The in vivo antibody response against exogenous antigens is not influenced by the mouse Bcg (Nramp1) gene.

The mouse Nramp1 (Bcg) gene on chromosome 1 exerts pleiotropic effects on macrophage function. The gene is known to affect presentation of mycobacteria, and other antigens in vitro, so that macrophages carrying the resistant Bcg allele better support the proliferation of antigen-specific T cells compared with macrophages of the sensitive phenotype. To determine whether the Bcg allele could affect in vivo the antibody response to antigens not related to mycobacterial infections, we tested the primary and secondary responses to sheep red blood cells (SRBC) and glycosylated bovine insulin (G-insulin) in two pairs of Bcg congenic strains: BALB/c (Bcgs) versus BALB/c.CD2 (Bcgr), and B10.A (Bcgs) versus B10Ar (Bcgr), and in C57BL/10ScSn (B10; Bcgs) and A/J (Bcgr) mice. Furthermore, the antigen-specific proliferative responses of T cells primed in vivo by protein antigens were also tested in Bcg congenic mice. We found no significant difference in in vivo antibody response either to SRBC or G-insulin between the Bcgr and Bcgs strains. The magnitude of in vitro antigen-specific proliferation of lymph node cells sensitized in vivo by hen egg lysozyme (HEL) or chicken ovalbumin (OVA) was also similar in Bcgs and Bcgr congenic mice. However, we have documented a higher antigen-presenting capacity of Bcgr macrophages in in vitro antigen-specific proliferation to OVA. Since the macrophages are the only cells in which the Nramp1 gene is expressed, we suggest that the activity of other types of antigen-presenting cells masks the effect of the Bcgr allele on antigen-presentation in vivo.     

The relative impact of bacterial virulence and host genetic background on cytokine expression during Mycobacterium avium infection of mice.

Resistance to Mycobacterium avium depends on both genetically encoded macrophage functions and acquired T-cell immunity. Cytokines may play a role in either type of resistance. We studied the expression of interleukin-2 (IL-2), IL-4 and interferon-gamma (IFN-gamma) in naturally susceptible BALB/c (Bcgs) and naturally resistant C.D2 (Bcgr) congenic mice infected with two strains of M. avium (one highly virulent and another of low virulence). We observed that cytokine expression patterns correlated better with the virulence of the micro-organism than with the genetic background of the host. The control of the infection by the low virulence strain in either mouse strain was associated with an increased expression of IFN-gamma and IL-2. Only Bcgs mice infected with a virulent strain of M. avium were unable to restrict bacterial growth. An increased expression of IL-4, early during infection, was detected in the course of the latter infection but played no role in determining the susceptibility to infection. Neutralization of IFN-gamma or IL-2 with specific monoclonal antibodies led to an exacerbation of the infection in Bcgr mice by the two strains of M. avium and in Bcgs mice infected with the low virulence strain of M. avium.     

Cytokine-mediated activation of macrophages from Mycobacterium bovis BCG-resistant and -susceptible mice: differential effects of corticosterone on antimycobacterial activity and expression of the Bcg gene (Candidate Nramp).

Previous work in our laboratory has shown that corticosterone increases the susceptibility of macrophages from Bcgs mice to the growth of Mycobacterium avium. The innate antimycobacterial activity of macrophages from Bcgr mice was not affected by corticosterone. In contrast to the differential effect of corticosterone on the antimycobacterial activity of the macrophages, corticosterone suppressed the production of tumor necrosis factor alpha and nitric oxide by macrophages from both Bcgr and Bcgs mice. The purpose of this investigation was to compare the effects of corticosterone on the antimycobacterial activity of macrophages from Bcgr and Bcgs mice that have been activated in vitro with recombinant gamma interferon or granulocyte-macrophage colony-stimulating factor. We found that macrophages from both strains of congenic mice responded equally to the activation stimuli. The capacity of the activated macrophages from Bcgs mice to suppress the growth of M. avium was inhibited by the addition of corticosterone to the cultures. The addition of NG-monomethyl-L-arginine to the cultures did not affect the capacity of resident splenic macrophages from Bcgr mice to limit the growth of M. avium. However, NG-monomethyl-L-arginine reduced the capacity of gamma interferon-activated, but not granulocyte-macrophage colony-stimulating factor-activated, macrophages to limit the growth of M. avium by macrophages from both Bcgr and Bcgs mice. The addition of corticosterone suppressed Nramp expression by macrophages from Bcgs mice. Nramp expression by macrophages from Bcgr mice was not affected by corticosterone.     

Pleiotropic effects of the Bcg gene: III. Respiratory burst in Bcg-congenic macrophages.

Macrophage respiratory burst, as assessed by H2O2 and O2- production, and HMS and chemiluminescence activity was investigated in a variety of conditions in macrophages from Bcg-congenic mice. Measurement of HMS and chemiluminescence in splenic macrophages challenged in vitro with BCG showed that the Bcgr cells were more stimulated by the challenge than their Bcgs counterparts. H2O2 production was measured in Bcgr and Bcgs splenic macrophages. PMA-triggering and LK-triggering were shown to stimulate a similar degree of H2O2 production Bcgr and Bcgs macrophages. In contrast, in vitro phagocytosis of BCG was shown to trigger superior production of H2O2 and O2- in the Bcgr splenic macrophages as compared to their Bcgs congenics. Finally, following the in vivo infection with BCG Montreal, Bcgr splenic macrophages were superior producers of H2O2 (both spontaneous and PMA-triggered) in the early phase of infection.     

Implication of phagosome-lysosome fusion in restriction of Mycobacterium avium growth in bone marrow macrophages from genetically resistant mice.

The ability of the host to resist infection to a variety of intracellular pathogens, including mycobacteria, is strongly dependent upon the expression of the Bcg gene. Mouse strains which express the resistance phenotype (Bcgr) restrict bacterial growth, whereas susceptible strains (Bcgs) allow bacterial growth. Expression of the Bcg allele is known to influence the priming of host macrophages (M phi s) for bactericidal function. In the present work, bone marrow-derived M phi s from congenic BALB/c (Bcgs) and C.D2 (BALB/c.Bcgr) mice were infected with the virulent strain Mycobacterium avium TMC 724 to define the mechanism involved in growth restriction of M. avium. By combining CFU measurements and ultrastructural analyses, we show that growth of this bacterium is restricted in marrow M phi s from resistant mice. Using acid phosphatase as a lysosomal marker, we provide evidence that the hydrolytic activity of M phi s, as measured by the capacity of lysosomes to fuse with and transfer active hydrolytic enzymes to phagosomes in which M. avium resides, is an expression of the Bcg gene and that this phenomenon is a key antibacterial activity responsible for growth restriction of M. avium: (i) the percentage of phagosome-lysosome fusions was twice as high in Bcgr M phi s as in Bcgs M phi s, and (ii) the percentage of intact viable bacteria residing in acid phosphatase-negative phagosomes was twice as low in Bcgr M phi s as in the Bcgs counterparts. These differences are not due to a lower activity of the enzyme in Bcgr M phi s. The mechanism by which the Bcg gene exerts control over the phagolysosomal fusion is discussed.     

Control of the Bcg gene of early resistance in mice to infections with BCG substrains and atypical mycobacteria.

The effect of the Bcg gene on the early host response to intravenous infection with a variety of BCG substrains and some atypical mycobacteria was investigated. The numbers of live bacilli of BCG Pasteur and BCG Tice recovered from the spleens of Bcgs mice (C57BL/6, B10.A and BALB/c) at 3 weeks following infection exceeded the bacterial dose injected, whereas the number of CFU recovered from the spleens of Bcgr mice (A/J, DBA/2 and C3H/HeN) did not exceed the number of CFU injected, thus following the pattern observed in Bcgr mice and Bcgs infected with BCG Montreal. BCG Russia failed to multiply in both test groups; however, the number of CFU recovered in Bcgr mice was significantly lower than in Bcgs mice. On the other hand, the presence of live bacilli in the spleens of either Bcgr or Bcgs mice injected with BCG Japan was undetectable in most cases. Involvement of the Bcg gene in the early resistance to infection with BCG Pasteur, BCG Russia, Mycobacterium kansasii and M. intracellulare was documented by the significant differences in the kinetics of infections in mice of the C.D2 (BALB/c-Bcgr) and BALB/c (Bcgs) congenic lines. In BCG Russia, M. intracellulare and M. fortuitum infections, the phenotypic expression of the Bcg gene resulted in a more rapid elimination of the bacteria in the spleens of Bcgr when compared with Bcgs mice. On the other hand, the hepatic granuloma formation correlated with bacterial load except when C.D2 mice were infected with a small dose of BCG Pasteur or M. kansasii where extensive granulomatous hepatitis developed although no bacterial multiplication occurred in the spleen. It is suggested that granuloma formation could depend of the properties of the mycobacteria as well as the genetic background of the host without implicating the bacterial burden.     

Role of iron in Nramp1-mediated inhibition of mycobacterial growth

Innate resistance to mycobacterial growth is mediated by a gene, Nramp1. We have previously reported that Nramp1 mRNA from macrophages of Mycobacterium bovis BCG-resistant (Bcgr) mice is more stable than Nramp1 mRNA from macrophages of BCG-susceptible (Bcgs) mice. Based on these observations and on reports that show that the closely related Nramp2 gene is a metal ion transporter, we evaluated the effect of iron on the growth of Mycobacterium avium within macrophages as well as on the stability of Nramp1 mRNA. The addition of iron to macrophages from Bcgs mice resulted in a stimulation of mycobacterial growth. In contrast, iron increased the capacity of macrophages from Bcgr mice to control the growth of M. avium. When we treated recombinant gamma interferon (IFN-gamma)-activated macrophages with iron, we found that iron abrogated the growth inhibitory effect of IFN-gamma-activated macrophages from Bcgs mice but that it did not affect the capacity of macrophages from Bcgr mice to control microbial growth. A more detailed examination of the effect of iron on microbial growth showed that the addition of small quantities of iron to resident macrophages from Bcgr mice stimulated antimicrobial activity within a very narrow dose range. The effect of iron on the growth inhibitory activity of macrophages from Bcgr mice was abrogated by the addition of catalase or mannitol to the culture medium. These results are consistent with an Fe(II)-mediated stimulation of the Fenton/Haber-Weiss reaction and hydroxyl radical-mediated inhibition of mycobacterial growth.     

Regulation of mycobacterial growth by the hypothalamus-pituitary-adrenal axis: differential responses of Mycobacterium bovis BCG-resistant and -susceptible mice.

The role of the hypothalamus-pituitary-adrenal (HPA) axis in regulating the growth of Mycobacterium avium in Mycobacterium bovis BCG-resistant and -susceptible congenic mice was evaluated. Restraint was used to activate the HPA axis, which resulted in an increase in the level of corticosterone in the plasma. Activation of the HPA axis increased the susceptibility of BALB/c.Bcgs mice to the growth of M. avium. In contrast, the growth of M. avium was not altered in BALB/c.Bcgr mice as a result of HPA activation. Adrenalectomy abolished the effect of HPA activation on mycobacterial growth, as did treatment of the mice with a glucocorticoid receptor antagonist, RU 486. Activation of the HPA axis also resulted in the increased susceptibility of splenic macrophages from Bcgs mice but not from Bcgr mice to M. avium growth in vitro. The production of tumor necrosis factor alpha and of reactive nitrogen intermediates by splenic macrophages from both strains of mice was suppressed as a result of HPA activation. The implications of these findings for resistance controlled by Bcg and for susceptibility to mycobacterial growth are discussed.     

Host and bacterial factors control the Mycobacterium avium-induced chronic peritoneal granulocytosis in mice.

Persistent peritoneal granulocytosis and elevated macrophage counts have been found in nine mouse strains from 8 to 90 days after infection with Mycobacterium avium. Peritoneal granulocytosis was higher in M. avium-resistant BALB/c. Bcgr (C.D2) mice, compared with congenic M. avium-susceptible BALB/c (Bcgs) animals. Although maximal granulocytosis values were not related to virulence of the inocula, the kinetics of the granulocytic response varied with the virulence of M. avium. Following infections by avirulent (rough) strains of M. avium, the peritoneal granulocytosis progressively declined in BALB/c and C3H/He mice. A similar decline in granulocyte number was observed in resistant C3H/He mice infected with virulent M. avium (smooth transparent strain). In both instances the decline in the peritoneal granulocytosis was associated with a progressive elimination of the inoculum. In the susceptible BALB/c mice, virulent M. avium strains induced progressive infection accompanied with a rapid decline in granulocyte number, whereas the infection with attenuated M. avium, which caused a chronic infection, induced persistent granulocytosis. The ability to recruit granulocytes following the intraperitoneal inoculation of a phlogistic substance (casein hydrolysate) was decreased in infected susceptible but not in infected resistant mice at 90 days of infection with virulent M. avium.     

Growth of Mycobacterium tuberculosis in BCG-resistant and -susceptible mice: establishment of latency and reactivation.

Growth of mycobacterial species is controlled by a gene, Bcg (candidate Nramp). Bcg acts at the macrophage level and is thought to control some aspect of macrophage priming for activation. Infection of Mycobacterium bovis BCG-susceptible (Bcgs) mice with several different mycobacterial species results in the growth of the microorganisms, while the growth of the same organisms is controlled in BCG-resistant (Bcgr) mice. The capacity of Bcg to control the growth of M. tuberculosis has not been extensively explored. The purpose of this investigation, therefore, was to compare the growth of M. tuberculosis in Bcgr and Bcgs mice. We found that the growth of tubercule bacilli was different in the lungs and spleens of Bcgr and Bcgs mice when they were inoculated with fewer then 10(3) CFU of the mycobacterium. The differences in growth were more easily distinguished in the lungs then in the spleens. The growth of the microorganisms in both strains of mice peaked between 35 and 43 days, and a latent infection was established by 65 days after infection. Activation of the hypothalamic-pituitary-adrenal axis resulted in reactivation of the growth of M. tuberculosis in both Bcgr and Bcgs mice. Greater numbers of tubercule bacilli were isolated from lungs than from spleens following reactivation. The utility of this mouse model in the study of the establishment of latency and reactivation of M. tuberculosis is discussed.     

T-cell immune responses in Mycobacterium avium-infected mice.

Mycobacterium avium infection was substantially more severe in C57BL/6 (Bcgs) than in (C57BL/6 x DBA/2)F1 hybrid (Bcgr) mice both in terms of bacterial growth in the spleens and lungs and in host survival. Prior Mycobacterium bovis BCG vaccination resulted in increased resistance as well as enhanced tuberculin hypersensitivity to both PPD-S (Mycobacterium tuberculosis) and PPD-A (M. avium). Mice heavily infected with M. avium were used as T-cell donors in an adoptive transfer system. Substantial resistance was observed for both recipient hosts regardless of the genotype of the donor strain. Transfer of resistance was ablated by treatment of the immune spleen cells with anti-Thy 1.2 monoclonal antibody and complement or by cyclophosphamide treatment. Spleen cells which were monodepleted of L3T4+ or Lyt-2+ T cells did not lose their ability to transfer resistance against a subsequent challenge. However, when these cells were doubly deleted, all resistance was ablated in both the BCG-susceptible and -resistant mice. The recipient host expressed a detectable adoptive immune response although the donor had been unable to reduce the growth of the primary M. avium infection in vivo.     

Regulation of Mycobacterium bovis BCG and foreign body granulomas in mice by the Bcg gene.

Natural bactericidal resistance to Mycobacterium bovis BCG is under the control of a single gene, designated Bcg. Lung granuloma formation in susceptible (Bcgs) and resistant (Bcgr) mice was studied in two sets of Bcg-congenic systems, namely, the BALB/c (Bcgs)-C.D2 (BALB/c.Bcgr) pair and the B10.A (Bcgs)-B10.Ar (Bcgr) pair, by using BCG as well as foreign body granuloma-inducing agents (dextran beads). Large granulomas of the lung induced by the intratracheal instillation of either BCG or dextran beads developed in Bcgs mice. In contrast, minimal inflammation was produced in Bcgr mice given BCG or dextran beads. Aqueous extracts prepared from pulmonary granuloma lesions induced by Bcgs mice by either BCG or dextran beads contained high levels of interleukin-1 (IL-1) activity but not interleukin-2 (IL-2) or interleukin-4 (IL-4) activity. Very low IL-1 activity was detected in extracts from Bcgr mice injected with BCG and dextran beads. The activity of IL-1 was correlated closely with the activity and size of the granulomatous inflammation in mice. These results suggest that pleiotropic effects of the Bcg gene are involved in the development of granulomas induced by either BCG or nonspecific foreign body agents (dextran beads) and that monokines participate in granuloma formation.     

Contribution of CD30/CD153 but not of CD27/CD70, CD134/OX40L, or CD137/4-1BBL to the optimal induction of protective immunity to Mycobacterium avium

A panel of monoclonal antibodies specific for CD27 ligand (CD70), CD30 ligand (CD153), CD134 ligand (OX40L), and CD137 ligand (4-1BBL) were screened in vivo for their ability to affect the control of Mycobacterium avium infection in C57Bl/6 mice. Only the blocking of CD153 led to increased mycobacterial burdens. We then used CD30-deficient mice and found an increase in the proliferation of two strains of M. avium in these mice as compared with control animals. The increased mycobacterial growth was associated with decreased T cell expansion and reduced interferon-gamma (IFN-gamma) responses as a result of reduced polarization of the antigen-specific, IFN-gamma-producing T cells. At late times but not early in infection, the lymphoid cuff surrounding granulomas was depleted in the CD30-deficient animals. This report expands our knowledge about tumor necrosis factor superfamily members involved in the immune responses to mycobacterial infection by identifying CD30-CD153 interactions as required for optimal immune control of M. avium infection.     

Evolution of cell types and T-cell subsets in the spleens of Mycobacterium bovis BCG-resistant and M. bovis BCG-susceptible strains of mice after infection with M. bovis BCG.

In mice, the early host response to intravenous infection with small doses of dispersed Mycobacterium bovis BCG is controlled by the Bcg gene. After infection with a low dose of M. bovis BCG, Lyt-1+ cells were generated in the spleens of BCG-susceptible mice (Bcgs) in parallel with an increase in the proportion of phagocytic cells. Very few changes occurred in the splenic cell types of BCG-resistant mice (Bcgr).     

Killing of Mycobacterium smegmatis by macrophages from genetically susceptible and resistant mice

The bactericidal function of macrophages was investigated in congenic mice expressing the phenotype of susceptibility (B10.A, Bcgs) or resistance (B10.ABcgr) to mycobacterial infection. When splenic and peritoneal macrophages from these two mouse strains were infected in vitro with Mycobacterium smegmatis, the Bcgr macrophages were shown to inactivate M. smegmatis more efficiently than their Bcgs congenic counterparts. The mechanisms of this superior antimycobacterial activity was studied further. Addition of catalase did not abolish killing to a significant degree in either allelic type of macrophage, suggesting that hydrogen peroxide production was not involved in the killing activity controlled by the Bcg gene. Activation of Bcgs macrophages by exposure to crude lymphokines rendered them equally as efficient as their Bcgr counterparts in their capacity to destroy M. smegmatis. This finding suggests that both the genetically resistant and susceptible macrophages have the potential to kill M. smegmatis in vitro. This potential is expressed constitutively by the Bcgr but not Bcgs macrophages and can be induced, by lymphokine treatment, in the Bcgs macrophages. In a final set of experiments, the macrophage killing of M. smegmatis was evaluated as a test system to type for the Bcg gene allelic type in vitro, using a set of AXB and BXA recombinant inbred strains of mice. Results obtained show that typing of AXB/BXA recombinant inbred strains for the trait of bactericidal activity vs. M. smegmatis in vitro revealed a perfect match with the strain distribution pattern of resistance/susceptibility to Mycobacterium bovis BCG in vivo.     

Cell-mediated immune response during primary Chlamydia pneumoniae infection

The development of Chlamydia pneumoniae-specific cell-mediated immunity was studied during a primary C. pneumoniae infection. The immune response was detected as positive lymphocyte proliferation and secretion of interferon gamma. C. pneumoniae-induced activation of both CD4(+) and CD8(+) T cells was detected in the early phase of infection, but activation of only CD4(+) T cells was detected in the later stage.     

Demonstration of acquired resistance in Bcgr inbred mouse strains infected with a low dose of BCG montreal.

The relationship between natural resistance to Mycobacterium bovis BCG, expressed by the Bcg gene, and the generation of acquired resistance to this infection in various selected inbred strains of mice was investigated. Consistent with previous findings, a low dose (approximately 10(4)) of BCG Montreal grew progressively in the spleens of inbred mouse strains previously designated susceptible to BCG (Bcgs), but grew poorly in resistant strains (Bcgr). In contrast, however, little difference was observed in the growth of the organism in the liver or lungs of these mice, whereas furthermore, all animals behaved as Bcgs when infected with the World Standard preparation of BCG, BCG Pasteur. Moreover, four strains tested (Bcgr; A/J, C3H/HeJ, and Bcgs; B10.A/J, BALB/c), all showed evidence of the generation of acquired resistance to a small inoculum of BCG Montreal, as demonstrated by their substantial protection against a subsequent intravenous challenge with virulent M. tuberculosis. These findings are interpreted as being inconsistent with the Bcg gene hypothesis and call into doubt the usage of the term Bcg as a gene designation.     

Role of gamma interferon and tumor necrosis factor alpha during T-cell-independent and -dependent phases of Mycobacterium avium infection.

To design an effective immunotherapy for Mycobacterium avium infections, the protective host response to the infection must be known. Here we analyzed the role of gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) in the innate and acquired responses to M. avium infections in mice. T-cell depletion studies showed that CD4+ T cells were required for control of the infection. CD(4+)-depleted mice showed enhanced bacterial proliferation and at the same time showed a reduction in the level of expression of both IFN-gamma and TNF-alpha mRNAs in spleen cells. In contrast, M. bovis BCG immunization restricted M. avium proliferation and at the same time promoted expression of the mRNAs for the two cytokines. In vivo depletion studies using specific monoclonal antibodies showed that both IFN-gamma and TNF-alpha are involved in an early protection possibly involving NK cells, and furthermore, IFN-gamma is involved in the later T-cell-protective response to infection. In vivo neutralization of IFN-gamma during M. avium infection also blocked the priming for enhanced TNF-alpha secretion triggered by endotoxin. Both cytokines were found to be involved in the resistance expressed in BCG-immunized animals and exhibited additive bacteriostatic effects in vitro on bone marrow-derived macrophages infected with different strains of M. avium. These data suggest that both cytokines act in an additive or synergistic fashion in the induction of bacteriostasis and that IFN-gamma is also involved in priming TNF-alpha secretion.     

Genetic resistance/susceptibility to mycobacteria: phenotypic expression in bone marrow derived macrophage lines.

Congenic strains of mice susceptible (B10A.Bcgs) or resistant (B10A.Bcgr) to BCG were established. Here we describe the model system which has been established to analyze the functional activities of macrophages in the two strains. We have immortalized bone marrow macrophages from B10A.Bcgs and B10A.Bcgr congenic strains of mice and derived cloned macrophage lines designated B10S and B10R, respectively. B10R and B10S cell lines exhibited surface markers and morphology typical of macrophages. B10S and B10R were similar in their phagocytic activity, in their level of c-fms, in their transforming growth factor beta (TGF beta) mRNAs expression, and in their expression of tumoricidal activity in response to interferon-gamma (IFN gamma) plus lipopolysaccharides (LPS). However, B10R macrophages expressed a higher level of la mRNA when activated with IFN gamma compared with B10S macrophages. Analysis of the bacteriostatic activity of the two cell lines revealed that B10R macrophages were much more active in inhibiting Mycobacterium smegmatis replication than B10S. To measure the intracellular destruction of bacilli, a bactericidal assay based on hybridization with an oligonucleotide probe specific for mycobacterial ribosomal RNA was designed. The results demonstrated that B10R macrophages were endowed with enhanced constitutive bactericidal activity as compared with B10S. In conclusion we have obtained macrophage lines from bone marrow of B10A.Bcgs and B10A.Bcgr mice that express to a similar extent functional and phenotypic characteristics of macrophages. However, we demonstrate that relative to B10S macrophages, the B10R macrophages have higher expression of la mRNA and that they are constitutively more active in expressing mycobactericidal activity.     

Studies on the development and role of some cell-mediated immune responses in experimental toxocarosis in mice

The studies were undertaken to investigate the development of some cell-mediated immune responses in experimental toxocarosis in mice and to assess the influence of these responses on the course of infection. Mice were infected orally with 350 eggs of Toxocara canis and reinfected with the same dose of parasites after 8 weeks. Groups of infected animals were killed each week of the experiments to obtain spleens, livers and brains for further studies. Lymphocytes from removed spleens were analysed by flow-cytometry for CD4 and CD8 expression and cultured in vitro to measure their responses to Concanavalin A and excretory-secretory (ES) antigen of T. canis in a lymphocyte transformation test. Pieces of livers were used to prepare paraffin sections to be stained later with haematoxylin and eosin, whereas whole brains of the infected animals were examined for the presence of parasite larvae. The results of the studies showed depression of T-cell responses to ConA in early stages of infection and significant increase in the blastogenic responses to the ES antigen from week 4 following infection. The depression of T-cell responses was accompanied by lowered CD4+/CD8+ ratio resulting from increased percentages of CD8+ T cells. Histopathological examination of liver sections revealed trapping of larvae in T. canis reinfected mice. The intensity of infection as measured by larval recoveries from the brains of mice increased gradually up to the 8th week of infection, but did not show significant changes after reinfection, testifying to the development of long-lasting protective immunity during primary infection.