Mycobacterium avium subspecies paratuberculosis causes Crohn's disease in some inflammatory bowel disease patients

Crohn’s disease(CD)is a chronic inflammatory condition that plagues millions all over the world.This debilitating bowel disease can start in early childhood and continue into late adulthood.Signs and symptoms are usually many and multiple tests are often required for the diagnosis and confirmation of this disease.However,little is still understood about the cause(s)of CD.As a result,several theories have been proposed over the years.One theory in particular is that Mycobacterium avium subspecies paratuberculosis(MAP)is intimately linked to the etiology of CD.This fastidious bacterium also known to cause Johne’s disease in cattle has infected the intestines of animals for years.It is believed that due to the thick,waxy cell wall of MAP it is able to survive the process of pasteurization as well as chemical processes seen in irrigation purification systems.Subsequently meat,dairy products and water serve as key vehicles in the transmission of MAP infection to humans(from farm to fork)who have a genetic predisposition,thus leading to the development of CD.The challenges faced in culturing this bacterium from CD are many.Examples include its extreme slow growth,lack of cell wall,low abundance,and its mycobactin dependency.In this review article,data from 60 studies showing the detection and isolation of MAP by PCR and culture techniques have been reviewed.Although this review may not be 100%comprehensive of all studies,clearly the majority of the studies overwhelmingly and definitively support the role of MAP in at least30%-50%of CD patients.It is very possible that lack of detection of MAP from some CD patients may be due to the absence of MAP role in these patients.The latter statement is conditional on utilization of methodology appropriate for detection of human MAP strains.Ultimately,stratification of CD and inflammatory bowel disease patients for the presence or absence of MAP is necessary for appropriate and effective treatment which may lead to a cure.

High prevalence of viable Mycobacterium avium subspecies paratuberculosis in Crohn's disease

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 CDassociated 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.

On deaf ears, Mycobacterium avium paratuberculosis in pathogenesis Crohn's and other diseases

The historic suggestion that Mycobacterium avium subsp. paratuberculosis(Map) might be a zoonotic pathogen was based on the apparent similarity of lesions in the intestine of patients with Crohn's disease(CD) with those present in cattle infected with Map, the etiological agent of Johne's disease. Reluctance to fully explore this possibility has been attributed to the difficulty in demonstrating the presence of Map in tissues from patients with CD. Advances in technology have resolved this problem and revealed the presence of Map in a significant proportion of patients with CD and other diseases. The seminal finding from recent investigations, however, is the detection of Map in healthy individuals with no clinical signs of disease. The latter observation indicates all humans are susceptible to infection with Map and lends support to the thesis that Map is zoonotic, with a latent stage of infection similar to tuberculosis, where infection leads to the development of an immune response that controls but does not eliminate the pathogen. This clarifies one of the reasons why it has been so difficult to document that Map is zoonotic and associated with the pathogenesis of CD and other diseases. As discussed in the present review, a better understanding of the immune response to Map is needed to determine how infection is usually kept under immune control during the latent stage of infection and elucidate the triggering events that lead to disease progression in the natural host and pathogenesis of CD and immune related diseases in humans.

Correlation between rpoB gene mutation in Mycobacterium avium subspecies paratuberculosis and clinical rifabutin and rifampicin resistance for treatment of Crohn’s disease

AIM： To investigate overlapping regions of the rpoB gene previously involved with rifamycin resistance in M. tuberculosis and seek correlation between rpoB mutations in dinical MAP strains with susceptibility to RIF and RFB. METHODS： We designed a molecular-based PCR method for the evaluation of rifabutin （RFB） and rifampicin （RIF） resistance based on probable determinant regions within the rpoB gene of MAP, including the 81 bp variable site located between nucleotides 1363 and 1443. The minimum inhibitory concentration （MIC） for RIF was also determined against 11 MAP isolates in attempt to seek correlation with rpoB sequences. RESULTS： We determined that MAP strain 18 had an MIC of 〉 30 mg/L and ≤ 5 mg/L for RIF and RFB respectively, and a significant and novel rpoB mutation C1367T, compared to an MIC of ≤ 1.0 mg/L for both drugs in the wild type MAP. The 30-fold increase in the MIC was a direct result of the rpoB mutation C1367T, which caused an amino acid change Thr456 to Ile456 in the drug＇s binding site. In addition, MAP strain 185 contained five silent rpoB mutations and exhibited an MIC comparable to the wild-type. Moreover, our in vitro selected mutation in MAP strain UCF5 resulted in the generation of a new resistant strain （UCF5-RIF16r） that possessed T1442C rpoB mutation and an MIC 〉 30 mg/L and 〉 10 mg/L for RIF and RFB respectively. Sequencing of the entire rpoB gene in MAP strains UCF4, 18, and UCF5-RIF16r revealed an rpoB mutation A2284C further downstream of the 81 bp variable region in UCF4, accounting for observed slight increase in MIC. In addition, no other significant mutations were found in strains 18 and UCF-RIF16r. CONCLUSION： The data clearly illustrates that clinical and in vitro-selected MAP mutants with rpoB mutations result in resistance to RIF and RFB, and that a single amino acid change in the beta subunit may have a significant impact on RIF resistance. Unconventional drug susceptibility testing such as our molecular approach will be beneficial for evaluation of antibiotic effectiveness. This molecular approach may also serve as a model for other drugs used for treatment of MAP infections.

Paratuberculosis Infection in Camel (<i>Camelus dromidarius</i>): Current and Prospective Overview

Camel (Camelus dromidarius) is an important source of meat and milk and an iconic animal of the Saudi Arabian heritage. The accumulative evidence indicated the spread of paratuberculosis infection in the camel herds. Despite the explicit studies on the details of the disease in camels and methods of its diagnosis, paratuberculosis infection in camels suffers from wide gap of knowledge of the disease pathogenesis, camel immune responses to the infection and factors that enhance camel’s resistance to the infection. The review discusses the current available information of the disease pathobiology and the approaches employed in the diagnosis of paratuberculosis infection in camel. Effective control of the disease in camel prompts for urgent innovation of the current approaches in the diagnosis. Efficient policies and inspection tools are becoming vital to tackle the possible threats of Crohn’s disease to the public health due to the meat and milk consumption.

Mycobacterium avium subspecies paratuberculosis in the causation of Crohn's disease

Mycobacterium avium subspecies paratuberculosis(MAP),originally called Johne’s bacillus was firstdescribed from Germany in 1895 as the cause of achronic inflammatory disease of the intestine in acow.As the 20th century progressed,clinical andsub-clinical MAP infection in farm animals inWestern Europe appeared to become moreprevalent.Among the early reviews available

Mycobacterium avium subspecies paratuberculosis in the etiology of Crohn's disease, cause or epiphenomenon?

The origin of inflammatory bowel disease is unknown. Attempts have been made to isolate a microorganism that could explain the onset of inflammation, but no pathological agent has ever been identified. Johne&#x02019;s disease is a granulomatous chronic enteritis of cattle and sheep caused by Mycobacterium avium subspecies paratuberculosis (MAP) and shows some analogies with Crohn&#x02019;s disease (CD). Several studies have tried to clarify if MAP has a role in the etiology of CD. The present article provides an overview of the evidence in favor and against the &#x0201c;MAP-hypothesis&#x0201d;, analyzing the methods commonly adopted to detect MAP and the role of antimycobacterial therapy in patients with inflammatory bowel disease. Studies were identified through the electronic database, MEDLINE, and were selected based on their relevance to the objective of the review. The presence of MAP was investigated using multiple diagnostic methods for MAP detection and in different tissue samples from patients affected by CD or ulcerative colitis and in healthy controls. On the basis of their studies, several authors support a close relationship between MAP and CD. Although increasing evidence of MAP detection in CD patients is unquestionable, a clear etiological link still needs to be proven.

The 19 kDa Protein from <i>Mycobacterium avium subspecies paratuberculosis</i>Is a Glycolipoprotein

This study characterizes the 19 kDa protein expressed by Mycobacterium avium subspecies paratuberculosis (MAP) as a glycolipoprotein, providing the foundation for future experiments regarding its antigenicity and role in disease pathogenicity. We have previously shown that a 4.8 kb insert from MAP will produce a 16 kDa recombinant protein when expressed in Escherichia coli and 19 kDa recombinant protein when expressed in M. smegmatis (smeg19K). The difference of 3 kDa in size of these expressed proteins may be related to post translational modifications that occur in Mycobacterium species. We hypothesized that smeg19K is a glycolipoprotein since BLAST analysis revealed approximately 76% amino acid identity between the MAP 19 kDa protein and a known lipoglycoprotein, the 19 kDa protein of M. tuberculosis. This prediction was confirmed by the following positive staining of smeg19K with Sudan Black 4B, a postelectrophoresis dye used to stain for lipids. Smeg19K has also stained positively for glycosylation with the lectin concavalin A, a highly specific stain for mannose residues. As expected, treatment with tunicamycin (an antibiotic known to inhibit N-glycosylation) and treatment with deglycosylation assay (non-specific for mannose), showed no reduction in size of 19 kDa glycolipoprotein.

Evaluation of “Indigenous Vaccine” Developed Using “Indian Bison Type” Genotype of <i>Mycobacterium avium subspecies paratuberculosis</i>Strain “S5” of Goat Origin in a Sheep Flock Endemic for Johne’s Disease: A Three Years Trial in India

“Indigenous vaccine” developed from native “Indian Bison Type” strain (“S5”) of Mycobacterium avium subspecies paratuberculosis (MAP) of “goat origin” was first time evaluated in a sheep flock of Bharat Merino breed located in Mannavanur town of Tamil Nadu in South India. Therapeutic efficacy of the vaccine was evaluated for 3 years between 2008 and 2010, on the basis of improvements in productivity (body weights, reproductive efficiency and survivability), physical condition, clinical symptoms (weakness, diarrhea, wool quality), immune response (sero-conversion) and infection load in feces (shedding). After immunization of the flock in 2008, the successive progenies of 112 and 53 lambs born in 2009 and 2010, respectively were vaccinated. Whereas, 40 lambs born to control animals were kept as unvaccinated controls. Though gain in body weights in vaccinated versus controls were not significant in 2008, growth rates were distinctly superior in Ist and IInd generations of vaccinated lambs. Reproductive performance (tupping percent) and survivability of lambs and adult sheep improved significantly. There was overall reduction in yearly morbidity (diarrhea) and mortality rates of the flock in post vaccination years. Shedding of MAP in feces was reduced in vaccinated sheep by 6.2%, 14.3% and 27.3% in 2008, 2009 and 2010 respectively, whereas shedding increased in the control sheep. Seromonitoring of the animals by “indigenous ELISA kit” showed enhanced “flock immunity” in successive generations. “Indigenous vaccine” reduced clinical disease and shedding and improved immunity and productivity of Bharat Merino flock, endemic for Johne’s disease.

Sero-Prevalence of Paratuberculosis (Johne's Disease) in Cattle Population of South-Western Bangalore Using ELISA Kit

Johne's disease or paratuberculosis is a chronic mycobacterial infection that affects cattle, sheep, goats and other ruminants, adversely, leading to huge economic losses throughout the world. The estimation of sero-prevalence of this disease in the cattle population of south-western Bangalore, Karnataka, using an immunological assay and statistical analyses, was the objective of this study. One of the diagnostic tools used to detect an antigen or an antibody in animal serum or milk is the Enzyme Linked Immuno-Sorbent Assay, which has been widely used in the research and diagnosis of animal and human diseases as its accuracy is of nanogram-picogram/milliltre level. In the present study, indirect-ELISA was used to diagnose and estimate the sero-prevalence of paratuberculosis in cattle showing diarrhoea and/or anaemia, at 5 local dairy farms in south-west Bangalore, India. Out of 350 bovine serum samples, 53 (15.14%) were positive, 55 milk samples out of 300 were found positive (18.33%) for antibody against Johne's disease by indirect ELISA. The positive samples were then confirmed by direct smear examination of dung by Ziehl-Neelsen staining. Statistical analyses were carried out to indicate the seroprevalence of Johne's disease in the cattle population of this region to be 15 ± 10%, taking a confidence interval of 95%. The results emphasize the need to prevent the further spread of infection to other susceptible animals and humans as the causative organism, Mycobacterium avium subsp. paratuberculosis is implicated in Crohn's disease, an irritable bowel syndrome in humans.

Mycobacterium avium subspecies paratuberculosis—An environmental trigger of type 1 diabetes mellitus

Type 1 diabetes mellitus (T1DM) is an autoimmune disease. The etiology of T1DM is incompletely understood but environmental agent(s) are thought to trigger T1DM in the genetically at-risk. Humans are widely exposed to Mycobacterium avium subspecies paratuberculosis (MAP), a proven multi-host chronic enteric pathogen that is mostly studied in ruminant animals and causes the inflammatory disease paratuberculosis or Johne’s disease. In humans, MAP is the putative cause of Crohn’s disease and has been linked to sarcoidosis, autoimmune thyroiditis, multiple sclerosis and autoimmune diabetes. The role of MAP as a trigger for T1DM was first postulated in 2005;subsequent studies suggest a link. This article discusses MAP, human exposure to MAP, genetic susceptibility to MAP and MAP in human disease including T1DM.

Evaluation of a Live Paratuberculosis Vaccine in Endemically Infected Flocks of Sheep and Goats in Greece

A large 7-year vaccination trial was conducted in 15 flocks of goats and 7 flocks of sheep, known to be infected with Mycobacterium avium subsp. paratuberculosis (MAP), in Northern Greece. A total of 3665 kids and 1685 lambs, 7 - 30 days old, were vaccinated during 1995-1999. Seven hundred and seventy-five kids and 413 lambs were kept as unvaccinated controls. For each trial, the Incidence Rate Ratio (IRR), with respective exact 95% confidence intervals, was calculated. All IRR point estimates for young animals were very large (from 5.68 to 11.78 for kids and from 4.28 to 10.08 for lambs), while none of the 95% confidence intervals included 1. The protective effect of vaccination was large and the difference in mortality among vaccinated and unvaccinated animals was more pronounced in young animals. The effect in adult animals was smaller than in young animals;it was, however, still considerable. Upon visual inspection of the K-M curves, it seems that for the young animal trials the vaccinated and control-group curves were diverging increasingly over time, which indicates that the gain from the vaccination (or the loss from non-vaccination) might increase over time during the trial.

Survival mechanisms of <i>Mycobacterium avium</i>subspecies <i>paratuberculosis</i>within host species and in the environment—A review

Mycobacterium avium subsp. paratuberculosis causes chronic inflammation of the intestine known as Johne’s disease (JD) in domestic and wild ruminants including primates. MAP has also been associated with inflammatory bowel disease (IBD) so called Crohn’s disease (CD) of human beings, which is incurable even after surgery. By virtue of the pasteurization resistant power, high endemicity of the infection in animals continues to be the permanent source of infection to human population. High bio-burden of MAP in wide range of biotic (animal hosts including human beings) and abiotic environment in each and every country where it has been investigated, serves a reminder about the survival abilities of the MAP in diverse range of environmental conditions. Ability of the MAP to evade immune system of the host and the temporal events during infection of the macro-phages, is an area of major concern and research activities as the pattern of distribution are quiet different from those of other pathogenic intracellular organisms. Moreover, the organism can survive over a wide range of environmental conditions such as high and low environmental temperatures, pasteurization, low pH, and high salt concentration etc. This superior survival efficiency from environmental degradation and dormancy within host allows the pathogen to be available for causing disease and pathogenicity in animals and human beings, when conditions are favorable. Perusal of literature reveal that, despite the availability of whole genome sequence of MAP, a very little is known about the replication, persistence and survival mechanisms of this pathogen. Therefore, this review tries to address the survival mechanisms of Mycobacterium avium subspecies paratuberculosis in the different host species and adverse environmental conditions in order to allow designing of more rational diagnostic and control procedures.

Bfra-loaded nanoparticles confer protection against paratuberculosis infection

Paratuberculosis is a chronic wasting disease of granulomatous enteritis in ruminants caused by Mycobacterium avium subsp.paratuberculosis(M.paratuberculosis,MAP)resulting in heavy economic losses to dairy industries worldwide.Currently,commercial vaccines were not effective in preventing pathogen shedding and were reported with serious side effects.To develop a novel and smarter paratuberculosis vaccine,we utilized PLGA nanoparticles encapsulating the Bfra antigen(Bfra-PLGA NPs).We observed that mice vaccinated with Bfra-PLGA NPs exhibited an enhanced secretory IFN-γ,CD4+T cells response and antibody IgG against MAP infection.In addition,secretions of the inflammatory cytokine TNF-αand IL-10 were increased following treatment with Bfra-PLGA NPs.A significant reduction in bacterial load was observed in the livers and spleens of animals vaccinated with Bfra-PLGA NPs.Furthermore,Bfra-PLGA NPs were effective to alleviate the pathological lesions of livers in mice.Overall,our approach provides a rational basis for employing PLGA nanoparticles to develop improved vaccines that induced protective immunity against paratuberculosis.

Pathophysiology of autism spectrum disorders:Revisiting gastrointestinal involvement and immune imbalance

Autism spectrum disorders(ASD)comprise a group of neurodevelopmental abnormalities that begin in early childhood and are characterized by impairment of social communication and behavioral problems including restricted interests and repetitive behaviors.Several genes have been implicated in the pathogenesis of ASD,most of them are involved in neuronal synaptogenesis.A number of environmental factors and associated conditions such as gastrointestinal(GI)abnormalities and immune imbalance have been linked to the pathophysiology of ASD.According to the March 2012 report released by United States Centers for Disease Control and Prevention,the prevalence of ASD has sharply increased during the recent years and one out of 88 children suffers now from ASD symptoms.Although there is a strong genetic base for the disease,several associated factors could have a direct link to the pathogenesis of ASD or act as modifiers of the genes thus aggravating the initial problem.Many children suffering from ASD have GI problems such as abdominal pain,chronic diarrhea,constipation,vomiting,gastroesophageal reflux,and intestinal infections.A number of studies focusing on the intestinal mucosa,its permeability,abnormal gut development,leaky gut,and other GI problem raised many questions but studies were somehow inconclusive and an expert panel of American Academy of Pediatrics has strongly recommended further investigation in these areas.GI tract has a direct connection with the immune system and an imbalanced immune response is usually seen in ASD children.Maternal infection or autoimmune diseases have been suspected.Activation of the immune system during early development may have deleterious effect on various organs including the nervous system.In this review we revisited briefly the GI and immune system abnormalities and neuropeptide imbalance and their role in the pathophysiology of ASD and discussed some future research directions.

NOD2 and ATG16L1 polymorphisms affect monocyte responses in Crohn's disease

AIM： TO assess whether polymorphisms in NOD2 and ATG16L1 affect cytokine responses and mycobacterium avium subspecies paratuberculosis （MAP） survival in monocytes from Crohn＇s disease （CD） patients METHODS： Monocytes were isolated from peripheral blood of CD patients of known genotype for common single nucleotide polymorphisms of NOD2 and ATG16L1, Monocytes were challenged with MAP and bacterial per- sistence assessed at subsequent time-points. Cytokine responses were assayed using a Milliplex multi-analyte profiling assay for 13 cytokines. RESULTS： Monooltes heterozygous for a NOD2 polymorphism （R702W, P268S, or 1007fs） were more permissive for growth of MAP （P = 0.045） than those without. There was no effect of NOD2 genotype on subsequent cytokine expression. The T300A polymorphism of ATG16L1 did not affect growth of MAP in our model （P = 0.175）, but did increase expression of cytokines interleukin （IL）-10 （P = 0.047） and IL-6 （P = 0.019）. CONCLUSION： CD-associated polymorphisms affected the eliminaUon of MAP from ex v/vo monooltes （NOD2）, or expression of certain oltokines （ATG16LI）, implying independent but contributory roles in the pathogenesis of CD.

Domino effect of hypomagnesemia on the innate immunity of Crohn's disease patients

Digestive diseases play major role in development and complications of other disorders including diabetes.For example,Crohn’s disease(CD)is an inflammatory bowel disease associated with Mycobacterium avium subspecies paratuberculosis.The inflammation is a complex process that involves the activity of both innate and adaptive immune responses.CD lesions are primarily due to T cell response,however;innate immune response has a significant role in initiating its pathogenesis.Toll-like receptors and NOD-like receptors promote the activity of nuclear factor(NF)-κB pathway for cytokines production.This results in the production of high levels of tumor necrosis factor-α,interleukin(IL)-1βand IL-6.Moreover,intestinal inflammation of CD is related to increased activity of NMDA receptors and the release of substance P.Imbalanced magnesium homeostasis in CD is a frequent finding in CD,Diabetes and others.The loss of such a major mineral affects many physiological processes in the body including its role as an immunomodulator.This review aims to(1)describe the significance of hypomagnesemia in the release of pro-inflammatory mediators in CD;(2)demonstrate effects of magnesium on pathways like NF-κB;(3)address the role of hypomagnesemia in the activity of CD;and(4)examine possible future research to establish a standard magnesium supplementation strategy;helping patients with CD or other disorders to maintain a sustained remission.

The many faces of Crohn’s Disease: Latest concepts in etiology

The notion that Crohn’s Disease (CD) occurs as a result of an aberrant reaction to the commensal microbiota in genetically susceptible hosts is widely regarded by physicians and scientists as fact. Yet although it is undisputed that Crohn’s Disease is immune-mediated, an aberrant reaction to one’s own native flora is far from proven. The aim of the current review is to present a summary of the known infectious causes of Crohn’s Disease, whilst highlighting the limitations of using outdated methods to attempt to classify the disease as a single entity. We propose a re-classification of Crohn’s Disease, and suggest that the disease is best conceptualized as a syndrome, an “umbrella-like” term comprising a group of diseases with varying infective etiologies, which clinically, endoscopically and histologically are indistinguishable from CD.

Mycobacterium avium subspecies hominissuis in Crohn’s disease:a case report

We have cultured Mycobacteria avium subspecies hominissuis(MAH)from the blood of a patient with Crohn’s disease.The patient is a 21 year-old-female with a diagnosis of Crohn’s disease for two years.She had been treated with corticosteroids and Humira for six months.A blood specimen was cultured in a specialized medium,and there was visible bacterial growth present in the liquid culture medium after eight weeks.PCR analysis of the bacterial growth and subsequent direct sequencing of the PCR amplicon confirmed the presence of MAH.The significance of this finding is discussed.