Specific Cationic Antimicrobial Peptides Enhance the Recovery of Low-Load Quiescent Mycobacterium tuberculosis in Routine Diagnostics.

The culture confirmation of Mycobacterium tuberculosis (MTB) remains the gold standard for the diagnosis of Tuberculosis (TB) with culture conversion representing proof of cure. However, over 40% of TB samples fail to isolate MTB even though many patients remain infectious due to the presence of viable non-culturable forms. Previously, we have shown that two short cationic peptides, T14D and TB08L, induce a hormetic response at low concentrations, leading to a stimulation of growth in MTB and the related animal pathogen Mycobacterium bovis (bTB). Here, we examine these peptides showing they can influence the mycobacterial membrane integrity and function through membrane potential reduction. We also show this disruption is associated with an abnormal reduction in transcriptomic signalling from specific mycobacterial membrane sensors that normally monitor the immediate cellular environment and maintain the non-growing phenotype. We observe that exposing MTB or bTB to these peptides at optimal concentrations rapidly represses signalling mechanisms maintaining dormancy phenotypes, which leads to the promotion of aerobic metabolism and conversion into a replicative phenotype. We further show a practical application of these peptides as reagents able to enhance conventional routine culture methods by stimulating mycobacterial growth. We evaluated the ability of a peptide-supplemented sample preparation and culture protocol to isolate the MTB against a gold standard routine method tested in parallel on 255 samples from 155 patients with suspected TB. The peptide enhancement increased the sample positivity rate by 46% and decreased the average time to sample positivity of respiratory/faecal sampling by seven days. The most significant improvements in isolation rates were from sputum smear-negative low-load samples and faeces. The peptide enhancement increased sampling test sensitivity by 19%, recovery in samples from patients with a previously culture-confirmed TB by 20%, and those empirically treated for TB by 21%. We conclude that sample decontamination and culture enhancement with D-enantiomer peptides offer good potential for the much-needed improvement of the culture confirmation of TB.

Assessment of the frequency of Mycobacterium bovis shedding in the faeces of naturally and experimentally TB infected cattle.

AIMS: To assess the prevalence of Mycobacterium bovis bacilli in faecal samples of tuberculous cattle, and to better understand the risk of environmental dissemination of bovine tuberculosis (TB) through the spreading of manure or slurry. METHODS AND RESULTS: Faecal samples were collected from 72 naturally infected cattle with visible lesions of TB that had reacted to the tuberculin skin test and 12 cattle experimentally infected with M. bovis. These were examined by microbial culture and PCR to assess the presence of M. bovis bacilli. There were no positive cultures from any naturally infected test reactor animal. A single M. bovis colony was cultured from a faecal sample from one of the experimentally infected animals. A single PCR positive result was obtained from the faecal sample of one naturally infected test reactor. CONCLUSIONS: The prevalence of M. bovis in the faecal samples of TB-infected cattle was extremely low. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that the risk of spreading TB through the use of slurry or manure as an agricultural fertilizer is lower than that suggested in some historical literature. The results could inform a reconsideration of current risk assessments and guidelines on the disposal of manure and slurry from TB-infected herds.

Increased viability but decreased culturability of Mycobacterium avium subsp. paratuberculosis in macrophages from inflammatory bowel disease patients under Infliximab treatment.

Mycobacterium avium subsp. paratuberculosis (MAP) has long been implicated as a triggering agent in Crohn's disease (CD). In this study, we investigated the growth/persistence of both M. avium subsp. hominissuis (MAH) and MAP, in macrophages from healthy controls (HC), CD and ulcerative colitis patients. For viability assessment, both CFU counts and a pre16SrRNA RNA/DNA ratio assay (for MAP) were used. Phagolysosome fusion was evaluated by immunofluorescence, through analysis of LAMP-1 colocalization with MAP. IBD macrophages were more permissive to MAP survival than HC macrophages (a finding not evident with MAH), but did not support MAP active growth. The lower MAP CFU counts in macrophage cultures associated with Infliximab treatment were not due to increased killing, but possibly to elevation in the proportion of intracellular dormant non-culturable MAP forms, as MAP showed higher viability in those macrophages. Increased MAP viability was not related to lack of phagolysosome maturation. The predominant induction of MAP dormant forms by Infliximab treatment may explain the lack of MAP reactivation during anti-TNF therapy of CD but does not exclude the possibility of MAP recrudescence after termination of therapy.

Prevalence of Mycobacterium avium subsp. paratuberculosis and Escherichia coli in blood samples from patients with inflammatory bowel disease.

Mycobacterium avium subsp. paratuberculosis (MAP) and adherent-invasive Escherichia coli (AIEC) have been implicated as primary triggers in Crohn's disease (CD). In this study, we evaluated the prevalence of MAP and E. coli (EC) DNA in peripheral blood from 202 inflammatory bowel disease (IBD) patients at various disease periods and compared against 24 cirrhotic patients with ascites (CIR) (non-IBD controls) and 29 healthy controls (HC). MAP DNA was detected by IS900-specific nested PCR, EC DNA by malB-specific nested PCR and AIEC identity, in selected samples, by sequencing of fimH gene. CD patients with active disease showed the highest MAP DNA prevalence among IBD patients (68 %). Infliximab treatment resulted in decreased MAP detection. CIR patients had high individual and coinfection rates (75 % MAP, 88 % EC and 67 % MAP and EC), whilst HC controls had lower MAP prevalence (38 %) and EC was undetectable in this control group. EC DNA prevalence in IBD patients was highly associated with CD, and 80 % of EC from the selected samples of CD patients analyzed carried the fimH30 allele, with a mutation strongly associated with AIEC. Our results show that coinfection with MAP and AIEC is common and persistent in CD, although the high MAP and EC detection in CIR patients suggested that colonization is, at least, partially dependent on increased gut permeability. Nevertheless, facilitative mechanisms between a susceptible host and these two potential human pathogens may allow their implication in CD pathogenesis.

Immunity, safety and protection of an Adenovirus 5 prime--Modified Vaccinia virus Ankara boost subunit vaccine against Mycobacterium avium subspecies paratuberculosis infection in calves.

Vaccination is the most cost effective control measure for Johne's disease caused by Mycobacterium avium subspecies paratuberculosis (MAP) but currently available whole cell killed formulations have limited efficacy and are incompatible with the diagnosis of bovine tuberculosis by tuberculin skin test. We have evaluated the utility of a viral delivery regimen of non-replicative human Adenovirus 5 and Modified Vaccinia virus Ankara recombinant for early entry MAP specific antigens (HAV) to show protection against challenge in a calf model and extensively screened for differential immunological markers associated with protection. We have shown that HAV vaccination was well tolerated, could be detected using a differentiation of infected and vaccinated animals (DIVA) test, showed no cross-reactivity with tuberculin and provided a degree of protection against challenge evidenced by a lack of faecal shedding in vaccinated animals that persisted throughout the 7 month infection period. Calves given HAV vaccination had significant priming and boosting of MAP derived antigen (PPD-J) specific CD4+, CD8+ IFN-γ producing T-cell populations and, upon challenge, developed early specific Th17 related immune responses, enhanced IFN-γ responses and retained a high MAP killing capacity in blood. During later phases post MAP challenge, PPD-J antigen specific IFN-γ and Th17 responses in HAV vaccinated animals corresponded with improvements in peripheral bacteraemia. By contrast a lack of IFN-γ, induction of FoxP3+ T cells and increased IL-1ß and IL-10 secretion were indicative of progressive infection in Sham vaccinated animals. We conclude that HAV vaccination shows excellent promise as a new tool for improving control of MAP infection in cattle.

Redefining Peptide 14D: Substitutional Analysis for Accelerated TB Diagnosis and Enhanced Activity against Mycobacterium tuberculosis.

Tuberculosis (TB) caused by Mycobacterium tuberculosis remains a predominant cause of mortality, especially in low- and middle-income nations. Recently, antimicrobial peptides have been discovered that at low concentrations could stimulate the growth of M. tuberculosis (hormetic response). In this study, such a peptide was used to investigate the effects on the time to positivity (TTP). A systematic substitution analysis of peptide 14D was synthesized using Spot synthesis technology, resulting in 171 novel peptides. Our findings revealed a spectrum of interactions, with some peptides accelerating M. tuberculosis growth, potentially aiding in faster diagnostics, while others exhibited inhibitory effects. Notably, peptide NH2-wkivfiwrr-CONH2 significantly reduced the TTP by 25 h compared to the wild-type peptide 14D, highlighting its potential in improving TB diagnostics by culture. Several peptides demonstrated potent antimycobacterial activity, with a minimum inhibitory concentration (MIC) of 20 µg/mL against H37Rv and a multidrug-resistant M. tuberculosis strain. Additionally, for two peptides, a strongly diminished formation of cord-like structures was observed, which is indicative of reduced virulence and transmission potential. This study underscores the multifaceted roles of antimicrobial peptides in TB management, from enhancing diagnostic efficiency to offering therapeutic avenues against M. tuberculosis.

Visualisation of Mycobacterium avium subsp. paratuberculosis in cultured cells, infected sheep and human tissue sections using fluorescent in situ hybridization (FISH).

We describe the development, testing and specificity of a modified oligonucleotide probe for the specific detection of Mycobacterium avium subsp. paratuberculosis (MAP) in culture and in infected tissue using fluorescent in situ hybridisation and confocal microscopy. The detection of MAP in both animal and human tissue using our modified probe allows for a more rapid diagnosis of MAP infection compared to the more often applied detection methods of culture and PCR and has the potential for quantification of cellular abundance. This approach would enable earlier treatment intervention and therefore the potential for reduced morbidity.

Cytokine expression in subjects with Mycobacterium avium ssp. paratuberculosis positive blood cultures and a meta-analysis of cytokine expression in Crohn's disease.

Objectives: 1) Culture Mycobacterium avium ssp. paratuberculosis (MAP)from blood, 2) assess infection persistence, 3) determine Crohn's disease (CD) cytokine expression, 4) compare CD cytokine expression to tuberculosis, and 5) perform a meta-analysis of cytokine expression in CD. Methods: The Temple University/Abilene Christian University (TU/ACU) study had a prospective case control design with 201 subjects including 61 CD patients and 140 non-CD controls. The culture methods included MGIT, TiKa and Pozzato broths, and were deemed MAP positive, if IS900 PCR positive. A phage amplification assay was also performed to detect MAP. Cytokine analysis of the TU/ACU samples was performed using Simple Plex cytokine reagents on the Ella ELISA system. Statistical analyses were done after log transformation using the R software package. The meta-analysis combined three studies. Results: Most subjects had MAP positive blood cultures by one or more methods in 3 laboratories. In our cytokine study comparing CD to non-CD controls, IL-17, IFNγ and TNFα were significantly increased in CD, but IL-2, IL-5, IL-10 and GM-CSF were not increased. In the meta-analysis, IL-6, IL-8 and IL-12 were significantly increased in the CD patients. Conclusion: Most subjects in our sample had MAP infection and 8 of 9 subjects remained MAP positive one year later indicating persistent infection. While not identical, cytokine expression patterns in MAP culture positive CD patients in the TU/ACU study showed similarities (increased IL-17, IFNγ and TNFα) to patterns of patients with Tuberculosis in other studies, indicating the possibilities of similar mechanisms of pathogen infection and potential strategies for treatment.

Genomic variations associated with attenuation in Mycobacterium avium subsp. paratuberculosis vaccine strains.

BACKGROUND: Mycobacterium avium subspecies paratuberculosis (MAP) whole cell vaccines have been widely used tools in the control of Johne's disease in animals despite being unable to provide complete protection. Current vaccine strains derive from stocks created many decades ago; however their genotypes, underlying mechanisms and relative degree of their attenuation are largely unknown. RESULTS: Using mouse virulence studies we confirm that MAP vaccine strains 316 F, II and 2e have diverse but clearly attenuated survival and persistence characteristics compared with wild type strains. Using a pan genomic microarray we characterise the genomic variations in a panel of vaccine strains sourced from stocks spanning over 40 years of maintenance. We describe multiple genomic variations specific for individual vaccine stocks in both deletion (26-32 Kbp) and tandem duplicated (11-40 Kbp) large variable genomic islands and insertion sequence copy numbers. We show individual differences suitable for diagnostic differentiation between vaccine and wild type genotypes and provide evidence for functionality of some of the deleted MAP-specific genes and their possible relation to attenuation. CONCLUSIONS: This study shows how culture environments have influenced MAP genome diversity resulting in large tandem genomic duplications, deletions and transposable element activity. In combination with classical selective systematic subculture this has led to fixation of specific MAP genomic alterations in some vaccine strain lineages which link the resulting attenuated phenotypes with deficiencies in high reactive oxygen species handling.

Discovery of Antimicrobial Peptides That Can Accelerate Culture Diagnostics of Slow-Growing Mycobacteria Including Mycobacterium tuberculosis.

Antimicrobial peptides (AMPs) can directly kill Gram-positive bacteria, Gram-negative bacteria, mycobacteria, fungi, enveloped viruses, and parasites. At sublethal concentrations, some AMPs and also conventional antibiotics can stimulate bacterial response increasing their resilience, also called the hormetic response. This includes stimulation of growth, mobility, and biofilm production. Here, we describe the discovery of AMPs that stimulate the growth of certain mycobacteria. Peptide 14 showed a growth stimulating effect on Mycobacteria tuberculosis (MTB), M. bovis, M. avium subsp. paratuberculosis (MAP), M. marinum, M. avium-intracellulare, M. celatum, and M. abscessus. The effect was more pronounced at low bacterial inocula. The peptides induce a faster transition from the lag phase to the log phase and keep the bacteria longer in the log phase before entering stationary phase when compared to nontreated controls. In some cases, an increase in the division rate was observed. An initial screen using MAP and a collection of 75 peptides revealed 13 peptides with a hormetic effect. For MTB, a collection of 25 artificial peptides were screened and 13 were found to reduce the time to positivity (TTP) by at least 5%, improving growth. A screen of 43 naturally occurring peptides, 11 fragments of naturally occurring peptides and 5 designed peptides, all taken from the database APD3, identified a further 44 peptides that also lowered TTP by at least 5%. Lasioglossin LL-III (Bee) and Ranacyclin E (Frog) were the most active natural peptides, and the human cathelicidin LL37 fragment GF-17 and a porcine cathelicidin protegrin-1 fragment were the most active fragments of naturally occurring peptides. Peptide 14 showed growth-stimulating activity between 10 ng/mL and 10 µg/mL, whereas the stability-optimised Peptide 14D had a narrow activity range of 0.1-1 µg/mL. Peptides identified in this study are currently in commercial use to improve recovery and culture for the diagnostics of mycobacteria in humans and animals.

Viable Mycobacterium avium subsp. paratuberculosis Colonizes Peripheral Blood of Inflammatory Bowel Disease Patients.

Pathobionts, particularly Mycobacterium avium subsp. paratuberculosis (MAP) and Escherichia coli isolates with adherence/invasive ability (AIEC) have been associated with inflammatory bowel disease (IBD), particularly Crohn's disease (CD). This study aimed to evaluate the frequency of viable MAP and AIEC in a cohort of IBD patients. As such, MAP and E. coli cultures were established from faecal and blood samples (with a total n = 62 for each) of patients with CD (n = 18), ulcerative colitis (UC, n = 15), or liver cirrhosis (n = 7), as well as from healthy controls (HC, n = 22). Presumptive positive cultures were tested by polymerase chain reaction (PCR), for a positive confirmation of MAP or E. coli identity. E. coli-confirmed isolates were then tested for AIEC identity using adherence and invasion assays in the epithelial cell line of Caco-2 and survival and replication assays in the macrophage cell line of J774. MAP sub-culture and genome sequencing were also performed. MAP was more frequently cultured from the blood and faecal samples of patients with CD and cirrhosis. E. coli presumptive colonies were isolated from the faecal samples of most individuals, in contrast to what was registered for the blood samples. Additionally, from the confirmed E. coli isolates, only three had an AIEC-like phenotype (i.e., one CD patient and two UC patients). This study confirmed the association between MAP and CD; however, it did not find a strong association between the presence of AIEC and CD. It may be hypothesized that the presence of viable MAP in the bloodstream of CD patients contributes to disease reactivation.

DNA-based detection of Mycobacterium avium subsp. paratuberculosis in domestic and municipal water from Porto (Portugal), an area of high IBD prevalence.

Mycobacterium avium subsp. paratuberculosis (MAP) may play a role in the pathology of human inflammatory bowel disease (IBD). Previously, we found a high frequency (98% in patients with active disease) of MAP DNA detection in the blood of Portuguese Crohn's Disease patients, suggesting this cohort has high exposure to MAP organisms. Water is an important route for MAP dissemination, in this study we therefore aimed to assess MAP contamination within water sources in Porto area (the residential area of our IBD study cohort). Water and biofilms were collected in a wide variety of locations within the Porto area, including taps connected to domestic water sources and from municipal water distribution systems. Baseline samples were collected in early autumn plus further domestic water samples in early winter, to assess the effect of winter rainfall. DNA was extracted from all 131 samples and IS900-based nested PCR used to assess the frequency of MAP presence. Our results show high MAP positivity in municipal water sources (20.7% of water samples and 41.4% of biofilm samples) and even higher amongst domestic sources (30.8% of water samples and 50% of biofilm samples). MAP positivity in biofilms correlated with positivity in water samples from the same sources. A significantly higher frequency of MAP-positivity was observed during winter rains as compared with samples collected in autumn prior to the winter rainfall period (61.9% versus 30.8%). We conclude that domestic and municipal water sources of Porto region have a high burden of MAP contamination and this prevalence increases with rainfall. We hypothesize that human exposure to MAP from local water supplies is commonplace and represents a major route for MAP transmission and challenge which, if positively linked to disease pathology, may contribute to the observed high prevalence of IBD in Porto district.

Repurposing Avermectins and Milbemycins against Mycobacteroides abscessus and Other Nontuberculous Mycobacteria.

Infections caused by nontuberculous mycobacteria (NTM) are increasing worldwide, resulting in a new global health concern. NTM treatment is complex and requires combinations of several drugs for lengthy periods. In spite of this, NTM disease is often associated with poor treatment outcomes. The anti-parasitic family of macrocyclic lactones (ML) (divided in two subfamilies: avermectins and milbemycins) was previously described as having activity against mycobacteria, including Mycobacterium tuberculosis, Mycobacterium ulcerans, and Mycobacterium marinum, among others. Here, we aimed to characterize the in vitro anti-mycobacterial activity of ML against a wide range of NTM species, including Mycobacteroides abscessus. For this, Minimum Inhibitory Concentration (MIC) values of eight ML were determined against 80 strains belonging to nine different NTM species. Macrocyclic lactones showed variable ranges of anti-mycobacterial activity that were compound and species-dependent. Milbemycin oxime was the most active compound, displaying broad-spectrum activity with MIC lower than 8 mg/L. Time kill assays confirmed MIC data and showed bactericidal and sterilizing activity of some compounds. Macrocyclic lactones are available in many formulations and have been extensively used in veterinary and human medicine with suitable pharmacokinetics and safety properties. This information could be exploited to explore repurposing of anti-helminthics for NTM therapy.

Presence of Infection by Mycobacterium avium subsp. paratuberculosis in the Blood of Patients with Crohn's Disease and Control Subjects Shown by Multiple Laboratory Culture and Antibody Methods.

Mycobacterium avium subspecies paratuberculosis (MAP) has long been suspected to be involved in the etiology of Crohn's disease (CD). An obligate intracellular pathogen, MAP persists and influences host macrophages. The primary goals of this study were to test new rapid culture methods for MAP in human subjects and to assess the degree of viable culturable MAP bacteremia in CD patients compared to controls. A secondary goal was to compare the efficacy of three culture methods plus a phage assay and four antibody assays performed in separate laboratories, to detect MAP from the parallel samples. Culture and serological MAP testing was performed blind on whole blood samples obtained from 201 subjects including 61 CD patients (two of the patients with CD had concurrent ulcerative colitis (UC)) and 140 non-CD controls (14 patients in this group had UC only). Viable MAP bacteremia was detected in a significant number of study subjects across all groups. This included Pozzato culture (124/201 or 62% of all subjects, 35/61 or 57% of CD patients), Phage assay (113/201 or 56% of all subjects, 28/61 or 46% of CD patients), TiKa culture (64/201 or 32% of all subjects, 22/61 or 36% of CD patients) and MGIT culture (36/201 or 18% of all subjects, 15/61 or 25% of CD patients). A link between MAP detection and CD was observed with MGIT culture and one of the antibody methods (Hsp65) confirming previous studies. Other detection methods showed no association between any of the groups tested. Nine subjects with a positive Phage assay (4/9) or MAP culture (5/9) were again positive with the Phage assay one year later. This study highlights viable MAP bacteremia is widespread in the study population including CD patients, those with other autoimmune conditions and asymptomatic healthy subjects.

Discovery of stable and variable differences in the Mycobacterium avium subsp. paratuberculosis type I, II, and III genomes by pan-genome microarray analysis.

Mycobacterium avium subsp. paratuberculosis is an important animal pathogen widely disseminated in the environment that has also been associated with Crohn's disease in humans. Three M. avium subsp. paratuberculosis genomotypes are recognized, but genomic differences have not been fully described. To further investigate these potential differences, a 60-mer oligonucleotide microarray (designated the MAPAC array), based on the combined genomes of M. avium subsp. paratuberculosis (strain K-10) and Mycobacterium avium subsp. hominissuis (strain 104), was designed and validated. By use of a test panel of defined M. avium subsp. paratuberculosis strains, the MAPAC array was able to identify a set of large sequence polymorphisms (LSPs) diagnostic for each of the three major M. avium subsp. paratuberculosis types. M. avium subsp. paratuberculosis type II strains contained a smaller genomic complement than M. avium subsp. paratuberculosis type I and M. avium subsp. paratuberculosis type III genomotypes, which included a set of genomic regions also found in M. avium subsp. hominissuis 104. Specific PCRs for genes within LSPs that differentiated M. avium subsp. paratuberculosis types were devised and shown to accurately screen a panel (n = 78) of M. avium subsp. paratuberculosis strains. Analysis of insertion/deletion region INDEL12 showed deletion events causing a reduction in the complement of mycobacterial cell entry genes in M. avium subsp. paratuberculosis type II strains and significantly altering the coding of a major immunologic protein (MPT64) associated with persistence and granuloma formation. Analysis of MAPAC data also identified signal variations in several genomic regions, termed variable genomic islands (vGIs), suggestive of transient duplication/deletion events. vGIs contained significantly low GC% and were immediately flanked by insertion sequences, integrases, or short inverted repeat sequences. Quantitative PCR demonstrated that variation in vGI signals could be associated with colony growth rate and morphology.

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.

Identification of members of Mycobacterium avium species by Accu-Probes, serotyping, and single IS900, IS901, IS1245 and IS901-flanking region PCR with internal standards.

From Mycobacterium avium species Mycobacterium avium subsp. paratuberculosis (n=961), Mycobacterium a. avium (n=677), Mycobacterium a. silvaticum (n=5), and Mycobacterium a. hominissuis (n=1566) were examined, and from Mycobacterium tuberculosis complex M. tuberculosis (n=2), Mycobacterium bovis (n=13), M. bovis BCG (n=4), and Mycobacterium caprae (n=10) were examined. From other mycobacterial species Mycobacterium intracellulare (n=60) and atypical mycobacteria (n=256) including Mycobacterium fortuitum, Mycobacterium chelonae, Mycobacterium scrofulaceum, Mycobacterium gastri and other species of conditionally pathogenic mycobacteria were analysed. The internal standard molecules corresponding to insertion sequences IS900, IS901, IS1245, and flanking region (FR300) of IS901 were produced by PCR of alfalfa genome segment and inserted into plasmid vector. The resulting recombinant plasmid molecules were used as internal standards in coamplification with a total of 4729 mycobacterial collection strains and field isolates between 1996 and 2003. The size differences between amplicons obtained from IS900 (258 bp), IS901 (1108 bp), IS1245 (427 bp), and FR300 (300 bp) and from corresponding internal standard molecules ISIS900 (591 bp), ISIS901 (1 336 bp), ISIS1245 (583 bp), and IS901 flanking region of 300 bp ISFR300 (488 bp), respectively, allowed easy discrimination. The internal amplicons were visible by naked aye on agarose gel when 10(1), 10(3), 10(2), and 10(2) molecules for ISIS900, ISIS901, ISIS1245, and ISFR300 were used in the PCR, respectively, when no bacterial DNA was added to the reaction. The system was tested to define the amount of internal standards that could be used in the PCR without affecting the amplification of the specific segment. Non-specific amplifications were observed in M. fortuitum with IS1245 PCR and mixed infections with M. a. avium and M. a. hominissuis from pigs and cattle were found. PCR results of typing were compared with serotyping and Accu-Probes analyses in selected field isolates.

Improved Culture Medium (TiKa) for Mycobacterium avium Subspecies Paratuberculosis (MAP) Matches qPCR Sensitivity and Reveals Significant Proportions of Non-viable MAP in Lymphoid Tissue of Vaccinated MAP Challenged Animals.

The quantitative detection of viable pathogen load is an important tool in determining the degree of infection in animals and contamination of foodstuffs. Current conventional culture methods are limited in their ability to determine these levels in Mycobacterium avium subspecies paratuberculosis (MAP) due to slow growth, clumping and low recoverability issues. The principle goal of this study was to evaluate a novel culturing process (TiKa) with unique ability to stimulate MAP growth from low sample loads and dilutions. We demonstrate it was able to stimulate a mean 29-fold increase in recoverability and an improved sensitivity of up to three logs when compared with conventional culture. Using TiKa culture, MAP clumping was minimal and produced visible colonies in half the time required by standard culture methods. Parallel quantitative evaluation of the TiKa culture approach and qPCR on MAP loads in tissue and gut mucosal samples from a MAP vaccine-challenge study, showed good correlations between colony counts (cfu) and qPCR derived genome equivalents (Geq) over a large range of loads with a 30% greater sensitivity for TiKa culture approach at low loads (two logs). Furthermore, the relative fold changes in Geq and cfu from the TiKa culture approach suggests that non-mucosal tissue loads from MAP infected animals contained a reduced proportion of non-viable MAP (mean 19-fold) which was reduced significantly further (mean 190-fold) in vaccinated "reactor" calves. This study shows TiKa culture equates well with qPCR and provides important evidence that accuracy in estimating viable MAP load using DNA tests alone may vary significantly between samples of mucosal and lymphatic origin.

A standardised restriction fragment length polymorphism (RFLP) method for typing Mycobacterium avium isolates links IS901 with virulence for birds.

A standardised method for PvuII-PstI-IS901 restriction fragment length polymorphism (RFLP) typing was developed and evaluated against 173 isolates of Mycobacterium avium subsp. avium and M. avium subsp. silvaticum originating from birds (N=46) and their aviaries (N=5), pigs (N=85), cattle (N=18), reference serotype strains (N=9), humans (N=7), a horse (N=1), a nutria (N=1), and strain M. avium subsp. avium ST 18 (formerly M. avium subsp. paratuberculosis ST 18). PvuII-IS1245 RFLP typing was also performed on all isolates. DNA was digested in parallel by restriction endonucleases PvuII or PstI and hybridised to standard probes prepared by PCR. DNA fingerprints were scanned by CCD camera and analysed by the Gel Compar (Applied Maths, Version 4.1, Kortrijk, Belgium) software using a standard isolate control profile. A total of 52 PvuII-PstI RFLP profiles was described including 25 PvuII RFLP profiles designated A to Y and 25 PstI RFLP profiles designated A1-L3. Profiles were found to be stable in vivo and in vitro after multiple subcultures. High IS901 copy number was associated with a "bird" PvuII-IS1245 RFLP profile and low IS901 copy number with M. avium subsp. avium isolates from humans and the nutria. A virulence assay of 100 IS901-positive isolates using intramuscular infections of pullets showed 83 isolates differentiated into 32 RFLP types to be virulent and 17 isolates differentiated into 12 RFLP types as nonvirulent. Attenuation of virulence for pullets could be attributed to either multiple in vitro subculture, polyclonal infection or human passage and was not related to IS901 or IS1245 profiles.

A 16 kb naturally occurring genomic deletion including mce and PPE genes in Mycobacterium avium subspecies paratuberculosis isolates from goats with Johne's disease.

In this study we characterise the genomic and transcriptomic variability of a natural deletion strain of Mycobacterium avium subspecies paratuberculosis (MAP) prevalent in Spanish Guadarrama goats. Using a pan-genome microarray including MAP and M. avium subspecies hominissuis 104 genomes (MAPAC) we demonstrate the genotype to be MAP Type II with a single deletion of 19 contiguous ORFs (16 kb) including a complete mammalian cell entry (mce7_1) operon and adjacent proline-glutamic acid (PE)/proline-proline-glutamic acid (PPE) genes. A deletion specific PCR test was developed and a subsequent screening identified four goat herds infected with the variant strain. Each was located in central Spain and showed epidemiological links suggestive of transmission between herds. A majority of animals infected with the variant manifested a paucibacillary form of the disease. Comparisons between virulent complete genome compliment strains isolated from multibacillary diseased goats and the MAP variant strain during entry into activated macrophages demonstrated an increased sensitivity in the variant to intracellular killing in human and ovine macrophages. As PPE and mce genes are associated with mycobacterial virulence and pathogenesis we investigated the interplay of these gene sets during cell entry using the MAPAC array. This showed significant differential transcriptome profiles compared to full genome complement MAP controls that included changes in other undeleted mce operons and PE/PPE genes, esx-like signalling operons and stress response/fatty acid metabolism pathways. This strain represents the first report of a MAP Type II genotype with significant natural genomic deletions which remains able to cause disease and is transmissible in goats.