REPORT : MODULATION OF MYCOBACTERIUM TUBERCULOSIS INFECTION BY PLASMODIUM IN THE MURINE MODEL

A large proportion of people with latent tuberculosis live in malaria-endemic areas, so co-infection with these two organisms is likely to be common. To determine whether there might be a biologic interaction between these two pathogens in vivo, we infected mice with Mycobacterium tuberculosis and then with a non-lethal strain of Plasmodium yoelii eight weeks later. Mice chronically infected with M. tuberculosis simulate the equilibrium between pathogen and host thought to exist in human latent infection. Co-infected mice were less able to contain growth of M. tuberculosis in lung, spleen, and liver (mean ± SEM log10 colony-forming units 5.50 ± 0.11 versus 5.12 ± 0.08, 4.58 ± 0.07 versus 4.13 ± 0.10, and 2.86 ± 0.10 versus 2.49 ± 0.10, respectively) and had increased mortality. In populations where both diseases are endemic, there may be implications for increased incidence of clinically detectable tuberculosis. More than one-third of the global population harbors the latent form of tuberculosis infection. During latency, Mycobacterium tuberculosis is postulated to exist in a dormant state where the host can effectively contain the pathogen. Some aspects of this equilibrium have been modeled using a strain of mouse C57BL/6 (B6), which is relatively resistant to infection with M. tuberculosis. One day after low-dose aerosol infection, 50−100 bacilli can be cultured from the lung of infected mice. The bacilli grow logarithmically in the lung reaching a plateau at 10–10 colony-forming units (CFU) within four weeks. Cell-mediated immune responses promoted by the release of Th1 cytokines interferon(IFN) and interleukin-12 (IL-12) effect containment of the infection, allowing B6 mice to survive for almost a year. Although there is a plateau in the bacterial growth, the murine host fails to eliminate the pathogen. B6 mice are highly susceptible to infection with a murine malaria parasite Plasmodium yoelii. Intraperitoneal infection using parasitized erythrocytes results in a steady increase in parasitemia, reaching up to 60% of the red blood cells. With a lethal P. yoelii strain, mice die within eight days of infection. With the non-lethal P. yoelii 17X strain, however, blood stage infection completely resolves and death is rarely seen. Infection with this and other strains of rodent malaria parasites (P. chabaudi chabaudi, P. vinckei vinckei, and P. berghei) is accompanied by initial Th1 type immune responses. A shift from the Th1 to Th2 type is thought to play a critical role in the resolution of infection. In this study, we sought to characterize the effect of infection with the nonlethal P. yoelii 17X during chronic infection with M. tuberculosis in the murine model (Figure 1A). Chronic infection with M. tuberculosis was established in 6−7-week-old female B6 mice (Charles River Laboratories, Wilmington, MA) using low-dose exposure to M. tuberculosis CDC1551 strain (five million CFU/mL to give 80−100 organisms in the lung at day 1 after a 30-minute whole body exposure in a Middlebrook chamber (Glas Col, Terre Haute, IN). Five mice were killed on day 1 to verify the number of bacteria in the lung as a result of aerosolization. Eight weeks later, the M. tuberculosis-infected mice were infected intraperitoneally with 10 non-lethal P. yoelii 17X−parasitized erythrocytes per mouse using previously described methods. Parasites were maintained by serial passage through mice. Parasitemia was determined by microscopic examination of 100 high-power fields in each Giemsa-stained thin blood smear every 2−4 days post-infection until resolution or death. In parallel, another group of mice was infected with either M. tuberculosis or non-lethal P. yoelii 17X alone. At 25 days post-infection with non-lethal P. yoelii 17X, mice from all groups were killed for bacterial counts and immune assays. Mice were maintained in microisolator cages in biosafety level 3 conditions and fed commercial mouse chow and water ad libitum. All animals were maintained in accordance with protocols approved by the Johns Hopkins University Institutional Animal Care and Use Committee. The CFU from the lung, spleen, and liver were determined after homogenization in phosphate-buffered saline (PBS)/ 0.05% Tween 80, plating serial dilutions on Middlebrook 7H10 agar supplemented with oleic acid, albumin, dextrose, catalase enrichment media (OADC; Becton Dickinson, Sparks, MD), 0.05% Tween 80 and 5% glycerol, and incubating at 37°C for 3−4 weeks to allow for the enumeration of colonies. Twenty-five days post-infection with non-lethal P. yoelii 17X, the bacterial burden of M. tuberculosis in coinfected mice was significantly higher in spleen, lung, and liver compared with mice infected with M. tuberculosis alone (P < 0.01) (Figure 1B). Our data suggest that the host containment mechanisms seen in chronic infection with M. tuberculosis were compromised in the co-infected experimental group at the time of sacrifice. Whole, formalin-fixed infected organs harvested from 2−3 mice per group showed qualitative differences in pigmentation and size. Groups infected with M. tuberculosis displayed grossly visible lesions in the lungs. We observed no qualitative difference in size of the tuberculous lesions between groups. The lungs of the co-infected group however, showed darker pigmentation resulting from the accumulation of hemozoin produced by the parasite during digestion of red blood cell hemoglobin (Figure 1C). Pigmentation was more pronounced in the lungs of the co-infected group compared with the malaria-only infected group. These findings were mirrored in the hemozoin pigmentation in the livers of the two groups infected with non-lethal P. yoelii 17X. The characteristic splenomegaly of the mice infected with non-lethal P. yoelii 17X also appeared to be accentuated in the spleens of the co-infected mice. Mice infected with both pathogens and Am. J. Trop. Med. Hyg. , 70(2), 2004, pp. 144–148 Copyright © 2004 by The American Society of Tropical Medicine and Hygiene