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Crohn's Disease Forum » Books, Multimedia, Research & News » Mycobacterium avium subsp. paratuberculosis survival during fermentation of milk


06-01-2012, 07:06 PM   #1
kiny
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Mycobacterium avium subsp. paratuberculosis survival during fermentation of milk

It's nothing new, MAP can survive pasteurization and very high and low temperatures. Just confirmation.

The part about the PH value is kind of interesting I think. Common probiotics can lower PH and that's why they can kill bacteria like salmonella.

http://www.ncbi.nlm.nih.gov/pubmed/22591549


Mycobacterium avium paratuberculosis (MAP), etiological agent of paratuberculosis in ruminants, is able to survive extreme conditions like very low pH (stomach), high temperature (pasteurization) or low temperature (refrigerated storage). Cheese, infant powder milk, cream and other milk and dairy products might thus be considered as possible sources of MAP for humans. The aim of this study was to investigate the survival of two MAP field isolates during fermentation of three different types of soured milk products (SMP; yogurt, acidophilus milk and kefir) under laboratory conditions. Pasteurized MAP-free milk was artificially contaminated with 10(6)MAPcells/mL and survival and absolute numbers of MAP were monitored during fermentation (4 or 16h) and after six weeks of storage at 4°C by culture and quantitative real time PCR (qPCR). Viability of MAP was determined by culture using Herrold's egg yolk medium and Middlebrook 7H10 with antibiotics, supplemented with Mycobactin J and incubated at 37°C for up to 12weeks. The absolute numbers of MAP were quantified by previously published qPCR assays targeting F57 and IS900 loci in MAP genome. We herein confirm that MAP can survive pH reduction, however, longer exposure to pH below 4 in SMP seems to be critical because it inhibits growth. Therefore, it is suggested that probiotic cultures that can decrease pH below 4 during fermentation could provide better inactivation of MAP in SMP.

Last edited by kiny; 06-01-2012 at 07:22 PM.
06-01-2012, 11:23 PM   #2
jmckinley
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Interesting! I have been reading some info about MAP causing/contributing to Crohn's. So how do they test for MAP if skin prick test isn't an accurate way to do it? And why would MAP affect some people in their lungs and some in their gut?
06-02-2012, 08:42 AM   #3
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The MAP test that's standardised now is called IS900 PCR. They let the samples culture for an extended period and then do the test on them. I made a thread about how they used nanosensors instead of IS900, which is far more precise and much faster than IS900 PCR.

Why MAP affects people in their lungs? Idk, where did you read that, I just know it causes Johne's disease in animals, which looks exactly like Crohn's disease in humans with very slight differences. Animals for example don't get strictures like humans, but they think that might be because animals like cows stand up all day and that influences if they can get strictures or not or perhaps cause they're still different from humans, they don't know, but outside of those differences, the disease looks completely the same.

There's a video online of a doc and he addresses the critiques of the MAP theory, and he shows 2 slides to a bunch of gastroentrologists, and he asked "which inflamed gut is from the cow and which is from the human?" and none of them could answer, both diseases look ridiculously similar, and might just be one and the same.
06-02-2012, 10:02 AM   #4
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The issue of MAP and Crohns has been discussed on and off for over a 100 years. It has re-emerged relatively recently after being dismissed but the theory, based on similar appearance to Johnes Disease in cattle.
There are many issues that are yet to be resolved;

-The pathology of Crohn’s disease is not identical to Johne’s disease.
• Pathology in Crohn’s disease commonly includes
fissuring ulceration, fistula formation and fibrosis,
whereas Johne’s disease in cattle does not.
• There is no caseation in the granulomata in
Crohn’s disease as in Johne’s disease.
• Causative agent is easy to recover in Johne’s
disease but not in Crohn’s disease.
• Crohn’s disease does not have any evidence of organism on histochemical examination.

-MAP is not reliably detected in 100% of Crohn’s disease patients.
• Histological examination rarely shows acid-fast
staining of MAP.
• MAP has been detected by faecal culture in
approximately only 5% of cases.
• MAP has been detected by blood culture in
approximately only 50% of cases.
• MAP has been detected by PCR in approximately
only 19-46% of cases.
• MAP has been detected by in situ hybridisation in only 40-92% of cases

-There is no apparent humoral response to MAP in Crohn’s disease patients.
• All bar one study have failed to find differences in
MAP reactive antibodies in Crohn’s disease
patients compared to healthy people.
• Cross reactivity was observed in patients with
tuberculosis or leprosy and in vaccinated people
and some ‘healthy’ controls.
• The results indicate a relatively weak recognition
of MAP in Crohn’s disease, which is only visible
if certain immunological targets are selected.
• Crohn’s disease patients have ‘leaky’ intestines and
thus elicit a response to normal intestinal flora, and bystander mycobacteria.

-Other microbial species have been detected in Crohn’s disease.
o M. avium complex
o Heliobacter sp,
o Listeria monocytogenes and
o E coli
o Bacteriodes vulgaris
and , measles virus.

The IS900 PCR assay has been around for over 10 years, and the issue is still far from clear.
Other issues include the small number of patients who contract Crohns relative to the total number consuming milk products. The fact that very young babies (note that all babies are born with sterile digestive tracts) have been diagnosed with Crohns is also incongruous, given the long incubation period of the bacteria.
Nonetheless, the theory will hang about until absolute evidence is gathered.....let's hope not another 100 years will pass!
06-02-2012, 03:11 PM   #5
jmckinley
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Thanks for the wealth of info! Reading about MAP is confusing.

Sorry, I was confused between this and MAI, (also known as Lady Windermere Syndrome). It is a different micobacterium species that causes lung problems. My son has Crohn's and was exposed to someone with MAI. In reading about it online, I came across a world of conflicting info on MAP.
06-02-2012, 03:44 PM   #6
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Isnt there a vaccine being developed in a London uni for this? I'm sure I read about people trying to raise money for it. If someone's got that far, are there any studies in humans or animals showing results in Crohn's?

P
06-02-2012, 04:05 PM   #7
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I don't know if MAP is a cause of Crohn's disease or not. Sarah was never tested for it but Matt was and his result came back as negative.

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06-07-2012, 07:46 PM   #8
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"Irish bulk raw and commercially
pasteurized milk
[81]. MAP DNA was detected in 12.9%
(50/389) of raw and 9.8% (35/357) of pasteurized milk
samples using immunomagnetic separation (IMS)‐PCR."

"Similar results were reported by Millar et al.
(1996) [82] who conducted an extensive study of retail
pasteurized milk in England and Wales and found that
7% of retail milk samples tested positive for MAP by
PCR
"

"in Switzerland, 19.7% of raw bulk‐tank milk
samples contained MAP DNA [83]."

"In a US study [75], viable MAP was found in 2.8%
(20/702) of retail milk samples
by two culture methods
and PCR."

"Similar results
were reported from the Czech Republic [73] using a
culture method. Viable MAP was present in 1.6% (4/244)
of commercially pasteurized (71.7° for 15 seconds) retail
milk samples collected from the country’s supermarkets
and stores."

"In addition, researchers in Argentina isolated
viable MAP from 2.9% (2/70) of commercially pasteurized
milk samples; one from pasteurized and the other from
ultra‐pasteurized (138° C for 30 seconds) milk [84]. Both
culture positive samples were also positive with IS900‐
PCR."

"Viable MAP was reported in 72% (13/18) of
commercially available pasteurized milk samples tested
in India [76]. The authors also found viable MAP in 56%
(5/9) of commercially available pasteurized milk
products."

"Researchers at the University of Guelph reported on an
investigation of the presence of MAP in pasteurized milk
obtained at retail outlets and dairy plants in southwest
Ontario [85]. From 710 milk samples tested, 110 (15%)
were positive by IS900 nested PCR."

http://cdn.intechopen.com/pdfs/26269..._and_water.pdf
06-08-2012, 07:34 AM   #9
Igor_Passau
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thank you Kiny!
06-08-2012, 06:13 PM   #10
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30 patients with crohn, ALL 30 were found to have MAP in spheroplast form, NONE of the controls did.


http://www.ncbi.nlm.nih.gov/pubmed/20857526

AIM:

To examine the detection rate of viable Mycobacterium avium subspecies paratuberculosis (MAP) in patients with inflammatory bowel disease [Crohn's disease (CD) and ulcerative colitis (UC)].


METHODS:

Thirty patients with CD (15 with at least one NOD2/CARD15 mutation), 29 with UC, and 10 with no inflammatory bowel disease (IBD). were tested for MAP by polymerase chain reaction (specific IS900 fragment) and blood culture.


RESULTS:


MAP DNA was detected in all original blood samples and 8-wk blood cultures (CD, UC and non-IBD). Positive MAP DNA status was confirmed by dot blot assays. All 69 cultures were negative by acid-fast Ziehl-Neelsen staining. Viable MAP, in spheroplast form, was isolated from the 18-mo blood cultures of all 30 CD patients, one UC patient, and none of the non-IBD controls. No association was found between positive MAP cultures and use of immunosuppressive drugs or CD-associated single nucleotide polymorphisms.


CONCLUSION:


MAP is widely present in our area and MAP DNA can be recovered from the blood of CD, UC and non-IBD patients. However, MAP spheroplasts were only found in CD patients.

06-09-2012, 08:21 PM   #11
kiny
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Hmm, the figure in India showed 72% of the milk supply has MAP in it, and although crohn is rising in India, it's still relatively low, so it didn't make sense.

But I went looking and talking to people, people in India boil milk, usually multiple times for several minutes, which should kill most of the bacteria instead of the few seconds at 75 degrees C. pasteurisation does. It tends to depend on the place in India too, some boil it once, others multiple times.
06-10-2012, 05:12 AM   #12
Igor_Passau
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What causes Crohn's disease?
The causation of Crohn's Disease has not been fully understood, nor recognised. As a consequence, conventional research and treatment are directed almost exclusively at suppressing the inflammation. This may help in the short term, but the disease almost invariably comes back. Most people with CD eventually have surgery, sometimes on more than one occasion. Epidemiological research has shown that the long term prospects for people with Crohns Disease have not improved significantly in 35 years.

Progress so far
Research we began and have continued since 1985, and which is now increasingly being taken up and confirmed by other research laboratories, shows that Crohn's Disease is largely caused by a bug called MAP (short for Mycobacterium avium subspecies paratuberculosis). The reliable scientific evidence for this has grown very strong.

MAP infection is widespread in the animal world.
•MAP is being transmitted to humans in milk and from exposure to environmental sources like contaminated waters.
• MAP in people is difficult to detect. The tests have to be done just right. When they are, almost everyone with Chrons Disease is found to be infected with MAP.
• MAP is what is called in microbiology a multi-host pathogen with the proven scientific ability to cause chronic inflammation of the intestine in many animals including primates. MAP is doing the same thing to people.


Modern Vaccines
MAP infections are difficult to eradicate. They are resistant to most antibiotics and drugs used to treat TB. In 1992, Prof Hermon-Taylor introduced a new treatment for Chron's Disease using a combination of two recently available drugs more active against MAP, called rifabutin and clarithromycin. They work in over 50% of people with active CD who can take them. Relapses sometimes occur. New anti-MAP treatments such as modern therapeutic vaccines are needed. Conventional vaccines make antibodies to prevent disease. They could not work in CD as the MAP bugs are already there inside cells. Modern vaccines make armies of hunter-killer cells which patrol the body getting rid of infected cells. So modern vaccines can be used to treat diseases caused by chronic infections.

A Crohn's cure?
Prof Hermon-Taylor and his team began seeking funding in 2001. Since then they have received and committed over £1.5 million. With this they have designed and delivered a state-of-the-art modern anti-MAP vaccine. It consists of a critically important cassette of MAP DNA in two harmless carrier viruses called Ad5 and MVA. These carriers are already working in approved clinical trials with other modern vaccines. In the Crohn's Disease vaccination treatment procedure, the Ad5 is given first and the MVA boost 6 weeks later. In multiple tests over the last two years, the Crohn's vaccine has consistently proved to be effective both in treating existing MAP infection and protecting against subsequent MAP infection, without any side effects.

What is still needed?
We have come a long way and are nearly there. The Crohn's treatment will move to clinical trials and market development over the next 3 years. Over this period there is an absolute scientific requirement to develop new quantitative tests for MAP in humans, new immunological tests for MAP in humans, and tests for the specific immune responses of people to the vaccine. Together these tests will establish proof of concept that anti-MAP vaccination can make people with Crohn's disease better, and it does so by depleting or eradicating the MAP infection. This final essential piece of scientific research will require £550,000.

http://freeola.com/crohns.php
06-10-2012, 05:17 AM   #13
PaulPhoenix
 
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I dont know how widespread MAP is in foods other than milk but I switched to UHT (longlife milk) anyway.

Are all foods contaminated or is milk the only one?
06-10-2012, 07:09 AM   #14
kiny
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I dont know how widespread MAP is in foods other than milk but I switched to UHT (longlife milk) anyway.

Are all foods contaminated or is milk the only one?
Well, the water supply is to begin with, so technically all the food that is made with water or came into contant with water could be contanimated.

In reality it seems that it's mostly milk, dairy in general and water (beef is also contaminated but less than milk, although many animals are culled while the farmer knows the animal has johne's disease. One study said that johne's is like aids amongs farmers, the farmer said: "you just don't talk about it"..)

Ice-cream is another product you should be careful of, since ice-cream often uses milk that is not pasteurized at all.

I personally avoid milk (if you do drink it I feel you should be boiling it) and all water is boiled for several minutes. Boiling water for a minute or more should be able to kill off a lot of MAP bacteria if the water is contaminated (and a huge percentage of tap and bottled water is worldwide).

Whey protein should be relatively safe, the process to make whey "should" have split the MAP off and the high temperatures involved "should" have killed a big percentage of MAP, but there are better alternatives like just pure protein or soy protein.


Right now they are culling (by killing them) animals in many Western countries, most farmers are asked to check for paratuberculosis (MAP) but many do not, and it's a question if culling those animals will even help if the environment as a whole is completely contaminated, they can find MAP in rivers and lakes (because it seeps into the ground from farms and ends up in the water), and in both bottled and tap water.

Here is a pamflet from Belgium (country I live in), given to farmers asking them to check their farms for paratuberculosis, they realise MAP could be causing crohn but most do not want to talk about it because they are scared of the economic backlash against dairy:


Last edited by kiny; 06-10-2012 at 07:40 AM.
06-10-2012, 07:46 AM   #15
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I'll add this study to this thread because I don't want to make 100 threads about the same thing.

It's a very significant study, since monkeys are much closer to us, and because up until that study Johne's disease was only found in cows, sheep, horses, etc.

Only study I know off that found Johne's disease in monkeys:


http://www.jstor.org/discover/10.230...21100843758861

"Mycobacterium paratuberculosis infection was documented in a colony of stumptail macaque monkeys (Macaca arctoides), with 29 (76%) of 38 monkeys infected and shedding organisms in feces. Thirteen deaths have occurred during the past five years. Animals without overt clinical disease were shedding as many as 2 x $10^6$ colony-forming units of M. paratuberculosis/g of feces. Intestinal tissues from animals dying of this disease contained up to $10^8$ colony-forming units of M. paratuberculosis/g of tissue. The clinical and pathological features of paratuberculosis in this species were comparable to those reported for paratuberculosis in ruminants and Mycobacterium avium infections in primates. By enzyme-linked immunosorbent assay, antibodies to M. paratuberculosis were found in 79%-84% of the animals. Antibodies could not be detected in six animals with clinical disease. These findings extend the natural host range of M. paratuberculosis to include nonhuman primates and add support to current suggestions that M. paratuber-culosis may be pathogenic for humans. "
06-10-2012, 07:52 AM   #16
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I'm not sure it contains much new information but the following was interesting.

http://wsutoday.wsu.edu/pages/public...cationID=30388

If it were the cause, for at least a proportion of Crohns' sufferers, immunosuppressants and steroids would hinder the immune system fighting MAP, wouldn't they?

And what's the relationship with the ecoli found in granuloma?

I'd love to know what stage the MAP vaccine is at and when they're moving on to human trials. There are enough celebrities and rich bussiness people with the disease. It's a pity one couldnt fund the vaccine. Imagine your legacy as having been a part of it, if it worked that is :-)
06-10-2012, 08:05 AM   #17
kiny
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I'm not sure it contains much new information but the following was interesting.

http://wsutoday.wsu.edu/pages/public...cationID=30388

If it were the cause, for at least a proportion of Crohns' sufferers, immunosuppressants and steroids would hinder the immune system fighting MAP, wouldn't they?

And what's the relationship with the ecoli found in granuloma?

I'd love to know what stage the MAP vaccine is at and when they're moving on to human trials. There are enough celebrities and rich bussiness people with the disease. It's a pity one couldnt fund the vaccine. Imagine your legacy as having been a part of it, if it worked that is :-)
Aye, there is a theory that immunosuppressants could in the long run make crohn worse since it's suppressing the immune response, especially since many crohn patients have MAP antibodies (this is one of they ways they check animals for MAP too, by looking if their body has antibodies).

Immunosuppressants like remicade for example cause apoptosis, which not only kills TNF-alpha but has shown to lower MAP, but how significant that is I don't know, since the moment you go off immunosuppressants, your body could be extremely vulnerable.

Some suggest E. Coli is the backdoor for MAP, since E. Coli causes permeability through inflammation and MAP could be taking advantage of it. E Coli and MAP have been found in crohn patients, one doesn't exclude the other.

I would like to know too about the vaccine. Animals never get cured of vaccinated because any cow or sheep with antibiotics or anything of the like would be ridiculously costful, and the animal would be useless since they are prohibited to sell animals like that and unlike MAP, antibiotics would easily be detected by authorities.
06-10-2012, 08:22 AM   #18
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Again, if it were a cause:

ingesting the bacteria isn't a good idea, so cut out non UHT milk, boiled etc

But assuming you have colonisation inside you, what are the options?

antibiotics,
Entereal diet to kill off their food (might explain why it works! Actually in John hunter's book I remember him saying milk was one of the two most common foods to cause relapse)
Probiotics (there's a new IBS one in the uk that might be worth a try)
Vaccine (wish they'd hurry)
SSI vaccine (not sure I understand MI of this!)

I have only very basic biology, but it seems to me there must be antigen(s) which are then painted by the immune system incorrectly identifying them as targets. So are they bacteria, bacterial waste products, cells in the gut?
06-10-2012, 08:41 AM   #19
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My understanding of biology isn't that good either yet, excess or disregulation of TNF-alpha interferes with the healing process and the excess is the cause of inflammation. http://www.mendeley.com/research/ref...ralpha/#page-1 The theory behind MAP is that the body of someone with croh is immune deficient http://www.springerlink.com/content/c313853j75872167/and once the pathogen (MAP, E. Coli) takes advantage of this situation, the body overreacts, the levels of TNF-alpha increase and it causes inflammation and MAP thrives in an environment where TNF-alpha and inflammation is high (that recent infliximab study shows that MAP and TNF-alpha levels are related, when they lower TNF-alpha, map was lowered and so were antibodies against MAP). The issue I have with all those immunosuppressants is that no one has a clue if this is a good idea, because it stops the inflammation, lowers TNF-alpha and it allows for healing, but what's the long term result on things like MAP and bacterial overgrowth, is it actually making things worse in the long run, is that why you see many people going on stronger and stronger drugs? I don't know, but much more research needs to be targeted at MAP instead of suppressing the immune system which is getting us nowhere.
06-10-2012, 09:11 AM   #20
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Wanted to add since you said antibiotics and I agree. Many biologists have warned though that those antibiotics need to be taken at the a high enough dosage, since many are using clarithromycin, bacteria become resistant to clarithromycin, really fast, if your dosage is too low you are basically wasting the antibiotic they say, since from that point on, clarithromycin will not be useful for those people anymore. That's part of the issue that borody and some others mentioned, if you underdosage the patient, you are not getting the right effect and the antibiotic will basically become useless since the bacteria will have become completely resistant to it.
06-10-2012, 09:54 AM   #21
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My Support Groups:
I remain convinced that MAP is what caused my problems. I went swimming in the sea in the summer of 1983, its near a river outfall & alongside that river are cattle fields.
The next day it was like a switch had been flipped. I'm sure I swallowed some sea water that day & the rest is history. There is nothing else I can pin it on. I also use UHT milk for cereals now.
Rgds
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