Rapid identification and differentiation of Mycobacterium avium subspecies paratuberculosis types by use of real-time PCR and high-resolution melt analysis of the MAP1506 locus

High-resolution melt (HRM) analysis can identify sequence polymorphisms by comparing the melting curves of amplicons generated by real-time PCR amplification. We describe the application of this technique to identify Mycobacterium avium subspecies paratuberculosis types I, II, and III. The HRM approach was based on type-specific nucleotide sequences in MAP1506, a member of the PPE (proline-proline-glutamic acid) gene family.

Occurrence of Mycobacterium avium subspecies paratuberculosis across host species and European countries with evidence for transmission between wildlife and domestic ruminants

BACKGROUND Mycobacterium avium subspecies paratuberculosis (Map) causes an infectious chronic enteritis (paratuberculosis or Johne's disease) principally of ruminants. The epidemiology of Map is poorly understood, particularly with respect to the role of wildlife reservoirs and the controversial issue of zoonotic potential (Crohn's disease). Genotypic discrimination of Map isolates is pivotal to descriptive epidemiology and resolving these issues. This study was undertaken to determine the genetic diversity of Map, enhance our understanding of the host range and distribution and assess the potential for interspecies transmission. RESULTS 164 Map isolates from seven European countries representing 19 different host species were genotyped by standardized IS900--restriction fragment length polymorphism (IS900-RFLP), pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphisms (AFLP) and mycobacterial interspersed repeat unit-variable number tandem repeat (MIRU-VNTR) analyses. Six PstI and 17 BstEII IS900-RFLP, 31 multiplex [SnaBI-SpeI] PFGE profiles and 23 MIRU-VNTR profiles were detected. AFLP gave insufficient discrimination of isolates for meaningful genetic analysis. Point estimates for Simpson's index of diversity calculated for the individual typing techniques were in the range of 0.636 to 0.664 but a combination of all three methods increased the discriminating power to 0.879, sufficient for investigating transmission dynamics. Two predominant strain types were detected across Europe with all three typing techniques. Evidence for interspecies transmission between wildlife and domestic ruminants on the same property was demonstrated in four cases, between wildlife species on the same property in two cases and between different species of domestic livestock on one property. CONCLUSION The results of this study showed that it is necessary to use multiple genotyping techniques targeting different sources of genetic variation to obtain the level of discrimination necessary to investigate transmission dynamics and trace the source of Map infections. Furthermore, the combination of genotyping techniques may depend on the geographical location of the population to be tested. Identical genotypes were obtained from Map isolated from different host species co-habiting on the same property strongly suggesting that interspecies transmission occurs. Interspecies transmission of Map between wildlife species and domestic livestock on the same property provides further evidence to support a role for wildlife reservoirs of infection.

Single nucleotide polymorphisms in the IS900 sequence of Mycobacterium avium subsp. paratuberculosis are strain type specific

Insertion sequence IS900 is used as a target for the identification of Mycobacterium avium subsp. paratuberculosis. Previous reports have revealed single nucleotide polymorphisms within IS900. This study, which analyzed the IS900 sequences of a panel of isolates representing M. avium subsp. paratuberculosis strain types I, II, and III, revealed conserved type-specific polymorphisms that could be utilized as a tool for diagnostic and epidemiological purposes.

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.

Genetic diversity of Mycobacterium avium isolates recovered from clinical samples and from the environment: molecular characterization for diagnostic purposes

Isolation of Mycobacterium avium complex (MAC) organisms from clinical samples may occur in patients without clinical disease, making the interpretation of results difficult. The clinical relevance of MAC isolates from different types of clinical samples (n = 47) from 39 patients in different sections of a hospital was assessed by comparison with environmental isolates (n = 17) from the hospital. Various methods for identification and typing (commercial probes, phenotypic characteristics, PCR for detection of IS1245 and IS901, sequencing of the hsp65 gene, and pulsed-field gel electrophoresis) were evaluated. The same strain was found in all the environmental isolates, 21 out of 23 (91.3%) of the isolates cultured from urine samples, and 5 out of 19 (26.3%) isolates from respiratory specimens. This strain did not cause disease in the patients. Testing best characterized the strain as M. avium subsp. hominissuis, with the unusual feature that 81.4% of these isolates lacked the IS1245 element. Contamination of certain clinical samples with an environmental strain was the most likely event; therefore, characterization of the environmental mycobacteria present in health care facilities should be performed to discard false-positive isolations in nonsterile samples, mainly urine samples. Molecular techniques applied in this study demonstrated their usefulness for this purpose.

Single-nucleotide polymorphism in two representative multidrug-resistant Mycobacterium bovis isolates collected from patients in a Spanish hospital harboring a human infection outbreak

Mycobacterium bovis is the etiological agent of tuberculosis in domestic and wild animals. Its involvement as a human pathogen has been highlighted again with the recent descriptions of transmission through dairy products (18), reactivation or primary infection in human immunodeficiency virus-infected patients (5), and association with meat industry workers, animal keepers, or hunters (3). Strains resistant to antituberculous drugs (M. bovis is naturally resistant to pyrazinamide) pose an additional risk (2). Several studies have demonstrated that mutations in target genes are associated with resistance to antituberculous drugs (4, 7, 10, 11, 16). However, most of them have been developed in Mycobacterium tuberculosis strains and limited data are available regarding M. bovis isolates. The aim of this study was to characterize by sequencing the main genes involved in antibiotic resistance in two multidrug-resistant (MDR) M. bovis isolates in a human outbreak detected in a hospital in Madrid that subsequently spread to several countries (5, 6, 15). The isolates were resistant to 11 drugs, but only their rpoB and katG genes have been analyzed so far (1, 14). We studied the first (93/R1) and last (95/R4) M. bovis isolates of this nosocomial outbreak, characterized by spoligotyping as SB0426 (hexacode 63-5F-5E-7F-FF-60 in the database at www.mbovis.org) (1, 13). Several genes involved in resistance to isoniazid (katG, ahpC, inhA, and the oxyR-ahpC intergenic region), rifampin (rpoB), streptomycin (rrs, rpsL), ethambutol (embB), and quinolones (gyrA) were studied. These genes, or fragments of genes, were amplified and sequenced as previously described (12). The sequence analysis revealed polymorphisms in five (ahpC, rpoB, rpsL, embB, and gyrA) out of nine analyzed genes (Table 1). Nucleotide substitutions in four genes cause a change in the encoded amino acid. Two additional synonymous mutations in ahpC and rpsL differentiated the first and last isolates from the outbreak.

Protection against Tuberculosis in Eurasian Wild Boar Vaccinated with Heat-Inactivated Mycobacterium bovis

Tuberculosis (TB) caused by Mycobacterium bovis and closely related members of the Mycobacterium tuberculosis complex continues to affect humans and animals worldwide and its control requires vaccination of wildlife reservoir species such as Eurasian wild boar (Sus scrofa). Vaccination efforts for TB control in wildlife have been based primarily on oral live BCG formulations. However, this is the first report of the use of oral inactivated vaccines for controlling TB in wildlife. In this study, four groups of 5 wild boar each were vaccinated with inactivated M. bovis by the oral and intramuscular routes, vaccinated with oral BCG or left unvaccinated as controls. All groups were later challenged with a field strain of M. bovis. The results of the IFN-gamma response, serum antibody levels, M. bovis culture, TB lesion scores, and the expression of C3 and MUT genes were compared between these four groups. The results suggested that vaccination with heat-inactivated M. bovis or BCG protect wild boar from TB. These results also encouraged testing combinations of BCG and inactivated M. bovis to vaccinate wild boar against TB. Vaccine formulations using heat-inactivated M. bovis for TB control in wildlife would have the advantage of being environmentally safe and more stable under field conditions when compared to live BCG vaccines. The antibody response and MUT expression levels can help differentiating between vaccinated and infected wild boar and as correlates of protective response in vaccinated animals. These results suggest that vaccine studies in free-living wild boar are now possible to reveal the full potential of protecting against TB using oral M. bovis inactivated and BCG vaccines

Infection of the central nervous system by Mycobacterium tuberculosis in patients infected with human immunodeficiency virus (the new neurotuberculosis)

Neurologic complications of HIV infection are numerous. This review focuses on the clinical presentation, diagnostic particularities and therapeutic issues of neurotuberculosis. The pertinent literature describing this important infection is succinctly summarized with a particular emphasis on the discussion of differences in clinical presentation between HIV-infected and -uninfected patients.

Viable mycobacterium avium subsp. paratuberculosis in retail artisanal coalho cheese from northeastern Brazil.

2014

Mycobacterium Bovis em queijo coalho artesanal em Parnaíba, Piauí.

O objetivo desse estudo foi detectar Mycobacterium bovis em queijo de coalho artesanal comercializado em Parnaíba, Piauí, por meio de cultivo microbiológico e pela Reação em Cadeia de Polimerase em Tempo Real.

Comparison of methods for processing drinking water samples for the isolation of Myocbacterium avium and intracellulare

Several protocols for isolation of mycobacteria from water exist, but there is no established standard method. This study compared methods of processing potable water samples for the isolation of Mycobacterium avium and Mycobacterium intracellulare using spiked sterilized water and tap water decontaminated using 0.005% cetylpyridinium chloride (CPC). Samples were concentrated by centrifugation or filtration and inoculated onto Middlebrook 7H10 and 7H11 plates and Lowenstein-Jensen slants and into mycobacterial growth indicator tubes with or without polymyxin, azlocillin, nalidixic acid, trimethoprim, and amphotericin B. The solid media were incubated at 32°C, at 35°C, and at 35°C with CO2 and read weekly. The results suggest that filtration of water for the isolation of mycobacteria is a more sensitive method for concentration than centrifugation. The addition of sodium thiosulfate may not be necessary and may reduce the yield. Middlebrook M7H10 and 7H11 were equally sensitive culture media. CPC decontamination, while effective for reducing growth of contaminants, also significantly reduces mycobacterial numbers. There was no difference at 3 weeks between the different incubation temperatures.

When and how to treat pulmonary non-tuberculous mycobacterial diseases

Nontuberculous mycobacteria are ubiquitous environmental organisms that have been recognised as a cause of pulmonary infection for over 50 years. Traditionally patients have had underlying risk factors for development of disease; however the proportion of apparently immunocompetent patients involved appears to be rising. Not all patients culture-positive for mycobacteria will have progressive disease, making the diagnosis difficult, though criteria to aid in this process are available. The two main forms of disease are cavitary disease (usually involving the upper lobes) and fibronodular bronchiectasis (predominantly middle and lingular lobes). For patients with disease, combination antibiotic therapy for 12-24 months is generally required for successful treatment, and this may be accompanied by drug intolerances and side effects. Published success rates range from 30-82%. As the progression of disease is variable, for some patients, attention to pulmonary hygiene and underlying diseases without immediate antimycobacterial therapy may be more appropriate. Surgery can be a useful adjunct, though is associated with risks. Randomised controlled trials in well described patients would provide stronger evidence-based data to guide therapy of NTM lung diseases, and thus are much needed.

A prime-boost immunisation regimen using recombinant BCG and Pr55 gag virus-like particle vaccines based on HIV type 1 subtype C successfully elicits Gag-specific responses in baboons

Mycobacterium bovis BCG is considered an attractive live bacterial vaccine vector. In this study, we investigated the immune response of baboons to a primary vaccination with recombinant BCG (rBCG) constructs expressing the gag gene from a South African HIV-1 subtype C isolate, and a boost with HIV-1 subtype C Pr55 gag virus-like particles (Gag VLPs). Using an interferon enzyme-linked immunospot assay, we show that although these rBCG induced only a weak or an undetectable HIV-1 Gag-specific response on their own, they efficiently primed for a Gag VLP boost, which strengthened and broadened the immune responses. These responses were predominantly CD8+ T cell-mediated and recognised similar epitopes as those targeted by humans with early HIV-1 subtype C infection. In addition, a Gag-specific humoral response was elicited. These data support the development of HIV-1 vaccines based on rBCG and Pr55 gag VLPs. © 2009 Elsevier Ltd. All rights reserved.

The role of inflammation in the pathogenesis of non-small cell lung cancer

The link between chronic immune activation and tumorigenesis is well established. Compelling evidence has accumulated that histologic assessment of infiltration patterns of different host immune response components in non-small cell lung cancer specimens helps identify different prognostic patient subgroups. This review provides an overview of recent insights gained in the understanding of the role played by chronic inflammation in lung carcinogenesis. The usefulness of quantification of different populations of lymphocytes, natural killer cells, macrophages, and mast cells within the tumor microenvironment in non-small cell lung cancer is also discussed. In particular, the importance of assessment of inflammatory cell microlocalization within both the tumor islet and surrounding stromal components is emphasized. Copyright © 2010 by the International Association for the Study of Lung Cancer.