Anti-Mycobacterium paratuberculosis (MAP) therapy for Crohn's disease: an overview and update.

The role of Mycobacterium avium subspecies paratuberculosis (MAP) in the pathogenesis of Crohn's disease (CD) has been strongly debated for many years. MAP is the known aetiological agent of Johne's disease, a chronic enteritis affecting livestock. At present, due to the paucity of high-quality data, anti-MAP therapy (AMT) is not featured in international guidelines as a treatment for CD. Although the much-quoted randomised trial of AMT did not show sustained benefits over placebo, questions have been raised regarding trial design, antibiotic dosing and the formulation used. There are several lines of evidence supporting the CD and MAP association with uncontrolled and controlled trials demonstrating effectiveness, including a retrospective review of cases treated at our own institution. Here, we provide an overview of the evidence supporting and refuting AMT in CD before focussing on updates of the current research in the field, including the ongoing trials with the novel RHB-104 formulation and the MAP vaccine trial. While controversial, gastroenterologists are often asked about long-term combination antibiotic therapy for CD. There has been broadcast and social media coverage surrounding this, particularly with regard to current trials. Although patients should not be deterred from treatments of proven effectiveness, this review aims to help with commonly asked questions and highlights our own approach for the use of anti-MAP in specific circumstances.

Safety and Immunogenicity of a Novel Recombinant Simian Adenovirus ChAdOx2 as a Vectored Vaccine.

Adenovirus vectored vaccines are a highly effective strategy to induce cellular immune responses which are particularly effective against intracellular pathogens. Recombinant simian adenovirus vectors were developed to circumvent the limitations imposed by the use of human adenoviruses due to widespread seroprevalence of neutralising antibodies. We have constructed a replication deficient simian adenovirus-vectored vaccine (ChAdOx2) expressing 4 genes from the Mycobacterium avium subspecies paratuberculosis (AhpC, Gsd, p12 and mpa). Safety and T-cell immunogenicity results of the first clinical use of the ChAdOx2 vector are presented here. The trial was conducted using a 'three-plus-three' dose escalation study design. We demonstrate the vaccine is safe, well tolerated and immunogenic.

The Consensus from the Mycobacterium avium ssp. paratuberculosis (MAP) Conference 2017.

On March 24 and 25, 2017 researchers and clinicians from around the world met at Temple University in Philadelphia to discuss the current knowledge of Mycobacterium avium ssp. paratuberculosis (MAP) and its relationship to human disease. The conference was held because of shared concern that MAP is a zoonotic bacterium that poses a threat not only to animal health but also human health. In order to further study this problem, the conferees discussed ways to improve MAP diagnostic tests and discussed potential future anti-MAP clinical trials. The conference proceedings may be viewed on the www.Humanpara.org website. A summary of the salient work in this field is followed by recommendations from a majority of the conferees.

Lamina propria macrophage phenotypes in relation to Escherichia coli in Crohn's disease.

BACKGROUND: Abnormal handling of E. coli by lamina propria (LP) macrophages may contribute to Crohn's disease (CD) pathogenesis. We aimed to determine LP macrophage phenotypes in CD, ulcerative colitis (UC) and healthy controls (HC), and in CD, to compare macrophage phenotypes according to E. coli carriage. METHODS: Mucosal biopsies were taken from 35 patients with CD, 9 with UC and 18 HCs. Laser capture microdissection was used to isolate E. coli-laden and unladen LP macrophages from ileal or colonic biopsies. From these macrophages, mRNA was extracted and cytokine and activation marker expression measured using RT-qPCR. RESULTS: E. coli-laden LP macrophages were identified commonly in mucosal biopsies from CD patients (25/35, 71 %), rarely in UC (1/9, 11 %) and not at all in healthy controls (0/18). LP macrophage cytokine mRNA expression was greater in CD and UC than healthy controls. In CD, E. coli-laden macrophages expressed high IL-10 & CD163 and lower TNFα, IL-23 & iNOS irrespective of macroscopic inflammation. In inflamed tissue, E. coli-unladen macrophages expressed high TNFα, IL-23 & iNOS and lower IL-10 & CD163. In uninflamed tissue, unladen macrophages had low cytokine mRNA expression, closer to that of healthy controls. CONCLUSION: In CD, intra-macrophage E. coli are commonly found and LP macrophages express characteristic cytokine mRNA profiles according to E. coli carriage. Persistence of E. coli within LP macrophages may provide a stimulus for chronic inflammation.

Mycobacterium avium Subspecies paratuberculosis: Human Exposure through Environmental and Domestic Aerosols.

Mycobacterium avium subspecies paratuberculosis (Map) causes Johne's disease in animals and is significantly associated with Crohn's disease (CD) in humans. Our previous studies have shown Map to be present in U.K. rivers due to land deposition from chronic livestock infection and runoff driven by rainfall. The epidemiology of CD in Cardiff showed a significant association with the River Taff, in which Map can be detected on a regular basis. We have previously hypothesized that aerosols from the river might influence the epidemiology of CD. In this preliminary study, we detected Map by quantitative PCR in one of five aerosol samples collected above the River Taff. In addition, we examined domestic showers from different regions in the U.K. and detected Map in three out of 30 independent samples. In detecting Map in river aerosols and those from domestic showers, this is the first study to provide evidence that aerosols are an exposure route for Map to humans and may play a role in the epidemiology of CD.

A rhodanine agent active against non-replicating intracellular Mycobacterium avium subspecies paratuberculosis.

BACKGROUND: Antibiotic therapy targeting chronic mycobacterial disease is often ineffective due to problems with the emergence of drug resistance and non-replicating persistent intracellular antibiotic resistant phenotypes. Strategies which include agents able to enhance host cell killing mechanisms could represent an alternative to conventional methods with the potential for host clearance if active against dormant phenotypes. Investigations of agents with potential activity against non-replicating mycobacteria however are restricted due to a need for assays that can assess bacterial viability without having to culture. RESULTS: This study describes the development and use of a pre16S ribosomal gene RNA/DNA ratio viability assay which is independent of the need for culture, supported by a novel thin layer accelerated mycobacterial colony forming method for determining viability and culturability of MAP in intracellular environments. We describe the use of these tools to demonstrate intracellular killing activity of a novel rhodanine agent (D157070) against the intracellular pathogen Mycobacterium avium subspecies paratuberculosis (MAP) and show that the culturability of MAP decreases relative to its viability on intracellular entry suggesting the induction of a non-culturable phenotype. We further demonstrate that D157070, although having no direct activity against the culturability of extracellular MAP, can bind to cultured MAP cells and has significant influence on the MAP transcriptome, particularly with respect of delta(L )associated genes. D157070 is shown to be taken up by bovine and human cells and able to enhance host cell killing, as measured by significant decreases in both culturability and viability of intracellular MAP. CONCLUSIONS: This work suggests that pre16srRNA gene ratios represent a viable method for studying MAP viability. In addition, the rhodanine agent D157070 tested is non-toxic and enhances cell killing activity against both growing and latent MAP phenotypes.

Mycobacterium avium subspecies paratuberculosis, Crohn's disease and the Doomsday scenario.

Johne's disease is chronic inflammation of the intestine caused by Mycobacterium avium subspecies paratuberculosis. Infection and disease are mainly in domestic livestock but can affect many species including primates. Johne's is a new disease which emerged at the turn of the 19th and 20th centuries and principally involved Europe and North America. It has since spread to former low incidence regions to become a global problem. Crohn's disease is a chronic inflammation of the intestine in humans which emerged in Europe and North America mid 20th century and increased to become a major healthcare problem. It has now spread to former low incidence regions. Infected animals shed Mycobacterium avium subspecies paratuberculosis in milk and into the environment. Human populations are widely exposed. Outcomes maybe influenced by microbial phenotype. Exposure to extracellular forms of these pathogens may confer some natural protection; exposure to intracellular forms which have passaged through milk macrophages or environmental protists may pose a greater threat to humans particularly individuals with an inherited or acquired susceptibility. Hot spots of human disease such as in Winnipeg which sits on rock at the junction of two rivers may result from local exposure to high levels of waterborne pathogens brought down from farmland. When appropriate methods are used most people with Crohn's disease are found to be infected. There are no data which demonstrate that these pathogens are harmless to humans. An overwhelming balance of probability and Public health risk favours the conclusion that Mycobacterium avium subspecies paratuberculosis is also pathogenic for people. A two tier co-operative pathogenic mechanism is proposed in Crohn's disease. Intracellular infection with the primary pathogen widely distributed throughout the gut causes an immune dysregulation and a specific chronic enteric neuropathy with loss of mucosal integrity. Segments of gross inflammatory disease result from the perturbed neuroimmune response to penetration into the gut wall of secondary pathogens from the lumen. These include both normal gut organisms and educated members of the enteric microbiome such as more aggressive E. coli. More new diseases may arise from failure to apply a range of remedial measures to this longstanding zoonotic problem.

Gut pathogens: invaders and turncoats in a complex cosmos.

Intestinal infections and diarrhoeal diseases of humans are under-reported yet each account worldwide for more deaths than those from Tuberculosis. For external gut pathogens to do this they have to penetrate, survive and prosper in an established and defended ecosystem comprising the human gut with a massive immune system, a structured tight mucosal lining and a lumen densely occupied by a huge community of diverse bacteria adapted to their environment and optimised in a balanced and mutually beneficial relationship with its host.

A novel multi-antigen virally vectored vaccine against Mycobacterium avium subspecies paratuberculosis.

BACKGROUND: Mycobacterium avium subspecies paratuberculosis causes systemic infection and chronic intestinal inflammation in many species including primates. Humans are exposed through milk and from sources of environmental contamination. Hitherto, the only vaccines available against Mycobacterium avium subspecies paratuberculosis have been limited to veterinary use and comprised attenuated or killed organisms. METHODS: We developed a vaccine comprising a fusion construct designated HAV, containing components of two secreted and two cell surface Mycobacterium avium subspecies paratuberculosis proteins. HAV was transformed into DNA, human Adenovirus 5 (Ad5) and Modified Vaccinia Ankara (MVA) delivery vectors. Full length expression of the predicted 95 kDa fusion protein was confirmed. PRINCIPAL FINDINGS: Vaccination of naïve and Mycobacterium avium subspecies paratuberculosis infected C57BL/6 mice using DNA-prime/MVA-boost or Ad5-prime/MVA-boost protocols was highly immunogenic resulting in significant IFN-gamma ELISPOT responses by splenocytes against recombinant vaccine antigens and a range of HAV specific peptides. This included strong recognition of a T-cell epitope GFAEINPIA located near the C-terminus of the fusion protein. Antibody responses to recombinant vaccine antigens and HAV specific peptides but not GFAEINPIA, also occurred. No immune recognition of vaccine antigens occurred in any sham vaccinated Mycobacterium avium subspecies paratuberculosis infected mice. Vaccination using either protocol significantly attenuated pre-existing Mycobacterium avium subspecies paratuberculosis infection measured by qPCR in spleen and liver and the Ad5-prime/MVA-boost protocol also conferred some protection against subsequent challenge. No adverse effects of vaccination occurred in any of the mice. CONCLUSIONS/SIGNIFICANCE: A range of modern veterinary and clinical vaccines for the treatment and prevention of disease caused by Mycobacterium avium subspecies paratuberculosis are needed. The present vaccine proved to be highly immunogenic without adverse effect in mice and both attenuated pre-existing Mycobacterium avium subspecies paratuberculosis infection and conferred protection against subsequent challenge. Further studies of the present vaccine in naturally infected animals and humans are indicated.

Mycobacterium avium subspecies paratuberculosis infection in cases of irritable bowel syndrome and comparison with Crohn's disease and Johne's disease: common neural and immune pathogenicities.

Mycobacterium avium subsp. paratuberculosis causes Johne's disease, a systemic infection and chronic inflammation of the intestine that affects many species, including primates. Infection is widespread in livestock, and human populations are exposed. Johne's disease is associated with immune dysregulation, with involvement of the enteric nervous system overlapping with features of irritable bowel syndrome in humans. The present study was designed to look for an association between Mycobacterium avium subsp. paratuberculosis infection and irritable bowel syndrome. Mucosal biopsy specimens from the ileum and the ascending and descending colon were obtained from patients with irritable bowel syndrome attending the University of Sassari, Sassari, Sardinia, Italy. Crohn's disease and healthy control groups were also included. Mycobacterium avium subsp. paratuberculosis was detected by IS900 PCR with amplicon sequencing. Data on the potential risk factors for human exposure to these pathogens and on isolates from Sardinian dairy sheep were also obtained. Mycobacterium avium subsp. paratuberculosis was detected in 15 of 20 (75%) patients with irritable bowel syndrome, 3 of 20 (15%) healthy controls, and 20 of 23 (87%) people with Crohn's disease (P = 0.0003 for irritable bowel syndrome patients versus healthy controls and P = 0.0000 for Crohn's disease patients versus healthy controls). One subject in each group had a conserved single-nucleotide polymorphism at position 247 of IS900 that was also found in isolates from seven of eight dairy sheep. There was a significant association (P = 0.0018) between Mycobacterium avium subsp. paratuberculosis infection and the consumption of hand-made cheese. Mycobacterium avium subsp. paratuberculosis is a candidate pathogen in the causation of a proportion of cases of irritable bowel syndrome as well as in Crohn's disease.

Replication and long-term persistence of bovine and human strains of Mycobacterium avium subsp. paratuberculosis within Acanthamoeba polyphaga.

Free-living protists are ubiquitous in the environment and form a potential reservoir for the persistence of animal and human pathogens. Mycobacterium avium subsp. paratuberculosis is the cause of Johne's disease, a systemic infection accompanied by chronic inflammation of the intestine that affects many animals, including primates. Most humans with Crohn's disease are infected with this chronic enteric pathogen. Subclinical infection with M. avium subsp. paratuberculosis is widespread in domestic livestock. Infected animals excrete large numbers of robust organisms into the environment, but little is known about their ability to replicate and persist in protists. In the present study we fed laboratory cultures of Acanthamoeba polyphaga with bovine and human strains of M. avium subsp. paratuberculosis. Real-time PCR showed that the numbers of the pathogens fell over the first 4 to 8 days and recovered by 12 to 16 days. Encystment of the amoebic cultures after 4 weeks resulted in a 2-log reduction in the level of M. avium subsp. paratuberculosis, which returned to the original level by 24 weeks. Extracts of resection samples of human gut from 39 patients undergoing abdominal surgery were fed to cultures of A. polyphaga. M. avium subsp. paratuberculosis detected by nested IS900 PCR with amplicon sequencing and visualized by IS900 in situ hybridization and auramine-rhodamine staining was found in cultures derived from 13 of the patients and was still present in the cultures after almost 4 years of incubation. Control cultures were negative. M. avium subsp. paratuberculosis has the potential for long-term persistence in environmental protists.

Detection and verification of Mycobacterium avium subsp. paratuberculosis in fresh ileocolonic mucosal biopsy specimens from individuals with and without Crohn's disease.

Mycobacterium avium subsp. paratuberculosis is a robust and phenotypically versatile pathogen which causes chronic inflammation of the intestine in many species, including primates. M. avium subsp. paratuberculosis infection is widespread in domestic livestock and is present in retail pasteurized cows' milk in the United Kingdom and, potentially, elsewhere. Water supplies are also at risk. The involvement of M. avium subsp. paratuberculosis in Crohn's disease (CD) in humans has been uncertain because of the substantial difficulties in detecting this pathogen. In its Ziehl-Neelsen staining-negative form, M. avium subsp. paratuberculosis is highly resistant to chemical and enzymatic lysis. The present study describes the development of optimized sample processing and DNA extraction procedures with fresh human intestinal mucosal biopsy specimens which ensure access to M. avium subsp. paratuberculosis DNA and maximize detection of these low-abundance pathogens. Also described are two nested PCR methodologies targeted at IS900, designated IS900[L/AV] and IS900[TJ1-4], which are uniquely specific for IS900. Detection of M. avium subsp. paratuberculosis in mucosal biopsy specimens was also evaluated by using mycobacterial growth indicator tube (MGIT) cultures (Becton Dickinson). IS900[L/AV] PCR detected M. avium subsp. paratuberculosis in 34 of 37 (92%) patients with CD and in 9 of 34 (26%) controls without CD (noninflammatory bowel disease [nIBD] controls) (P = 0.0002; odds ratio = 3.47). M. avium subsp. paratuberculosis was detected by IS900[L/AV] PCR in MGIT cultures after 14 to 88 weeks of incubation in 14 of 33 (42%) CD patients and 3 of 33 (9%) nIBD controls (P = 0.0019; odds ratio = 4.66). Nine of 15 (60%) MGIT cultures of specimens from CD patients incubated for more than 38 weeks were positive for M. avium subsp. paratuberculosis. In each case the identity of IS900 from M. avium subsp. paratuberculosis was verified by amplicon sequencing. The rate of detection of M. avium subsp. paratuberculosis in individuals with CD is highly significant and implicates this chronic enteric pathogen in disease causation.

Monitoring bacterial pathogens in the environment: advantages of a multilayered approach.

The application of advanced and highly sensitive molecular techniques to the detection of specific bacteria in the freshwater environment is limited, in the first instance, by sampling strategy and sample quality. Further combinations of molecular methods and techniques from apparently unrelated disciplines will ultimately shape the monitoring techniques of the future.

Use of bioinformatics to predict a function for the GS element in Mycobacterium avium subspecies paratuberculosis.

Mycobacterium avium subsp. Paratuberculosis (MAP) is a member of the Mycobacterium avium complex (MAC) and causes the inflammatory bowel disease, Johne's disease, in livestock. MAP has also been implicated as the causative agent of a similar disease, Crohn's disease, in humans. One of three major genetic differences between MAP and non-pathogenic MAC is the 6496-bp GS element. Based on the output from freely available protein sequence and structural bioinformatics tools, and the close homology of GS genes with the SER2 region of the closely related Mycobacterium avium subsp. Avium (MAA), we predict that GS encoded enzymes are involved in the biosynthesis of GDP-fucose, and the addition to, and modification of fucose on, the oligosaccharide moiety of GPL. GPL is a major constituent of the cell wall of the MAC and has immunomodulatory properties. Therefore, the enzymes involved in its synthesis may provide novel drug targets against MAP and other pathogenic MAC members.

Characterization of IS900 loci in Mycobacterium avium subsp. paratuberculosis and development of multiplex PCR typing.

Mycobacterium avium subsp. paratuberculosis is a pathogen that causes chronic inflammation of the intestine in many animals, including primates, and is implicated in Crohn's disease in humans. It differs from other members of the M. avium complex in having 14-18 copies of IS900 inserted into conserved loci in its genome. In the present study, genomic DNA flanking 14 of these insertions was characterized and homologues in the Mycobacterium tuberculosis and M. avium subsp. avium genomes were identified. These included regions encoding a sigma factor (sigJ) at locus 3, a nitrate reductase (nirA) at locus 4, a transcription regulator (tetR) and polyketide synthase at locus 6, and a 6-O-methylguanine methyltransferase at locus 9. In addition, locus numbers were assigned to 9 of 15 RFLP bands previously described. IS900 insertion at 7 of the 14 characterized loci was into the RBS of a gene substituting an RBS encoded by IS900 sited two bases closer to the initiation codon. IS900 insertion at five loci interrupted an ORF at the target site, one of which encoded a homologue of the immunodominant mycobacterial DesA1 protein. Eleven of eighty-one M. avium subsp. paratuberculosis isolates lacked the insertion site at locus 6 together with flanking genomic DNA. This region was also absent from seven reference strains of M. avium subsp. avium, from one M. avium subsp. silvaticum and from six other mycobacterial species. A multiplex PCR of IS900 loci (MPIL) typing method was developed which was able to discriminate 10 different types of M. avium subsp. paratuberculosis from the panel of 81 isolates with consistent differences between those of bovine and ovine origin. Nine MPIL types corresponded with a single PstI/Bst:EII RFLP type, suggesting that this method may be applicable to typing of M. avium subsp. paratuberculosis directly from a sample without the need for culture. The remaining MPIL type corresponded with seven PstI/BstEII RFLP types. Further resolution of these may come from sequencing the remaining four uncharacterized IS900 loci.

A low G+C content genetic island in Mycobacterium avium subsp. paratuberculosis and M. avium subsp. silvaticum with homologous genes in Mycobacterium tuberculosis.

The technique of representation difference analysis PCR has been applied to find genes specific to Mycobacterium avium subsp. paratuberculosis. This generated a 671 bp fragment which was used to isolate a larger genetic element found in the enteric pathogens M. avium subsp. paratuberculosis and M. avium subsp. silvaticum but which was absent from the very closely related and relatively benign M. avium subsp. avium. This element, designated GS, is greater than 6.5 kbp in length and has a G+C content 9 mol% lower than other genes from this species. There is a previously uncharacterized insertion sequence associated with one end. The GS element encodes five ORFs in M. avium subsp. paratuberculosis and M. avium subsp. silvaticum, all of which have counterparts encoded in Mycobacterium tuberculosis. Database searches revealed homologues for these ORFs in a number of bacterial species, predominantly Gram-negative organisms, including a number of enteric pathogens. These homologous genes encode functions related to LPS or extracellular polysaccharide biosynthesis. This element has a number of features in common with pathogenicity islands such as its low G+C content, an association with a putative insertion sequence and a grouping of genes of related function with a possible link to virulence. No direct link to pathogenicity has been shown but GS may belong to a group of related 'genetic islands' and represents the first such element to be identified in mycobacteria.

IS900 targets translation initiation signals in Mycobacterium avium subsp. paratuberculosis to facilitate expression of its hed gene.

The Mycobacterium avium subsp. paratuberculosis (formerly Mycobacterium paratuberculosis) atypical insertion sequence, IS900, encodes a novel gene on the complementary strand to the putative transposase, p43. This gene requires a promoter, ribosome binding site (RBS) and termination codon to be acquired upon insertion into the M. avium subsp. paratuberculosis genome and hence is designated the hed (host expression-dependent) gene of IS900. Analysis of IS900 insertion sites suggests that this element targets translation initiation signals in M. avium subsp. paratuberculosis, specifically inserting between the RBS and start codon of a putative gene sequence. This aligns the hed initiation codon adjacent to a functional RBS and possibly downstream of an active promoter, driving expression of Hed protein. We have confirmed this unique targeting process by detecting expression of hed in M. avium subsp. paratuberculosis at the level of transcription by reverse transcription-PCR. Further, two Hed-specific antibodies detected Hed translation products in Western blots of protein extracts from M. avium subsp. paratuberculosis. A recombinant form of Hed expressed and purified from Escherichia coli will facilitate studies of IS900 transposition and will also be assessed as a diagnostic antigen for M. avium subsp. paratuberculosis disease. Implications of IS900 insertion in M. avium subsp. paratuberculosis pathogenicity are discussed.