Analysis of parental strains

Previous studies by other groups have already reported the differences in resistance to M. tuberculosis among the inbred strains of mice (Sever & Youmans 1957, Gray 1958, Lynch et al 1965, Nikonenko et al 1985, Musa et al 1987). The data presented by those groups are somewhat inconsistent in respect to the strain distribution pattern as well as the criteria used to determine susceptibility. Attempts to identify genes in those studies have led to the view that a multigenic inheritance pattern is involved in tuberculosis resistance (Buschman et al 1988). No linkages to mouse chromosomes have yet been reported for susceptibility to M. tuberculosis, although Bcg (also known as Nrampl) on chromosome 1 controls early growth of BCG strains. We have compared survival times of several inbred strains of immunocompetent mice after infection with the laboratory strain of M. tuberculosis (Erdman strain). Mice were infected with 1 million colony-forming units (CFU) intravenously. C57BL/6J, C57BL/10J, C57BL/10SnDem as well as 129/SvJ survived the infection for more than 20 weeks, whereas median survival times (MSTs) of BALB/cJ, DBA/2J and several substrains of C3H did not exceed 10 weeks. Among the susceptible strains C3H and DBA/2 carry the resistant allele of Nrampl, which confers the ability to control early replication of avirulent M. bovis BCG (Skamene et al 1982, Vidal et al 1993), and thus comply with criteria discussed above. C3H and B6 strains were chosen for subsequent analysis as a susceptible and resistant parental strain, respectively.

To characterize the infection in C57BL/6J and C3HeB/FeJ parental strains, we determined the growth of M. tuberculosis in lungs, livers and spleens after intravenous infection at weekly intervals (Fig. 1). Initially, the bacteria grew progressively in the spleens and livers of both strains and the CFU did not differ significantly between the two at one week post-infection. Few bacteria were detected in the lungs of either strain at that time point. Two weeks after infection the growth pattern in the spleens and livers differed between C3H and B6, both quantitatively and qualitatively. Whereas the growth of M. tuberculosis in the spleens and livers of the susceptible (C3H) mice continued at about the same doubling time up to four weeks after infection, the numbers of the bacteria in the spleens and livers of the resistant mice decreased as compared to the Day 7 time point. In the lungs, the bacterial loads at two weeks after infection were similar in both strains. While they remained at the same low level in the lungs of resistant mice within the first four weeks of infection and slowly increased during next six months of experiment, the bacterial growth in the lungs of susceptible mice was more rapid than in the other organs. The bacterial load in the lungs exceeded that in

FIG. 1. Kinetics of the growth of Mycobacterium tuberculosis Erdman in the organs of C57BL/6J (B6) and C3HeB/FeJ (C3H) mice after infection with 106 colony-forming units (CFU) intravenously.

Days after infection

FIG. 1. Kinetics of the growth of Mycobacterium tuberculosis Erdman in the organs of C57BL/6J (B6) and C3HeB/FeJ (C3H) mice after infection with 106 colony-forming units (CFU) intravenously.

the spleens and livers by the fifth week after infection. Mice of the C3HeB/FeJ substrain died within five to seven weeks after infection. Histopathological analysis revealed macroscopic lesions in the lungs, kidneys and hearts of those mice. No visible pathology in the same organs of the B6 mice sacrificed at the same time was observed.

Under microscopical examination acid-fast bacilli (AFB) were detected in the spleens, livers, lungs, kidneys and hearts of both resistant and susceptible mice at various times after infection in the lungs two weeks after infection (Fig. 2). AFB were localized within separate foci associated with mononuclear infiltrates and the numbers of AFB per focus were somewhat greater in the susceptible mice. By the fifth week of infection, the lungs of C3H mice displayed prominent areas of necrosis containing the AFB. The bacteria were also spread throughout the interstitial lung tissue. The lungs of the B6 mice showed interstitial infiltration with macrophages and lymphocytes as well as isolated foci of AFB associated mostly with large epithelioid-like cells. At six months after infection, the alveolar space in the lungs of the resistant mice was greatly reduced due to the massive interstitial mononuclear infiltrates, although no necrotic lesions were observed. The infected cells contained few bacteria and were surrounded by numerous mononuclear cells.

Overall the data in our model are consistent with the following four inferences. (i) Early growth of virulent M. tuberculosis is equal in both strains, and hence there is no discernible difference in the efficiency of natural resistance against the virulent M. tuberculosis between the two strains. (ii) Differences in CFU emerge

FIG. 2. Acid-fast fluorescent staining of the organs of mice infected with Mycobacterium tuberculosis Erdman. (A) and (B) are lungs of B6 and C3H, respectively, two weeks after infection. Magnification = X 400.

at the time that immune responses develop. The resistant B6 mice control the growth of M. tuberculosis in lungs, liver and spleen, at times when M. tuberculosis growth in C3H is progressive in all organs, but is most rapid in lungs. (iii) The inability to control bacterial growth is reflected in disorganized granuloma formation in the lungs, liver, kidney and heart of C3H mice. (iv) The resistant strain does develop a chronic disease with the slow progressive development of the lung pathology, while the susceptible C3HeB/FeJ strain succumbs to fulminating disseminated tuberculosis. Therefore, our model deals with the mechanisms involved in the control of the disease progression and is not likely to address the natural resistance mechanisms preventing the disease at the onset of infection.

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