Evidence of zoonotic leprosy in Pará, Brazilian Amazon, and risks associated with human contact or consumption of armadillos.

Mycobacterium leprae (M. leprae) is a human pathogen and the causative agent for leprosy, a chronic disease characterized by lesions of the skin and peripheral nerve damage. Zoonotic transmission of M. leprae to humans by nine-banded armadillos (Dasypus novemcinctus) has been shown to occur in the southern United States, mainly in Texas, Louisiana, and Florida. Nine-banded armadillos are also common in South America, and residents living in some areas in Brazil hunt and kill armadillos as a dietary source of protein. This study examines the extent of M. leprae infection in wild armadillos and whether these New World mammals may be a natural reservoir for leprosy transmission in Brazil, similar to the situation in the southern states of the U.S. The presence of the M. leprae-specific repetitive sequence RLEP was detected by PCR amplification in purified DNA extracted from armadillo spleen and liver tissue samples. A positive RLEP signal was confirmed in 62% of the armadillos (10/16), indicating high rates of infection with M. leprae. Immunohistochemistry of sections of infected armadillo spleens revealed mycobacterial DNA and cell wall constituents in situ detected by SYBR Gold and auramine/rhodamine staining techniques, respectively. The M. leprae-specific antigen, phenolic glycolipid I (PGL-I) was detected in spleen sections using a rabbit polyclonal antibody specific for PGL-I. Anti-PGL-I titers were assessed by ELISA in sera from 146 inhabitants of Belterra, a hyperendemic city located in western Pará state in Brazil. A positive anti-PGL-I titer is a known biomarker for M. leprae infection in both humans and armadillos. Individuals who consumed armadillo meat most frequently (more than once per month) showed a significantly higher anti-PGL-I titer than those who did not eat or ate less frequently than once per month. Armadillos infected with M. leprae represent a potential environmental reservoir. Consequently, people who hunt, kill, or process or eat armadillo meat are at a higher risk for infection with M. leprae from these animals.

Presence of Mycobacterium leprae DNA and PGL-1 antigen in household contacts of leprosy patients from a hyperendemic area in Brazil.

Leprosy is a highly infectious disease endemic to underdeveloped countries. In Maranhão State, Northeastern Brazil, the hyperendemic rate of 56.11 cases/100,000 inhabitants increased the necessity of better understanding the epidemiological profile of this population, particularly regarding efficient methods for evaluating individuals residing with diagnosed patients to understand disease transmission and the risk of infection. In this study, we examined the percentage of contacts with positive indices for Mycobacterium leprae DNA and phenol-glycolipid-1 antigen (PGL-1). PGL-1 was analyzed by an enzyme-linked immunosorbent assay, the ML-Flow test, and polymerase chain reaction of oral and nasal secretions of 808 leprosy contacts from Maranhão. PGL-1 was detected in 14.0% of patients and differed by operational classification of the index case (P < 0.05). Seropositive results of ML-Flow were 15.0% and identified individuals with and without Bacillus Calmette-Guérin vaccine scars. Molecular diagnosis detected M. leprae DNA in 5.6% of oral samples and 4.6% of nasal tissues, and 87% of subjects resided with high bacillary load patients. This study reinforces the efficacy of combining molecular and serological techniques to identify potential bacillus carriers in the asymptomatic stage of infection, such as in household contacts, highlighting the importance of these meth-ods for monitoring hyperendemic populations.

Phenolic glycolipid-1 (PGL-1) in Buruli ulcer lesions. First demonstration by immuno-histochemistry.

Buruli ulcer, caused by Mycobacterium ulcerans, is emerging as the third most common mycobacterial disease after leprosy and tuberculosis in some tropical regions. Although a toxin of the polyketide family is central to the pathogenesis of the disease, there are still several parameters that need clarification. Among them and of crucial interest are the curative drug treatment and the test for early detection of the disease. In this study, we used mouse monoclonal antibodies, raised against synthetic sugars of the terminal trisaccharide of M. leprae PGL-1, to detect the immunoreactivity of this antigen in tissue infected with M. ulcerans. Thirty specimens of skin tissue from Buruli ulcer patients (3 plaques, 10 nodules, 1 ulcerated nodule, 7 deep ulcer beds and 9 ulcers in healing) were obtained from Ghana. Eighty-three percent of the submitted cases were compatible with the lesions of Buruli ulcer. AFB were positive in 33% of plaques, 40% of nodules, 44% of actives ulcers and 22% of the ulcer in healing stage. Immunohistochemically, phenolic glycolipid-1 (PGL-1) was detected in all AFB-positive cases. This observation implies that Mycobacterium ulcerans may express an M. leprae PGL-1-like substance and should tentatively emulate research to further characterize such a substance. The search for an early diagnostic tool for the Buruli disease may benefit from such investigations.

Recognition of lipid antigens by sera of mice infected with Mycobacterium lepraemurium.

Lipids extracted from mouse tissues infected with Mycobacterium lepraemurium (MLM) were analyzed by thin-layer chromatography. Although the extracted lipids were heterogeneous in polarity, the lipids of intermediate polarity were the ones that predominated. All of the lipids of intermediate polarity were glycosylated species. There were also lipids of low and high polarity, the latter being glycolipids. Compared to lipids extracted from normal tissue (mostly to lipids of high and low polarity), all of the additional lipids extracted from the infected tissue corresponded to lipids present in the purified bacteria. Enzyme-linked immunoassays (ELISAs) were then performed with the whole lipids extracted from purified bacilli, the lipids of high, intermediate and low polarity, and the sera from 20 normal and 20 MLM-infected mice. Lipids of intermediate polarity were specifically recognized by MLM-infected mice. Neither sera (diluted 1:500) from normal mice nor infected mice reacted with the lipids of high or low polarity, but a higher concentration (sera diluted 1:100) of some sera from mice in both groups reacted significantly with these lipids. In the ELISAs the whole-lipid extract and the lipids of intermediate polarity were similarly recognized by the sera of the infected mice. Thus, as observed in human leprosy, the mycobacterial disease in the mouse (murine leprosy) is also accompanied by the development of antibodies to the glycolipids of the infecting microorganism.

Structural elucidation and antigenicity of a novel phenolic glycolipid antigen from Mycobacterium haemophilum.

The structure of a novel antigenic glycolipid that distinguishes the opportunistic pathogen Mycobacterium haemophilum from all other mycobacteria was established by a series of degradation reactions leading to products that were analyzed by gas/liquid chromatography-mass spectrometry. The complete structure of the oligosaccharide unit was determined as 2,3-di-O-CH3-alpha-L-Rhap(1----2)3-O-CH3-alpha-L-Rhap(1----4 )-2,3-di-O-CH3-alpha-L-Rhap(1----. The lipid portion of the phenolic glycolipid was composed of two component phenolphthiocerols differing by two methylene groups, as determined by analysis of their per-O-trideuteriomethylated derivatives. The diol unit of the phenolphthiocerols has a threo relative configuration. The absolute stereochemistry of the asymmetric centers of the phenolphthiocerols is uncertain, but the centers are probably 3R, 4S, 9R, and 11R as found for phthiocerol A from Mycobacterium tuberculosis. The hydroxyl functions of the branched glycolic chain are esterified to a complex mixture of multi-methyl branched mycocerosic acids, C27, C30, C32, C34, and C37 with molecular weights (as methyl esters) of 424, 466, 494, 522, and 564, respectively. The stereochemistry of the methyl branches of the mycocerosates have R absolute configuration. The glycolipid is highly antigenic and appears to be specific for M. haemophilum. There are intriguing similarities between the product from M. haemophilum and the well-known phenolic glycolipid I of Mycobacterium leprae, a matter that is discussed.

Characteristic new members of the phthiocerol and phenolphthiocerol families from Mycobacterium ulcerans.

Diacyl phthiodiolone A and phenolphthiodiolone A lipids were isolated from two strains of Mycobacterium ulcerans. The diol units of the phthiodiolone A and phenolphthiodiolone A components were shown to have erythro stereochemistry by infrared spectroscopy and proton nuclear magnetic resonance of an acetal derivative. This stereochemistry is shared only by related diols from M. marinum, the diols from M. bovis, M. kansasii, M. leprae and M. tuberculosis having threo stereochemistry.

Relationships between structure and biological activity of the mycolic acid-containing glycolipids from Nocardia asteroides "sensu stricto" and related species.

To reveal the taxonomical situation of Nocardia asteroides "sensu stricto", we compared the mycolic acid and mycolic acid-containing glycolipid composition and their granulomagenic activities in mice. The major glycolipids were glucose mono- and dimycolate, trehalose mono- and dimycolate and several unknown glycolipids, commonly, although the relative amount differed from strain to strains. On the other hand, molecular species composition of mycolic acids differed distinctively among the three closely related species: N. asteroides "sensu strico", N. farcinica and N. nova. GC/MS analysis showed the most abundant species of mycolic acids were C50(52) in N. asteroides, C54(52) in N. farcinica and C58(56) in N. nova, respectively with a different alpha-alkyl branch. Glucose mycolate and trehalose dimycolate possessing C50 mycolic acid showed a strong activity for granuloma formation in mice.

Specificity and antigenicity of mycoside G and other glycolipids from Mycobacterium marinum.

The purpose of this work was to examine the immunologic properties of the phenolicglycolipid produced by M. marinum (mycoside G). Cell mass processed with organic solvents yielded a lipid extract containing mycoside G and four other glycolipid fractions (G2 to G5). No mycoside G was found in the Mycobacterium marinum type strain. Neither mycoside G, nor fractions G2, G3 and G4 were immunogenic in the rabbit whereas fraction G5 was very immunogenic. The immune serum raised in the rabbit showed that fraction G5 is species specific since it was consistently detected in all Mycobacterium marinum strains examined so far.

Serological specificity of Mycobacterium tuberculosis glycolipids.

Six glycolipid fractions can be extracted from lipid crude extracts of Canetti type strains of M. tuberculosis. Among these fractions are two phenolglycolipids. The major component is a triglycosyl phenolphthiocerol dimycocerosate (PGL-Tb 1) and the second is a monoglycosyl diacyl phenolphthiocerol identical to mycoside B of M. bovis. Similar glycolipid compounds can also be found in wild strains of M. tuberculosis recently isolated from tuberculous patients. One of these compounds has been identified as PGL-Tb 1.

Inflammation induced by a glycolipid fraction from Mycobacterium leprae.

1. The inflammatory properties of a glycolipid fraction isolated from human recovered Mycobacterium leprae were investigated. The inflammatory reaction induced in mouse lung by the inoculation of the glycolipid fraction adsorbed to charcoal particles was characterized by a large influx of macrophages at various stages of maturation and of epithelioid cells around the particles. 2. When injected as an aqueous emulsion into the footpad of mice, the same fraction evoked a dose-dependent massive influx of mononuclear (MN) cells. The inflammatory reaction reached a peak at 6 days. The minimal effective dose of glycolipid was 0.1 micrograms. 3. The kinetics of inflammatory cell migration was studied by total and differential counts of leucocytes that migrated to the peritoneal cavity of mice inoculated intraperitoneally with the glycolipid fraction. This fraction initially induced intense polymorphonuclear (PMN) migration, which was later reduced, with a simultaneous increase in MN cells. 4. Adherent peritoneal cells (APC) incubated with glycolipid released one or more soluble factor(s) which induce active PMN and MN cell chemotaxis in vivo as well as in vitro. Thus, the MN cells may be attracted to the site of glycolipid inoculation by factor(s) released through the interaction of macrophages with the glycolipid fraction. 5. The present results demonstrate that a glycolipid containing trehalose and mycolic acid isolated from M. leprae reproduces some aspects of the fundamental lesion of leprosy.

Inflammation induced by a glycolipid fraction from Mycobacterium leprae

1. The inflammatory properties of a glycolipid fraction isolated from human recovered Mycobacterium leprae were investigated. The inflammatory reaction induced in mouse lung by the inoculation of the glycolipid fraction adsorbed to charcoal particles was characterized by a large influx of macrophages at various stages of maturation and of epithelioid cells around the particles. 2. When injected as aqueous emulsion into the footpad of mice, the same fraction evoked a dose-dependent massive influx of mononuclear (MN) cells. The inflammatory reaction reached a peak at 6 days. The minimal effective dose of glycolipid was 0.1 microng. 3. The kinetics of inflammatory cell migration was studied by total and differential counts of leucocytes that migrated to the peritoneal cavity of mice inoculated intraperitoneally with the glycolipid fraction. This fraction initially induced intense polymorphonuclear (PMN) migration, which was later reduced, with a simultaneous increase in MN cells. 4. Adherent peritoneal cells (APC) incubated with glycolipid released one or more soluble factor9s) which induce active PMN and MN cell chemotaxis in vivo as well as in vitro. Thus, the MN cells may be atracted to the site of glycolipid incolulation by factor(s) released through the interaction of macrophages with the glycolipid fraction. 5. the present results demonstrate that a glycolipid containing trehalose and mycolic acid isolated from M. leprae reproduces some aspects of the fundamental lesion of leprosy

Detection of trehalose monomycolate in Mycobacterium leprae grown in armadillo tissues.

Trehalose-6-monomycolate (TMM) was isolated from the lipids of armadillo-derived Mycobacterium leprae. Only meagre amounts of this glycolipid were recovered, but its structure was unequivocally established. Only alpha-mycolates were detected in the TMM by 252Cf plasma desorption mass spectrometry. Electron impact mass spectrometry showed the alpha branch to be principally C20. Trehalose dimycolate (cord factor) was not detectable. Since we have also found TMM in M. lepraemurium and in every Mycobacterium species so far examined, we suggest that this glycolipid is truly ubiquitous amongst mycobacteria.

Isolation of the Mycobacterium leprae-specific glycolipid antigen, phenolic glycolipid-I, from formalin-fixed human lepromatous liver.

A Mycobacterium leprae-specific phenolic glycolipid antigen was purified from Formalin-fixed liver preserved from an advanced lepromatous leprosy patient. Its chemical and immunological properties were compared with those of phenolic glycolipid-I obtained from M. leprae-infected armadillo liver. Based on the findings that the glycolipids from the two sources have the same thin-layer chromatographic properties, infrared absorption spectrum, sugar composition, and seroreactivity, we conclude that large quantities of the phenolic glycolipid-I antigen are produced in human lepromatous leprosy lesions and that Formalin-fixed lepromatous livers and spleens from the prechemotherapeutic era are suitable sources of the glycolipid.

Further specific extracellular phenolic glycolipid antigens and a related diacylphthiocerol from Mycobacterium leprae.

Mycobacterium leprae in infected armadillo tissue produces extracellular phthiocerol-containing lipids in amounts well in excess of the bacterial mass. The principal component (1.38 mg in 1 g of liver, wet weight, containing 3.7 X 10(10) M. leprae bacilli) consists of a mixture of two phthiocerol homologs, 3-methoxyl-4-methyl-9, 11-dihydroxyoctacosane and 3-methoxyl-4-methyl-9, 11-dihydroxytriacontane, (formula: see text); in which the hydroxyl functions are acylated by a mixture of three 'mycocerosic acids': 2,4,6,8-tetramethylhexacosanoate, 2,4,6,8-tetramethyloctacosanoate, and 2,4,6,8-tetramethyltriacontanoate. The structures were established by saponification of the native lipid, direct probe electron impact- or chemical ionization-mass spectrometry of the phthiocerol or its permethylated derivative, and gas-liquid chromatography-electron impact-mass spectrometry of the methyl esters of the fatty acids. In addition to the previously reported M. leprae-specific triglycosylphenolicdiacyl phthiocerol (Hunter, S. W., Fujiwara, T., and Brennan, P. J. (1982) J. Biol. Chem. 257, 15072-15078), the extracellular products contain small amounts (about 60 micrograms/g of infected liver, wet weight) of two other phenolic glycolipids, one of which (Phenolic Glycolipid III) has been structurally elucidated, (formula: see text); assuming certain enantiomeric configurations for the sugar substituents; the R-acyl functions are identical with those in the diacylphthiocerol. Phenolic Glycolipid-III reacts in enzyme-linked immunosorbent assays with sera from patients with leprosy and with rabbit antisera raised against whole M. leprae. The phthiocerol-containing lipids may be synonymous with the electron transparent capsules of M. leprae, and their unreactive state may confer on them the role of passive protectors of the bacillus.