Detection of Mycobacterium avium subsp. paratuberculosis in Crohn’s Disease Patients and Ruminants Intestine by In Situ Hybridization

Mycobacterium avium subsp. paratuberculosis is an acid fast rod, extremely slow growing and mycobactin-dependent for its in vitro growth. This mycobacterium is the etiological agent of the chronic enteritis that affects domestic and wild ruminants, well-known as paratuberculosis or Johne’s disease. In humans, a possible M. avium subsp. paratuberculosis infection has been suggested to be involved in the pathogenesis of Crohn’s disease [1, 2], sarcoidosis; and recently sarcoidosis-like multisystem autosomal-dominant granulomatous disorder (Blau syndrome) [3, 4]. Crohn’s disease encompasses a spectrum of clinical and pathological patterns manifested by focal, asymmetric, transmural, and, chronic granulomatous inflammation affecting the gastrointestinal tract with the potential for systemic and extraintestinal complications. The etiology of Crohn’s disease is thought to be multifactorial, involving an interaction between genetic susceptibility, environmental triggers, and immune-mediated tissue injury. In this regard, there is considerable suggestive evidence to support an environmental factor, such as M. avium subsp. paratuberculosis infection. This evidence includes similarity of the macro and microscopic injuries, bacteriological isolation and molecular biology tests which have allowed the detection of this mycobacterium in intestine, milk and peripheral blood of


Introduction
Mycobacterium avium subsp.paratuberculosis is an acid fast rod, extremely slow growing and mycobactin-dependent for its in vitro growth.This mycobacterium is the etiological agent of the chronic enteritis that affects domestic and wild ruminants, well-known as paratuberculosis or Johne's disease.In humans, a possible M. avium subsp.paratuberculosis infection has been suggested to be involved in the pathogenesis of Crohn's disease [1,2], sarcoidosis; and recently sarcoidosis-like multisystem autosomal-dominant granulomatous disorder (Blau syndrome) [3,4].Crohn's disease encompasses a spectrum of clinical and pathological patterns manifested by focal, asymmetric, transmural, and, chronic granulomatous inflammation affecting the gastrointestinal tract with the potential for systemic and extraintestinal complications.The etiology of Crohn's disease is thought to be multifactorial, involving an interaction between genetic susceptibility, environmental triggers, and immune-mediated tissue injury.In this regard, there is considerable suggestive evidence to support an environmental factor, such as M. avium subsp.paratuberculosis infection.This evidence includes similarity of the macro and microscopic injuries, bacteriological isolation and molecular biology tests which have allowed the detection of this mycobacterium in intestine, milk and peripheral blood of

In situ hybridization (ISH)
The biotinylated probe used consists in 25 bases: TAGGACTGGTCGGCTGCAAGGTAG.This sequence belongs to the region 639-664 of the 3' chain of IS900.Sections of 2 m of thickness were placed in positive charged and pre-cleaned slides (FisherBiotech, USA).The slides were baked for 30 min at 60°C and submerged 5 min in xylol, rehydrated through graded alcohol (2 x 100%, 96%, 70%, 3 min each step) and air dried.Sections were incubated with proteinase K (DakoCytomation, USA) for 10 min at 42°C.Sections were washed twice with distilled water and H 2 O 2 0.3% were added and placed 5 min at room temperature (RT).By capillarity, 5-10 l of the probe at 17.04 g/ml was added (depending on the large of the sample).Slides were placed in termoblock for 10 min at 94.5°C.Hybridization was allowed overnight at 37°C.Sections were washed 4 times with Tris Buffered Solution (TBS, pH 7.5).Immediately, 50 l of the stringent wash solution (0.015 M NaN 3 ) were added on each slide and they were placed in termoblock, 30 min at 40°C and Finally TBS-washing for 4 times.

Signal detection
Two systems of signal detection were used.Catalyzed signal amplification system (CSA).50 l of primary streptavidin-HRP was added and slides were placed in termoblock 20 min at 37°C.Slides were washed 4 times with TBS.Then, the slides were incubated with a drop of Biotinyl-tyramide 20 min at RT. Slides were washed 4 times with TBS and a drop of secondary streptavidin-HRP was added to the sections and incubated 15 min at RT. Reaction was revealed with 20 l of 1:50 dilution of diaminobenzidine tetrahydrochloride (DAB).The reaction was observed in the microscope and it was stopped rinsing slides with distilled water.

Alkaline peroxidase system (AP)
Alkaline streptavidin phosphatase (DakoCytomation, USA) was added and incubated 30 min at 37°C.Slides were washed 4 times with TBS and the reaction was revealed adding 2 drops of N2 N-dimethylformamide (NBT/BCIP) to the 1.7%.Every 10 min the reaction was observed in the microscope and finally stopped rinsing slides with distilled water.Finally the reaction was counterstained with light green at 3%.Slides were rinsed with alcohol 96%; air dried and submerged in xylol to adhere them a cover slip with entellan resin.The results were submitted to a difference between proportions test in the program Statistica 6.0 (Statsoft, Tulsa Ok.EU), to verify the difference between groups A and B, as well as between groups C and D, with a confidence level of 95%.A p value of less than 0.05 was considered to be significant.

Results
In the ruminant groups A and B, as expected, all the samples were positive by in situ hybridization.In humans (groups C and D), overall positivity for M. avium subsp.paratuberculosis by in situ hybridization was 64.28% (n= 9).According to the groups distribution, all 7 patients (100%) in group C were positive for M. avium subsp.paratuberculosis by in situ hybridization, compared to 3/8 (37.5%) patients in group D; this difference was statistically significant (p= 0.0239).Of the 8 patients in which more than one sample was analyzed, 3 of them were positive, 2 (25%) showed one positive and one negative sample and in 3 cases (37.5%) all were negative.After in situ hybridization M. avium subsp.paratuberculosis was observed intracellularly within macrophages, epithelioid cells and giant cells in the intestinal mucosa and submucosa.In groups A, B and C the positive signal was observed within granulomas, whereas in group D it was seen within little macrophages that contribute to the inflammatory infiltrate.The positive signals were granular and intracytoplasmatic.In group A, the cases in group A with abundant intracellular Mycobacteria; the reaction coalesces and looks diffuse in the cytoplasm.

Discussion
Crohn's disease is a chronic inflammatory bowel disease of as yet unknown and possibly heterogeneous etiology.Diet, infections, immune dysregulation, and other unidentified environmental factors, all working under the influence of a genetic predisposition, have been suspiciously regarded.Among these, one of the most enduring hypotheses has been that M. avium subsp.paratuberculosis could be the causative agent of Crohn's disease.By bacteriological isolation, serology and techniques of molecular biology several authors have been able to detect the presence of M. avium subsp.paratuberculosis in a great proportion of patients with Crohn's disease [2,3,[5][6][7].Even more, some authors have reported clinical remission of Crohn's disease patients who received anti-mycobacterial therapy which included macrolide antibiotics [8].However, early results were inconsistent so that the role of this chronic enteric pathogen in the pathogenesis of Crohn's disease is not yet clearly established.The disparity in the earlier results can be due to several factors; among them the application of different methodologies.Successful isolations have been achieved by bacteriologists in veterinary laboratories since human medicine laboratories do not routinely culture mycobactin dependent mycobacteria, and therefore, don't necessary have adequate media for the isolation of these very difficult and slow growing organisms.In addition, when culture has been successful, the primary isolation consists on ZN-negative cell wall deficient forms which require long incubation times; which can extend for years to revert to bacillary forms [1].In this study all cases showed a granular and intracytoplasmatic stain pattern, as expected for intracellular bacteria like mycobacteria and Chlamydophila in agreement with the previous work described by Hulten et al and Meijer et al. [12,[14][15].In the veterinary group A, the tissues were from multibacillary Johne's disease, with the labeled probe therefore hybridizing to the bacillary forms of M. avium subsp.paratuberculosis.In group B, as well as in the cases of Crohn's disease in groups C and D; the labeled probe hybridized in situ to the ZN-negative form of these versatile pathogens showing that this technique is capable of recognizing the cell wall deficient phenotype.Therefore, the protocol of in situ hybridization used identifies both forms of M. avium subsp.paratuberculosis.The enzymatic digestion treatment is apparently able to permeabilise the mycobacterial cellular wall without altering intestine tissue architecture in ruminants and humans.The identification, by in situ hybridization of positive cases to M. avium subsp.paratuberculosis in groups B, C and D is consistent with the findings of the study of Hulten et al in which they identify spheroplasts of this mycobacterium in paucibacillary cases of Johne´s disease in ovines [14], as well as their later study when they demonstrated the presence of spheroplasts in 7 of 37 (18.91%) of the Crohn's disease patients [15].In the same way, Sechi et al demonstrated by in situ hybridization, the presence of M. avium subsp.paratuberculosis spheroplasts in 35 of 48 (72.91%) of Crohn's disease patients, despite the fact that on this occasion they did not find M. avium subsp.paratuberculosis by PCR IS900 in the same samples [17].However, these authors did identify M. avium subsp.paratuberculosis by PCR in subsequent work using improved sample processing procedures [18].Although some literature classifies patients with Crohn's disease according to the fistulous or obstructive phenotype, in our present series of clinical cases we observed that the same patient can demonstrate both phenotypes in different intestinal segments.The value of this 126 method of classification is in our view uncertain.Most of the studies of the molecular pathology in Crohn's disease do not differentiate between patients with the granulomatous and non granulomatous presentation.The results obtained in the present study indicate that a greater proportion of patients with granulomatous disease are M. avium subsp.paratuberculosis positive by in situ hybridization.This observation agrees with those of Hulten et al.However; we found the positive in situ hybridization signal particularly in relation to the granulomas in Crohn's disease patients.Greenstein suggested that the identification of M. avium subsp.paratuberculosis DNA in the tissues of patients with Crohn's disease cannot be taken as proof of a causal association between this mycobacterium with Crohn's disease.It could be a coincidence of M. avium subsp.paratuberculosis DNA being in the intestinal lumen of people who had ingested cow's milk containing these pathogens [19].In the present study using an in situ diagnostic procedure we were able to confirm the presence of M. avium subsp.paratuberculosis within the inflamed gut wall and related to the granulomatous lesions.In this situation M. avium subsp.paratuberculosis is most unlikely to be a transient food-borne contaminant.It is much more likely that is well characterized multi-host chronic enteric pathogen is related to the causation of the chronic inflammatory intestinal disease.The exact mechanism is not know but one theory is that M. avium subsp.paratuberculosis either can infect the host and cause a primary infection responsible for the development of the disease, or it is a secondary opportunistic infection that could perpetuate the cycle of inflammation and cytokine release.Regarding the Crohn's disease pathogenesis, there is no doubt that Crohn's disease is a result of immune dysregulation, where an excessive TH1-driven, cell-mediated immune response is elicited and persists.In this context, chronic intracellular M. avium subsp.paratuberculosis infection may be the trigger for this excessive TH1 immune response that results in the clinical, endoscopic, radiological, and histological manifestation known as Crohn's disease.Even if M. avium subsp.paratuberculosis is not causally linked with Crohn's disease, the presence of DNA from M. avium subsp.paratuberculosis and other bacteria within the mucosa might have secondary clinical implications.Some studies have proved that bacterial DNA has immunomodulatory activity by signaling via pattern recognition receptors such as toll-like receptor 9 on host epithelial cells [20].Further studies are needed, and it is imperative that researchers adopt standardized methods and techniques, including appropriate controls, and search for intracellular bacteria other than M. avium subsp.paratuberculosis.The availability of these tests could influence the therapeutic approach of these patients; antimycobacterial therapy may include macrolide antibiotics which have been beneficial for remission of intestinal lesions [8].Although previous epidemiological studies have suggested that the incidence of inflammatory bowel disease is lower in Latin American populations, recent studies have demonstrated that the incidence is increasing and may be higher than suspected.For example, Appleyard et al showed that in Puerto Rico, an Hispanic predominant population; the total incidence of IBD increased significantly between 1996 and 2000 (3.07/100,000 to 7.74/100,000; p <0.001), being significantly higher for Crohn's disease (four-fold increase, p < 0.01), but not for ulcerative colitis (1.7-fold increase) [10].Puerto Ricans are genetically complex and comprised of various proportions of Native American, African, and European genetic origins, as our Mexican population.Although the reasons for this are unclear, the data suggest several possible explanations such as changes in health care system, movement www.intechopen.comDetection of Mycobacterium avium subsp.paratuberculosis in Crohn's Disease Patients and Ruminants Intestine by In Situ Hybridization 127 from rural areas to urban cities, a more Westernized and fairly high carbohydrates and fat diet, and decrease in the incidence of intestinal parasites; which have a protective effect via regulation of the immune response.

Conclusion
In conclusion, despite the obvious need to evaluate a greater number of Mexican patients with and without inflammatory bowel disease these initial results remark the exposure of the Mexican population to M. avium subsp.paratuberculosis and provide further evidence in support of the zoonotic features of this agent.Taking on account the public health relevance of the issue as well as its economic impact, wider studies are needed in Mexico.
Fig. 1.Case of paratuberculosis from group A. ISH, DAB.40x.Positive reaction inside macrophages in ileal submucosa.
www.intechopen.comDetection of Mycobacterium avium subsp.paratuberculosis in Crohn's Disease Patients and Ruminants Intestine by In Situ Hybridization 125