Thermostable antigens of malignant tumors and normal tissues of experimental animals]

Gel precipitation and immunoelectrophoresis tests tests demonstrated the presence of thermostable antigens in dimethylbenzanthracene-induced malignant tumours of the muscle tissues of Wistar rats. These antigens were relatively tissue-specific for the tumours, amniotic fluid and the uterus on the one hand, and for the lung, spleen, and the serum on the other hand. The thermostable antigens of the tumours and the amniotic fluid had the electrophoretic mobility in the zone of alpha and alpha1-globulins.     

Molecular Detection of Mycobacterium bovis and Mycobacterium bovis BCG (Pasteur) in Soil

PCR primers specific for the Mycobacterium tuberculosis complex were used to detect the presence of Mycobacterium bovis BCG (Pasteur) in soil microcosms and Mycobacterium bovis in environmental samples taken from a farm in Ireland with a history of bovine tuberculosis. M. bovis genes were detected in soil at 4 and 21 months after possible contamination. Gene levels were found in the range of 1 × 10³ to 3.6 × 10³ gene copies g of soil⁻¹, depending on the sampling area. Areas around badger setts had the highest levels of detectable genes and were shown to have the highest levels of gene persistence. M. bovis-specific 16S rRNA sequences were detected, providing evidence of the presence of viable cells in Irish soils. Studies of DNA turnover in soil microcosms proved that dead cells of M. bovis BCG did not persist beyond 10 days. Further microcosm experiments revealed that M. bovis BCG survival was optimal at 37°C with moist soil (−20 kPa; 30% [vol/wt]). This study provides clear evidence that M. bovis can persist in the farm environment outside of its hosts and that climatic factors influence survival rates.     

Molecular detection of Mycobacterium bovis and Mycobacterium bovis BCG (Pasteur) in soil

PCR primers specific for the Mycobacterium tuberculosis complex were used to detect the presence of Mycobacterium bovis BCG (Pasteur) in soil microcosms and Mycobacterium bovis in environmental samples taken from a farm in Ireland with a history of bovine tuberculosis. M. bovis genes were detected in soil at 4 and 21 months after possible contamination. Gene levels were found in the range of 1 x 10(3) to 3.6 x 10(3) gene copies g of soil(-1), depending on the sampling area. Areas around badger setts had the highest levels of detectable genes and were shown to have the highest levels of gene persistence. M. bovis-specific 16S rRNA sequences were detected, providing evidence of the presence of viable cells in Irish soils. Studies of DNA turnover in soil microcosms proved that dead cells of M. bovis BCG did not persist beyond 10 days. Further microcosm experiments revealed that M. bovis BCG survival was optimal at 37 degrees C with moist soil (-20 kPa; 30% [vol/wt]). This study provides clear evidence that M. bovis can persist in the farm environment outside of its hosts and that climatic factors influence survival rates.     

Antigens common to human malignant tumors and certain species of microorganisms

Agglutination, gel precipitation and immunoelectrophoresis demonstrated the presence of antigens common for human malignant tumors of different sites and BCG and Listeria monocytogenic microorganisms. The radioimmunoassay of sera in the agglutination test showed that these sera reacted with tumor cells rather than with cells from normal human tissues. The common antigen had the electrophorectic mobility in the beta-globulin zone.     

Shared antigens between human malignant melanoma cells and Mycobacterium bovis (BCG).

This study was undertaken to investigate the antigenic relationships between human malignant melanoma cells and Mycobacterium bovis (BCG). Rabbits were immunized with sonicates of BCG or with malignant melanoma cells from different patients and the resulting antisera were tested for their capacity to bind radiolabeled soluble extracts prepared from BCG and melanoma cells. The binding of antibodies to radiolabeled antigens was studied by precipitation of radiolabeled antigen-antibody complexes by anti-rabbit immunoglobulin. Antibodies in sera from rabbits immunized with either BCG (anti-BCG) or melanoma cells (anti-melanoma) bound both the labeled BCG and melanoma antigens. Control antisera, from rabbits immunized with human acute or chronic lymphatic leukemia cells or with normal human spleen cells, did not bind significant amounts of radiolabeled BCG. Antibodies in sera from rabbits immunized with normal spleen cells bound small but significant amounts of radiolabeled melanoma antigens. Binding by anti-BCG and anti-melanoma to the radiolabeled antigens was studied before and after absorption of antisera with cells from human melanoma, leukemia, guinea pig hepatoma, and normal human spleen cells. Inhibition studies using unlabeled BCG extracts also were carried out. The absorption and inhibition studies confirmed that the binding reactions were specific and that antigens from five melanoma patients shared antigenic determinants with BCG.     

Susceptibility of raccoons (Procyon lotor) to infection with Mycobacterium bovis

Tuberculosis due to Mycobacterium bovis infection is endemic in white-tailed deer (Odocoileus virginianus) in the northeastern portion of the lower Michigan peninsula (USA). Various wild carnivores and omnivores, including raccoons (Procyon lotor), are infected with M. bovis within the endemic area. To investigate the pathogenesis of tuberculosis in raccoons and the likelihood of M. bovis transmission from infected raccoons to other susceptible hosts, we experimentally inoculated raccoons with single oral doses of M. bovis (ranging from 30 to 1.7 x 10(5) colony forming units [CFU]), five daily oral doses of M. bovis (ranging from 10 to 1 x 10(5) CFU), or a single intravenous (i.v.) dose of 1 x 10(5) CFU of M. bovis, from November 1998 through December 2000. Granulomatous lesions consistent with tuberculosis, or tissue colonization with M. bovis, were seen in one of five raccoons in the single low oral dose group, one of five raccoons in the multiple low oral dose group, two of five raccoons in the multiple medium oral dose group, five of five raccoons in the multiple high oral dose group, and five of five raccoons in the i.v. inoculated group. In oral inoculated raccoons, lesions were most common in the tracheobronchial and mesenteric lymph nodes and lung. Excretion of M. bovis in saliva or nasal secretions was noted in all i.v. inoculated raccoons and two of five multiple low oral dose raccoons. Mycobacterium bovis was not isolated from urine or feces from any experimentally inoculated raccoons. The need for multiple large oral doses to establish infection, and the low number of orally inoculated raccoons that excreted M. bovis in nasal secretions or saliva, suggest that wide-spread tuberculosis among raccoons is unlikely.     

Aerosol exposure of white-tailed deer (Odocoileus virginianus) to Mycobacterium bovis

Tuberculosis due to Mycobacterium bovis affects both captive and free-ranging Cervidae in the United States. Various animal models have been developed to study tuberculosis of both humans and animals. Generally, tuberculosis is transmitted by aerosol and oral routes. Models of aerosol exposure of large animals to M. bovis are uncommon. In order to develop a reliable method of aerosol exposure of white-tailed deer (Odocoileus virginianus) to M. bovis, 12 healthy white-tailed deer, aged 8-10 mo, were infected by aerosol exposure to 2 x 10(5) to 1 x 10(6) colony forming units (CFU) (high close, n = 4) of M. bovis or 6 x 10(2) to 1.6 x 10(3) CFU (low dose, n = 8) of M. bovis. Tuberculous lesions were more widely disseminated in (leer receiving the high dose, while lesions in deer receiving the low dose were more focused on the lungs and associated lymph nodes (tracheobronchial and mediastinal). Aerosol delivery of M. bovis to white-tailed deer results in a reliable manner of experimental infection that may be useful for studies of disease pathogenesis, immune response, mycobacterial shedding, and vaccine efficacy.     

Phenotypic expression of genetically-controlled natural resistance to Mycobacterium bovis (BCG)

Mycobacterium bovis (BCG), when maintained in vitro, readily incorporates [3H]uracil, the RNA precursor. The rate of [3H]uracil incorporation into bacilli is sharply reduced when the BCG is phagocytized by murine adherent resident peritoneal macrophages and subsequently released by the lysis of monolayers. Macrophages derived from mouse strains that are innately resistant to BCG infection in vivo (Bcgr) are able to inhibit the [3H]uracil incorporation into the bacilli in a significantly more effective way than macrophages from BCG-susceptible (Bcgs) strains. This difference is best demonstrated with a low rate of infection (BCG: macrophage ratio between 1:1 and 2:1), and is most pronounced at 4 to 5 days after in vitro infection of macrophage monolayers. In vivo interaction of BCG with peritoneal macrophages in situ results in the same pattern of enhanced inhibition of [3H]uracil incorporation by Bcgr macrophages. The use of Bcg-congenic mouse strains has confirmed that the Chromosome 1 Bcg (Ity, Lsh) locus is regulating the antimycobacterial activity of macrophages. We conclude that the resident macrophage is the cell population that expresses the phenotype of genetically determined resistance to BCG infection.     

Transmissible modification induced in Mycobacterium tuberculosis and Mycobacterium bovis in vitro]

A biologically active material (fraction "S") is isolated from cultures of scotochromogenic mycobacteria. Mycobacterium tuberculosis, or Mycobacterium bovis by disrupting the cells, sedimentation through 2.2 M sucrose, and ultrafiltration. The fraction "S" induces the modification of tubercle bacilli into non acid-fast bacteria forming smooth colonies on nutritive glycerol agar within 24-36 h of incubation. Three new phenotypes are thus obtained; two proved to be stable upon subculturing. Frequently the phenomenon occurs with a very large part of the Koch's bacillus population exposed to the inducing agent effect. It can be reproduced with crude preparations of DNA obtained from the fraction "S." It is inhibited by concanavalin A. The observed modification does not correspond to a transfer of characteristics of the inducing agent from the donor mycobacteria; furthermore it can be manifested even in the strain used for the preparation of the fraction "S."     

Electron microscopy of Mycobacterium leprae passed in laboratory animals

The ultrastructure of M. leprae was studied in the process of its intraplantar passage from man to mice (passages 1-8). Changes in the morphology of M. leprae, observed in the course of 3 passages, were established. From the phase of dormant forms (passage 1) the bacteria became adapted to the new host by passage 3, which was confirmed by the absence of differences in the ultrastructure of cells by passages 3-8. The possibilities of the phenotypic variability of M. leprae in the process of in vivo passage should be taken into consideration when they are used for the production of specific biopreparations, experimental modeling and the screening of antileprous preparations.     

Monoglycosyldiacylphenol-phthiocerol of Mycobacterium tuberculosis and Mycobacterium bovis

The structure of a minor glycolipid of M. tuberculosis (strain Canetti) is shown to be 2-O-methyl-alpha-L-rhamnosyldiacylphenol-phthiocerol. A similar compound with non-methylated rhamnose as sugar moiety was also detected. In the course of this work, the structure of mycoside B from Mycobacterium bovis was reexamined, and was shown to be identical to that of the 2-O-methylrhamnosyldiacylphenol-phthiocerol of the Canetti strain, while it was described as a 2-O-methyl-beta-D-rhamnosyl derivative in the literature. This result is in agreement with the known close relationship between M. tuberculosis and M. bovis. Careful examination of chromatographic fractions containing the above mentioned lipids showed that the occurrence of mycoloyl residues in some phenol-phthiocerol glycolipids, postulated in the literature, was likely to be due to the presence of glycerol monomycolate contaminants.     

Immune responses of white-tailed deer (Odocoileus virginianus) to Mycobacterium bovis BCG vaccination

The objective was to evaluate cellular immune response of captive white-tailed deer (Odocoileus virginianus) to live Mycobacterium bovis bacille Calmette Guerin (BCG) vaccination and to determine diagnostic implications of these responses. In vitro proliferative and interferon-gamma (IFN-gamma) responses to M. bovis purified protein derivative (PPD) were detected beginning 9 days postvaccination. Responses to Mycobacterium avium PPD, however, generally exceeded responses to M. bovis PPD. Interferon-gamma responses to M. avium PPD were not detected prior to vaccination nor in nonvaccinated deer, suggesting that vaccination with BCG boosted prior quiescent M. avium-sensitized cells. Both CD4+ and gammadelta T cells from vaccinated deer proliferated in response to M. bovis PPD stimulation. Intradermal administration of M. bovis PPD resulted in increases in skin thickness of vaccinated deer beginning 24 hr postinjection. Such early reactions were characterized by edema and minimal mononuclear cell infiltration, whereas later reactions (i.e., 72 hr postinjection) were more typical of delayed type hypersensitivity. Upon in vitro activation with pokeweed mitogen, CD44 expression increased and CD62L expression decreased on lymphocytes from deer regardless of vaccination status. Likewise, M. bovis PPD stimulation of lymphocytes from vaccinated deer resulted in increases in CD44 expression and decreases in CD62L expression. These findings demonstrate the potential of BCG vaccination to elicit strong cell-mediated immune responses and appropriate alterations in CD44 and CD62L expression with in vitro stimulation of white-tailed deer lymphocytes. In relation to M. bovis diagnosis, vaccination of white-tailed deer with BCG can induce skin test responses that classify the animal as a tuberculosis reactor. In contrast, BCG vaccination will likely not interfere with tuberculosis testing by the IFN-gamma assay.