Benefits and costs of ecological restoration: Rapid assessment of changing ecosystem service values at a U.K. wetland.

Restoration of degraded land is recognized by the international community as an important way of enhancing both biodiversity and ecosystem services, but more information is needed about its costs and benefits. In Cambridgeshire, U.K., a long-term initiative to convert drained, intensively farmed arable land to a wetland habitat mosaic is driven by a desire both to prevent biodiversity loss from the nationally important Wicken Fen National Nature Reserve (Wicken Fen NNR) and to increase the provision of ecosystem services. We evaluated the changes in ecosystem service delivery resulting from this land conversion, using a new Toolkit for Ecosystem Service Site-based Assessment (TESSA) to estimate biophysical and monetary values of ecosystem services provided by the restored wetland mosaic compared with the former arable land. Overall results suggest that restoration is associated with a net gain to society as a whole of $199 ha(-1)y(-1), for a one-off investment in restoration of $2320 ha(-1). Restoration has led to an estimated loss of arable production of $2040 ha(-1)y(-1), but estimated gains of $671 ha(-1)y(-1) in nature-based recreation, $120 ha(-1)y(-1) from grazing, $48 ha(-1)y(-1) from flood protection, and a reduction in greenhouse gas (GHG) emissions worth an estimated $72 ha(-1)y(-1). Management costs have also declined by an estimated $1325 ha(-1)y(-1). Despite uncertainties associated with all measured values and the conservative assumptions used, we conclude that there was a substantial gain to society as a whole from this land-use conversion. The beneficiaries also changed from local arable farmers under arable production to graziers, countryside users from towns and villages, and the global community, under restoration. We emphasize that the values reported here are not necessarily transferable to other sites.

Mycobacterium recombinant vaccines.

Intracellular diseases are difficult to treat and constitute a major problem for modern medicine. In this type of diseases, a TH-1 immune response favors protection, while a TH-2 response is detrimental to the host. Current vaccines are using antigens to initiate an immune response regardless of its nature and its mechanism. New vaccines are designed to combine selected antigens with potent adjuvants to stimulate the appropriate pathway of the immune system and deliver a lasting protective immunity. The Mycobacterium recombinant vaccine system for treatment of intracellular diseases utilizes antigen delivery systems in the form of non pathogenic Mycobacterium strains, genetic transfer systems in the form of cloning and expression vectors, and related technologies to provide products containing non toxic immuno-regulating Mycobacterium adjuvants, non toxic immuno-stimulating exogenous antigens specific for a variety of diseases, and non toxic amounts of cytokines that boost the TH-1 pathway. The cloning and expression Mycobacterium vectors include both pAL5000-based extra-chromosomal and D29-based integrative vectors.

Intrinsic resistance of Mycobacterium tuberculosis to clarithromycin is effectively reversed by subinhibitory concentrations of cell wall inhibitors.

Subinhibitory concentrations of bacitracin, vancomycin and other inhibitors of cell wall synthesis reversed to varying extents the intrinsic resistance of Mycobacterium tuberculosis to clarithromycin. Ethambutol reversed clarithromycin resistance in all of the M. tuberculosis strains studied regardless of their susceptibility to this drug.

An epidemic of tuberculosis with a high rate of tuberculin anergy among a population previously unexposed to tuberculosis, the Yanomami Indians of the Brazilian Amazon.

A survey of an emerging tuberculosis epidemic among the Yanomami Indians of the Amazonian rain forest provided a unique opportunity to study the impact of tuberculosis on a population isolated from contact with the tubercle bacillus for millennia until the mid-1960s. Within the Yanomami population, an extraordinary high prevalence of active tuberculosis (6.4% of 625 individuals clinically examined) was observed, indicating a high susceptibility to disease, even among bacille Calmette-Guérin-vaccinated individuals. Observational studies on cell-mediated and humoral immune responses of the Yanomami Indians compared with contemporary residents of the region suggest profound differences in immunological responsiveness to Mycobacterium tuberculosis infection. Among the Yanomami, a very high prevalence of tuberculin skin test anergy was found. Of patients with active tuberculosis, 46% had purified protein derivative of tuberculosis reactions <10 mm; similarly 58% of recent bacillus Calmette-Guérin vaccines exhibited skin test reactions <5 mm. The Yanomami also had higher titers of antibodies against M. tuberculosis glycolipid antigens (>70%) than the control subjects comprised of Brazilians of European descent (14%). The antibodies were mostly of the IgM isotype. Among the tuberculosis patients who also produced IgG antibodies, the titers of IgG4 were significantly higher among the Yanomami than in the control population. Although it was not possible to analyze T-cell responses or patterns of lymphokine production in vitro because of the remoteness of the villages from laboratory facilities, the results suggest that the first encounter of the Yanomami Indian population with tuberculosis engenders a diminished cell-mediated immune response and an increased production antibody responses, relative to other populations with extensive previous contact with the pathogen. These findings suggest that tuberculosis may represent a powerful selective pressure on human evolution that over centuries has shaped the nature of human immune responses to infection.

Mycobacteriophage D29 contains an integration system similar to that of the temperate mycobacteriophage L5.

A mycobacteriophage D29 DNA fragment cloned in pRM64, a shuttle plasmid that transforms Mycobacterium smegmatis, was sequenced. The determined sequence was 2592 nucleotides long and had a mean G+C content of 63.7 mol%, similar to that of mycobacterial DNA. Four ORFs were identified: one with strong homology to dCMP deaminase genes; one homologous to mycobacteriophage L5 gene 36, whose function is unknown; one encoding a possible excisase; and one encoding an integrase. The intergenic region between the putative excisase gene and the integrase gene had a lower than average G+C content and showed the presence of the same attP core sequence as mycobacteriophage L5. Transformation experiments using subclones of pRM64 indicated that the integrase gene and all the intergenic region were essential for stable transformation. A subclone containing the integrase gene and the core attP sequence was able to transform but recombinants were highly unstable. Southern analysis of total DNA from cells transformed with pRM64 and its derivatives showed that all the plasmids were integrated at one specific site of the bacterial chromosome. A recombinant exhibiting a high level of resistance to the selective drug kanamycin had two plasmids integrated at different sites. These results demonstrated that the D29 sequences contained in pRM64 were integrative, indicating that the generally hold view of D29 as a virulent phage must be reviewed.

Circulating immune complexes in leprosy sera: demonstration of antibodies against mycobacterial glycolipidic antigens in isolated immune complexes.

Circulating immune complexes (CIC) were assayed in sera of leprosy patients. Using an immunoassay for two mycobacterial antigens--phenolic glycolipid-I (PGL-I) and glycolipid IV (SL-IV)--sera from 65 patients with leprosy (38 lepromatous, 18 borderline, and 9 tuberculoid) were studied. The CIC were isolated by polyethylene glycol (PEG) precipitation, washed, treated with an acid buffer, neutralized, and tested using an enzyme-linked immunosorbent assay (ELISA). We demonstrated that CIC could contain IgG and IgM antibodies reacting against PGL-I and SL-IV. The high levels of antibodies in the precipitable CIC showed concordance with high levels in the original sera, although some patients presented high levels of precipitable CIC in the absence of high titers of antibodies in their sera. It was concluded that some of the CIC observed in patients with leprosy were composed of IgG and IgM immunoglobulins against specific mycobacterial antigens.

Isolation and characterization of serologically reactive lipooligosaccharides from Mycobacterium tuberculosis.

Two major highly polar antigenic glycolipids were isolated from recent isolates of Mycobacterium tuberculosis from a wide range of geographical origins. The occurrence of these polar glycolipids was demonstrated by isolation, purification and chromatographic characterization and/or serological procedures in 12 strains. Based on their chromatographic properties, these polar glycolipids belong to the lipooligosaccharide family. Preliminary data on the use of these newly described antigens in the serodiagnosis of tuberculosis is presented.

Comparison of bis-di-octadecylamide of trehalose dicarboxylic acid (BDA.TDA) with glycolipid SL-IV as ELISA antigens for the serodiagnosis of leprosy.

Two glycolipids--one synthetic and non-natural (BDA.TDA), the other natural and Mycobacterium tuberculosis species-specific (SL-IV)--were tested to determine their serological activity in sera obtained from leprosy patients, and to determine their discriminating ability in the detection of disease. The ELISA results obtained in the IgG antibody class show that both were useful substances capable of detecting multibacillary and paucibacillary disease in about 2 out of 3 leprosy patients. When these antigens were tested in parallel, the sensitivity of the ELISA test was increased by 10% without a decrease in specificity.

Thin-layer chromatography systems for the identification of Mycobacterium tuberculosis, M. bovis BCG, M. kansasii, M. gastri and M. marinum.

Knowledge of mycobacterial glycolipid antigens and the study of their specificity have resulted in their utilization as species markers. We describe a thin-layer chromatography method which could serve as a useful adjunct for the identification of Mycobacterium tuberculosis, M. bovis BCG, M. kansasii, M. gastri and M. marinum.

Relationships between titers of antibodies immunoreacting against glycolipid antigens from Mycobacterium leprae and M. tuberculosis, the Mitsuda and Mantoux reactions, and bacteriological loads: implications in the pathogenesis, epidemiology and serodiagnosis of leprosy and tuberculosis.

Analysis of cell-mediated immunity [(CMI) as judged from the Mantoux, Fernandez, and Mitsuda reactions and the presence of granulomas in biopsy material] against humoral immunity (measurements of anti-PGL-I, PGL-Tb1, and SL-IV IgG and IgM antibody titers by ELISA) were performed in selected human populations. The investigations yielded data indicating that humoral (B-cell) responses preceded protective CMI in both tuberculosis and leprosy. The B-cell responses were unrelated to (unfavorable) cell-mediated delayed-type hypersensitivity (DTH). Notwithstanding the difficulty in inferring sequential events from studies in humans, it was shown that in humoral responses there was an initial rise of specific IgM immunoglobulins that switched afterward to IgG production during subclinical tuberculosis and leprosy infections. In patent tuberculosis disease the IgM-to-IgG switch was observed in the majority of patients; in patent leprosy disease the switch was impaired in the majority of patients. The clinical, immunological, and laboratory data indicated that the B-cell responses were suppressed as protective CMI was re-established in the patients during the protracted subclinical infection. According to the data, the diagnosis of subclinical tuberculosis and leprosy may be accomplished using ELISA. The yearly risk of tuberculosis in apparently healthy persons but with significant antibody titers was estimated at 44%; the yearly risk for leprosy has not yet been established. The clinical, epidemiologic, and diagnostic implications of these findings are discussed.

DOT-ELISA for detection of phenolic glycolipid PGL-Tb1 and diacyl-trehalose antigens of Mycobacterium tuberculosis.

A DOT-ELISA method for detection of 2,3-diacyl-trehalose (DAT, previously referred to as SL-IV antigen) and triglycosyl phenol phthiocerol di-mycocerosate (PGL-Tb1) antigens from Mycobacterium tuberculosis is described. The method enabled the detection of both antigens in 14 clinical isolates of M. tuberculosis from different geographic origins; the presence of the glycolipids was confirmed by chemical analysis. It was therefore concluded that the synthesis of both of these compounds is characteristic of the species.

Identification of sulpholipid I by thin-layer chromatography in the rapid identification of Mycobacterium tuberculosis.

A simple, rapid and reliable thin-layer chromatography method for the detection of the 2,3,6,6'-tetraacyl trehalose-2'-sulphate (sulpholipid I) from Mycobacterium tuberculosis was described. The method was found to be satisfactory as an aid in the rapid identification of M. tuberculosis in clinical laboratories.

Cloning and expression of Mycobacterium aurum carotenogenesis genes in Mycobacterium smegmatis.

This report describes the first successful transfer and complete expression of clustered mycobacterial genes controlling a biosynthetic pathway (carotenogenesis) in a homologous system. A genomic library of pigmented Mycobacterium aurum A+ (yellow-orange) DNA was constructed in shuttle vector pHLD-69. The colourless mutant A11 and the brick-red mutant NgR9 derived from M. aurum A+ were electroporated with the plasmid library. Among the transformants, colonies different in colour from the recipient mutants were detected, and were cloned. One of the clones from the transformed A11 mutant had a yellow-orange phenotype, and was designated A11T; one of the clones from the NgR9 (brick-red) mutant had a yellow-orange phenotype and was designated NgR9T. The carotenoid pigments from the A11T and NgR9T clones were analyzed and in both the end product of carotenogenesis in M. aurum (leprotene) was detected. A11T and NgR9T harboured the same recombinant plasmid (Cl) containing a 11-kb M. aurum fragment. pCl was used to transform the colourless Mycobacterium smegmatis MC2-155 strain. All the transformants were pigmented. A colony (MC2-T) was arbitrarily chosen and leprotene was detected. It was therefore concluded that M. aurum genes involved in carotenogenesis had been cloned, and were expressed not only in M. aurum mutants, but also in M. smegmatis.

Mycobacterial flora of the skin in leprosy.

The presence of mycobacteria on the skin of healthy people and in leprosy lesions has been documented previously. The present study observed the mycobacterial flora on the hands (by the hand-washing method) and fingers (by the inoculated culture medium using scraped material obtained during the preparation of slit-skin smears) in 89 untreated leprosy patients. We also evaluated the slit-skin smears from fingers for the diagnosis of leprosy. In 16 patients (17.9%) mycobacteria were cultured from scrapings and hand washings. The frequency of isolates from lepromatous (LL) leprosy cases (52.9%) was significantly higher than from tuberculoid (TT) leprosy cases (5.2%). It was observed that Mycobacterium avium and M. scrofulaceum were the only opportunistic mycobacteria isolated from multibacillary patients, and two hypotheses are discussed to explain these findings. The slit-skin smears from fingers were as satisfactory as smears from other sites for the diagnosis of leprosy, but they were less satisfactory for estimating the morphological index.

Fourth report of the cooperative, open-ended study of slowly growing mycobacteria by the International Working Group on Mycobacterial Taxonomy.

The open-ended study of the International Working Group on Mycobacterial Taxonomy is an ongoing project to characterize slowly growing strains of mycobacteria that do not belong to well-established or thoroughly characterized species. In this fourth report we describe two numerical taxonomic clusters that represent subspecies or biovars of Mycobacterium simiae, one cluster that encompasses the erstwhile type strain of the presently invalid species "Mycobacterium paraffinicum," one cluster that is phenotypically very similar to Mycobacterium avium and Mycobacterium intracellulare but may be a separate genospecies, one cluster that appears to be phenotypically distinct from M. avium but reacts with a nucleic acid probe specific for M. avium, and three tentatively defined clusters in proximity to a cluster that encompasses the type strain of Mycobacterium malmoense. Of special practical interest is the fact that one of the latter three clusters is composed of clinically significant scotochromogenic bacteria that can be misidentified as the nonpathogenic organism Mycobacterium gordonae if insufficient biochemical tests are performed.

A new insight into the mycobacterial cell envelope architecture by the localization of antigens in ultrathin sections.

In an attempt to have a better insight into the mycobacterial cell envelope architecture, various subcellular fractions of Mycobacterium avium were prepared and characterized chemically and ultrastructurally. The various fractions corresponding to the mycobacterial "capsular material", outer layer, cell wall skeleton, cytoplasmic membrane, and cytosol as well as intact bacteria were then used to raise antisera in rabbits. The antisera so raised were then used to immunolabel the intact bacteria prior to embedding in epon. In parallel studies, bacteria were processed by a novel gelatin-uranyl acetate-low temperature Lowicryl HM20 embedding which preserved mycobacterial antigens, permitting to immunolabel antigens on ultrathin sections. Immunolabelling of epon-embedded intact bacteria showed that in the tripartite structure of the bacterial cell envelope, the middle electron-transparent layer acted as a barrier, not permitting the antibodies to penetrate into deeper structures. Immunolabelling of ultrathin sections showed that mycobacteria were surrounded by a "capsule" containing specific surface antigens with a glycocalyx-like topography, and that the intermediate electron transparent layer which separated the surface amphiphils from the inner arabinogalactan-peptidoglycan layer, was a virtual no man's land as it only seldom contained a single gold particle irrespective of the various antisera used. Furthermore, location of various layers in the cell envelope of M. avium using antisera raised against the subcellular fractions prepared was in agreement with chemical and ultrastructural data. A cell envelope model compatible with chemical, ultrastructural and immunolabelling data is proposed and its validity discussed.

Use of the SL-IV and the PGL-Tb1 glycolipid antigens in ELISA for the diagnosis of tuberculosis in AIDS patients.

At a predetermined specificity of 100.0%, the sensitivity of ELISA using the PGL-Tb1 and SL-IV antigens and IgG assays was 35.0% for the diagnosis of tuberculosis in AIDS patients (44.1% when tuberculosis was diagnosed before AIDS, 21.7% when AIDS was diagnosed before tuberculosis). Serial assays in sera collected from 11 AIDS patients before tuberculosis was diagnosed indicated that significant antibody titres were detected 10 months before the onset of clinical tuberculosis. Consequently, it was proposed that serodiagnosis using the glycolipid specific antigens should help in deciding on preventive antituberculosis treatment in these patients.