Reassortant analysis of guinea pig virulence of pichinde virus variants

The new world arenavirus Pichinde (PIC) is the basis of an accepted small animal model for human Lassa fever. PIC (Munchique strain) variant P2 is attenuated in guinea pigs, whereas variant P18 is extremely virulent. Previous sequence analysis of the S segments of these two viruses indicated a small number of possible virulence markers in the glycoprotein precursor (GPC) and nucleoprotein (NP) genes. In order to determine the role of these S segment genes in guinea pig virulence in this system, we have generated reassortant viruses. When tested in outbred guinea pigs, the reassortant containing the S segment from the virulent parent P18 (S18L2) caused significantly higher morbidity than the reciprocal reassortant. This increased morbidity was associated with higher viral titers in serum and spleen. However, the S18L2 reassortant was not as fully virulent in this system as the P18 parent, indicating a role for L segment genes in virulence.     

Sensitization and recall of anti-Brucella immunity in Guinea pig macrophages by attenuated and virulent Brucella

Peritoneal macrophages from guinea pigs vaccinated with strain Rev I of Brucella melitensis were only moderately activated thereby to limit, in an in vitro system, the intracellular growth of Rev I bacilli. Nevertheless, the appropriate memory cells had been primed, as demonstrated by the observation that reinfection of animals with virulent B. melitensis followed by intraperitoneal inoculation of mineral oil called forth macrophages in immunized guinea pigs which inhibited strongly the intracellular growth of brucellae. These macrophages slowed the growth of brucellae in the absence of immune serum. The intensity of the recall response was related to the challenge route and to the virulence of the challenge strain. After equal doses of attenuated or virulent brucellae, resistance was highest in macrophages recalled by the virulent strain. An important basis for the attenuation of the Rev I strain may lie in its initially low degree of macrophage activation during primary infection, although still retaining the capacity to prime stem cells. This property is associated with a protein found in fraction I, because 600 μg/ml in Freund's adjuvant primed guinea pigs so that challenge by strain 6015 evoked activated macrophages. This was seen microscopically as a reduced spread of infection in and amongst the macrophage population. Immune serum further reduced this spread and limited the number of viable intracellular brucellae.     

Pathogenesis of a Pichinde Virus Strain Adapted to Produce Lethal Infections in Guinea Pigs

A model for studying the pathogenesis of virulent arenavirus infection was developed by adapting Pichinde virus to produce lethal infections of inbred guinea pigs. This adapted Pichinde virus retained low virulence for primates, thus potentially reducing the biohazard to investigators. Whereas all inbred (strain 13) guinea pigs were infected and killed by 3 plaque-forming units or more of adapted Pichinde virus injected subcutaneously, outbred (Hartley strain) guinea pigs were relatively resistant. All infected, inbred guinea pigs died at 13 to 19 days after inoculation, with viremias in excess of 5 log₁₀ plaque-forming units/ml, severe lymphopenia (<1,000/mm³), and elevated serum glutamic oxaloacetic acid transaminase levels. Immunofluorescent antibody examination of tissues and infectivity titrations of tissue homogenates obtained at 3- to 4-day intervals demonstrated significant viral replication in all visceral tissues examined, but not in brain. Livers of all moribund guinea pigs contained moderate to severe hepatocellular necrosis and diffuse fatty change. Splenic red pulp and adrenal cortical tissues were engorged with blood and contained necrotic foci. Pancreatic acinar tissues were atrophied and vacuolated; lung sections typically contained areas of moderate to severe interstitial pneumonia. Inflammatory cells were conspicuously absent from all lesions. The virological and pathological features of adapted Pichinde infection in guinea pigs are remarkably similar to those described for Lassa virus infections in rhesus monkeys and humans, suggesting that this model might provide insight into the pathogenesis and treatment of Lassa fever in humans.     

In vitro responses of guinea pig peritoneal macrophages to Legionella pneumophila.

Transmission and scanning electron microscopy were used to study the phagocytosis of virulent and avirulent strains of Legionella pneumophila. The interaction between L. pneumophila and peritoneal macrophages from normal guinea pigs or from animals that had survived infection was studied. The virulent strains survived and proliferated within the phagocyte after ingestion by either type of macrophage, whereas the avirulent strain of bacteria was killed by normal macrophages. Although the addition of immune serum enhanced phagocytosis, the outcome was the same as with normal serum.     

Differential nitric oxide (NO) production by macrophages from mice and guinea pigs infected with virulent and avirulent Legionella pneumophila serogroup 1

l-arginine-dependent reactive nitrogen intermediates have been identified as macrophage cytotoxic effector molecules against intracellular pathogens. To determine its role, ex vivo production of NO by peritoneal macrophages of C3H/HeN mice and Dunkin–Hartley guinea pigs infected intraperitoneally with a virulent and isogenic avirulent Legionella pneumophila serogroup 1 strain was compared with bacterial clearance from the lungs. While the virulent strain was cleared from mice lungs, the guinea pigs died within 96 h. In vivo infection with both strains resulted in the production of NO by mouse peritoneal macrophages ex vivo. In contrast, guinea pig macrophages did not produce detectable NO. In addition, infection by the avirulent strain led to the production of significantly more NO by mouse macrophages than the virulent parent strain, irrespective of stimulation with lipopolysaccharide (LPS) and/or interferon-gamma (IFN-γ). These results suggest that resistance to Leg. pneumophila infection may depend on the production of NO by host macrophages.     

Genome comparison of virulent and avirulent strains of the Pichinde arenavirus

A virulent (P18) strain of the Pichinde arenavirus produces a disease in guinea pigs that somewhat mimics human Lassa fever, whereas an avirulent (P2) strain of this virus is attenuated in infected animals. It has been speculated that the composition of viral genomes may confer the degree of virulence in an infected host; the complete sequence of the viral genomes, however, is not known. Here, we provide for the first time genomic sequences of the S and L segments for both the P2 and P18 strains. Sequence comparisons identify three mutations in the GP1 subunit of the viral glycoprotein, one in the nucleoprotein NP, and five in the viral RNA polymerase L protein. These mutations, alone or in combination, may contribute to the acquired virulence of Pichinde virus infection in animals. The three amino acid changes in the variable region of the GP1 glycoprotein subunit may affect viral entry by altering its receptor-binding activity. While NP has previously been shown to modulate host immune responses to viral infection, we found that the R374 K change in this protein does not affect the NP function of suppressing interferon-beta expression. Four out of the five amino acid changes in the L protein occur in a small region of the protein that may contribute to viral virulence by enhancing its function in viral genomic RNA synthesis.     

Metabolism of platelet-activating factor in neutrophils isolated from Pichinde virus-infected guinea pigs.

Metabolism of platelet-activating factor (PAF) was studied in cultured polymorphonuclear neutrophils (PMNs) obtained from Pichinde virus-infected strain 13 guinea pigs. Neutrophils obtained from control and infected guinea pigs on postinoculation days 10 and 14 were incubated with [3H]lyso-PAF, [3H]PAF, or [3H]acetate. After incubation for 1 h at 37 degrees C, formation of [3H]acyl-PAF from either [3H]lyso-PAF or [3H]PAF increased significantly in PMNs from infected guinea pigs compared to control PMNs. Furthermore, total radioactivity from [3H]lyso-PAF or [3H]PAF was higher in PMNs from infected animals than in those from controls. However, compared to PAF production by control PMNs, production of PAF by infected PMNs was unchanged. These results suggest that PMNs may not be the major source of increased blood PAF levels during Pichinde viral disease.     

Immune responses induced in cattle by virulent and attenuated Mycobacterium bovis strains: correlation of delayed-type hypersensitivity with ability of strains to grow in macrophages

Comparison of immune responses induced in cattle by virulent and attenuated strains of Mycobacterium bovis will assist in identifying responses associated with resistance or susceptibility to disease. Four strains of M. bovis, one which is virulent in guinea pigs (WAg201) and three which are attenuated in guinea pigs (an isoniazid-resistant strain [WAg405], ATCC 35721, and BCG) were compared for their abilities to induce immune responses in cattle and to grow in bovine lung alveolar macrophage cultures. Extensive macroscopic lesions were found only in cattle inoculated with the virulent M. bovis strain. Strong antibody responses to M. bovis culture filtrate, as well as persistently high levels of gamma interferon and interleukin-2 released from purified protein derivative (PPD)-stimulated peripheral blood lymphocyte cultures, were observed in the cattle inoculated with the virulent strain compared to those inoculated with the attenuated strains. All cattle inoculated with the virulent strain or two of the attenuated strains (WAg405 and ATCC 35721) elicited strong delayed-type hypersensitivity responses to PPD in skin tests, while animals inoculated with BCG induced only a weak response. The three strains which produced strong skin test responses proliferated well in bovine alveolar macrophages and induced high levels of proinflammatory cytokine mRNAs compared to BCG. Our study showed that skin test responsiveness to PPD correlated with the ability of the strains to grow in alveolar macrophages rather than to their pathogenicity in cattle.     

Modulation of splenic macrophages,and swine leukocyte antigen (SLA) and viral antigen expression following African swine fever virus (ASFV) inoculation

Summary Expression of viral and major histocompatibility complex (MHC) antigens and localization of T cells and macrophages was studied in frozen tissue sections of spleens taken from normal pigs or from pigs inoculated with highly virulent Lisbon 60 (L60), or with moderately virulent Dominican Republic 1978 (DR-II), African swine fever virus (ASFV) isolates. Splenic sections from L60 inoculated pigs exhibited a large decrease in macrophage staining, whereas DR-II infected animals appeared more intensely stained in the macrophage sheath arteries. Class I and class II MHC expression was decreased in spleens from pigs infected with either isolate at 3 day post inoculation (DPI). This was reversed in DR-II inoculated pigs at 4 DPI. Splenic tissue sections from L60 inoculated pigs exhibited only a marginal increase in SLA expression at a later time, 6 DPI. We suggest that the recovery of SLA expression during infection of pigs with ASFV is associated with survival or replacement of macrophages in the spleen leading to an effective immune response against the virus.     

Inhibition of mouse peritoneal macrophage DNA synthesis by infection with the arenavirus Pichinde.

Macrophage DNA synthesis and proliferation occur during the development of cell-mediated immunity and in the early nonspecific reaction to infection. Arenaviruses have a predilection for infection of cells of the reticuloendothelial system, and in this study we have examined the effect of the arenavirus Pichinde on macrophage DNA synthesis. We have found that infection of mouse peritoneal macrophages with Pichinde caused a profound dose-dependent inhibition of the DNA synthesis induced by macrophage growth factor-colony stimulating factor. At a multiplicity of inoculum of 5, there is a 75 to 95% inhibition of DNA synthesis. Viable virus is necessary for inhibition since Pichinde inactivated by heat or cobalt irradiation had no effect. Similarly, virus pretreated with an antiserum to Pichinde was without inhibitory effect. Inhibition was demonstrated by measuring DNA synthesis spectrofluorometrically as well as by [3H]thymidine incorporation. The inhibition of DNA synthesis was not associated with any cytopathology. There was no evidence that the inhibition was due to soluble factors, such as prostaglandins or interferon, released by infected cells. These studies demonstrate, for the first time in vitro, a significant alteration in macrophage function caused by infection with an arenavirus. It is possible that inhibition of macrophage proliferation represents a mechanism by which some microorganisms interfere with host resistance.     

Classical swine fever: morphological and morphometrical study of pulmonary intravascular macrophages.

To gain further insight into the pathogenesis of classical swine fever (CSF), the changes induced by hog cholera (HC) virus in pulmonary intravascular macrophages (PIMs) were examined. Twelve pigs were inoculated by the intramuscular route with a virulent strain of HC virus (Quillota strain) and killed in groups of three at 4, 7, 10 and 14 days post-inoculation. Immunohistochemical and ultrastructural examination revealed HC virus infection in endothelial cells, PIMs, and interstitial and alveolar macrophages. In addition to viral replication, a predominant feature was the secretory activation of PIMs, characterized by expanded rough endoplasmic reticulum and hyperplastic Golgi complexes. The results obtained suggest that macrophage activation and the subsequent release of pro-inflammatory mediators play an important role in the pathogenesis of CSF. Copyright Harcourt Publishers Ltd.     

Altered Cytokine Production and Impaired Antimycobacterial Immunity in Protein-Malnourished Guinea Pigs

Protein malnutrition leads to multiple detrimental alterations of host immune responses to mycobacterial infection. In this study, we demonstrated that splenocytes from low-protein (LP) guinea pigs vaccinated 6 weeks previously with attenuated Mycobacterium tuberculosis H37Ra failed to control the accumulation of virulent M. tuberculosis H37Rv in cocultured autologous peritoneal macrophages, despite the fact that they were able to control the accumulation of virulent tubercle bacilli in cocultured syngeneic peritoneal macrophages from normally nourished guinea pigs as successfully as did those from high-protein (HP) counterparts. Vaccine-induced growth control of virulent M. tuberculosis H37Rv in these cocultures appeared to be mediated by CD4 lymphocytes but not CD8 cells. Tuberculin (purified protein derivative [PPD])-induced lymphoproliferation was markedly impaired in vaccinated LP guinea pigs, and the depletion of CD4 lymphocytes significantly decreased lymphocyte proliferation whereas CD8 cell depletion did not. Protein malnutrition also impaired the abilities of cells from vaccinated LP guinea pigs to produce cytokines, including interferon, tumor necrosis factor alpha (TNF-α) and transforming growth factor beta (TGF-β), in response to PPD, despite the demonstration of higher serum levels of TNF-α and TGF-β after an intravenous injection of PPD into LP guinea pigs. In contrast, peritoneal macrophages from protein-malnourished guinea pigs produced a higher level of TGF-β 4 days after infection in vitro with M. tuberculosis H37Rv than did those from protein adequate controls. These results suggest that dietary protein malnutrition impairs vaccine-induced resistance to M. tuberculosis, in part, by altering the cytokine profile to favor macrophage deactivation.     

Glutamine synthetase GlnA1 is essential for growth of Mycobacterium tuberculosis in human THP-1 macrophages and guinea pigs

To assess the role of glutamine synthetase (GS), an enzyme of central importance in nitrogen metabolism, in the pathogenicity of Mycobacterium tuberculosis, we constructed a glnA1 mutant via allelic exchange. The mutant had no detectable GS protein or GS activity and was auxotrophic for L-glutamine. In addition, the mutant was attenuated for intracellular growth in human THP-1 macrophages and avirulent in the highly susceptible guinea pig model of pulmonary tuberculosis. Based on growth rates of the mutant in the presence of various concentrations of L-glutamine, the effective concentration of L-glutamine in the M. tuberculosis phagosome of THP-1 cells was approximately 10% of the level assayed in the cytoplasm of these cells (4.5 mM), indicating that the M. tuberculosis phagosome is impermeable to even very small molecules in the macrophage cytoplasm. When complemented by the M. tuberculosis glnA1 gene, the mutant exhibited a wild-type phenotype in broth culture and in human macrophages, and it was virulent in guinea pigs. When complemented by the Salmonella enterica serovar Typhimurium glnA gene, the mutant had only 1% of the GS activity of the M. tuberculosis wild-type strain because of poor expression of the S. enterica serovar Typhimurium GS in the heterologous M. tuberculosis host. Nevertheless, the strain complemented with S. enterica serovar Typhimurium GS grew as well as the wild-type strain in broth culture and in human macrophages. This strain was virulent in guinea pigs, although somewhat less so than the wild-type. These studies demonstrate that glnA1 is essential for M. tuberculosis virulence.     

Fate of Legionella pneumophila Philadelphia-1 strain in resident, elicited, activated, and immune peritoneal macrophages of guinea pigs.

Legionella pneumophila is known to grow intracellularly in resident peritoneal macrophages of guinea pigs. The present study was done to determine what kinds of macrophage stimulants are able to activate guinea pig macrophages to inhibit intracellular growth of the organism. Peritoneal macrophages were harvested from healthy guinea pigs, from guinea pigs injected intraperitoneally with proteose peptone (PP) or thioglycolate medium, from guinea pigs injected intraperitoneally with live Mycobacterium bovis BCG or killed Propionibacterium acnes (Corynebacterium parvum), and from guinea pigs surviving infection with live L. pneumophila. After in vitro phagocytosis, the L. pneumophila CFU in each well were counted on charcoal-yeast extract agar plates. In the macrophages elicited by PP or thioglycolate medium, the organism grew as well as it did in resident macrophages. In BCG-activated and immune macrophages, growth was inhibited almost completely. In P. acnes-activated macrophages, the initial growth of L. pneumophila was inhibited to some extent, but its growth reached the same level as in the resident and PP-induced macrophages after 3 or 4 days of culture. In the lethal challenge experiments in vivo, the superior protection provided by BCG over P. acnes was ascertained and the importance of macrophages in resistance to L. pneumophila was confirmed. Difference of activation by BCG and P. acnes in relation to the inhibition of intracellular growth of L. pneumophila in guinea pig macrophages is discussed.     

Interactions Between Macrophages of Guinea Pigs and Salmonellae II. Phagocytosis of Salmonella typhimurium by Macrophages of Normal Guinea Pigs

Peritoneal macrophages from normal guinea pigs were allowed to phagocytize Salmonella typhimurium in vitro. The extent of phagocytosis was determined by quantitative viable counts of the bacteria released after lysis of the phagocytes with sodium deoxycholate. It was shown that the avirulent strain RIA of salmonellae was more susceptible to ingestion by macrophages than the virulent strain SR-11. The presence of immune serum in the phagocytic mixture greatly enhanced the extent with which strain SR-11 was phagocytized. Also, the virulent bacteria recovered from infected mice exhibited a greater resistance to phagocytosis than those maintained in artificial media.     

Cytotoxicity of extracellular Legionella pneumophila.

Legionella pneumophila, the causative agent of Legionnaire's disease and Pontiac fever, is known to produce a cytopathic effect on macrophages. The capacity of extracellular L. pneumophila to mediate toxicity for guinea pig peritoneal macrophages and J774 mouse macrophages was assessed. Extracellular organisms were found to be capable of mediating toxicity; however, toxic activity appeared to require close proximity with the mononuclear cell surface. Serogroup 1 strains grown on supplemented Mueller-Hinton agar exhibited variable expression of toxic activity. One strain positive on supplemented Mueller-Hinton agar was cytotoxic and unable to replicate in J774 macrophages but remained virulent for guinea pigs at high doses.     

Interactions Between Salmonellae and Macrophages of Guinea Pigs IV. Relationship Between Migration Inhibition and Antibacterial Action of Macrophages

The in vitro macrophage migration inhibition test was used to detect the development of delayed-type hypersensitivity in guinea pigs infected with Salmonella typhimurium. Four different preparations from supernatants of S. typhimurium cultures were used as the antigens in this test. They included the concentrated bacterial antigens, the high-molecular-weight (>50,000) antigens, the ammonium sulfate-precipitated antigens, and the ribonuclease-treated antigens. All four antigen preparations were shown to inhibit the migration of peritoneal macrophages of salmonella-infected (immune) guinea pigs from capillary tubes, in comparison with cells of normal control animals. By use of the high-molecular-weight antigens and the ammonium sulfate-precipitated antigens, the production of the migration inhibition factor(s) was elicited from cultures of lymphocytes obtained from the peripheral blood of immune guinea pigs. The activity of the migration inhibition factor(s) was demonstrated by its ability to inhibit the migration of peritoneal macrophages of normal guinea pigs from capillary tubes. In contrast, normal peritoneal macrophages exposed to products of antigen-stimulated immune lymphocytes did not exhibit an enhanced phagocytic or bactericidal action against virulent S. typhimurium as compared with those of the normal control. The present study indicated that the bacterial antigens responsible for the elicitation of the production of the migration inhibition factor from lymphocytes of immune guinea pigs are inactivated by proteolytic enzymes, but not by ribonuclease, and have molecular weights of >50,000.     

Effects of diet and genetics on Mycobacterium bovis BCG vaccine efficacy in inbred guinea pigs.

Strain 2 and strain 13 guinea pigs were vaccinated with Mycobacterium bovis BCG and placed on low-protein or protein-adequate diets. Five weeks later all animals were infected by the respiratory route with virulent Mycobacterium tuberculosis H37Rv organisms. Four weeks postchallenge, guinea pigs were skin tested with purified protein derivative and sacrificed. Protein deficiency resulted in significant reductions in body weight and thymus weight and in an impairment in the ability to control the M. bovis BCG vaccine organisms and to mount delayed hypersensitivity reactions. Protein deficiency also adversely affected the efficacy of the BCG vaccine as demonstrated by the numbers of virulent organisms recovered in spleens and lungs. Strain differences were observed in the number of leukocytes, thymus weight, and the responsiveness of blood lymphocytes to purified protein derivative stimulation. In general, strain 13 guinea pigs responded more dramatically to dietary insult than did their strain 2 counterparts. Protein deprivation completely abolished BCG vaccine protection in the lungs and spleens of strain 13 animals and significantly reduced the protection afforded to strain 2 animals. In both strains, the BCG vaccine protected normally nourished guinea pigs. There was no significant difference between strains with respect to susceptibility to pulmonary infection with virulent mycobacteria. Thus, diet and genetic pedigree each had a significant influence on BCG vaccine efficacy.     

Mycobacterium bovis BCG vaccination augments interleukin-8 mRNA expression and protein production in guinea pig alveolar macrophages infected with Mycobacterium tuberculosis

Alveolar macrophages are likely the first cell type to encounter Mycobacterium tuberculosis in a pulmonary infection, resulting in the production of chemokines. In order to evaluate this response, alveolar macrophages harvested from nonvaccinated and Mycobacterium bovis BCG-vaccinated guinea pigs were infected in vitro with live M. tuberculosis H37Ra or H37Rv (multiplicity of infection, 1:1) or cultured with lipopolysaccharide (10 micro g/ml) for 3, 12, and 24 h. Interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) mRNA expression was determined by real-time PCR. Culture supernatants were assayed for guinea pig IL-8 protein by using a human IL-8 enzyme-linked immunosorbent assay kit. Alveolar macrophages harvested from BCG-vaccinated guinea pigs produced significantly more mRNA and protein for IL-8 than alveolar macrophages harvested from nonvaccinated guinea pigs at 12 and 24 h poststimulation or postinfection. Infection with attenuated M. tuberculosis (H37Ra) stimulated alveolar macrophages isolated from BCG-vaccinated guinea pigs to produce significantly more IL-8 mRNA than did alveolar macrophages infected with a virulent strain (H37Rv) at 12 and 24 h postinfection. Significant MCP-1 mRNA production was also detected in stimulated or infected alveolar macrophages; however, prior vaccination did not significantly affect levels of MCP-1 mRNA. Alveolar macrophages isolated from BCG-vaccinated guinea pigs produced significantly more IL-8 mRNA and protein when stimulated for 24 h with heat-killed H37Ra, heat-killed H37Rv, and H37Rv cell wall, but not mannose-capped lipoarabinomannan (ManLAM), than did cells stimulated with media alone. These observations indicate that prior vaccination may alter very early events in the M. tuberculosis-infected lung.     

Effect of Mycobacterium bovis BCG vaccination on interleukin-1 beta and RANTES mRNA expression in guinea pig cells exposed to attenuated and virulent mycobacteria

The effect of Mycobacterium bovis BCG vaccination on interleukin-1 beta (IL-1 beta) or regulated-upon-activation, normally T-cell-expressed and -secreted chemokine (RANTES) mRNA expression in guinea pig spleen cells stimulated with concanavalin A, lipopolysaccharide (LPS), phorbol myristate acetate (PMA) plus ionomycin, or purified protein derivative (PPD) was studied in vitro. Similarly, peritoneal exudate cell-derived macrophages from na?ve and BCG-vaccinated guinea pigs were infected with M. bovis BCG, Mycobacterium avium, the attenuated Mycobacterium tuberculosis H37Ra strain, or virulent strains H37Rv and Erdman of M. tuberculosis. Total RNA was subjected to Northern blot analysis using probes generated from guinea pig IL-1 beta or RANTES cDNA. Although IL-1 beta and RANTES mRNA could be detected in the spleen cells from na?ve animals stimulated with LPS or PMA plus ionomycin, the levels were significantly enhanced after BCG vaccination. mRNA expression was also elevated in macrophages infected with live mycobacteria after BCG vaccination. However, macrophages infected with the virulent H37Rv strain of M. tuberculosis showed 75 to 90% reductions in IL-1 beta expression and 25 to 60% reductions in RANTES mRNA expression compared with macrophages infected with the attenuated H37Ra strain. The IL-1 beta mRNA levels peaked as soon as 1 h after PPD stimulation and 4 h after M. tuberculosis H37Rv infection of macrophages. In contrast, RANTES mRNA expression was delayed until 48 h after infection. These results indicate that molecular mediators produced in response to various stimuli associated with protective immunity against mycobacteria are upregulated after BCG vaccination; however, a significantly weaker response was observed with virulent M. tuberculosis. These initial studies indicate that BCG vaccination has a positive effect on IL-1 beta and RANTES mRNA expression by host cells in a highly relevant animal tuberculosis model.