New Skin Test for Detection of Bovine Tuberculosis on the Basis of Antigen-Displaying Polyester Inclusions Produced by Recombinant Escherichia coli

The tuberculin skin test for diagnosing tuberculosis (TB) in cattle lacks specificity if animals are sensitized to environmental mycobacteria, as some antigens in purified protein derivative (PPD) prepared from Mycobacterium bovis are present in nonpathogenic mycobacteria. Three immunodominant TB antigens, ESAT6, CFP10, and Rv3615c, are present in members of the pathogenic Mycobacterium tuberculosis complex but absent from the majority of environmental mycobacteria. These TB antigens have the potential to enhance skin test specificity. To increase their immunogenicity, these antigens were displayed on polyester beads by translationally fusing them to a polyhydroxyalkanoate (PHA) synthase which mediated formation of antigen-displaying inclusions in recombinant Escherichia coli. The most common form of these inclusions is poly(3-hydroxybutyric acid) (PHB). The respective fusion proteins displayed on these PHB inclusions (beads) were identified using tryptic peptide fingerprinting analysis in combination with matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). The surface exposure and accessibility of antigens were assessed by enzyme-linked immunosorbent assay (ELISA). Polyester beads displaying all three TB antigens showed greater reactivity with TB antigen-specific antibody than did beads displaying only one TB antigen. This was neither due to cross-reactivity of antibodies with the other two antigens nor due to differences in protein expression levels between beads displaying single or three TB antigens. The triple-antigen-displaying polyester beads were used for skin testing of cattle and detected all cattle experimentally infected with M. bovis with no false-positive reactions observed in those sensitized to environmental mycobacteria. The results suggested applicability of TB antigen-displaying polyester inclusions as diagnostic reagents for distinguishing TB-infected from noninfected animals.     

Serodiagnosis of environmental mycobacterial infections

To demonstrate the usefulness of enzyme-linked immunosorbent assay for serodiagnosis of mycobacterioses due to environmental mycobacteria we utilized a panel of glycolipid antigens selective for Mycobacterium avium-intracellulare, Mycobacterium kansasii, Mycobacterium xenopi, Mycobacterium scrofulaceum and Mycobacterium gordonae. The levels of circulating antibodies were determined against the environmental mycobacteria, and Mycobacterium tuberculosis in human immunodeficiency virus-negative and -positive patient sera. The method used immunomagnetic separation of the antigens, with covalent immobilization of antibodies to superparamagnetic amine and carboxyl terminated particles in solutions of the specific antigens. Enzyme-linked immunosorbent assay was performed on 195 patient sera: 34 with infections due to environmental mycobacteria, 114 with tuberculosis, 47 with other respiratory diseases. There were 46 human immunodeficiency virus-1 infected individuals. Among the 34 infections due to environmental mycobacteria, 9 patients were singularly infected with an environmental mycobacterium, and 25 co-infected with both M. tuberculosis and an environmental mycobacterium. Sensitivity, specificity and false positivity ranges were determined for each of the volunteer groups: tuberculosis positive, human immunodeficiency virus negative; tuberculosis positive, human immunodeficiency virus positive; those with infections due to individual environmental mycobacteria (such as M. scrofulaceum and M. kansasii); and those with other respiratory diseases. We demonstrate that such multiple assays, can be useful for the early diagnosis of diverse environmental mycobacterial infections to allow the start of treatment earlier than henceforth.     

Cell-mediated immunity in experimental allergic encephalomyelitis: cross reactivity between myelin basic protein and mycobacteria antigens.

Guinea pigs injected with Freund's incomplete adjuvant emulsified with guinea pig spinal cord, purified guinea pig myelin basic protein, or human myelin basic protein showed dermal reactivity to both of the basic proteins as well as to mycobacteria antigens. Animals receiving only mycobacteria antigens expressed dermal reactivity to the sensitizing antigen in addition to basic protein. This cross reactivity may help explain the role of mycobacteria in inducing and protecting against EAE, and may have important implications concerning human demyelinating diseases.     

Distribution of Environmental Mycobacteria in Karonga District, Northern Malawi

The genus Mycobacterium includes many species that are commonly found in the environment (in soil and water or associated with plants and animals), as well as species that are responsible for two major human diseases, tuberculosis (Mycobacterium tuberculosis) and leprosy (Mycobacterium leprae). The distribution of environmental mycobacteria was investigated in the context of a long-term study of leprosy, tuberculosis, Mycobacterium bovis BCG vaccination, and the responses of individuals to various mycobacterial antigens in Karonga District, northern Malawi, where epidemiological studies had indicated previously that people may be exposed to different mycobacterial species in the northern and southern parts of the district. A total of 148 soil samples and 24 water samples were collected from various locations and examined to determine the presence of mycobacteria. The detection method involved semiselective culturing and acid-fast staining, following decontamination of samples to enrich mycobacteria and reduce the numbers of other microorganisms, or PCR with primers specific for the mycobacterial 16S rRNA gene, using DNA extracted directly from soil and water samples. Mycobacteria were detected in the majority of the samples, and subsequent sequence analysis of PCR products amplified directly from soil DNA indicated that most of the products were related to known environmental mycobacteria. For both methods the rates of recovery were consistently higher for dry season samples than for wet season samples. All isolates cultured from soil appeared to be strains of Mycobacterium fortuitum. This study revealed a complex pattern for the environmental mycobacterial flora but identified no clear differences between the northern and southern parts of Karonga District.     

Comparative Study of 14C-Labeled Purified Protein Derivative from Various Mycobacteria: II. Skin Cross-Reactivity in Sensitized Guinea Pigs

A study on skin cross-reactivity between stabilized ¹⁴C-labeled mycobacterial antigens, namely tuberculin purified protein derivative (PPD; from Mycobacterium tuberculosis), PPD-A (M. avium), PPD-Y (M. kansasii), PPD-G (M. scrofulaceum), PPD-B (M. intracellulare), and PPD-F (M. fortuitum), has been carried out in groups of guinea pigs sensitized with one of the following heat-killed mycobacteria: M. tuberculosis, M. avium, M. kansasii, M. scrofulaceum, M. intracellulare, or M. fortuitum. For each type of sensitization, the average response for the corresponding PPD antigen was higher than the average response for any of the other antigens. However, the responses to the heterologous PPD antigens were not necessarily significantly different among themselves, and the significant differences of the heterologous PPD antigens were distributed differently according to the type of sensitization. Therefore, ¹⁴C-PPD antigens skin cross-reacted in guinea pigs essentially in the same manner as reported by others for nonradioactive PPD antigens.     

Protective immunity to Mycobacterium tuberculosis infection by chemokine and cytokine conditioned CFP-10 differentiated dendritic cells

Background

Dendritic cells (DCs) play major roles in mediating immune responses to mycobacteria. A crucial aspect of this is the priming of T cells via chemokines and cytokines. In this study we investigated the roles of chemokines RANTES and IP-10 in regulating protective responses from Mycobacterium tuberculosis (M. tb) 10 kDa Culture Filtrate Protein-10 (CFP-10) differentiated DCs (CFP10-DCs).

Methods and Findings

Infection of CFP10-DCs with mycobacteria down-modulated RANTES and IP-10 levels. Pathway specific microarray analyses showed that in addition to RANTES and IP-10, mycobacteria infected CFP10-DCs showed reduced expression of many Th1 promoting chemokines and chemokine receptors. Importantly, T cells co-cultured with RANTES and IP-10 conditioned CFP10-DCs mediated killing of mycobacteria from infected macrophages. Similarly, T cells recruited by RANTES and IP-10 conditioned CFP10-DCs mediated significant killing of mycobacteria from infected macrophages. IFN-gamma treatment of CFP10-DCs restored RANTES and IP-10 levels and T cells activated by these DCs mediated significant killing of virulent M. tb inside macrophages. Adoptive transfer of either RANTES and IP-10 or IL-12 and IFN-gamma conditioned CFP10-DCs cleared an established M. tb infection in mice. The extent of clearance was similar to that obtained with drug treatment.

Conclusions

These results indicate that chemokine and cytokine secretion by DCs differentiated by M. tb antigens such as CFP-10 play major roles in regulating protective immune responses at sites of infection.     

Mycobacteria and Fungi in Moisture-Damaged Building Materials

In contrast to the growth of fungi, the growth of mycobacteria in moisture-damaged building materials has rarely been studied. Environmental mycobacteria were isolated from 23% of samples of moisture-damaged materials (n = 88). The occurrence of mycobacteria increased with increasing concentrations of fungi. Mycobacteria may contribute to indoor exposure and associated adverse health effects.     

Microscopy,Culture, and Quantitative Real-Time PCR Examination Confirm Internalization of Mycobacteria in Plants

The environment is a reservoir of nontuberculous mycobacteria and is considered a source of infection for animals and humans. Mycobacteria can persist in different types of environments for a relatively long time. We have studied their possible internalization into plant tissue through intact, as well as damaged, root systems of different types of plants grown in vitro and under field conditions. The substrate into which plants were seeded was previously contaminated with different strains of Mycobacterium avium (10⁸ to 10¹⁰ cells/g of soil) and feces from animals with paratuberculosis. We detected M. avium subsp. avium, hominissuis, and paratuberculosis in the stems and leaves of the plants by both culture and real-time quantitative PCR. The presence of mycobacteria in the plant tissues was confirmed by microscopy. The concentration of mycobacteria found inside plant tissue was several orders of magnitude lower (up to 10⁴ cells/g of tissue) than the initial concentration of mycobacteria present in the culture medium or substrate. These findings led us to the hypothesis that plants may play a role in the spread and transmission of mycobacteria to other organisms in the environment.     

Real-Time Bioluminescence Imaging of Mixed Mycobacterial Infections

Molecular analysis of infectious processes in bacteria normally involves construction of isogenic mutants that can then be compared to wild type in an animal model. Pathogenesis and antimicrobial studies are complicated by variability between animals and the need to sacrifice individual animals at specific time points. Live animal imaging allows real-time analysis of infections without the need to sacrifice animals, allowing quantitative data to be collected at multiple time points in all organs simultaneously. However, imaging has not previously allowed simultaneous imaging of both mutant and wild type strains of mycobacteria in the same animal. We address this problem by using both firefly (Photinus pyralis) and click beetle (Pyrophorus plagiophthalamus) red luciferases, which emit distinct bioluminescent spectra, allowing simultaneous imaging of two different mycobacterial strains during infection. We also demonstrate that these same bioluminescence reporters can be used to evaluate therapeutic efficacy in real-time, greatly facilitating our ability to screen novel antibiotics as they are developed. Due to the slow growth rate of mycobacteria, novel imaging technologies are a pressing need, since they can they can impact the rate of development of new therapeutics as well as improving our understanding of virulence mechanisms and the evaluation of novel vaccine candidates.     

Profiling serum antibodies to Mycobacterium tuberculosis proteins in rhesus monkeys with nontuberculous Mycobacteria

Recent evidence indicates that the prevalence of diseases caused by nontuberculous mycobacteria (NTM) has been increasing in both human and animals. In this study, antibody profiles of NTM in rhesus monkeys (Macaca mulatta) were determined and compared with those of monkeys infected with Mycobacterium tuberculosis complex (MTBC). Antibodies against 10 M. tuberculosis proteins, purified protein derivative (PPD), and mammalian old tuberculin (MOT) were detected in 14 monkeys naturally infected with NTM by indirect ELISA. Sera from 10 monkeys infected with MTBC and 10 healthy monkeys were set as controls. All antigens showed high serological reactivities to MTBC infections and low reactivities in healthy monkeys. NTM infections showed strong antibody responses to MOT and PPD; moderate antibody responses to 16kDa, U1, MPT64L, 14kDa, and TB16.3; and low antibody responses to 38kDa, Ag85b, CFP10, ESAT-6, and CFP10-ESAT-6. According to the criteria of MTBC, only CFP10, ESAT-6, and CFP10-ESAT-6 showed negative antibody responses in all NTM infections. Taken together, these results suggest that positive results of a PPD/MOT-based ELISA in combination with results of antibodies to M. tuberculosis-specific antigens, such as CFP10 and ESAT-6, could discriminate NTM and MTBC infections. Two positive results indicate an MTBC infection, and a negative result for an M. tuberculosis-specific antigen may preliminarily predict an NTM infection.     

First Detection of Mycobacteria in African Rodents and Insectivores, Using Stratified Pool Screening

With the rising number of patients with human immunodeficiency virus (HIV)/AIDS in developing countries, the control of mycobacteria is of growing importance. Previous studies have shown that rodents and insectivores are carriers of mycobacteria. However, it is not clear how widespread mycobacteria are in these animals and what their role is in spreading them. Therefore, the prevalence of mycobacteria in rodents and insectivores was studied in and around Morogoro, Tanzania. Live rodents were trapped, with three types of live traps, in three habitats. Pieces of organs were pooled per habitat, species, and organ type (stratified pooling); these sample pools were examined for the presence of mycobacteria by PCR, microscopy, and culture methods. The mycobacterial isolates were identified using phenotypic techniques and sequencing. In total, 708 small mammals were collected, 31 of which were shrews. By pool prevalence estimation, 2.65% of the animals were carriers of mycobacteria, with a higher prevalence in the urban areas and in Cricetomys gambianus and the insectivore Crocidura hirta. Nontuberculous mycobacteria (Mycobacterium chimaera, M. intracellulare, M. arupense, M. parascrofulaceum, and Mycobacterium spp.) were isolated from C. gambianus, Mastomys natalensis, and C. hirta. This study is the first to report findings of mycobacteria in African rodents and insectivores and the first in mycobacterial ecology to estimate the prevalence of mycobacteria after stratified pool screening. The fact that small mammals in urban areas carry more mycobacteria than those in the fields and that potentially pathogenic mycobacteria were isolated identifies a risk for other animals and humans, especially HIV/AIDS patients, that have a weakened immune system.     

Prevalence of mycobacterial sensitivity in Montreal children

The prevalence of sensitivity to atypical mycobacteria was found to be low in 2152 Montreal first-grade children tested with tuberculin and one of four atypical antigens randomly allocated. Forty-nine had positive reactions to an atypical antigen. PPD-G produced more reactions than PPD-A, -B or -K. In Greater Montreal there was a higher prevalence of sensitivity to atypical mycobacteria than in the two suburban areas studied. The range of this prevalence, standardized for the type of antigen, was from 0.86 to 2.92% according to region. The highest prevalence of sensitivity to any single atypical antigen, 5.3%, was found for PPD-G in Greater Montreal.Tuberculous infection was found in 1.35% of the children. Small tuberculin reactions (5 to 9 mm to stabilized PPD 5 TU) require clarification by differential tuberculin testing. More of them are caused by M. tuberculosis than by atypical mycobacteria in this particular age group and region.Routine BCG immunization is not indicated for the particular population studied; when given to individuals at risk, its effectiveness should not be impaired by atypical sensitivity at the low level found locally. Future BCG plans require more epidemiologic data.     

Evidence that the capsule around mycobacteria grown in axenic media contains mycobacterial antigens: implications at the level of cell envelope architecture

The intracellular growth of pathogenic mycobacteria has been linked to the presence of an electron transparent zone (ETZ or capsule), which surrounds the phagocytized bacteria and prevents the diffusion of lysosomal enzymes in infected macrophages. Recently, it was suggested that this capsule may be a bacterial structures, even being present in test tube-grown pathogenic mycobacteria (FEMS Microbiol. Lett. 1988, 56, 225-230). In the present paper, we show that under special fixation and embedding conditions, this capsule was clearly observed among 7 strains of mycobacteria grown in axenic media and also in M. leprae extracted and purified from experimentally infected armadillo or nude mice. In the case of bacteria treated likewise but subject to a prior dehydration step, this capsular structure disappeared suggesting its lipidic nature. Ultrathin sections of M. intracellular after immunolabelling showed for the first time that this capsule obtained mycobacterial antigens confirming its mycobacterial origin. It is suggested that the mycobacterial capsule may be formed of inert lipids, in which surface antigens are embedded.     

Environmental Factors Affecting the Occurrence of Mycobacteria in Brook Waters

To evaluate the impact of environmental factors on the occurrence of environmental mycobacteria, viable counts of mycobacteria were measured in samples of brook water collected from 53 drainage areas located in a linear belt crossing Finland at 63° north latitude. The numbers of mycobacteria were correlated with characteristics of the drainage area, climatic parameters, chemical and physical characteristics of the water, and counts of other heterotrophic bacteria in the water. The numbers of mycobacteria in the water ranged from 10 to 2,200 CFU/liter. The counts correlated positively (P < 0.001) with the presence of peatlands, precipitation data, chemical oxygen demand, water color, and concentrations of Fe, Al, Cu, Co, and Cr. The mycobacterial counts correlated negatively (P < 0.001) with water pH, whereas other heterotrophic bacterial counts lacked any correlation with pH. A linear regression model with four independent variables (i.e., peatlands in the drainage area, chemical oxygen demand, concentration of potassium, and pH) explained 83% of the variation in mycobacterial counts in brook waters. Our results suggest that acidification may enhance the growth of environmental mycobacteria.     

Azurophil Granule Proteins Constitute the Major Mycobactericidal Proteins in Human Neutrophils and Enhance the Killing of Mycobacteria in Macrophages

Pathogenic mycobacteria reside in, and are in turn controlled by, macrophages. However, emerging data suggest that neutrophils also play a critical role in innate immunity to tuberculosis, presumably by their different antibacterial granule proteins. In this study, we purified neutrophil azurophil and specific granules and systematically analyzed the antimycobacterial activity of some purified azurophil and specific granule proteins against M. smegmatis, M. bovis-BCG and M. tuberculosis H37Rv. Using gel overlay and colony forming unit assays we showed that the defensin-depleted azurophil granule proteins (AZP) were more active against mycobacteria compared to other granule proteins and cytosolic proteins. The proteins showing antimycobacterial activity were identified by MALDI-TOF mass spectrometry. Electron microscopic studies demonstrate that the AZP disintegrate bacterial cell membrane resulting in killing of mycobacteria. Exogenous addition of AZP to murine macrophage RAW 264.7, THP-1 and peripheral blood monocyte-derived macrophages significantly reduced the intracellular survival of mycobacteria without exhibiting cytotoxic activity on macrophages. Immunofluorescence studies showed that macrophages actively endocytose neutrophil granular proteins. Treatment with AZP resulted in increase in co-localization of BCG containing phagosomes with lysosomes but not in increase of autophagy. These data demonstrate that neutrophil azurophil proteins may play an important role in controlling intracellular survival of mycobacteria in macrophages.     

A broad set of different llama antibodies specific for a 16 kDa heat shock protein of Mycobacterium tuberculosis

Background

Recombinant antibodies are powerful tools in engineering of novel diagnostics. Due to the small size and stable nature of llama antibody domains selected antibodies can serve as a detection reagent in multiplexed and sensitive assays for M. tuberculosis.

Methodology/Principal Findings

Antibodies for Mycobacterium tuberculosis (M. tb) recognition were raised in Alpaca, and, by phage display, recombinant variable domains of heavy-chain antibodies (VHH) binding to M. tuberculosis antigens were isolated. Two phage display selection strategies were followed: one direct selection using semi-purified protein antigen, and a depletion strategy with lysates, aiming to avoid cross-reaction to other mycobacteria. Both panning methods selected a set of binders with widely differing complementarity determining regions. Selected recombinant VHHs were produced in E. coli and shown to bind immobilized lysate in direct Enzymelinked Immunosorbent Assay (ELISA) tests and soluble antigen by surface plasmon resonance (SPR) analysis. All tested VHHs were specific for tuberculosis-causing mycobacteria (M. tuberculosis, M. bovis) and exclusively recognized an immunodominant 16 kDa heat shock protein (hsp). The highest affinity VHH had a dissociation constant (KD) of 4×10⁻¹⁰ M.

Conclusions/Significance

A broad set of different llama antibodies specific for 16 kDa heat shock protein of M. tuberculosis is available. This protein is highly stable and abundant in M. tuberculosis. The VHH that detect this protein are applied in a robust SPR sensor for identification of tuberculosis-causing mycobacteria.     

Confinement-Induced Drug-Tolerance in Mycobacteria Mediated by an Efflux Mechanism

Tuberculosis (TB) is the world’s deadliest curable disease, responsible for an estimated 1.5 million deaths annually. A considerable challenge in controlling this disease is the prolonged multidrug chemotherapy (6 to 9 months) required to overcome drug-tolerant mycobacteria that persist in human tissues, although the same drugs can sterilize genetically identical mycobacteria growing in axenic culture within days. An essential component of TB infection involves intracellular Mycobacterium tuberculosis bacteria that multiply within macrophages and are significantly more tolerant to antibiotics compared to extracellular mycobacteria. To investigate this aspect of human TB, we created a physical cell culture system that mimics confinement of replicating mycobacteria, such as in a macrophage during infection. Using this system, we uncovered an epigenetic drug-tolerance phenotype that appears when mycobacteria are cultured in space-confined bioreactors and disappears in larger volume growth contexts. Efflux mechanisms that are induced in space-confined growth environments contribute to this drug-tolerance phenotype. Therefore, macrophage-induced drug tolerance by mycobacteria may be an effect of confined growth among other macrophage-specific mechanisms.     

Mycobacterial Ecology of the Rio Grande

This is the first study to characterize the environmental conditions which contribute to the presence and proliferation of environmental mycobacteria in a major freshwater river. Over 20 different species of environmental mycobacteria were isolated, including the pathogenic M. avium and M. kansasii. Species of the rapidly growing M. fortuitum complex were the most commonly isolated mycobacteria, and one-third of all isolates were not identified at the species level, even by 16S sequencing. PCR restriction analysis of the hsp65 gene was more accurate and rapid than biochemical tests and as accurate as yet less expensive than 16S sequencing, showing great promise as a new tool for species identification of environmentally isolated mycobacteria. Total environmental mycobacteria counts positively correlated with coliform and Escherichia coli counts and negatively correlated with chemical toxicity and water temperature. Environmental mycobacteria can survive in the alkaline conditions of the river despite previous reports that especially acidic conditions favor their presence. A representative river isolate (M. fortuitum) survived better than E. coli O157:H7 at pHs below 7 and above 8 in nutrient broth. The river strain also retained viability at 8 ppm of free chlorine, while E. coli was eliminated at 2 ppm and above. Thus, in vitro studies support environmental observations that a variety of extreme conditions favor the hardy environmental mycobacteria.