate (but ineffective) immune response to harmful bacteria that are introduced into the gut. In these scenarios, tissues of the gut become damaged and inflamed as ancillary events to the main battle, the attempt of the immune and inflammatory cells to eliminate the offending bacteria. The bacteria that have been implicated to date include certain soil Mycobacteria and variant of the normal flora of the gut, Escherichia coli. The cause (or causes) of CD is not known, and the disease is complex. It is pos- sible that CD is a syndrome with many different origins. Considering the diverse range of symptoms in different patients, CD could result from different infectious agents and/or different underlying genetic or immune factors.
Soil contains a wide variety of habitats and ecological niches that teem with life, playing host to as many as 10 million bacteria, fungi, and parasites per gram. MAP belongs to a large and diverse Mycobacteria family of bacteria, many of whose members, some 40 different species, are ubiquitous in the environment. Most of these Mycobacteria are not pathogenic for animals or humans, but live out their lives contributing to the richness and fertility of croplands or pastures. Some, like M. kansasii, M. abscissium, or M scrofulaceum, live primarily in the soil, but under the right circumstances can become "opportunistic pathogens" and cause disease if introduced into mammalian tissues inappropriately or inadvertently.
On the other end of the family spectrum are several mycobacterial species that are not generally found in soil and that are highly adapted to life inside mammalian hosts. These Mycobacteria are facultative intracellular pathogens that spend much of their life within mammalian white blood cells called macrophages, the very cells that should be the first line of defense against infection. They can cause serious, life-threatening chronic illnesses in both animals and humans and have been acknowledged public health threats for over a century. They belong to the M. tuberculosis complex (M. tuberculosis, M. bovis, M. africanum, M. microti and M. canetti) and are the etiologic agents of contagious and fatal tuberculosis (TB) disease in animals (from M. bovis) or humans (from M. tuberculosis). Human TB can be acquired from infected animals (a zoonosis) or from other infected humans through contaminated respiratory secretions. So pathogenic are these agents that 2 billion of the 6 billion people in the world are infected with M. tuberculosis (1 of every 3 people), and 2-3 million deaths from TB are recorded each year.
Species of another mycobacterial family, M. avium Complex (MAC), represent another threat to human health. These bacteria are found in the environment in soil and water, but certain MAC members have specifically adapted to life within particular mammalian or avian hosts. The MAC family evolved two distinct subfamilies that dis- tinguish themselves both genetically and phenotypically (6) (see Figure 2, Page 22).
M. avium subspecies avium (MAA) has evolved to be more like the soil Mycobacteria that can become opportunistic pathogens as described above.
MAA strains cluster genetically into a group of free-living bacteria that replicate in the environment, especially in water. But MAA can also infect birds and human lungs, causing a transient low-pathology infection or triggering a chronic inflammation that is ultimately fatal. For example, MAA can cause a potentially fatal pneumonia in immunocompromised hosts, and is one of the more intractable opportunistic pathogens of Human Immunodeficiency Virus (HIV)-infected individuals.
Mycobacterium avium subspecies paratuberculosis, or MAP, comprises a The question the colloquium
second genetic subgroup that can survive in soil or water for months to years.
addressed is whether MAP
It is not clear whether MAP can actually replicate in the environment because has also evolved to infect
it is unable to produce mycobactin, an essential iron transport chemical human gastrointestinal tissue,
synthesized by every other Mycobacterium sp. Instead, MAP has learned to and whether, once there,
successfully infect and replicate inside the same white blood cells of mam- it can cause disease.
mals as M. tuberculosis, macrophages. Rather than targeting the respiratory tract, however, MAP has adapted to the gastrointestinal tract of ruminants and other animals and can cause a slowly developing, but eventually fatal, inflammatory disorder called Johne's disease.
Johne's disease is a $1.5 billion a year cattle industry problem in the U.S. (7).
Johne's disease is found in livestock herds around the globe, and though the condi- tion is easy to identify once symptoms arise, it is challenging to diagnose in the early stages of infection. Healthy but infected animals transmit the organism through milk and manure, invisibly spreading the infection to young animals. As a result of this silent transmission, it is difficult to eliminate MAP from individual herds.
Although MAP, the cause of Johne's disease, has a broad host range, the most commonly infected animals are ruminants, such as cattle, sheep, goats, or deer, both wild and domestic (8). Johne's disease is more common in the more closely confined dairy cattle than in beef cattle, however, where higher animal density and more extensive premise contamination means greater contact with the organism.
While there is evidence for host predisposition for certain strains of MAP, it is believed that all strains are capable of causing infection and pathology.
MAP has a broad geographic distribution, and Johne's disease has been reported on every continent in virtually every country that has animal agriculture and laboratory diagnostic capability. In 1996, 3-10% of U.S. dairy cattle were infected with MAP, and 22% of U.S. dairy herds had a MAP prevalence of >10% (9). A similar survey in 2007 reported that 68% of U.S. dairy herds are infected with MAP. In Europe, MAP infection rates among dairy cattle range from 0% now in Sweden and Norway, where new infections are actively culled, to 50% in the Netherlands and 80% in Denmark. Infection rates vary widely across the various cattle-growing regions of Australia. Few countries are free of MAP and Johne's disease, but the reported prevalence of infected animals in any country is only as good as the diligence with which the disease is surveyed. Although difficult early diagnosis certainly contributes to the uncertainty of the actual figures, a recent increase in the number of infected herds is noticeable, including in countries that previously had low infection rates. A study ending in the fall of 2008 is expected to reveal much higher U.S. domestic MAP infection rates than previously reported (10).
MAP is shed from infected animals in feces, and infection transmission between and among animals is similar to other fecal-orally-transmitted diseases.
There is an age-dependent susceptibility to MAP infection, and most new infec- tions happen early in the animal's life. Infection of newborns likely happens through transmission in utero (before birth), by contaminated colostrum or milk from fecal-contaminated teats or a MAP-contaminated birthing environment.
Infected animals are the only source of MAP in nature, since the pathogen does not generally replicate outside of mammalian cells. When found in soil or water samples, one can assume that the environment was contaminated by MAP in feces deposited by an infected animal (11). MAP shed by infected animals can persist in the environment for years, and soil is another possible source of new animal infec- MYCOBACTERIUM AVIUM PARATUBERCULOSIS: INFREQUENT HUMAN PATHOGEN OR PUBLIC HEALTH THREAT?
tions, although more research is needed to clarify the role of MAP-contaminated water and soil on animal infection rates. The second source of infection, one more The high variability in Johne's
amenable to experimentation, is the transmission of MAP from dam to offspring disease infection rates in
through nursing from contaminated teats or contaminated milk.
various geographic areas
presents an opportunity to
learn more about the factors
Johne's disease in cattle is characterized by chronic or intermittent diarrhea, that maintain MAP infection
weight loss, decline in milk production, and, eventually, death. Animals usually and Johne's disease in cattle
become infected with MAP prior to six months of age, but the signs of Johne's disease are not likely to be observed until two or more years after the initial studies should be designed to
infection. This extended delay between infection and onset of illness makes the make use of data on infection
disease difficult to manage in cattle herds, since infected non-symptomatic cattle rates and information
can contaminate the livestock areas and spread the infection to other animals.
regarding hygiene practices.
There are several types of diagnostic tests available for Johne's disease; some detect the presence of MAP-specific antibody (serology), some depend on identifi- cation of the organism itself (fecal, tissue or environmental sample culture; PCR to detect MAP-specific nucleic acids) (12). Additional support of a positive Johne's disease diagnosis can be obtained from assessing the clinical condition of the animal and from microscopically examining tissue samples for characteristic pathologic lesions and/or the presence of the organism itself, by culture or PCR.
In general, accuracy of diagnosis is affected by stage of the infection, host species, and MAP strain differences (6). Laboratory diagnostic tests for Johne's disease have high specificity (low false-positive results), but low sensitivity (per- centage of MAP-infected animals that test positive). ELISA, a commonly used immunological technique to detect MAP-specific antibodies in blood, detects only about 30% of infected animals when they are still in the preclinical disease phase. In the very early stages of infection, before animals begin to shed MAP in feces or develop an immune response to infection, all animals usually test negative. As the infection progresses and clinical symptoms appear, most of the tests become positive. Diagnostics work best to confirm MAP infection in animals with clinical signs of disease, i.e., diarrhea and/or weight loss.
In the absence of sensitive and accurate diagnostics for early infection, animals are culled when they begin to exhibit decreased milk production, a practice that may result in unnecessary destruction of cattle and economic losses. The lack of tools for early diagnosis also cripples the ability to interrupt the chain of MAP transmission, critical for limiting loss of livestock and, if MAP is a human pathogen, for limiting human exposure.
A number of different control measures are available to limit spread of MAP within herds of cattle and other livestock. Animals become infected with MAP by ingesting the organism through contaminated feed or water. Young animals can MYCOBACTERIUM AVIUM PARATUBERCULOSIS: INFREQUENT HUMAN PATHOGEN OR PUBLIC HEALTH THREAT?
acquire MAP by consuming tainted milk or colostrum. Newborns may become infected in utero, but the significance of this route of infection has been difficult A number of methodological
to ascertain.
and technical improvements
are needed to refine existing
Most dairy cattle herds have frequent turnover (35-40% per year), so preventing veterinary diagnostic tests for
the introduction of new animals with uncertain MAP credentials to an uninfected Johne's disease and to develop
herd is critical. Control measures for MAP infection include (13): new molecular and antigenic
targets using MAP gene
Segregating birthing facilities from the general herd premises and maintaining sequences or other approaches
to improve speed, accuracy,
and sensitivity of diagnosis in
Bottle-feeding colostrum from test-negative cows, the earliest stages of infection.
These improvements will
Using milk replacer (pasteurized) instead of herd milk (raw), facilitate cattle and other
domestic animal disease
Diagnostic test surveillance for the infection and culling test-positive animals, management, interrupt the
MAP transmission cycle, and
Certifying MAP-free herds to provide sources of her replacement, and stem the financial losses due to
culling that the dairy industry
Vaccinating against MAP.
currently experiences. If MAP
is a human pathogen, then a
Although anti-MAP vaccines attenuate severity of Johne's disease in domestic better diagnostic for early-
cattle, sheep, goats and deer, and may reduce MAP shedding by up to 90%, stage Johne's disease will help
vaccination does not protect against all new cases of infection by MAP (7). It may, to reduce MAP in the food
paradoxically, facilitate transmission of MAP within a herd and to other herds, supply and environment and
by diminished culling of infected animals. Until otherwise certified as MAP-free, may help prevent development
MAP-vaccinated animals should be regarded as potentially infected with MAP.
of human disease.
There is evidence that the physical control measures described above can reduce the prevalence of Johne's disease (14), but regional disparities in effective- ness do exist. This may be because most control programs are voluntary, in part due to the costs of replacing culled animals. Considering this, and the current limitations in testing and control measures, eradication of MAP and Johne's dis- ease does not appear to be a practical target.
Unless MAP is demonstrated to cause disease in humans, however, there is no immediate imperative for widespread mandatory cattle culling. If MAP is not a pathogen of humans, but only animals, then economic considerations, the effec- tiveness of other transmission control measures, and local policy considerations will help inform the need for culling infected animals.
Better methods to determine
Cattle are the source of milk on the shelves of our supermarkets and the source the true prevalence of MAP in
of beef in our freezers. Cattle herds in the U.S. that are plagued with MAP infections foods are needed, as well as
and Johne's disease in increasing numbers should be a public health concern until methods to determine whether
the controversy over MAP as an etiologic agent of human disease is resolved.
MAP in food is viable and
infectious, the infectious dose
for animals, if not humans, and
A recent study using culture-dependent methods detected viable MAP in 2.8% the dose that can cause
of homogenized milk cartons sampled from supermarket shelves in the U.S. disease in animals and in
Molecular detection methods, which detect the presence of MAP nucleic acids, humans. More research is
detected MAP in 64% of samples (15). Other studies detected MAP in samples of needed to determine which
soft cheese (16, 17). There is some evidence that MAP can be found in meat, but processes, if any, are effective
research in this area is scanty (18, 19). Overall, different levels of MAP contamina- at killing MAP in food
tion of milk and food products have been noted in the U.S. and a variety of products. More research is
countries around the world. MAP has also been identified in other environmental also needed to identify
sources in addition to food, including river water and municipal water (20).
whether MAP can be carried
away from MAP-contaminated
Prevention of MAP in the food chain could be assured by only sourcing raw farms and pastures to potable
products from animal herds free of MAP. A formal regulatory policy for mandatory water supplies via surface
screening for MAP in consumer foods should only be considered, however, when water and dust, and whether
focused research studies determine the actual risks of human exposure from this these MAP are sources
source. Knowing the incidence of MAP in the food chain is of no use unless we of infection and disease in
also have a detailed understanding of the infectious MAP dose from these sources animals or humans.
and an understanding of the consequences of infection.
Standard HTST pasteurization reduces the number of culturable MAP cells in milk products, but does not eradicate MAP (21, 22). "Light pasteurization" (thermization), a process that is less rigorous than regular pasteurization, permits development of higher densities of MAP cells. Successful methods for reducing MAP in milk may include: Ultra-high temperature pasteurization (UHT milk), Dehydration followed by rehydration, Freezing before pasteurization, Gamma irradiation, Centrifugation, and Mycobacteriophages (viruses that infect and kill mycobacteria).
Public Health Concerns
Scientists and public health authorities do not know whether meat and milk products contaminated with MAP expose the public to any risk of illness. When seen on a global scale, the potential implications of such a scenario are staggering.
Responsibility for managing exposure and risk from MAP in consumer goods in the U.S., in this case, would fall to the Department of Agriculture, the Food and Drug Administration, the Centers for Disease Control and Prevention, and other federal agencies.
The question of whether MAP-infected animals are fit for human consumption depends on whether MAP is a zoonotic pathogen. Although there is evidence for public exposure to MAP in consumer goods, the biological significance of MAP presence in milk, cheese, and meat is not known, and it is also not known whether eating these products leads to MAP infection, or whether MAP infection necessarily leads to disease.
The appropriateness of taking precautionary measures in the face of a plausible but uncertain risk to human health by exposure to MAP in food sources is a hotly contested debate. What is the level of evidence that must be met in order to take action to limit human exposure to MAP? In the face of current evidence, at least minimal actions, like removing clinically diseased animals from the food chain, are considered appropriate.
At the heart of the controversy of MAP as an etiologic agent of CD is whether MAP can initiate human infection at all. MAP can be detected in the human body, and researchers and clinicians have published this fact in a number of case reports in the scientific literature. Moreover, persons with CD are seven-fold more likely than the general population to have MAP associated with their disease (23). However, there has been no definitive causal relationship established between MAP and a specific disease process in humans similar to that which has been established in animals (Johne's disease).
The current methods for isolating MAP from human intestinal tissue for authoritative identification are invasive and are generally obtained from individuals suffering the later stages of CD. The relationship between MAP and the initial stages of CD cannot be extrapolated from testing such late disease-stage tissue samples, and the role of MAP in the initiation of the infection is unclear. The question that needs to be answered is whether MAP is: A "professional" human pathogen (pathogenic to otherwise healthy individuals), MYCOBACTERIUM AVIUM PARATUBERCULOSIS: INFREQUENT HUMAN PATHOGEN OR PUBLIC HEALTH THREAT?
A co-conspirator with other pathogenic bacteria (either one or both partners pathogenic only in the presence of the other, similar to the "enhancement Efficient and effective
effect" seen in certain virus pairs), clinical methods for
sub-speciation of MAP
An opportunistic pathogen (a pathogen in only diseased, genetically-susceptible into ruminant-specific
or immune-compromised individuals), or strains would enable the
identification of sources
A harmless commensal organism that is simply a bystander, with no role in of human MAP and assist
any human disease process.
in the epidemiology of
MAP is unequivocally pathogenic in animals and may also be pathogenic in humans, but until sensitive and specific diagnostics are discovered and distributed for widespread use, the controversy over MAP and its role in any disease, including CD, will continue.
It is possible that MAP plays a role not only in CD and other gastrointestinal dysfunctions, but also in a variety of other diseases. MAP has been detected in tissues outside the gastrointestinal tract, in a patient with HIV, in lymph tissue, and in breast milk from a lactating mother.
Although the numbers of MAP organisms isolated from any tissue, gastroin- testinal or other, has been invariably small, there is precedence for paucibacillary (exhibiting few bacteria) disease caused by other Mycobacteria. Tuberculoid leprosy, caused by M. leprae, is a very good example of a mycobacterial disease in which extensive tissue pathology, immunologic in nature, is observed in the absence (or sparse presence) of the inciting bacteria. Since MAP is not widely recognized as a pathogen, physicians do not consider MAP during differential diagnosis, and pathological tissues are seldom tested for the presence of MAP.
Compounding this problem is the absence of non-invasive and specific diagnos- tics available to test for MAP in humans, even if it were suspected.
The noted differences between bovine MAP strains and human MAP strains pose another barrier to our understanding the possible role of MAP in human disease. If MAP is transmitted from infected livestock to humans through the consumption of contaminated food, then testing for the presence of bovine MAP strains in humans could be useful for determining the epidemiology of MAP disease. However, species-specific strain identification is infrequently performed, so it is not known whether ovine strains can lead to human illness.
The limited number of studies looking for MAP in CD tissue revealed the prevalence of MAP to be between 2% and 20% in various control groups, as well 25-35% in persons with CD ( The prevalence of MAP in the general population is currently unknown because the main method for isolation of MAP is intestinal tissue biopsy, and healthy people are not routinely subjected to this procedure. Prevalence and incidence of MAP infection in the general population MYCOBACTERIUM AVIUM PARATUBERCULOSIS: INFREQUENT HUMAN PATHOGEN OR PUBLIC HEALTH THREAT?
awaits the identification and development of specific and sensitive non-invasive diagnostics for this organism.
diagnostics with high
Infections with MAP must be distinguished from infections caused by other specificity for MAP will need
agents, since identifying the correct etiologic agent is the basis for understanding to be developed for
the epidemiology of the disease and its public health implications.
epidemiology studies in
humans to assess incidence
and prevalence of MAP
For every condition MAP causes in animals, there may also be a parallel condi- infections and determine
tion in humans. Moreover, it is possible that MAP can cause disease similar to that the public health importance
caused by its close mycobacterial family members or distant cousins, although of these infections.
evidence for MAP infections of other organs (i.e., lungs) is certainly more limited than for CD. MAP has been implicated in scrofula, ulcerative colitis, sarcoidosis, and type 1 diabetes, and there are also isolated reports of MAP disseminating outside the gastrointestinal tract, causing bacteremia (24).
Individual research laboratories have developed a number of different assays and techniques for diagnosing MAP disease in animals and humans, including bacterial culture, PCR techniques to identify cultured bacteria or to identify MAP DNA in tissues, immunological techniques (cell proliferation and serologic assay), and histopathological examination of tissue using special stains. Samples for diagnos- ing MAP infection include blood cells or serum, stool, breast milk, biopsies of bowel tissue, and tissue from bowel resection. Culture, stain, or DNA assays are Scientists and clinicians in
most frequently performed on tissue samples, generally from patients with late- different labs should be
stage disease, and the ability to do these tests on other samples, such as blood, encouraged to collaborate to
are limited to just a few research labs (24, 25). Immunologic assays have not been sufficiently sensitive for diagnosis of early Johne's disease, where MAP is the reproducible assays, to make
definitive causative agent, so it is not a surprise that serologic studies to identify MAP infection of healthy humans or patients with gastrointestinal or other disor- and to clarify the possible
ders are not demonstrating differences in these populations.
connection between the
presence of MAP in tissues
From a regulatory and commercial perspective, there are no approved tests for and disease.
diagnosing MAP infection in humans. GenProbe's Amplified™ MTD test is often used for "diagnosing" MAP, but this product is not specific for MAP and will also be positive for other members of the broad Mycobacteria family. Unfortunately, the most definitive research techniques for diagnosing MAP infection are not widely used in clinical laboratories, so isolation and identification of MAP is spotty across a wide distribution of interested parties.
Despite decades of research, there are no universally effective vaccines against other Mycobacteria sp., such as M. tuberculosis (TB) or M. leprae (leprosy). Like these well-known pathogens, it may be possible to develop a MAP vaccine using post-genomic techniques, but so far these efforts have not been fruitful for any of the Mycobacteria. M. tuberculosis, M. leprae, M. avium, and MAP (26) genomes MYCOBACTERIUM AVIUM PARATUBERCULOSIS: INFREQUENT HUMAN PATHOGEN OR PUBLIC HEALTH THREAT?
have been sequenced, and comparative analysis might provide direction for MAP vaccine design. Until MAP is decisively identified as a human pathogen, however, Once MAP is definitively
vaccine developers are likely to direct their efforts elsewhere.
identified as a human
pathogen, the research
community must develop,
If MAP is a human pathogen and causes human disease, it is one of the characterize, and standardize
few in which Koch's postulates were satisfied before the associated disease in appropriate animal models
humans was described. MAP can be isolated from ruminants with Johne's that can address critical
disease, identified genetically as MAP, and can be re-infected into the same research questions about
ruminant class (or different ruminants) to cause the same disease. MAP unequivo- cally causes Johne's disease.
immunological processes
and can demonstrate
If any human gastrointestinal syndrome is equivalent to Johne's disease, then candidate vaccine efficacy.
ruminants could help in the identification of diagnostics and drugs for MAP. These animal models will
However, the link between any gastrointestinal disease and Johne's disease is still be necessary to power the
highly contentious, and the pathologies of these diseases and Johne's disease, vaccine development process.
while sharing many characteristics, also have certain characteristics that are distinct. Veterinary experience with Johne's disease identified potential ways to diagnose of human MAP infections, but none of these tests have yet been validated, and they are not routinely available in clinical laboratories. Since it is impractical to treat MAP infections in animals, no guidance for treatment of human MAP infections is found in veterinary literature.
Small animal models of CD are available, but the extent of their relevance to human disease remains uncertain.
Suitable in vitro techniques or
animal models that can be
used to accurately predict the
If MAP is an infectious cause of human disease, there is only one therapeutic effectiveness of prophylactics,
regimen that could address cure: antibiotic therapy. The only antimicrobial thera- diagnostics, and antimicro-
pies currently recommended for potential MAP infections use drugs presumed to bials useful in MAP infections
have activity against MAP (macrolides, rifamycins, clofazimine, and ethambutol), all in humans will be an impor-
of which actually have limited potency against MAP. They were selected for clinical tant research agenda. MAP
evaluation because they worked in a small number of CD patients whose disease can infect and cause disease
was presumed to be initiated by MAP. To date, there are no antibiotics that have in primates. Primate research
been specifically developed for treatment of MAP infections.
has the potential to contribute
substantially to the under-
As is the case with other mycobacterial diseases (including M. avium pneumonias, standing of pathogenicity of
TB, and leprosy), multi-drug therapy with agents from different antibiotic classes may MAP and risk of MAP expo-
be necessary to eliminate MAP infections and avoid development of MAP resist- sure of humans, as well as
ance. There is recent evidence that existing anti-inflammatory agents may also affect development of new anti-MAP
MAP directly. This possibility should be considered in clinical study design and analy- prophylaxis and therapies.
sis, since most patients with suspected MAP infections are treated for their inflammatory disorder before (and sometime during) treatment with antibiotics.
Table 2. Clinical similarities between CD and Johne's Diseasea
Intermittent diarrhea Ileac region mass Quiescent periods a Table abstracted from Chiodini, 1989 (31)b Domestic animals generally fail to display chronic painc Vomiting (regurgitation) is uncommon in ruminants, although they eructate (move ingesta from their stomach into their mouth for repeated mastication, commonly called "chewing their cud") MYCOBACTERIUM AVIUM PARATUBERCULOSIS: INFREQUENT HUMAN PATHOGEN OR PUBLIC HEALTH THREAT?
Researchers and physicians debate whether MAP, which causes the gastroin- Research should be targeted
testinal inflammation, diarrhea, and weight loss of Johne's disease in animals, may to identify novel anti-
also be to blame for CD. Since Johne's disease shares a number of similarities mycobacterials that act
with CD, a connection between infected livestock and a disease in humans is specifically and synergisti-
conceivable. In fact, a correlation between CD and Johne's disease was first cally against MAP. This will
suggested in 1913, nearly 100 years ago. Dalziel (27) described several patients require better methods to
with chronic intestinal enteritis that, although very similar to intestinal TB, was culture MAP, identification
believed to be a new disorder. He compared this new disease to a disease in of drug targets that are spe-
cattle described first in 1894, now known as paratuberculosis, ".in which the cific for MAP, development
histological characters and naked-eye appearances are as similar as may be to of high throughput screens
those we have found in man. In many cases the absence of acid-fast bacilli to evaluate existing drug
would suggest a clear distinction, but the histological characters are so similar compound libraries to
as to justify a proposition that the disease may be the same." relationships, and creation
Evidence of MAP as an etiologic agent includes the similarity of CD to Johne's of new chemical entities
disease caused by MAP (Table 2), the detection of MAP in the bowels, blood, or that are safe and effective
breast milk of some CD patients (24, 25, 28, 29), the effectiveness of antimicrobial for MAP disease.
drugs in certain MAP-positive and MAP-negative CD patients (30), and the seven-fold higher incidence of MAP in CD patients compared to the population in general (23).
Since MAP can be found in soil, it is not a mystery how grazing ruminants can be infected. But how are CD patients exposed? MAP has been traced to potable water from streams and rivers adjacent to MAP-infected dairy herds. MAP transmission could also occur by ingestion of contaminated milk or milk products or meat from Johne's-infected animals. Molecular analysis of MAP isolates from humans and animals show remarkable similarity. Exposure to a potential enteropathogenic bacterium through the commercial food supply is a troubling prospect.
Susceptibility to CD is multigenetic and can be conferred by any of a number of different genes. A recent investigation (4, 5) compared the gene expression pat- terns of healthy individuals with those of CD patients and was able to identify nine genes linked to susceptibility to this disease. One follow-up study (3) implicated certain immune pathways as genetically deficient in CD patients and suggested that defects in the immune response to intracellular bacteria in particular may be respon- sible for CD. The pathways that are associated with increased susceptibility to intracellular pathogens have not, however, been specifically investigated for MAP.
Genetic susceptibility traits and exposure to bacteria, however, are not sufficient to predict the development of CD in an individual. Other non-genetic factors appear to be at work.
Figure 2. Farm to Fork transmission of MAP: possible route of human exposure
The principle hurdle in diagnosis of CD is simply recognizing the possibility that CD There is an urgent need to find
is in the differential, since disease symptoms vary from patient to patient, and CD the underlying cause of CD in
symptoms are difficult to distinguish from other gastrointestinal disorders, such as order to design therapies that
Inflammatory Bowel Disease (IBD). Diagnosis of CD is currently made by combining can cure this disease. Fully
clinical observations with radiologic, endoscopic, and histological findings. Because exploring the potential links
of the range of symptoms, CD can be a difficult condition to diagnose with certainty.
between CD and MAP is
critical to rule in or rule out
CD can be stratified according to age at the onset of symptoms, anatomical MAP as a pathogen of humans
location of the diseased tissue, and the behavior of the disease (including and as a public health threat
inflammatory, structuring, or fistulizing forms).
transmitted through the
commercial food supply.
The prevalence of CD varies from nation to nation and often varies even among the various ethnic groups within a country. The annual incidence of CD is Investigation of the genes
6-10/100,000 and the prevalence of CD is about 20 times the incidence, or responsible for CD
approximately 150/100,000 people in the U.S. ( Prevalence is roughly susceptibility could enable
250/100,000 in the Canadian province of Manitoba. CD appears to be increasing scientists to understand
among Asian populations, and researchers note that individuals of Native American the underlying host issues
heritage experience lower rates of CD than other ethnic groups.
in development of CD,
and could generate further
research into therapies
There are currently no treatments that cure CD. Medical treatment for CD can that address a specific
be divided into treatments to induce remission of clinical symptoms and treatments genetic defect, resulting in
to maintain remission. Treatment approaches are also different for fistulizing and individualized therapy and
non-fistulizing CD. The primary goal of CD treatment today is to control the disease facilitating the current trend
by increasing the length and frequency of disease-free remissions. Because the for personalized medicine.
current CD therapies address only the symptoms of disease, at least 80-90% of CD patients have to undergo surgery at least once for their condition during their lifetimes, and 50% of CD patients undergo a second surgery.
Table 1 lists all the current anti-inflammatory agents, biologics, and other treat- ments for CD, duration of treatment, and wholesale costs for a 30-day supply.
Retail costs will be substantially higher, and costs for administering IV infusions are higher yet because hospital or clinic costs must also be incorporated into the final cost. There are many treatments of varying efficacy, and the majority of these therapies treat the symptoms of a runaway inflammatory response, not Off-label use of existing
the etiologic agent of the disease, if there is one.
approved antibiotics for other
conditions may
Treatments for maintaining remission of CD symptoms include, in order of be applicable to CD, and
effectiveness (most effective to least effective): immunomodulatory agents (i.e., clinical research on these
azathioprine, 6-mercaptopurine, and methotrexate); anti-tumor necrosis factor-alpha drugs and drug combinations
drugs (anti-TNF alpha); and the antibiotics ciprofloxacin and metronidazol. Steroids should continue. Research
can rapidly induce remission of CD symptoms, but steroids are rarely able to should also, however, be
maintain remission and can be quite toxic if administered for long periods of time.
targeted to identify novel
The long-term outcomes that could be achieved using newer approaches and antimycobacterials that act
treatments (anti-TNF alpha drugs) will require time to assess, as most of these specifically against MAP and
biologics have been approved only in the last year or so. One major impediment that, if MAP is discovered to
to their widespread use is, however, cost (see Table 1, page 11).
be the (or one) etiologic agent,
are efficacious for CD.
If CD is caused by MAP or any other microorganism, appropriate antibiotic ther- apy will be critical for its cure. The only antibacterial drugs approved for use in CD in the U.S. were selected because they worked in a small number of patients (30).
There are no antibiotics available today that have been developed specifically for treatment of MAP-induced CD. As described in MAP in Human Infections, it is anticipated that combination drug therapy will ultimately be recommended for CD, if MAP is an etiologic agent, to reduce the probability of MAP drug resistance.
Most drugs used for treating CD are associated with significant side effects and toxicities, some life-threatening, and all require long-term monitoring of the patient.
Development of MAP-specific
immunologic assays will be
necessary to distinguish
Evidence for a possible link between MAP and CD includes similarity of between MAP and other
disease patterns with Johne's disease, immune response data, the results of Mycobacteria family mem-
tissue sample analysis, and the effectiveness of antimycobacterial drugs. bers, many of whom are
Epidemiologic pattern similarities between Johne's disease (known to be present in soil and water,
caused by MAP) and CD include: and many of which confound
current screening techniques
Triggering event is in early in life.
for such public health hazards
as TB in broad populations in
Prolonged period of time between trigger and clinical disease (incubation period).
the US and around the world.
Validation of immunological
Clinical disease onset commonly after sexual maturity.
assays will be essential
and could be problematic,
Both diseases follow a normal distribution pattern for onset.
since there is no gold
standard technique against
Main target organ is the ileum.
which a new method can be
measured and current human
Host response for both is chronic granulomatous inflammation.
MAP diagnostic tests are
Immune Responses to MAP in CD
The results of immunological testing of CD patients are mixed; some CD patients respond specifically to MAP antigens, while others do not (32). Consistent and repro- ducible immune responses to MAP by patients with CD would be a strong indication that MAP plays a direct role in this condition, but the lack of a consistent immune response would not necessarily mean the lack of an association. For example: If MAP causes CD and susceptibility to MAP depends upon diminished and dysfunctional immune responses, then immune cells of a CD patient could be insensitive to MAP. Immune responses to MAP in cattle are only evident at late stages of disease, when the clinical signs and symptoms of disease are over- whelming. So impaired early immune responses to MAP may be the norm.
Testing of CD patients may be confounded by the use of immunomodulatory therapies to ameliorate symptoms of disease, which would also dampen the development of antigen-reactive T cells or production of antibodies.
Since CD symptoms vary, it is also possible that not all CD, but only a portion of CD, is caused by MAP and the rest is caused by a different etiologic agent(s). An immunological survey of all CD patients in a geographic region looking for those who had immunologic responses to MAP might demonstrate that only a segment of the CD population had MAP antibodies. This would be expected with multiple etiologies, but could be misinterpreted as lack of MYCOBACTERIUM AVIUM PARATUBERCULOSIS: INFREQUENT HUMAN PATHOGEN OR PUBLIC HEALTH THREAT?
This figure was published in Microbes and Infection, 9, Mangalakumari Jeyanathan, Odette Boutros-Tadros, Jasim Radhi, Makeda Semret, Alain Bitton, Marcel A. Behr, Visualiza-tion of Mycobacterium avium in Crohn's tissue by oil-immersion microscopy, page 1572, Copyright Elsvier (2007).
causality if the other agents are unknown. Moreover, antibodies are only an indirect indication of exposure and do not necessarily relate to disease.
Investigators involved in MAP
or CD research must develop
It also follows that, if CD is in part controlled by genetic susceptibility, and agree upon standards for
randomly surveying a population for MAP antibodies could identify healthy indi- proficiency testing in
viduals who had experienced and controlled MAP infection or (unless the molecular methods of MAP
antigen was specific only to MAP) any other Mycobacteria in the past. This detection, so that results from
would also lead (and has led) to confusion about a MAP etiology of CD.
different labs can be
Tissue Samples from CD Patients and MAP
Nucleic acid amplification methods (which rely on the presence of DNA or RNA) can detect MAP in diseased tissues obtained from CD patients and patients with ulcerative colitis, but not consistently. These variable results could be due to at least four possibilities: (1) distribution and number of bacteria may vary among individuals experiencing CD symptoms or vary with the specific symptoms (differences in sensi- tivity to infective doses); (2) people may have different infection rates in different parts of the country (geographical differences); (3) different methods may be used by different investigators, some more sensitive or robust than others (methodological differences); or (4) only a portion of CD is caused by MAP (etiologic differences).
Due to the exacting requirements for growth of MAP for in vitro culture, it is difficult to detect MAP by culture-dependent techniques. It is critical to expand the successes achieved in some laboratories to other laboratories involved in determi- nation of CD etiology. Until more consistent cultivation methods are developed, however, molecular methods are the most efficient surrogate for detecting MAP.
There are reports of the successful treatment of CD, including remission and sustained response, using drugs selected for their antimicrobial activity against M. avium organisms. The only reported controlled clinical trial of combination antibiotic treatment in CD, using these drugs, showed a significant short-term advantage of anti-MAP therapy over steroid therapy, but did not show long-term effects in the majority of patients (32).
Prior clinical trials with anti-tuberculous therapies used to combat M. tuber- culosis infections (the front-line TB drugs rifampin, isoniazid, ethambutol, and pyrazinamide) did not show a benefit for CD patients. These drugs, however, are very specific for M. tuberculosis, and even M. avium is far less susceptible to any of these drugs than M. tuberculosis. So these results were not unexpected.
No studies have systematically tested for the presence of MAP in CD patients before and after treatment with anti-M. avium therapies, so it is not known whether these drugs actually reduce the numbers of MAP bacteria in CD patients.
The reported success of these drugs in treating CD disease could be due to their effects on MAP, or could be due to other activities of the drugs, or to their effects on other bacteria.
Table 3. Pros and Cons of the Association Between MAP and CD
Facts that SUPPORT the existence of a link
Facts that DO NOT support the existence
between MAP and Crohn's disease:
of a link between MAP and CD:
MAP causes a severe and fatal Dairy farmers and others who may have gastrointestinal disease in ruminants greater exposure to MAP than the general (Johne's Disease) that has clinical and population did not experience higher rates pathological similarities to CD (7).
of CD in one study (33).
MAP has been detected in milk, cheese, meat, There are some dissimilarities in the and water targeted for human consumption, clinical and pathological presentation of signifying a possible route of exposure to CD and Johne's disease (31).
MAP for the general public (15-20).
Cell-mediated immune responses to Genes associated with CD suggest that MAP or MAP antigens have not been inappropriate response to an intracellular demonstrated in CD patients.
pathogen may trigger this disease, and MAP is an intracellular pathogen (3, 5).
There have been no systematic studies, but CD has not yet been reported to worsen Increased serological responses to MAP with progressive immunocompromise, have been detected in CD patients.
such as happens with M. tuberculosis infections (exacerbated by HIV or MAP has been detected using molecular and anti-TNF therapy) (34).
histopathological techniques in tissues from CD patients, including blood (24, 25).
In a controlled clinical trial, CD patients undergoing six months of antibiotic therapy MAP has been observed in tissues of CD did not maintain a sustained response to patients by Ziehl-Neelsen staining (35).
the drugs, and the relapse rate after two years was similar in treated and control MAP has been grown from various tissues groups (30).
and fluids taken from CD patients (24, 25).
One clinical study identified a significant and prolonged response to antimicrobial therapy in patients with CD, another study demonstrated a short-term benefit to antimicrobial therapy (30).
CD patients have a seven-fold more likely chance of having MAP in their tissues than the general population (23).
Considering reports of success in using antibiotic therapy to treat CD in some patients, it is perplexing that more physicians aren't actively pursuing antimicrobial Despite the difficult nature
drug therapies for their CD patients and more research is not happening to discover of MAP culture, attempts to
new and better (more specific) drugs to treat this disease. It is likely that the current develop consistent and
lack of evidence-based studies on the MAP causality of CD is the basis for this reproducible methods to
reluctance. On the positive side of antibiotic therapy, most antibacterial drugs are isolate and culture MAP
generally well tolerated compared to the anti-inflammatory drugs used to suppress from tissues of CD patients
CD symptoms. Side effects are well characterized through trials for other infections continues to be an important
and are not notably different in CD patients. On the other hand, use of antibiotics for research activity, the results
a disease without a known infectious etiology could induce drug resistance in MAP of which will eventually
or other bacteria, adding more antimicrobial resistance genes to the pool of genes inform many of the
circulating in the population. Despite these reservations, physicians are encouraged to continue helping CD patients with whatever tools they deem necessary.
regarding the diagnosis and
treatment of CD.
There has been no systematic and widespread effort to explore possible links between exposure to MAP bacteria (in food or elsewhere) and the subsequent devel- opment of CD disease. In the U.S., CD is not reported to public health agencies, so public documents on the actual prevalence of CD over time are extrapolated from academic and clinical publications in various populations. There is both insufficient data on the occurrence of MAP in the environment and an absolute requirement for a widely available, sensitive, and specific human MAP diagnostic(s) to undertake an epidemiological study of sufficient scope to discover a possible link.
More research support and substantial additional research effort by both scien- tists and clinicians is necessary before we will know whether CD has an infectious etiology, and whether MAP is the culprit. It is possible that a subset of CD patients have MAP disease; future research must be directed toward establishing whether there is a strong and reproducible relationship between MAP and CD.

The prospect that MAP could play a role in the incitement or development of CD is a sobering one, and once the situation becomes clearer, there could be impor- tant changes in store for agriculture, food safety, and public health. It is in the best interest of the public that the possible connection between CD and MAP be Colloquium participants strongly urge scientists to carry out rigorous, exacting research and epidemiological studies on MAP and CD and to develop reproducible diagnostics for MAP in order to provide the data needed to achieve consensus on this matter in the scientific and regulatory communities.
There are no easy answers to the problem of identifying the role of MAP in CD or other human infections; no single "home run" study will fill the many gaps in the collective scientific knowledge. Instead, researchers must tackle the problem from multiple directions.
The two broad areas of uncertainty are: The transmissibility of MAP from food and the environment to humans, and The susceptibility of the human body to MAP infection and disease.
To clear up these uncertainties, a strong international research collaboration must be forged with a variety of stakeholders, all with common interests in solving the puzzle—microbiologists and mycobacteriologists, veterinarians, food microbiologists and regulators, epidemiologists, research scientists, product developers and regulators, domestic food-source animal industries, public health officials, clinicians, gastroenterologists, and patient populations. If MAP is associ- ated with human disease and is in the U.S. food supply, this will be a public health concern to rival that of TB in the early part of the 20th century.
Transmissibility of MAP to Humans
MAP exists in Johne's-infected livestock, in livestock waste, on farms, and possi- bly in the food and water supplies (see MAP in the Food Supply, above), but it is far from clear whether humans can become infected with MAP upon exposure to these sources. And if they become infected with MAP, do humans develop disease? Of primary importance to the issue of MAP transmissibility is whether or not the MAP strains isolated from humans can be traced back to MAP strains isolated from animals. Are they related genetically? To determine this will require develop- ment of robust and highly specific MAP speciation techniques and the ability to MYCOBACTERIUM AVIUM PARATUBERCULOSIS: INFREQUENT HUMAN PATHOGEN OR PUBLIC HEALTH THREAT?
isolate MAP from human tissues. Genetic similarities and differences may also identify potential virulence factors in human MAP isolates.
Direct assessment of transmissibility will be difficult. A potential source of MAP exposure for humans is the consumption of contaminated milk from Johne's dis- ease-infected cows, but it is unethical to conduct a prospective study to clarify this possibility (i.e., humans fed deliberately contaminated milk). Retrospective studies, which require subjects to think back on past exposure to milk, are not particularly illuminating, since subjects must recall when and how much milk was consumed, and researchers are unable to confirm whether or not the milk drunk in the past was contaminated at the time of consumption. The best direct answer to MAP transmission by contaminated milk may be to employ an animal model. Johne's disease in goats, whose pathophysiologic symptoms are the most similar of the ruminants to human CD, could be developed into a surrogate animal model for this purpose. The incidence of Johne's disease in groups of animals fed contaminated or MAP-free milk (or other foods or water) could then be compared, and the dose of MAP that is infective by this route could be established. Moreover, having a well-characterized animal model would allow researchers to determine the relative infectivity and pathogenicity of MAP strains isolated from humans.
MAP transmissibility to humans from environmental sources, particularly from MAP-infected livestock, could possibly be uncovered and evaluated by epidemio- logic studies that look at populations living in geographical regions with a low rate of reported Johne's disease, including Sweden, Iceland, and the eastern regions of Australia, compared to geographic regions with a high rate of Johne's disease. For these studies to occur, sensitive and specific MAP diagnostics must first be devel- oped and standardized, and for these studies to be informative, simultaneous (or contemporaneous) MAP surveys of cattle and other ruminants in the area must also be performed. MAP contamination of milk and water from the specific region will also be important to assess. If MAP infection rates are absent or significantly lower in low Johne's disease regions, and are high in high Johne's disease regions, then a strong inference could be made that humans contract MAP (directly or indirectly) from infected livestock. However, this will be complicated by the extensive regional and international trade of foods, making it challenging to assure that the foods consumed originated from the place where the CD patients live.
If it is established that humans exposed to MAP in their environments become infected (i.e., diagnostics detect MAP), all the stakeholders will need to know the health consequences of exposure in order to gauge the possible risks of disease from MAP. These studies will not be easy, because all measurements will be an indirect indication (or surrogate marker) of infection or disease—symptoms or immunological reactions to MAP. In some instances, new animal models may be needed to address questions of MAP pathogenesis and therapy.
Human Immune Responses to MAP
One way to assess exposure (but not necessarily infection or disease) is to determine human MAP immunological reactions in the general population or in specific disease populations like CD. Onset of immune responses to most Mycobacteria, especially the intracellular pathogens, takes months. Because of their intracellular habitat, these pathogens are nearly impossible to eradicate, although they can be immunologically contained. The immune response acceler- ates and expands to include all manner of reactions and immune factors over time, and tissue destruction in both TB and leprosy is largely due to the constant anti- genic challenge of immune cells and resulting immunologic chaos.
To understand the interaction of human immune system following exposure to MAP, it would be helpful to first have an indication of the timing, type, and dura- tion of inflammatory and immune events in an animal model that is undergoing disease reasonably similar to CD. That would at least narrow down the types of immune responses that should be investigated with priority in humans.
A basic understanding of the ongoing MAP-specific cellular and humoral immune interactions that constitute a response to MAP in humans could facilitate identifica- tion of specific immunological responses that occur in CD patients versus healthy individuals, or that occur in different stages or types of CD. Of course, it will not be useful to survey all CD patients for MAP responses if MAP causes only a por- tion of CD cases; in that event, sensitive and specific human diagnostics will need to be identified, developed, and standardized so that patients can be segregated in to MAP-infected CD and non-MAP CD. It may then be possible to identify MAP- specific human immune reactions that can be developed into additional diagnostics to detect early symptoms of relapse or verify cure. It may also be possible to cor- relate human MAP-specific immune reactions to bacillary load in the infected tissues or to other markers of disease.
What if MAP is not the sole agent of CD? What if the relevant aspect of MAP infection is its ability to modulate human immune response(s) to other pathogens? Or to divert the proper immune reaction for a different bacterium to an ineffective one, thus allowing the second pathogen to gain a foothold in tissues that it is not normally allowed to penetrate? Some understanding of the "typical" human immune reaction to MAP may enable scientists to recreate the more complicated CD induction scenario (more than one pathogen necessary to create disease) in appropriate animal models of CD.
There are reports that a percentage of healthy humans are positive for MAP serology. If screening diagnostic tests are highly specific for MAP, these healthy MAP-exposed individuals could provide a window into the "normal" MAP response. Research including these subjects could provide information on immuno-dominant MAP antigens, as well as a comparison with immune responses observed in CD patients.
At the moment, and unfortunately, human MAP infection is most obvious and easiest (although not easy) to identify in CD patients with symptoms of disease.
These patients are generally being treated with powerful anti-inflammatory agents or immune suppressive therapies. There may be no way to assess a "typical" anti-MAP immune response in CD patients unless clinicians are able to identify MAP-infected persons when they first seek medical care and are not yet on thera- peutics that suppress immune cells. Good MAP diagnostics will be essential. If diagnostics are not yet available, good animal models that mimic CD could be help- ful, with the caveat that immune systems and immune reactivity differ between species. With an animal model, however, MAP can ethically be administered by itself or co-administered with other pathogens to determine outcomes.
The antimicrobial agents available to treat MAP infections or CD were not devel- oped specifically to treat MAP and appear inadequate to provide significant long-term results for patients. Clinical research to establish effective therapeutic regimens with existing antibiotics is encouraged. Further research should also be directed toward identifying new and effective antimicrobials for treating MAP infection.
Other Research Priorities
The National Research Council has made recommendations for research examin- ing possible links between CD and MAP (2003). Recommendations 17 through 25 in the report are particularly compelling and require follow-up. In addition, two new reports will be released by agencies in the U.S. Federal Government in 2008, and these reports may contain additional Research Recommendations: Development of an Action Plan to Address Surveillance, Epidemiologic, Laboratory and Environmental Issues Related to Disease Caused by Nontuberculous Mycobacteria. Centers for Disease Control and Prevention External Consultation. May 2007 – April 2008.
National Advisory Committee on Microbiological Criteria for Foods. Assessment of Food as a Source of Exposure to Mycobacterium avium subspecies paratuberculosis (MAP). FINAL DRAFT REPORT. September 24, 2007 Development of Reproducible Analytical Techniques for MAP
There is a conspicuous and fundamental barrier to accomplishing the research on MAP as an etiologic agent of CD: the lack of a reproducible, sensitive, and specific diagnostic(s) for MAP. New tools to identify and isolate MAP are critical to every recommended activity outlined in this document, and without them researchers cannot move forward. Public health laboratories and the U.S. Centers for Disease Control and Prevention laboratories have made it clear that they cannot grow MAP, which hinders diagnosis and reporting. Research would also benefit from more sensitive methods for identifying MAP when it is isolated, and for speciating MAP isolates from infected animals and humans.
A gold-standard test for MAP that researchers and clinicians alike can turn to may stratify testing results, clearly separating individuals with MAP infections from uninfected CD (or other) patients, and thus clarify the role of MAP in CD. Research that uses unreliable, inconsistent technologies for detecting MAP will always be open to criticism and negative scrutiny.
Inter-laboratory collaborations to establish reproducible, standardized techniques for detecting MAP are strongly encouraged. Research in this area often requires the comparison of assay results from different laboratories. Therefore, standardized tech- niques that produce the same results regardless of the service provider are critical.
Once better methods have been developed, a network of proficiency testing for laboratories that offer MAP diagnostics should be established. A strong, unbiased, external laboratory proficiency evaluation program or quality assessment program is critical to establishing and maintaining confidence in MAP research and clinical testing.
A task force should be established to move forward and develop more specific recommendations for developing improved methods for MAP detection.
Eventually, interested parties will be forced to address the problem of which specimen types are most appropriate for testing for MAP infections in humans.
Blood, intestinal mucosal tissue, stool, and colonic exudate are all possibilities for testing purposes. Better assays are also needed for detecting MAP in environ- mental (water and soil) samples, as well as food.
A Note About Resolving Causality
There are certain details to consider in attempting to find a causal link between MAP and CD. For example, if MAP is associated with CD-affected tissues, the possibility that MAP is an opportunist taking advantage of inflammation caused by another organism must be considered and investigated. In this scenario, anti-MAP therapies would alleviate the secondary MAP infection, but not the underlying inflammation due to a different pathogen, as yet identified.
Another imperative in defining causality is the use of appropriate controls. When- ever researchers test tissue for the presence of MAP, they should also check for the presence of other organisms in order to bolster confidence in the results.
CD is likely to be a multifactorial disease, created by the confluence of many different phenomena, and the dividing line between CD and other similar diseases, like ulcerative colitis and IBD, is sometimes difficult to discern. Researchers need to clearly define the conditions of study participants in order to make the conclu- sions of the work as unambiguous as possible.
Research to discover and standardize diagnostics that are both sensitive and specific for MAP in animals and in humans and can determine the source of MAP cultured from human tissue is imperative. Virtually all the research topics that will clarify the role of MAP in CD rely on this.
Research must address the issue of MAP transmissibility and determine whether or not the MAP strains isolated from animals or food are genetically identical to the MAP strains isolated from humans.
Research should also examine the potential virulence factors in human MAP isolates and determine whether MAP can be transmitted from human to human.
Researchers must develop better animal models for evaluating MAP effects on human hosts and for evaluating the effectiveness of potential therapies for MAP infection.
A policy for regularly screening foods for MAP should not be put into place until focused research studies can determine the actual risks of exposure and disease.
Identifying novel MAP-specific antimicrobials and effective antibiotic treatment regimens for MAP infections is a research priority.
A Word on the Precautionary Principle
The Precautionary Principle ( as a formal concept evolved from German social and legal tradition in the early 20th century. It is underpinned by common sense aphorisms that pre-date the term, such as "an ounce of prevention is worth a pound of cure," "better safe than sorry," and "look before you leap." The Precautionary Principle is also considered to have evolved from the ancient medical principle, "first, do no harm," as it applies to institutions and institutional decision-making processes, rather than individuals.
The 1998 Wingspread Statement on the Precautionary Principle summarizes the principle this way: "When an activity raises threats of harm to human health or the environment, precautionary measures should be taken, even if some cause and effect relationships are not fully established scientifically" (Science and Environmental Health Network). In deciding how to apply the principle, one uses cost-benefit analysis, assessing both the opportunity cost of not acting and the option value of waiting for further information before acting. In modern policy making, there is often an irreducible conflict between different interests, so the debate is necessarily political. This is no more evident than in the debate about MAP and CD.
When should policy makers evaluate the Precautionary Principle as it relates to the role of MAP in CD? There are indications that MAP is pathogenic for humans, and MAP is found in U.S. cattle that supply food for human consumption and in milk and perhaps other foods on the supermarket shelf. A significant number of U.S. citizens, more than 500,000, are affected by CD.
The colloquium participants were not prepared to recommend as a group that public health authorities move today to mitigate the public's exposure to Johne's disease-infected animals by enacting mandatory agriculture and food safety regula- tory policies to eliminate potential routes of exposure to MAP.
It is important, however, for all regulatory bodies concerned with agriculture and public health to consider this report, and the other Federal reports that will be published later this year, in light of the Precautionary Principle. The science and clinical communities will continue to identify gaps in the current knowledge relevant to MAP exposure and, with appropriate funds and collaborations, begin to provide data that will inform these deliberations. But the decision to invoke the Precautionary Principle should be a political debate at the highest levels of our federal institutions charged with guarding the public health, and the timing of this debate should be now.
1. Crohn, B.B., L. Ginzburg, G.D. Oppenheimer. 1984 Regional 10. Veterinary Services Report, due 2008, Johne's Disease on U.S.
ileitis. A pathological and clinical entity. (Landmark article 1932).
Dairy Farms, 1991-2007. United States Department of Agriculture.
JAMA 251: 251:73-79.
11. Whittington, R.J., D.J. Marshall, P.J. Nichols, et al., 2004. Sur- 2. Ashford, D., B. Imhoff, F. Angulo, and the FoodNet working vival and dormancy of Mycobacterium avium subspecies group, CDC, Atlanta, GA. Over a Half-Million Persons May Have paratuberculosis in the environment. Appl Environ Microbiol Crohn's Disease in the United States. Infectious Diseases Society of America. San Francisco, CA, October 2001.
12. Collins, M.T., I.A. Gardner, F.B. Garry, A.J. Roussel, S.J. Wells.
2006. Consensus recommendations on diagnostic testing for the 3. Ferwerda, G., B.J. Kullberg, D.J. deJong, et al. 2007.
detection of paratuberculosis in cattle in the United States. J Am Mycobacterium paratuberculosis is recognized by Toll-like Vet Assoc 229:1912-1919.
receptors and NOD2. J. Leukoc Biol 82:1011-1018.
13. USDA. 2005. Dairy 2002 Johne's disease follow-up study: Man- 4. Wellcome Trust Case Control Consortium. 2007. Genome-wide agement practices and within-herd prevalence. APHIS Info Sheet, association study of 14,000 cases of seven common diseases and Veterinary Services. Centers for Epidemiology and Animal Health.
5. Parkes, M., J.C. Barrett, N.J. Prescott, et al. 2007. Sequence 14. Goodger, W.J., M.T. Collins, K.V. Nordlund, C. Eisele, J. Pelletier, variants in the autophagy gene IRGM and multiple other replicating C.B. Thomas and D.C. Sockett. 1996. Epidemiologic study of loci contribute to Crohn's disease susceptibility. Nature Gen on-farm management practices associated with prevalence of Mycobacterium paratuberculosis infections in dairy cattle. J Am Vet Assoc 208, 1877-1881.
6. Turenne, C.Y., R. Wallace, Jr., and M.A. Behr. 2007.
Mycobacterium avium in the post-genomic era. Clin Micro Rev 15. Corti, S. and R. Stephan. 2002. Detection of Mycobacterium avium subspecies paratuberculosis specific IS900 insertion sequences in bulk-tank milk samples obtained from different 7. Harris, N.B., R.G. Barletta. 2001. Mycobacterium avium regions throughout Switzerland. BMC Microbiol. 2, 15.
subsp. paratuberculosis in Veterinary Medicine. Clin Micro Rev 16. Clark, D.L., J.L. Anderson, J.J. Koiziczkowski and J.L.E. Ellingson.
8. Chiodini, R.J., H.J. Van Kruiningen, R.S. Merkal. 1984. Ruminant 2006. Detection of Mycobacterium avium subspecies paratuberculo- paratuberculosis (Johne's disease): the current status and future sis genetic components in retail cheese curds purchased in prospects. Cornell Vet 74:218-262.
Wisconsin and Minnesota by PCR. Mol. Cel. Probes 20, 197-202.
9. Committee on Diagnosis and Control of Johne's Disease, 17. Donaghy, J.A., N.L., Totton, and M.T. Rowe. 2004. Persistence National Research Council. 2003. Diagnosis and control of Johne's of Mycobacterium paratuberculosis during manufacture and disease. The U.S. National Academy of Sciences Board on Agricul- ripening of cheddar cheese. Appl Environ Microbiol 70, ture and Natural Resources Report. National Academy Press, MYCOBACTERIUM AVIUM PARATUBERCULOSIS: INFREQUENT HUMAN PATHOGEN OR PUBLIC HEALTH THREAT?
18. Jaravata, C.V., W.L. Smith, G.J. Rensen, J. Ruzante, and J.S.
27. Dalziel, T.K. 1913. Chronic interstitial enteritis. Br. Med. J.
Cullor. 2007. Survey of ground beef for the detection of Mycobac- terium avium paratuberculosis. Foodborne Pathog Dis 4, 103-106.
19. Abubakar, I., D.J. Myhill, A.R. Hart, et al. 2007. A case-control 28. Bull, T.L, E.J. McMinn, K. Sidi-Boumedine, et al. 2003.
study of drinking water and dairy products in Crohn's disease— Detection and verification of Mycobacterium avium subspecies further investigation of the possible role of Mycobacterium avium paratuberculosis in fresh ileocolonic mucosal biopsies from subspecies paratuberculosis. Am J Epidemiol 165:776-783.
individuals with and without Crohn's disease. J Clin Micro 20. Pickup, R.W., G. Rhodes, T.J. Bull, S. Arnott, K. Sidi-Boumedine, 29. Autschbach, F., S. Eisold, U. Hinz, et al. 2005. High prevalence M. Hurley and J. Hermon-Taylor. 2006 Mycobacterium avium subsp.
of Mycobacterium avium subspecies paratuberculosis IS900 DNA in paratuberculosis in Lake Catchments, in river water abstracted for gut tissues from individuals with Crohn's disease. Gut 54:944-949.
domestic use, and in effluent from domestic sewage treatment works: Diverse opportunities for environmental cycling and human exposure. Appl Environ Microbiol 72:4067-4077.
30. Selby, W., P. Pavli, B. Crotty, et al. 2007. Two-year combination antibiotic therapy with clarithromycin, rifabutin, and clofazimine for Crohn's disease. Gastroenterol 132:2313-2319.
21. Chiodini, R.J. and J. Hermon-Taylor. 1993. The thermal resistance of Mycobacterium paratuberculosis in raw milk under 31. Chiodini RJ. 1989. Crohn's disease and the mycobacterioses: a conditions simulating pasteurization. J Vet Diagn Invest 5:629-631.
review and comparison of two disease entities. Clin Microbiol Rev 22. Donaghy, J.A., M. Linton, M.F. Patterson, and M.T. Rowe. 2007.
Effect of high pressure and pasteurization on Mycobacterium avium 32. Marks, D.J., M. W. Harbord, R. MacAllister, 2006. Defective subsp. paratuberculosis in milk. Lett Appl Microbiol 45:154-159.
acute inflammation in Crohn's disease: a clinical investigation.
23. Feller, M., K. Huwiler, R. Stephen, et al. 2007. Mycobacterium avium subspecies paratuberculosis and Crohn's disease: a systematic review and meta-analysis. Lancet Infec Dis 7:607-613.
33. Jones, P.H., T.B. Farver, B. Beaman, et al. 2006.Crohn's disease in people exposed to clinical cases of bovine paratuberculosis.
24. Chamberlin, W., G. Ghobrial, M. Chehtane, S.A. Naser. 2007.
2006. Epidemiol Infect 134:49-56.
Successful treatment of a Crohn's disease patient infected with bacteremic Mycobacterium paratuberculosis. Am J Gastroenterol.
34. Sartor, R.B. 2005. Does Mycobacterium avium subspecies paratuberculosis cause Crohn's disease? Gut 54:896-898.
25. Chamberlin, W. and S. A. Naser. 2007. Blood Cultures of 19 Crohn's Disease Patients. Am J Gastroenterol. (In press) 35. Jeyanathan, M., O. Boutros-Tadros , J. Radhi, M. Semret , A. Bitton, and M.A. Behr. 2007. Visualization of Mycobacterium 26. Li, L., J.P. Bannantine, Q. Zhang, A. Amosin, B.J. May, D. Alt, avium in Crohn's tissue by oil-immersion microscopy. Microb and N. Banerji, S. Kanjilal and V. Kapur. 2005. The complete genome Infect 9: 1567-1573.
sequence of Mycobacterium avium subspecies paratuberculosis.


Microsoft word - 3295.doc

LIGHT DIAGNOSTICS™ Cat. No. 3295 FOR IN VITRO DIAGNOSTIC USE  Authorised. Representative: Millipore (UK) Ltd. Fleming Road, Kirkton Campus Livingston EH54 7BN UK. Tel +44 1506 404000 Fax +44 1506 404073 28820 Single Oak Drive Temecula, CA 92590 • USA & Canada Phone: +1(800) 437-7500 • Fax: +1 (951) 676-9209 • ISO Registered Worldwide • •

Laboratory testing of cyp2d6 alleles in relation to tamoxifen therapy

ACMG STANDARDS AND GUIDELINES ©American College of Medical Genetics and Genomics laboratory testing of CYP2D6 alleles in relation to tamoxifen therapy Elaine Lyon, PhD, FACMG1, Julie Gastier Foster, PhD, FACMG2,3, Glenn E. Palomaki, PhD4, Victoria M. Pratt, PhD, FACMG5, Kristen Reynolds, PhD6, M. Fernanda Sábato, MS7,

Copyright © 2008-2016 No Medical Care