Immune response to persistent mycobacterial infection in mice.

Mycobacterium marinum has been recommended as a possible model of M. leprae for use in laboratory studies of antileprosy immunity. M. marinum introduced into the footpads of normal mice underwent a steady decline in viability, with less than 1% survival after a 30-day period. Small numbers of viable bacilli were recovered from the footpads of these mice up to 12 months later. Similarly, mice infected with M. simiae exhibited bacterial populations that persisted for up to 18 months with little change in viability. Injection of M. simiae into the footpads was followed by an extensive redistribution of the organisms in the tissues. Eventally, bacterial counts for footpads and draining lymph nodes stabilized, with small numbers of bacilli still present in the footpads 18 months later. Persistent growth, with little sign of any immune response, was also observed in mice infected with several strains of M. avium, as well as with one strain of M. intracellulare. Other strains of M. intracellulare, as well as M. vaccae and M. nonchromogenicum, failed to establish persistent infections in normal mice, regardless of whether they were introduced by an intravenous or subcutaneous (footpad) route. The relevance of these findings is discussed in relation to antileprosy immunity in experimental animals and in humans.

Mycobactericidal activity of glutaraldehyde solutions.

Aqueous solutions of alkaline glutaraldehyde (buffered at pH 8.5) inactivated a standard suspension of Mycobacterium tuberculosis H37Rv faster than the corresponding acid (pH 3.7 preparation. Quantitative differences in the rate of inactivation of eight other species of Mycobacterium were determined using a 1% solution of alkaline glutaraldehyde and inactivation of residual glutaraldehyde with 1% sodium bisulfite solution. Variations in the rate of kill were observed between the various mycobacterial species tested, but such differences were probably not sufficiently large to be of practical importance. A 2% alkaline glutaraldehyde solution inactivated 10(5) viable M. tuberculosis cells present on the surface of porcelain penicylinders within 5 min at 18 degrees C. This rate of inactivation was faster than in the acidic solution.

Distribution of mycobacteria grown in vivo in the organs of intravenously infected mice.

Suspensions of 35-day-old Mycobacterium tuberculosis H37Rv prepared from stirred liquid cultures and injected intravenously into CD-1 mice accumulated in the lungs at a significantly higher concentration that that seen with logarithmically growing cells. Mice were infected with logarithmic 8-day-old or stationary phase 35-day-old suspensions of H37Rv, and 24 hours later, the bacilli within pooled lung and splenic homogenates were recovered by differential centrifugation. The bacilli were then homogenized in Tween saline and injected intravenously into fresh mice. The partitioning of the 4 inocula into the lungs and spleens of the secondary recipients was compared to that for the original suspensions grown in vitro. There was a significant increase in the number of lung-adapted H37Rv that could again be recovered from the lungs of the secondary recipients compared to that observed for the corresponding splenic preparations. This effect was not due to bacterial clumping or to size differences in the organisms grown in vivo. Homogenation of H37Rv with normal lung increased the relative accumulation of viable bacilli in the lungs compared to the spleens of recipient mice.

Growth of Mycobacterium marinum in the footpads of T-cell-depleted mice.

Mycobacterium marinum strains 1218 and 1219 were inoculated into the hind footpads of T-cell-depleted specific pathogen-free C57B1/6 mice, and the growth and survival of the organisms at the site of injection, the draining popliteal lymph node, and the spleen and lung were quantitated for up to 70 days. T-cell depletion largely ablated the normal cell-mediated antituberculous response to the M. marinum population. The mice were able to control the further growth of the inoculum within the footpad only after it had reached 5 to 10 times that present in the normal controls. The high temperature-adapted strain (37 C; strain no. 1218) induced an increasing infection in the liver, spleen, and lungs of the THXB mice, and the infection eventually spread to the opposite footpad and to the tail skin. Strain 1219 gave rise to considerable systemic involvement in the THXB host despite its inability to survive at 37 C, but the size of the splenic and lung populations was considerably lower than in the 1218-infected animals. Both M. marinum infections persisted in the tissues of the T-cell-depleted mice with no indication of a cell-mediated immune response. Footpad swelling in the M. marinum-infected mice was not greatly reduced by T-cell depletion, and, if anything, tended to persist at high levels long after the swelling of the control feet had gone into a decline. On the other hand, incorporation of tritiated thymidine by cells within the infected footpads, the draining lymph node, and the spleen was considerably reduced in the T-cell-depleted host compared with control values. Late in the infection, there was a significant increase in the amount of label taken up by the cells in the footpads of the T-cell-depleted host.

Growth and immunogenicity of photochromogenic strains of mycobacteria in the footpads of normal mice.

Specific pathogen-free CD-1 mice were infected subcutaneously in the footpad with mycobacterium kansasii, three strains of M. marinum, and two strains of M. simiae-habana, and the growth of the organisms in the footpad, the draining popliteal lymph node, and the lung and spleen was followed quantitatively for up to 60 days. The ability of a footpad inoculum of M. marinum to spread to the lungs and spleen correlated with the ability of the organism to survive and multiple at 37 C in vitro cultures. The amount of footpad swelling which developed in the M. kansasii- and M. marinum-infected mice varied depending upon the strain of organism and the size of the original footpad inoculum. Injection of dead M. marinum into the footpad also induced an extensive amount of swelling which varied with the strain used, as well as being dose dependent. M. marinum- and BCG-vaccinated mice were protected against a later footpad challenge with M. marinum or the highly mouse virulent M. tuberculosis strain ERDMAN. The significance of this finding is discussed in relation to cross-protection studies using a variety of mycobacteria in the footpad infection model.

Relative immunogenicity of streptomycin-susceptible and -resistant strains of BCG. II. Effect of the route of inoculation on growth and immunogenicity.

Normal CD-1 mice were infected intravenously, subcutaneously, or aerogenically with live bacille Calmette Guerin (BCG) Tice or BCG streptomycin resistant (SM-res) and growth of the organisms in the footpad, the draining popliteal lymph node, the blood, lung, liver, and spleen was followed for as long as 50 days. The vaccinated mice were then challenged on day 50 with 10-5 viable Mycobacterium tuberculosis Erdman organisms introduced intravenously or subcutaneously. The growth of the Erdman challenge was followed in the appropriate organs for the next 20 days. Measurement of tuberculin hypersensitivity was carried out by footpad tests. The BCG Tice introduced aerogenically or subcutaniously into normal mice induced degrees of antituberculous resistance equivalent to those seen earlier in intravenously infected mice. The BCG SM-res was still nonimmunogenic when introduced subcutaneously or by the aerogenic route. Suspension of the organisms in sterile mineral oil before their injection into the footpad slowed their rate of inactivation and marginally increased the immune response seen later in the host. Introduction of BCG SM-res into T-cell depleted mice by the 3 inoculation routes was associated with no marked improvement in the survival of this organism in vivo, suggesting that BCG SM-res is inactivated in vivo by a nonimmunologically mediated mechanism.

Effect of T-cell depletion on the growth of BCG in the mouse footpad.

The growth of Mycobacterium bovis (BCG Montreal) and M. tuberculosis Erdman was determined in normal and T-cell depleted (THXB) mice when injected subcutaneously into a hind footpad. The bacilli multiplied only to a limited extent within the footpad itself but the infection quickly spread to the draining popliteal lymph node to eventually reach the liver, spleen and lung. The amount of systemic growth seen in the THXB mice was 10-100 times greater than in the normal controls, all of which developed a tuberculin hypersensitivity and an immune response in 14-18 days. T-cell depletion completely inhibited the expression of tuberculin sensitivity by the infected host as well as ablating the antituberculous response against both the vaccinating BCG population and a superinfecting Erdman challenge inoculum. Incorporation studies in the THXB mice indicated a striking reduction in cell division within the draining lymph node but there was an unexpected elevation in the level of incorporation by the lung cells as the BCG infection progressed. The significance of these findings is discussed in relation to the possible use of the BCG footpad model for studies of leprosy immunity.

Relative immunogenicity of streptomycin-sensitive and -resistant strains of BCG.

Normal, specific pathogen-free mice were vaccinated intravenously with increasing amounts of a streptomycin-resistant variety of BCG Tice (BCG SM(R)). The behavior of BCG SM(R) in the lungs, liver, and spleen was followed quantitatively for up to 50 days. One or two intravenous doses of 10(6) viable organisms were steadily eliminated from the tissues without producing detectable tuberculin sensitivity or raising resistance to a subsequent challenge with Mycobacterium tuberculosis strain Erdman. But mice receiving six weekly injections of 10(6) viable BCG SM(R) or a single injection of 10(6) BCG SM(R) by the intravenous route did develop effective levels of antituberculous resistance. Heat inactivation of the BCG SM(R) inoculum removed the organism's protective activity which could, however, be restored by incorporation of the organisms into Freund adjuvant. The ability of living BCG SM(R) to induce an effective antituberculous resistance when introduced into the tissues in an appropriate manner is discussed in terms of the mechanism of antituberculous immunity.

Determination of drug resistance of Mycobacterium tuberculosis cultures.

A total of 3,303 strains of Mycobacterium tuberculosis were tested for sensitivity to streptomycin (SM), isoniazid (INH), and p-aminosalicylic acid (PAS) by the Steenken modified minimal inhibitory concentration (MIC) test. A simultaneous double blind comparison was carried out on 277 selected strains by the Steenken MIC test and the Canetti proportion method. Agreement between the results for the two tests was 82% for SM, 95% for INH, and 89% for PAS. A small number of strains appeared to be sensitive when tested by one method but resistant by the other. MIC determinations were carried out on 83 strains by using Steenken-Smith, Lowenstein-Jensen, and Middlebrook 7H10 media containing a more extended range of concentrations of the test drugs. The MIC values for both SM and dihydrostreptomycin increased on Steenken-Smith medium compared with the other two. INH did not show any medium effect, whereas PAS showed increased MIC values in 7H10 agar. The significance of the comparisons of the MIC values on the various media is discussed in terms of possible changes in the drug sensitivity testing methods used at present in this laboratory.