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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.

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 &lsquo, three-plus-three&rsquo, dose escalation study design. We demonstrate the vaccine is safe, well tolerated and immunogenic.

Background and Aim Escherichia coli can be isolated from lamina propria macrophages in Crohn's disease (CD), and their intramacrophage persistence may provide a stimulus for inflammation. To further determine the contributions of macrophage dysfunction and E. coli pathogenicity to this, we aimed to compare in vitro functioning of macrophages from patients with CD and healthy controls (HC) in response to infection with CD-derived adherent-invasive E. coli (AIEC) and less pathogenic E. coli strains. Methods Monocyte-derived macrophages were cultured from patients with CD and HC. Intramacrophage survival of E. coli strains (CD-derived adherent-invasive [AI] and non-AI strains and laboratory strain K-12) was compared. Macrophage cytokine release (tumor necrosis factor alpha [TNFα], interleukin [IL]-23, IL-8 and IL-10) and monocyte phagoctyosis and respiratory burst function were measured after E. coli infection. For CD patients, laboratory data were correlated with clinical phenotype, use of immunomodulation, and CD risk alleles (NOD2, IL-23R, ATG16L1 and IRGM). Results Attenuated TNFα and IL-23 release from CD macrophages was found after infection with all E. coli strains. There was prolonged survival of CD-derived AIEC, CD-derived non-AIEC and E. coli K-12 in macrophages from CD patients compared to within those from HC. No abnormality of monocyte phagocytosis or respiratory burst function was detected in CD. Macrophage dysfunction in CD was not influenced by phenotype, use of immunomodulation or genotype. Conclusions CD macrophage responses to infection with E. coli are deficient, regardless of clinical phenotype, CD genotype or E. coli pathogenicity. This suggests host immunodeficiency is an important contributor to intramacrophage E. coli persistence in CD.

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 subsp. paratuberculosis from infected animals enters surface waters and rivers in runoff from contaminated pastures. We studied the River Tywi in South Wales, United Kingdom, whose catchment comprises 1,100 km 2 containing more than a million dairy and beef cattle and more than 1.3 million sheep. The River Tywi is abstracted for the domestic water supply. Between August 2002 and April 2003, 48 of 70 (68.8%) twice-weekly river water samples tested positive by IS 900 PCR. In river water, the organisms were associated with a suspended solid which was depleted by the water treatment process. Disposal of contaminated slurry back onto the land established a cycle of environmental persistence. A concentrate from 100 liters of finished water tested negative, but 1 of 54 domestic cold water tanks tested positive, indicating the potential for these pathogens to access domestic outlets. In the separate English Lake District region, with hills up to 980 m, tests for M. avium subsp. paratuberculosis in the high hill lakes and sediments were usually negative, but streams and sediments became positive lower down the catchment. Sediments from 9 of 10 major lakes receiving inflow from these catchments were positive, with sediment cores indicating deposition over at least 40 to 50 years. Two of 12 monthly 1-liter samples of effluent and a single 100-liter sample from the Ambleside sewage treatment works were positive for M. avium subsp. paratuberculosis . Since Lake Ambleside discharges into Lake Windermere, which is available for domestic supply, there is a potential for these organisms to cycle within human populations.

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 IS 900 PCR with amplicon sequencing and visualized by IS 900 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.

Summary Mycobacterium avium subspecies paratuberculosis infection in domestic livestock is widespread in many countries throughout the world. Studies in Europe and the USA show that M. avium subspecies paratuberculosis can be cultured from retail pasteurized cow’s milk and that these organisms are being transmitted to humans by this route. Most people with chronic inflammation of the intestine of the Crohn’s disease type are infected with these chronic enteric pathogens. The production and consumption of cow’s milk has increased in China and so also has the incidence of Crohn’s disease. The present preliminary investigation was carried out to determine whether M. avium subspecies paratuberculosis is present in the intestinal tissues of Chinese patients with Crohn’s disease who have never left China. Archival paraffin-embedded surgical pathology blocks from patients having surgery for Crohn’s disease (CD) or for cancer (nIBD) in China were studied. M. avium subspecies paratuberculosis was detected by nested IS900 PCR with Southern blotting and amplicon sequencing. The intestinal tissues of 9 of 13 (69.2% )C D patients and 2 of 14 (14.3%) nIBD patients were IS900 PCR positive (P = 0.0063; odds ratio = 13.5). These initial studies suggest that people in China are exposed to M. avium subspecies paratuberculosis and that as in other countries, the infection is significantly associated with Crohn’s disease. M. avium subspecies paratuberculosis in dairy herds and retail milk in China needs to be investigated.

In South Wales, United Kingdom, a populated coastal region lies beneath hill pastures grazed by livestock in which Mycobacterium avium subsp. paratuberculosis is endemic. The Taff is a spate river running off the hills and through the principal city of Cardiff. We sampled Taff water above Cardiff twice weekly from November 2001 to November 2002. M. avium subsp. paratuberculosis was detected by IS 900 PCR and culture. Thirty-one of 96 daily samples (32.3%) were IS 900 PCR positive, and 12 grew M. avium subsp. paratuberculosis bovine strains. Amplicon sequences from colonies were identical to the sequence with GenBank accession no. X16293 , whereas 16 of 19 sequences from river water DNA extracts had a single-nucleotide polymorphism at position 214. This is consistent with a different strain of M. avium subsp. paratuberculosis in the river, which is unculturable by the methods we used. Parallel studies showed that M. avium subsp. paratuberculosis remained culturable in lake water microcosms for 632 days and persisted to 841 days. Of four reservoirs controlling the catchment area of the Taff, M. avium subsp. paratuberculosis was present in surface sediments from three and in sediment cores from two, consistent with deposition over at least 50 years. Previous epidemiological research in Cardiff demonstrated a highly significant increase of Crohn's disease in 11 districts. These bordered the river except for a gap on the windward side. A topographical relief map shows that this gap is directly opposite a valley open to the prevailing southwesterly winds. This would influence the distribution of aerosols carrying M. avium subsp. paratuberculosis from the river.

Mycobacterial interspersed repetitive units (MIRU) comprise short tandem repeat structures found at multiple loci throughout the Mycobacterium tuberculosis genome and have been used for typing these pathogens. We have identified MIRU at 18 conserved loci throughout the common portions of the Mycobacterium avium subspecies paratuberculosis (MAP) and M. avium subspecies avium (MAA) genomes. Six of these loci were found to differ between MAA and MAP in the number of tandem repeat motifs occurring at each MIRU locus. Locus specific PCR at 4 of these loci segregated MAP into two major groups, which could be differentiated from ovine-pigmented strains of MAP and the MAP vaccine strain 316F. The same PCR differentiated MAA into five MIRU profiles. PCR at either MIRU locus 1 or MIRU locus 4 distinguished between MAP and all other M. avium complex (MAC) tested. PCR at both loci 1 and 4 also distinguished MAP from Mycobacterium intracellulare. MIRU typing may provide an additional simple and rapid procedure for the differentiation between MAP and other MAC.

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 IS 900 , designated IS 900 [L/AV] and IS 900 [TJ1-4], which are uniquely specific for IS 900 . Detection of M. avium subsp. paratuberculosis in mucosal biopsy specimens was also evaluated by using mycobacterial growth indicator tube (MGIT) cultures (Becton Dickinson). IS 900 [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 IS 900 [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 IS 900 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.

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 6,496-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.

We have recently described the GS element, found in Mycobacterium avium subsp. paratuberculosis (MAP), Mycobacterium avium subsp. silvaticum (MAS) and some isolates of Mycobacterium avium subsp. avium serotype 2 (MAAs2), which contains a set of genes of low GC% content, putatively associated with the biosynthesis, modification and transference of fucose to cell wall glycopeptidolipids. Here we describe a further gene of low GC% content (mpa), within the GS element in MAP. mpa is a putative acetyltransferase with homology to genes directly responsible for host specificity and virulence in Salmonella typhimurium and Shigella flexneri. Unlike other GS genes, strong homologues of mpa have not been found in related species, including Mycobacterium tuberculosis (MTB). In MAP, mpa encodes an ORF of 445aa, however, in MAS and MAAs2 mpa contains a single inserted copy of a novel insertion sequence. This element (IS1612) has two sets of inverted repeats at each terminus and encodes two ORFs with good homologies to transposase and helper proteins of IS21 (E. coli) and IS1415 (R. erythropolis). Sequence comparisons between mpa in MAP and MAS indicate the target site for IS1612 is duplicated on insertion to give a direct repeat at each end of the element. Immediately, downstream of the mpa gene in both MAP and MAS are a group of three genes with good homology to the daunorubicin resistance cluster. This cluster has a high GC% content which suggests a 'border' for the GS element. A short motif present at the beginning of this cluster matches with an inverted repeat of this motif at the beginning of the first gene in the GS element. This encapsulates the whole of this group of low GC% genes in MAP and further suggests its cassette-like nature. Homologues of the GS element in MTB show a marked similarity of organisation, suggesting a parallel role for these genes in both pathogens.

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/BstEII 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.

Enterostatins [Val-Pro-Asp-Pro-Arg (VPDPR), Val-Pro-Gly-Pro-Arg (VPGPR), and Ala-Pro-Gly-Pro-Arg (APGPR)] are pentapeptides derived from the NH2-terminus of procolipase after tryptic cleavage and belong to the family of gut-brain peptides. Although enterostatin-like immunoreactivities exist in blood, brain, and gut, and exogenous enterostatins decrease fat appetite and insulin secretion in rats, the roles of these peptides in human obesity remain to be examined. To determine whether VPDPR and APGPR secretion is altered in obesity, serum VPDPR and APGPR levels were measured in 38 overnight-fasted subjects (body mass index, 17.9–54.7 kg/m2) before and after a meal. The mean fasting VPDPR in the serum of lean subjects was significantly lower than that in obese subjects [lean = 603 ± 86 nmol/L (n = 17); obese, 1516 ± 227 nmol/L (n = 21); P = 0.0023]. In addition, the rise in serum APGPR after a meal (postmeal/fasting ratio) was significantly higher in lean than in obese subjects [lean, 1.71 ± 0.24 (n = 17); o...

Enterostatins belong to a family of peptides (e.g., Val-Pro-Asp-Pro-Arg, VPDPR; Ala-Pro-Gly-Pro-Arg, APGPR; and Val-Pro-Gly-Pro-Arg, VPGPR) derived from the tryptic cleavage of amino-terminal pentapeptide from procolipase. Pharmacologic studies have suggested a role for these peptides in appetite regulation and insulin secretion. Studies into the distribution of enterostatins or the role of endogenous peptides have not been possible until now due to the lack of a suitable method for assay. Using two polyclonal antibodies raised against VPDPR and APGPR and different chromatographic methods, we have examined the nature and distribution of enterostatin-like immunoreactivity in human cerebrospinal fluid. The results reported here show for the first time the presence of enterostatin-like immunoreactivity in the human cerebrospinal fluid. Further characterization of cerebrospinal fluid enterostatin-like immunoreactivity revealed that it is not due to APGPR, VPGPR, or VPDPR but to another peptide similar to VPDPR.

Thirty one cultures were established in MG3 medium from the intestinal tissues of 29 patients, including 18 with Crohn's disease, five with ulcerative colitis, and six non-inflammatory bowel disease controls. All cultures grew either acid fast bacilli or uncharacterized spheroplasts. Pellets from these cultures were coded and assayed blind for M paratuberculosis and M avium subsp silvaticum using IS900- and IS902-PCR (polymerase chain reaction) assays, respectively. IS900 and IS902 are multicopy DNA insertion elements specific for these two organisms. Six Crohn's disease cultures and a single non-inflammatory bowel disease control were positive for M paratuberculosis. A further six cultures were positive for M avium subsp silvaticum, of which two each were from Crohn's disease, ulcerative colitis, and non-inflammatory bowel disease controls. The intensity of the IS900-PCR signals indicated very low numbers of M paratuberculosis organisms and bore no relation to visible spheroplastic or bacillary mycobacterial growth. The results suggest that M paratuberculosis isolated from man exists in a form which hardly replicates if at all when cultured in MG3 medium in vitro, and are consistent with the involvement of this known animal enteric pathogen in a proportion of chronic enteritis in man.

Crohn's disease has long been suspected of having a mycobacterial cause. Mycobacterium paratuberculosis is a known cause of chronic enteritis in animals, including primates, but may be very difficult to detect by culture. IS900 is a multicopy genomic DNA insertion element highly specific for M paratuberculosis. A polymerase chain reaction (PCR) based on the 5' region of IS900 and capable of the specific detection of a single M paratuberculosis genome was developed. This was applied to DNA extracts of full thickness samples of intestine removed at surgery from 40 patients with Crohn's disease, 23 patients with ulcerative colitis, and 40 control patients without inflammatory bowel disease. Stringent precautions were taken that excluded contamination artefact. M paratuberculosis was identified in 26 of 40 (65%) Crohn's disease, in 1 of 23 (4.3%) ulcerative colitis, and in 5 of 40 (12.5%) control tissues. Positive samples from Crohn's disease were from both the small intestine and colon, those from control tissues were from the colon those from control tissues were from the colon only. All PCR internal control reactions were negative. The presence of M paratuberculosis in a small proportion of apparently normal colonic samples is consistent with a previously unsuspected alimentary prevalence in humans. The presence in two thirds of Crohn's disease tissues but in less than 5% of ulcerative colitis tissues is consistent with an aetiological role for M paratuberculosis in Crohn's disease.

An insertion sequence element of Mycobacterium avium subsp. silvaticum was isolated and its complete nucleotide sequence determined. IS902 is 1470 bp in size and is repeated 10-12 times per genome. An open reading frame of 1200 bp was identified, encoding a protein product of Mr 43932. This protein is highly similar to the predicted proteins of IS900 of Mycobacterium paratuberculosis, IS116 of Streptomyces clavuligerus and IS110 of Streptomyces coelicolor. IS902 lacks terminal inverted repeats and flanking direct repeats but displays insertion site specificity.

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.

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.

DNA probes that hybridize to a mycobacterial insertion sequence, IS900, present in multiple copies in the genome of Mycobacterium paratuberculosis were found to be highly specific for M. paratuberculosis. DNA sequences derived from IS900 were used to prepare DNA primers for detection and identification of M. paratuberculosis by the polymerase chain reaction. Highly specific direct detection of M. paratuberculosis DNA in feces from cattle with Johne's disease was obtained. The polymerase chain reaction test had a sensitivity equal to or greater than that obtained by standard culture techniques and was much more rapid, taking only hours compared with 6 to 12 weeks for culture.

BackgroundMycobacterium 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.MethodsWe 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 findingsVaccination 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/significanceA 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 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 IS 900 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 IS 900 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.

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.

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.

Introduction Abnormal intestinal macrophage function which could allow persistence of candidate bacterial triggering agents such as E coli and Mycobacterium avium subspecies paratuberculosis (MAP), has been implicated in the pathogenesis of Crohn9s disease (CD).1 Prolonged survival of E coli in CD-derived peripheral blood monocytes (PBM) has been demonstrated,2 however current methods of determining MAP infection are difficult and time-consuming. The application of a luminescent MAP strain in a novel rapid technique may improve survival assays.3 Aims Determine the sensitivity and accuracy of a method to quantify luminescent MAP and develop an intracellular survival assay. Monitor cytokine profiles induced during MAP infection in CD and healthy controls (HC). Methods Quantitative real-time PCR (qRT PCR) was used to calibrate a standard curve of luminescence from MAP ‘Lux’ 19 698 strain. Monocytes from CD and HC were isolated and infected with MAP lux 19 698 (multiplicity of infection 4:1) for up to 4 weeks. Luminescence was recorded at intervals to determine killing activity compared to medium only controls. Intracellular uptake was visualised using an anti-MAP antibody and confocal microscopy. Cytokine release (TNFα, IL10, IL6 and IL23) from PBM was measured using ELISA pre and 24 h post infection. Results A close linear relationship (R 2 = 0.9999) was seen between luminescence and number of MAP organisms by qRT PCR. Sensitivities were also similar. Monocytes from 8 CD patients and 7 HC infected with MAP lux 19 698 assayed over a mean of 15 days (range 8 to 27) showed some killing during the experiment time course. However a majority of MAP survived intracellularly (visualised by confocal microscopy) in all samples and the degree of killing was not significantly different between CD and HC. Cytokine profiling showed only a proportion of all subject9s (CD or HC) PBM responded to MAP infection. Of these responders, there was a difference in cytokine profiles between CD and HC and in particular there was a strong trend to greater IL6 response in CD. Conclusion This study provides initial validation for a luminescent MAP intracellular survival assay. Consistent, persistent survival of MAP was demonstrated in both CD and controls. Alterations in cytokine responses to MAP infection between CD and normals suggest that this agent is perceived and processed differently by CD monocytes.

Introduction Intracellular Escherichia coli isolates are found in up to 60% of gut biopsies from patients with Crohn9s disease (CD). Although adherent invasive E coli (AIEC) are described, other intracellular E coli are frequently isolated but specific pathogenic determinants for these have not been identified by conventional techniques. DNA microarray and Matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) mass spectrometry enable rapid and accurate DNA and proteomic analysis of bacteria. Aims To investigate for virulence and antimicrobial resistance (AMR) genes in, and analyse the structure of CD-related intracellular E coli isolates. Methods 17 E coli strains were isolated using gentamicin protection assay from gut biopsies of 17 CD patients (ileal n=7, colonic n=10). DNA was extracted for analysis by DNA microarray (Prodigy) of 392 virulence and AMR genes. DNA PCR was performed for an additional 18 virulence genes. For Bruker Biotyper MALDI-TOF sample preparation, several colonies of each bacterial strain were spotted onto target plate and prepared as described previously. 10 replicates of the mass/charge spectra for each strain were compared to spectra from a bacterial database. Results Virulence factors were identified by DNA microarray in a number of E coli strains including senB (encodes enterotoxin), Iha (encodes an adhesin) Sat (toxin of uropathogenic E coli ), iron regulation genes and microcin synthesis genes. DNA PCR revealed additional virulence factors including ibeA (associated with E coli invasion in neonatal meningitis) in 2 strains. A minority of strains were positive for AMR genes relating to β-lactams, sulphonamides, tetracycline and streptomycin. MALDI-TOF analysis confirmed the bacteria as E coli and clustered the Crohn9s disease E coli strains together and separate from other E coli strains on the Bruker database. There was no clustering within CD strains in relation to disease or biopsy site or immunomodulation. Conclusion Although CD appears to be at least in part a host immune disorder, pathogenic factors of various organisms may still play a role in pathogenesis. A number of genes encoding for virulence factors have been identified in a proportion of CD-related E coli strains which warrant further investigation (eg, whole gene sequencing and functional assays). Most strains had no antimicrobial resistance genes. Further MALDI-TOF comparison of CD-related bacterial structure with commensal and IBD-related bacteria may be revealing.

Trypsinogen was identified in cerebrospinal fluid (CSF), where it has not previously been reported and its activation state in experimental subarachnoid haemorrhage (SAH) in rats and in neurosurgical patients was determined. Trypsinogen activation peptide (TAP) release provided an equimolar marker for trypsinogen. Total TAP was significantly reduced to 26% of the baseline level (P

Summary: 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.

Mycobacterium paratuberculosis was first described by Johne and Frothingham in 18951 as the cause of a chronic inflammatory disease of the intestine in a German cow. The organism was called Johne's bacillus, and the illness (in which millions of acid fast mycobacteria were visible in the diseased tissues), Johne's disease. In 1901 Thomas Dalziel, a surgeon at the Western Infirmary in Glasgow, operated on a colleague with chronic inflammation of the intestine. He was aware of the description of Johne's disease and of the subsequent bacteriological research in the field. He collected other cases and published his observations in the BMJ in 1913.2 He wrote that the “histological characters” of the disease he had described in humans were so similar to those of Johne's disease that the diseases may be the same. Dalziel's dilemma was that he could not see acid fast mycobacteria in the diseased intestine in humans.3 M paratuberculosis belongs to the M avium-intracellulare group.4 The term paratuberculosis suggests close similarity to M tuberculosis , but in truth it is very different. Unlike M tuberculosis , it can survive in the environment and is highly resistant in vivo to most standard antituberculous drugs. M paratuberculosis cannot be reliably detected by culture in the laboratory; different subtypes of the organism with different preferred hosts5 6 range from very slow growing to unculturable. M paratuberculosis can cause chronic inflammation of the intestine in many species of animals including primates.7 8 9 The disease in animals shows a broad range of histopathological types, from a pluribacillary (lepromatous) form with millions of typical acid fast bacilli visible in the tissues, to a paucimicrobial (tuberculoid) form in which M paratuberculosis cannot be seen in the tissues and cannot be detected by culture but in which there is …

Fifty-two patients with severe Crohn's disease were enrolled in this study. Six (11.5%) were intolerant of the medication and had to be excluded. The remaining 46 patients were treated with rifabutin in combination with a macrolide antibiotic (clarithromycin or azithromycin). Patients were treated for a mean of 18.7 (range 6-35) months and followed up for 25.1 (range 7-41) months. Of the 19 patients who were steroid dependent at the start of this study, only two continued to require steroids when treatment was established. A reduction in the Harvey-Bradshaw Crohn's disease activity index occurred after 6 months' treatment (P = 0.004, paired Wilcoxon test) and was maintained at 24 months (P < 0.001). An improvement in inflammatory parameters was observed as measured by a reduction in erythrocyte sedimentation rate (P = 0.009) and C-reactive protein (P = 0.03) at 18 months compared with pretreatment levels, and an increase in serum albumin at 12 months (P = 0.04). When subsets of the study population were analysed, patients with pan-intestinal disease achieved better remission at 2 years than did those with less extensive involvement (P = 0.04, Mann-Whitney U-test). No difference in treatment response by age, disease duration, the presence of granulomas on histology, or the occurrence of drug-induced side-effects, was observed. These data suggest that treatment with rifabutin and clarithromycin or azithromycin may result in a substantial clinical improvement in Crohn's disease and justify the conduct of a randomized controlled trial.

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.

Using an assay for measurement of released type 1-prophospholipase A2 (type 1-proPLA2) propeptides (PROP assay), we have shown that human granulocytes, but not lymphocytes or macrophages, abundantly express this 'pancreatic' type 1-proPLA2 zymogen. Stimulation with tumour necrosis factor-alpha (TNF-alpha) and other cytokines results in the immediate release from granulocytes of a mixture of free propeptides and type 1-proPLA2 precursor. We also found that granulocytes contain an approximately 29 kDa trypsin-like endogenous type 1-proPLA2 activator. PROP assay and TAP (trypsinogen activation peptide) assay of plasma samples accurately predicts the segregation of acute pancreatitis into three clearly defined categories of severity--mild, intermediate and severe--at the time of first hospital admission and over the next few hours of observation. Mild and intermediate pancreatitis are associated with a degree of granulocyte stimulation limited to the release of the unactivated type 1-proPLA2 precursor. Progression to severe disease is accompanied by the activation of granulocyte type 1-proPLA2, apparently carried to completion. This identifies the approximately 29 kDa endogenous activator of type 1-proPLA2 in granulocytes as a critical mediator at a threshold stage in acute pancreatitis, which marks the transition from uncomplicated pancreatitis to the potentially lethal disease. Specific inhibitors of this key regulatory enzyme modelled on the P3-P1 domain of the type 1-proPLA2 activation peptide would seem to be promising candidates for a new class of chemotherapeutic agents.

objective: To establish a ELISA assay to measure release of type 1-phospholipase A2 propeptide from activated granulocytes. Human type 1-prophospholipase A2 (1-proPLA2) is biosynthesized and stored as inactive zymogen. Activation involves tryptic-like cleavage at the N-terminus, with equimolar release of the heptapeptide DSGISPR. Methods: Using antibodies directed to the carboxyterminus of synthetic DSGISPR we developed a sensitive solid-phase ELISA specific for the released propeptide that accurately reports the activation of 1-proPLA2. The presence of the 1-proPLA2 precursor itself can be determined by trypsinization of the sample and subsequent assay for free DSGISPR. Results: Using this ELISA, we demonstrated the presence of immunoreactive DSGISPR and its 14 kDa 1-proPLA2 like precursor in human granulocytes, but their absence in human macrophages and lymphocytes. Stimulation of cultured granulocytes with 1 pM of TNFa or GM-CSF caused rapid release of DSGISPR and precursor into the surrounding medium. The immunoreactive signal coeluted with standard synthetic DSGISPR on G50 Sephadex chromatography. Conclusion: Release of DSGISPR immunoreactivity appears to be a specific consequence of granulocyte activation of potential relevance to the clinical pathophysiology of conditions like acute lung injury.

Introduction A well established expression of the proposed defect in innate immunity in Crohn9s disease (CD) is the prolonged survival of strains of Escherichia coli in peripheral blood monocytes/macrophages (PBM) from patients with CD compared to those from healthy controls (HC). Altered handling of E coli in CD has been demonstrated in PBM and in-situ in CD by laser dissection of E coli infected macrophages. Several of the gene polymorphisms conferring an increased risk of CD occur in genes involved in bacteria-immune cell interactions. The aim of this study was to determine the influence of these polymorphisms on the interaction of E coli with CD-derived monocyte / macrophages. Methods PBM9s and lamina propria macrophages were obtained from 43 and 17 CD patients respectively (mean age 39 years / range 20–70 years). PBM9s were challenged with E coli and then the response of these PBM9s measured by levels of bacterial killing, using the gentamicin protection assay. Phagocytosis and reactive oxygen radicals (ROR9s) production were measured by flow cytometry using the phagotest and phagoburst assays. Cytokine (TNFα, IL10, IL23) production was measured by ELISA. In parallel experiments, TNFα, IL10 and CD163 expression was measured by qRT-PCR in laser dissected E coli laden and unladen lamina proprial macrophages. All patients were genotyped for the three common NOD-2 SNP9s (G908R, L1007fs and R702W), and the autophagy related SNP9s (ATG16L1 and IRGM). Genotypes and monocyte/macrophage markers were compared. Results Presence of the ATG16L1 risk variant was strongly associated with a reduced E coli induced TNFα expression in CD PBM9s (p=0.0017) and increased levels of ROR9s on uptake of CD derived E coli (p=0.0027). Presences of IRGM risk variant was associated with increased IL10 expression from PBM9s. NOD-2 mutations were not associated with a difference in any variable studied. No genotype altered survival of E coli in PBM at 2 or 4 h after infection or in-situ lamina propria macrophage cytokine expression. Conclusion These results show that defective autophagy modifies the handling of intracellular bacteria in CD but appears unlikely to be the main cause of prolonged bacterial survival. Further studies are needed to elucidate the nature of this innate defect and it9s role in CD pathogenesis.

The effect of a satiating meal on the serum and urinary concentrations of procolipase propeptide (Ala-Pro-Gly-Pro-Arg, APGPR) immunoreactivity, as measured by enzyme linked immunosorbent assay (ELISA) specific for free APGPR, has been studied in normal and morbidly obese human subjects. The normal subjects displayed a biphasic response with coordinate increases in both serum and urine APGPR immunoreactivity both occurring within the first two hours after the meal. In two of three of the morbidly obese subjects, this early rise in APGPR concentration in urine was not seen but was followed by a slow rise in urinary APGPR immunoreactivity at four to six hours. In both the normal and obese groups, the urinary immunoreactive signal was found to coelute with synthetic APGPR on gel chromatography. In rats, procolipase propeptide (Val-Pro-Asp-Pro-Arg, VPDPR) specifically inhibits fat intake early in the postprandial period when given peripherally or centrally. This study suggests that in humans APGPR reaches the circulation shortly after feeding and is excreted in the urine. These findings are consistent with the hypothesis that human procolipase propeptide may also act as a satiety signal. In addition the late appearance of the peptide in some of the morbidly obese patients could be associated with perturbation of appetite control in these subjects.

Summary An enzyme-linked immunosorbent assay for trypsinogen activation peptide (TAP) was used to measure urinary TAP levels in standard feline models of acute oedematous pancreatitis and acute haemorrhagic pancreatitis. It has been shown that the extent of pancreatic damage as assessed histologically is significantly greater in the model of acute haemorrhagic pancreatitis. This increase in damage has been found to be associated with a significantly greater increase in the excretion of urinary TAP.

Introduction Whist the exact cause of Crohn9s disease (CD) remains unclear, an innate defect in the handling of intracellular bacteria appears to be involved. Evidence for this comes from 1. genome wide association studies implicating a role for genes involved in bacteria-immune cell interactions including NOD-2 and the autophagy related genes, ATG16L1 and IRGM, 2. an observed defect in phagocyte recruitment and 3. the consistent demonstration of viable E coli within lamina propria macrophages in CD. It is not known if this latter observation is due to increased uptake of E coli or a failure to achieve effective phagolysosomal killing of bacteria once within the cell. Methods To examine phagocytosis and oxidative burst activity of phagocytic cells in peripheral blood of 16 healthy individuals and 16 patients with CD. Phagocytic activity was assessed by the number of cells taking up fluorescently tagged E coli (Phagotest) and the potential role of defective reactive oxygen radical (ROR) production involved in bacterial killing (Bursttest). This is measured by the conversion of non-fluorescent dihydrorhodamine 123 to the fluorescent rhodamine 123 by ROR9s. The leucocytes were challenged with three strains of E coli, a heat killed sample, a laboratory strain (K12) and a strain isolated from a patient with CD. Results were obtained using florescent markers measured by flow cytometery. Results Using the phagotest, to measure the uptake of bacteria no difference was seen between CD and controls and did not vary according to strain of E coli used. However, although the number of monocytes expressing ROR9s was similar in both groups, their was a significantly level of ROR9s produced in cells from CD compared to controls when viable bacteria were used (E coli K12: control 98.5±7.4 vs CD 68.1±13.2, p=0.04, and the CD isolate: control cells 101.1±7.4 vs CD 59.2±12.2, p=0.010). No difference was seen in the granulocytes between CD and controls for either phagocytosis or ROR9s. Conclusion The ability of monocytes from CD patients to engulf E coli appears to be normal. However, there is good evidence of an impaired capacity to kill these bacteria as measured by reduced levels of ROR9s. These results points to either an inability to complete the process of phagolysosome formation or defects in the enzyme pathways involved in formation of reactive oxygen products that are induced by phagocytosis and support the concept of impaired innate immunity in CD.

Trypsinogen activation peptides (TAP) were quantified by radioimmunoassay in blood, urine, and peritoneal exudate of rats with experimental pancreatitis. Forty-four animals were studied, comprising a control group and four different induction techniques (cerulein, cerulein plus either 2- or 10-min intraductal glycodeoxycholic acid [GDOC] infusion, and cerulein plus intraductal GDOC with enterokinase [EK]). Significantly higher TAP concentrations were found at 6 h (or at death) in plasma and ascites of all pancreatitis groups compared with controls. TAP quantitation in hourly urine samples demonstrated significantly higher concentrations from the third hour onward in the most severe groups and from the fourth hour onward in the cerulein-treated rats. All nonsurviving rats had a plasma TAP of greater than 2.5 nM/L, whereas only 1 of 34 surviving animals had such a concentration (p less than 0.001). A significant stepwise increase in total TAP in ascites was found when comparing the cerulein group, the two GDOC groups, and the EK group (p less than 0.001). Chromatography of samples with a high TAP content demonstrated comigration with synthetic TAP. We conclude that free TAP are present in blood, urine, and peritoneal exudate of rats with experimental pancreatitis of different pathogenesis and that the amount of TAP may be indicative of the severity of the disease process.