Antibody responses in resistant and susceptible inbred mice infected with Trypanosoma congolense.

Antibody responses were evaluated in inbred mice previously shown to be susceptible (A/J) or resistant (C57BL/6J and B6AF1 hybrid) to infections with relatively avirulent Trypanosoma congolense. Titres and the isotype distribution antibodies specific for the trypanosome variant surface glycoprotein (VSG) were determined by indirect immunofluorescence in sera of mice after primary infections with Trypanosoma congolense and after challenge infections with the same variant following drug cure. The results of these investigations showed that, during active infection, resistant mice made relatively strong VSG-specific IgM antibodies. This isotype also predominated in challenge infections with the homologous variant following drug cure. In contrast, A/J mice made little or no VSG-specific antibody on first exposure to T. congolense. However, these animals were able to produce substantial amounts of protective VSG-specific IgG antibody after multiple-challenge infections with the homologous variant. Substantial titres of VSG-specific antibodies in resistant mice did not influence the numbers of trypanosomes in the first parasitaemic peak as initial parastiaemias were similar in both C57BL/6J and A/J mice. However, C57BL/6J mice cleared parasites in this peak, whereas A/J mice did not. Mice of both strains immunized by infection cure were equally effective in clearing parasites when challenged with homologous trypanosomes. It is clear from the results of this study that antibody is not the sole factor contributing to murine resistance to African trypanosomes.     

Differences in resistance to Trypanosoma musculi infection among strains of inbred mice.

Inbred strains of mice were inoculated with Trypanosoma musculi, and the course of the ensuing parasitemia was followed. The mouse strains fell into three groups: those displaying high and moderate (fivefold less) parasitemia and C57BL/6 (B/6) mice which had exceptionally low infections. To gain insight concerning the mechanisms responsible for interstrain variations in infections, several types of experiments were performed. Comparison of the ability of spleen cells from the various strains to provide the growth-promoting substances required by T. musculi for growth in culture revealed that B/6 cells were deficient; this suggested one mechanism for regulating parasite infections. Exposure of C3H (high parasitemia) and B/6 mice to graded levels of ionizing radiation revealed that B/6 mice have much greater innate resistance to infection than do C3H mice. The effects of treating mice with silica dust or mercaptoethanol indicated that relative resistance to infection is not primarily associated with macrophage activity or limited growth-promoting substances. We conclude that variations in immune responsiveness to parasite antigens (probably not associated with the H-2 complex), possibly in concert with variations in a non-immunological mechanism, account for interstrain variation in resistance to T. musculi infections.     

Genetic resistance to Trypanosoma congolense infections in mice.

The mechanisms of genetic resistance or "trypanotolerance" to infection with Trypanosoma congolense were investigated in two strains of mice. One strain C57BL, is outstandingly resistant to most stabilates of T. congolense and can survive for over 80 days, whereas CFLP, in common with most other strains, generally succumbs in less than 20 days. Evaluation of several pathophysiological and immunological parameters showed that after infection both strains initially developed similar levels of parasitemia, anemia, biochemical derangement, and immunosuppression. The most outstanding difference was after day 8 postinfection, when the susceptible strain (CFLP) sustained high levels of parasitemia (10(9) trypanosomes per ml) until death 2 to 4 days later, whereas the resistant strain (C57BL) showed a marked decrease to less than 10(6) trypanosomes per ml by day 10 postinfection. Clear evidence that this was associated with the presence of trypanocidal antibody in the resistant mice was provided by the results of an infectivity neutralization test on serum collected from each strain at 10 days postinfection. Chronically infected C57BL mice showed declining waves of parasitemia and a slow restoration of most hematological and biochemical indexes to near normal levels by 80 days postinfection, although at this stage they remained immunosuppressed.     

Interleukin-1 and interleukin-2 production in resistant and susceptible inbred mice infected with Trypanosoma congolense.

Human adherent cells, obtained by EDTA reversible adherence to plastic, are potent effectors in cell-mediated cytotoxicity. Spontaneous cytotoxicity in a 2-hr assay against K562 target cells was shown to be largely mediated by contaminating natural killer (NK) cells. Treatment of adherent cells with NK-specific monoclonal antibody anti-Leu-11 plus complement abolished almost completely the spontaneous cytotoxicity. Spontaneous cytotoxicity by adherent cells was also reduced when the phorbol ester PMA was present in the assay. On the other hand, PMA induced a cytotoxic response in NK-cell depleted adherent cells after prolonged 18 hr incubation. The cell population responsible for this dichotomous effect of PMA on adherent cell-mediated cytotoxicity was shown to be monocytes, as revealed by monoclonal antibody treatment. Pure NK cell preparations were not affected by PMA in their cytolytic capacities. Reactive oxygen species are not involved in NK-cell mediated cytotoxicity, while PMA stimulated the monocytes to exert cytolysis and suppressed NK cells by the generation of these highly toxic oxygen products. Hydrogen peroxide especially seemed to be the mediator in this oxygen-dependent monocyte-mediated cytotoxicity and NK-cell suppression.     

Trypanosoma congolense: an in vitro assay to distinguish drug-resistant from drug-sensitive populations

An in vitro assay to distinguish drug-resistant from drug-sensitive populations ofTrypanosoma congolense has been developed. The incorporation of radiolabelled hypoxanthine by procyclic trypanosomes in vitro was measured after 48 h exposure to different concentrations of trypanocides. In the presence of either isometamidium chloride (Samorin) or diminazene aceturate (Berenil), the ability of procyclics of a drug-sensitive stock (TREU 1627) to incorporate hypoxanthine at 28° C was impaired to a much greater extent than that of procyclics of a drug-resistant stock (TREU 1467), when compared with control organisms grown in the absence of drugs. Serum from a rabbit given 1 mg/kg Samorin also inhibited incorporation of radiolabel in TREU 1627 procyclics more severely than in TREU 1467 procyclics, although the difference between stocks was not substantial. When used with cultured bloodstream forms maintained at 35° C, the assay could distinguish the stocks in the presence of Samorin, but no difference was detected between the populations in their incorporation of hypoxanthine after exposure to Berenil.     

Susceptibility of inbred mice to Rickettsia parkeri

Rickettsia parkeri, a member of the spotted fever group Rickettsia, is the causative agent of American boutonneuse fever in humans. Despite the increased recognition of human cases, limited information is available regarding the infection of invertebrate and vertebrate hosts for this emerging tick-borne disease. Toward the development of a viable transmission model and to further characterize the pathology associated with R. parkeri infection, inbred mouse strains (A/J, BALB/c, C3H/HeJ, and C3H/HeN) were intravenously and intradermally inoculated with 10(5) low-passage-number R. parkeri (Portsmouth strain), and infection, gross pathology, and histopathology were scored. Additionally, a quantitative real-time PCR (qPCR) was performed to estimate rickettsial load in heart, lung, spleen, and liver tissues of infected mice at 19 days postinoculation. Of the A/J, BALB/c, and C3H/HeN mice, none displayed universal pathology consistent with sustained infection. Compared to age-matched control mice, the intravenously inoculated C3H/HeJ mice exhibited marked facial edema and marked splenomegaly upon gross examination, while the intradermally inoculated mice developed characteristic eschar-like lesions. The C3H/HeJ mice also exhibited the greatest concentrations of rickettsial DNA from heart, lung, liver, and spleen samples when examined by qPCR. The similarity of the pathology of human disease and sustained infection suggests that the C3H/HeJ strain of mice is a promising candidate for subsequent experiments to examine the tick transmission, dissemination, and pathology of R. parkeri rickettsiosis.     

Susceptibility and resistance of inbred mice to Paracoccidioides brasiliensis.

Nine different inbred strains of mice inoculated intraperitoneally with yeast cells of Paracoccidioides brasiliensis showed significantly varying patterns of susceptibility. The A/SN strain was found to be the most resistant, while BIOD2/nSn, BIO.A and BIOD2/oSn the most susceptible strains. These susceptibility differences were not dependent on the size of challenge inocula and sex of animals. All strains studied showed a mean survival time proportional to the size of inocula used. Although almost all infected male mice presented a shorter survival time when compared with females, significant mortality differences between sexes were found only in two of the strains studied, namely BALB/c and BIOD2/nSn. The H-2 region did not influence the susceptibility pattern since the A/SN and BIO.A strains share the same H-2 haplotype and were respectively highly resistant and susceptible to P. brasiliensis. Furthermore, the presence of C5 and unresponsiveness to lipopolysaccharide had no influence on the mortality data observed. Specific antibodies were detected only in a small number of animals and titres were consistently low, appearing later in the resistant (A/SN) than in a susceptible strain (BIO.A). Omentum, spleen and liver were the most affected organs in both strains, but the susceptible mice had more granulomatous lesions and earlier dissemination of the fungus.     

Susceptibility and resistance of inbred mice to Giardia muris.

The susceptibility and resistance of inbred mice to Giardia muris was studied during the acute and elimination phases of infection. The infection in susceptible A/J and C3H/He mice was characterized by a short latent period, high cyst output during the acute phase of infection, and prolonged periods of cyst release. In contrast, resistant B10.A and DBA/2 mice had a longer latent period, a lower cyst output during the acute phase, and relatively rapid resolution of infection. The trait of susceptibility and resistance during both acute and elimination phases of the infection was found to be under complex multigenic control as determined by examination of the response of F1 hybrid mice and backcross analyses. The genes controlling this trait did not appear to be linked to the H-2 locus. In addition, the control of the response of mice to G. muris during the acute phase of infection was probably mediated by mechanisms independent from those controlling the response during the elimination phase of infection.     

Susceptibility of inbred mouse strains to infection with Serpula (Treponema) hyodysenteriae.

Several inbred strains of mice were inoculated with Serpula (Treponema) hyodysenteriae B204 to determine susceptibility to infection. Challenge doses of 10(7) or 10(8) spirochetes induced cecal lesions in C3H/HeJ mice and other C3H strains of mice. However, more than a 100-fold difference existed between the dose required to induce lesions in 50% of the infected C3H/HeJ mice (8.3 x 10(7)) and that required to induce them in 50% of the infected C3H/HeN mice (5 x 10(5)). C3H/HeJ mice lack a splenocyte mitogenic response to Escherichia coli lipopolysaccharide but exhibited a mitogenic response comparable to those of other C3H strains of mice when stimulated with S. hyodysenteriae endotoxin (butanol-water extract). Different inbred strains exhibited different susceptibilities to infection, with the strain C3H/HeN being the most susceptible on the basis of colonization and development of macroscopic cecal lesions. The ity gene had no apparent effect on susceptibility of mice challenged with S. hyodysenteriae. The involvement of the H-2 haplotype with susceptibility is unclear, but the mice bearing H-2k were more susceptible than mice with the H-2b, H-2d, or H-2q haplotype. These data support the hypothesis that the host's responsiveness to lipopolysaccharide influences the susceptibility to infection with S. hyodysenteriae. However, differences in susceptibility between inbred mice exist independent of the lps locus, suggesting that there are other inherent differences between mouse strains that affect susceptibility to infection by S. hyodysenteriae.     

Activity rhythms in inbred mice. I. Genetic analysis with recombinant inbred strains

Rhythm of activity of inbred mice was recorded automatically by a set of actographs. This rhythm was characterized by six indices in the temporal and frequency domains. Two methods of genetic analysis were applied to these indices using parental strains BALB/c and C57BL/6, the reciprocal F₁'s, and the seven recombinant inbred strains (RI). Findings on the F₁'s show no maternal effect but indicate dominance and heterosis. The RI method successfully rejects the hypothesis of a monogentic correlate for all measures. In line with F₁ data, it demonstrates the presence of a polygenetic correlate: at least one other locus is involved in each of the six outcome parameters.This study was supported by CNRS (URA 1294, affilieé Inserm), MEN (UFR Biomédicale des Saints-Pères, Paris V), Foundation pour la Recherche Medicale.     

Impaired Kupffer cells in highly susceptible mice infected with Trypanosoma congolense

In highly susceptible BALB/c mice infected with Trypanosoma congolense, the total number of Kupffer cells in the liver remains constant; however, their mean size increases fivefold towards the terminal stage. About 25% of Kupffer cells undergo apoptosis. We suggest that development of an impairment of the macrophage system might be a major mechanism for inefficient elimination of trypanosomes.     

Susceptibility of CXB recombinant inbred mice to murine plasmodia

The genetic control of susceptibility to two species of murine malarial parasites was investigated with recombinant inbred (RI) mouse strains as a model system. Initially, the nonlethal Plasmodium yoelii 17X strain was cloned by limiting dilution to minimize parasite variability. This cloned P. yoelii was used to infect C57BL and BALB/c mice, strains which are the progenitors of the CXB RI strains. Since these two strains displayed consistent differences in the kinetics of parasitemia, the seven CXB RI strains were compared for their susceptibility to the same parasite. The RI strains varied considerably when infected with P. yoelii and could be divided into susceptible and resistant groups based on mortality observed with this normally mild infection. This suggests a complex, multigenic inheritance determining susceptibility to this parasite. However, when the susceptible and resistant CXB RI mice were infected with another, unrelated plasmodial species, Plasmodium chabaudi adami, all the mice showed identical patterns of disease. Since susceptibility to different murine plasmodia does not cosegregate in the CXB RI mice, different mechanisms of resistance may be required for different plasmodial species.     

Susceptibility of inbred mice to rickettsiae of the spotted fever group.

A mouse strain susceptible to lethal infection with Rickettsia conorii was required for testing vaccine efficacy and for studying the immunology and pathogenesis of infection. Among 20 strains of inbred mice inoculated intraperitoneally with the Malish strain of R. conorii, the C3H/HeJ mouse strain was the most susceptible, with a 50% lethal dose of approximately 10 PFU. Infection of all mouse strains resulted in a measurable antibody response; the highest titers correlated with the greatest degree of rickettsial replication as measured by plaque assay of infected spleen homogenates. Inoculation of C3H/HeJ mice with 5.0 log10 organisms of strain Malish by the subcutaneous route did not result in lethal infection. The Casablanca and Moroccan strains of R. conorii were not lethal for C3H/HeJ mice and, in addition, produced plaques in L-929 cells morphologically distinct from those produced by the Malish strain. The only other spotted fever group rickettsia tested which produced a lethal infection in C3H/HeJ mice was Rickettsia sibirica. Sublethal infection with any of the spotted fever rickettsiae tested protected against lethal infection with R. conorii. These data established a lethal challenge system for examining the protective efficacy of spotted fever immunogens and presented evidence of biological variation among strains of R. conorii.     

Low-Dose Intradermal Infection with Trypanosoma congolense Leads to Expansion of Regulatory T Cells and Enhanced Susceptibility to Reinfection

BALB/c mice are highly susceptible to experimental intraperitoneal Trypanosoma congolense infection. However, a recent report showed that these mice are relatively resistant to primary intradermal low-dose infection. Paradoxically, repeated low-dose intradermal infections predispose mice to enhanced susceptibility to an otherwise noninfectious dose challenge. Here, we explored the mechanisms responsible for this low-dose-induced susceptibility to subsequent low-dose challenge infection. We found that akin to intraperitoneal infection, low-dose intradermal infection led to production of interleukin-10 (IL-10), IL-6, IL-12, tumor necrosis factor alpha (TNF-α), transforming growth factor β (TGF-β), and gamma interferon (IFN-γ) by spleen and draining lymph node cells. Interestingly, despite the absence of parasitemia, low-dose intradermal infection led to expansion of CD4⁺ CD25⁺ Foxp3⁺ cells (T regulatory cells [Tregs]) in both the spleens and lymph nodes draining the infection site. Depletion of Tregs by anti-CD25 monoclonal antibody (MAb) treatment during primary infection or before challenge infection following repeated low-dose infection completely abolished the low-dose-induced enhanced susceptibility. In addition, Treg depletion was associated with dramatic reduction in serum levels of TGF-β and IL-10. Collectively, these findings show that low-dose intradermal infection leads to rapid expansion of Tregs, and these cells mediate enhanced susceptibility to subsequent infection.     

Susceptibility and resistance of inbred mice to Paracoccidioides brasiliensis

Nine different inbred strains of mice inoculated intraperitoneally with yeast cells of Paracoccidioides brasiliensis showed significantly varying patterns of susceptibility. The A/SN strain was found to be the most resistant, while BIOD2/nSn, BIO.A and BIOD2/oSn the most susceptible strains. These susceptibility differences were not dependent on the size of challenge inocula and sex of animals. All strains studied showed a mean survival time proportional to the size of inocula used. Although almost all infected male mice presented a shorter survival time when compared with females, significant mortality differences between sexes were found only in two of the strains studied, namely BALB/c and BIOD2/nSn. The H-2 region did not influence the susceptibility pattern since the A/SN and BIO.A strains share the same H-2 haplotype and were respectively highly resistant and susceptible to P. brasiliensis. Furthermore, the presence of C5 and unresponsiveness to lipopolysaccharide had no influence on the mortality data observed. Specific antibodies were detected only in a small number of animals and titres were consistently low, appearing later in the resistant (A/SN) than in a susceptible strain (BIO.A). Omentum, spleen and liver were the most affected organs in both strains, but the susceptible mice had more granulomatous lesions and earlier dissemination of the fungus.     

Susceptibility of human lymphocyte populations to infection by herpes simplex virus.

The antibody response to diphtheria toxoid by cultured tonsil cells was suppressed by herpes simplex virus during its inductive stage. Since only T lymphocytes readily supported virus replication, this immunosuppression may be attributed to a selective effect of the virus on this population of cells.     

Antibody responses to ganglio-series gangliosides in different strains of inbred mice.

We studied antibody responses after immunization with ganglio-series gangliosides against 10 strains of inbred mice, including Balb/c, C57BL/6, A/J, C3H/HeN, C3H/HeJ, CBA/N, AKR/N, NZB/N, DBA/2 and nu/nu Balb/c. Twelve gangliosides having NeuAc as their sialic acid moiety (GM4, GM3, GM2, GM1, GD3, O-Ac-GD3, GD2, GD1a, GD1b, GT1a, GT1b and GQ1b), four gangliosides having NeuGc (GM3, GM2, GM1 and GD3) and four asialo-gangliosides (GA4, GA3, GA2 and GA1) were injected intravenously adsorbed to Salmonella minnesota. The antibody titers of the mice sera were determined by an enzyme-linked immunosorbent assay and an immune adherence assay. Antibody responses were found to depend not only on the ganglioside used as an immunogen but also on the mouse strain. Gangliosides having a trisaccharide sequence (NeuAc alpha 2----8NeuAc alpha 2----3Gal-) such as GD3, GD2, GD1b, GT1a and GQ1b, in particular O-Ac-GD3, induced high-titer antibody responses, whereas those having a disaccharide sequence (NeuAc alpha 2----3Gal-) such as GM4, GM3, GM2, GM1, GD1a and GT1b induced low-titer antibody responses. On the other hand, gangliosides with NeuGc developed minimum titers. In contrast, asialogangliosides induced much higher responses than the corresponding gangliosides. The differences in ceramide portions of these gangliosides did not appear to be involved in inducing antibody responses. Mice could be divided into three groups according to the magnitude of their antibody responses: Group 1, those that produce the highest antibody responses (C3H/HeN and A/J); Group 2, those that demonstrate moderate antibody titers (Balb/c, C57BL/6, DBA/2 and nu/nu Balb/c); and Group 3, those that make minimum responses (AKR/N, C3H/HeJ, CBA/N and NZB/N). The pattern of reactivity to the various gangliosides was similar in all the strains tested.     

Susceptibility of different strains of mice to hepatic infection with Plasmodium berghei.

Despite the low susceptibility of BALB/c mice to hepatic infection by Plasmodium berghei, this animal model is routinely used to investigate the basic biology of the malaria parasite and to test vaccines and the immune response against exoerythrocytic (EE) stages derived from sporozoites. A murine model in which a large number of EE parasites are established would be useful for furthering such investigations. Therefore, we assayed six mouse strains for susceptibility to erythrocytic and hepatic infections. The administration of 50 sporozoites by intravenous inoculation was sufficient to establish erythrocytic infections in five of five C57BL/6 mice compared with 10,000 sporozoites required to infect 100% of BALB/c mice. To assay for hepatic infections, mice received an intravenous inoculum of 10(6) sporozoites, and liver sections for light microscopy and histology were obtained at 29 and 44 h postinoculation. EE parasites were visualized by immunofluorescence, using an antibody to a P. falciparum heat shock protein. The mean number of EE parasites per 100 cm2 for C57BL/6 and A/J strains was significantly higher than that for BALB/c (2,190 +/- 260, 88 +/- 38, and 6 +/- 2, respectively). The proportion of inoculated sporozoites transforming into liver schizonts was 8.2% in C57BL/6 and < 1% in C3H/HeJ, DBA/1, and Swiss CD-1/ICR mice. Nonspecific inflammatory infiltrates around EE parasites were less prevalent in liver sections from C57BL/6 mice than in those from BALB/c mice, which contributed to the decrease in developing EE stages in BALB/c mice. These data indicate that the C57BL/6-P. berghei system is preferable for investigating the biology and immunology of liver stage parasites.