Augmented production of gamma interferon in mice experimentally infected with Toxoplasma gondii

The gamma interferon (IFN-gamma) production and the cell populations participating to this production were examined in Toxoplasma-infected mice. When spleen cells from Toxoplasma-infected mice were cultured with Concanavalin A (Con A) or OK-432, a Streptococcal preparation, they produced significantly high levels of IFN-gamma as compared with that of noninfected mice. Such enhanced IFN titers were observed as early as at 5 days postinfection, reached at the maximum levels on 20 days around and declined gradually thereafter. Treatment of spleen cells from the infected mice with either monoclonal anti-Thy-1.2 antibody plus complement or macrophage-blocking agents virtually abolished the IFN production. The spleen cells producing IFN-gamma were more susceptible to the treatment with monoclonal anti-Lyt-1.2 than anti-Lyt-2.2 antibodies, suggesting that CD4+ T cells are main producers of this lymphokine. When mice infected with Toxoplasma 10 days previously were injected with lipopolysaccharide (LPS), a well-known inducer of IFN-alpha/beta, the sequential production of IFN-alpha/beta and IFN-gamma was induced in their circulation.     

Regulated upon activation normal T-cell expressed and secreted (RANTES) contributes to abortion caused by Brucella abortus infection in pregnant mice

Brucella abortus (B. abortus) is a facultative intracellular pathogen that can survive inside macrophages and trophoblast giant cells, and the causative agent of brucellosis. In the present study, we found that production of regulated upon activation normal T-cell expressed and secreted (RANTES) due to B. abortus infection contributes to abortion in pregnant mice. B. abortus infected pregnant interferon-gamma (IFN-gamma) knockout mice died within 15 days of infection, but non-pregnant IFN-gamma knockout mice were still alive. With infection by wild type B. abortus, a large amount of RANTES production was observed in pregnant IFN-gamma knockout mice, and induction of RANTES was also observed in normal pregnant mice infected with the wild type, but not in those infected with the intracellular replication-defective mutant. Production of RANTES and IFN-gamma were inhibited in mice inoculated with the respective RANTES or IFN-gamma antibody. Neutralization of RANTES, induced by B. abortus infection, served to prevent abortion. These results indicate that the production and function of RANTES are correlated with IFN-gamma in pregnant mice infected with B. abortus.     

The role of CD4(+) or CD8(+) T cells in the protective immune response of BALB/c mice to Neospora caninum infection

The role of CD4(+) or CD8(+) T cells in the immune response of BALB/c mice against Neospora caninum infection was examined by using anti-CD4 and/or anti-CD8 monoclonal antibodies (mAbs). Mice were intraperitoneally inoculated with anti-CD4 and/or anti-CD8 mAbs before and after infection with N. caninum and observed for 30 days after infection. Most of the anti-CD4 mAb-treated mice and all of the anti-CD4 and anti-CD8 mAbs-treated mice died within 30 days post-infection (p.i.). In contrast, 100% of PBS-treated mice and 70% of anti-CD8 mAb-treated mice survived more than 30 days. When compared with phosphate-buffered saline (PBS)-treated mice, the weight of mice treated with mAbs tended to decrease. From these results CD4(+) T cells, but not CD8(+) T cells, have an important role for protection of mice against N. caninum infection. Serum antibody levels to N. caninum in infected-mice treated with anti-CD4 mAb or a mixture of anti-CD4 and anti-CD8 mAbs were lower than those in the infected mice treated with anti-CD8 mAb or PBS. The mice treated with anti-interferon-gamma (IFN-gamma) mAb produced high antibody levels to N. caninum, but all mice died within 18 days p.i. These results indicated that IFN-gamma is an important cytokine for protection against N. caninum infection at the early stage of infection. However, since CD4(+) T cells against N. caninum were essential to the production of specific antibody, these antibodies might have important roles in host protection at the later stage of infection.     

Equine herpesvirus-1 infection induces IFN-gamma production by equine T lymphocyte subsets

A commercial bovine IFN-gamma-specific monoclonal antibody was used to measure antigen-specific IFN-gamma production by equine lymphocytes. Paired PBMC samples were collected from six ponies prior to and 10 days after challenge infection with equine herpesvirus-1 (EHV-1). Each sample was stimulated in vitro with EHV-1, virus-free medium, or PMA and ionomycin, and labelled with monoclonal antibodies specific for various equine lymphocyte subset markers. Following fixation, intracellular IFN-gamma was detected using a FITC-conjugated bovine IFN-gamma-specific monoclonal antibody. In vitro restimulation of PBMC with EHV-1 induced IFN-gamma production by a significantly higher percentage of total (CD5(+)) T lymphocytes, and CD4(+) and CD8(+) T lymphocyte subsets among post-EHV-1 infection PBMC samples compared to pre-infection samples. This response was associated with an increase in virus-specific CTL activity, a critical immune effector for the control of EHV-1 infection and disease. No significant increase in IFN-gamma production by B lymphocytes was observed. These data demonstrate that EHV-1 challenge infection of ponies results in increased production of IFN-gamma by virus-specific T lymphocytes, and that this response can be quantitated using flow cytometry.     

Tumor necrosis factor alpha and gamma interferon are required for the development of protective immunity to secondary Corynebacterium pseudotuberculosis infection in mice

The production and role of endogenous cytokines during the course of secondary Corynebacterium (C.) pseudotuberculosis infection were investigated in mice. When immunized mice were challenged on day 28 after primary infection, tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) were found to appear at 3 hr and to reach the maximum at 24 hr after challenge. Spleen cells of mice primarily infected from 2 to 8 weeks before produced a significant amount of TNF-alpha and IFN-gamma when stimulated with formalin-killed bacteria. However, they could not produce detectable amounts of IL-4. The administration of anti-TNF-alpha monoclonal antibody (MAb) and IFN-gamma MAb increased bacterial proliferation in the organs of immune mice and exacerbated the secondary infection. Injection of anti-CD4 MAb alone or anti-CD4 plus anti-CD8 MAbs resulted in significantly increased mortality and a marked suppression of bacterial elimination as well as cytokine production of secondarily infected mice, while the treatment with anti-CD8 MAb alone showed no effect on either the resistance or cytokine production of mice. These results suggest that CD4, probably Th1 T cells, play an important role for establishment of protective immunity against secondary C. pseudotuberculosis infection by secreting TNF-alpha and IFN-gamma.     

Toxoplasma gondii induction of interleukin-12 is associated with acute virulence in mice and depends on the host genotype

The intracellular parasite Toxoplasma gondii can influence host resistance by modulating immune functions in various cell types. The stimulation of interleukin (IL)-12 production in macrophages, dendritic cells and neutrophils by T. gondii has been implicated to be important for skewing anti-parasite immunity early after infection as well as in mediating the pathologic effects induced by the parasite. The present study demonstrates secretion of IL-12 p40 and the bioactive p70 heterodimer by inflammatory macrophages following exposure to live Toxoplasma or tachyzoite lysate. Parasite induction of IL-12 occurred in a dose-dependent manner. Predigestion of T. gondii lysate with proteinase K abrogated its IL-12 inducing activity, thus indicating that a parasite protein(s) triggers this response. Macrophages from various mouse inbred strains showed a differential responsiveness: cells from T. gondii-susceptible mice released more IL-12 upon toxoplasmic challenge than those from resistant mice, although the infection rate and intracellular parasite growth were similar. In triggering macrophage production of IL-12, tachyzoites proved superior to bradyzoites prepared from the same T. gondii isolate. Furthermore, parasites of a mouse-virulent isolate became less efficient inducers of IL-12 following attenuation. The parallel loss in macrophage stimulation in vitro and acute virulence in vivo suggests a linkage of both parasite capacities. Together with the correlation on host side between the genotype-dependent mouse susceptibility to infection and cellular responsiveness to the parasite trigger, these findings indicate that an overproduction of parasite-induced IL-12 might represent a basic mechanism of T. gondii pathogenicity.     

Toxoplasma gondii rhomboid protein 1 (TgROM1) is a potential vaccine candidate against toxoplasmosis

Infection with the intracellular protozoan parasite Toxoplasma gondii causes serious public health problems and is of great economic importance worldwide. The rhomboid proteins which are responsible for adhesion and invasion of host cells have been suggested as vaccine candidates against toxoplasmosis. A DNA vaccine (pVAX-ROM1) encoding T. gondii rhomboid protein 1 (TgROM1) gene was constructed and the immune response and protective efficacy of this vaccine against lethal challenge in BALB/c mice were evaluated. The results indicated that specific antibody and lymphocyte proliferative responses were elicited in mice receiving pVAX-ROM1. The production levels of IFN-γ, IL-2, IL-4, and IL-10, as well as the percentage of CD4(+) cells in mice vaccinated with pVAX-ROM1 were significantly increased respectively, compared to controls receiving either pVAX1 alone or PBS. After lethal challenge, the mice immunized with pVAX-ROM1 showed an increased survival time compared with the mice in the controls. Our data suggested that a DNA vaccine pVAX-ROM1 encoding T. gondii rhomboid protein 1 triggered strong humoral and cellular responses, and prolonged survival time against T. gondii infection in BALB/c mice.     

Prevalence of Toxoplasma gondii in dogs from Colombia, South America and genetic characterization of T. gondii isolates

The prevalence of Toxoplasma gondii in 309 unwanted dogs from Bogotá, Colombia, South America was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT) and found in 52 (16.8%) of 309 dogs with titers of 1:20 in 20, 1:40 in six, 1:80 in 17, 1:160 in three, 1:320 in three, 1:1280 or higher in three. Some organs obtained after necropsy of dogs (hearts, tongues and brains, either separately or pooled) were used in bioassays carried out in mice (37 samples, of which 20 were assayed with separate organs and 17 were assayed with pooled organs), cats (pooled organs from six) and pooled organs of two dogs both in mice and cat. Mice receiving dog tissues were examined for T. gondii infection. Feces of cats that received dog tissues were examined for oocyst shedding. In total, T. gondii was isolated from tissues of 20 dogs (16 by bioassays in mice, 3 by bioassay in cats and 1 by bioassay in mice and cat). All infected mice from 7 of 17 isolates bioassayed in this host died of toxoplasmosis during primary infection. Only 10 of the 20 dogs whose tissues were bioassayed separately induced infections in mice. Interestingly, dog organs varied in their capacity to induce T. gondii infection in mice, hearts and tongues producing more positive results than the brain. The 20 T. gondii isolates obtained from seropositive dogs were PCR-RFLP genotyped using polymorphisms at 10 nuclear markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, a new SAG2 and an apicoplast marker Apico. Ten genotypes were revealed. These genotypes are different from the three predominant Types I, II and III lineages that are widely spread in North America and Europe. A new allele denoted u-3 at PK1 locus was identified in three isolates. This result supports previous findings that T. gondii population is highly diverse in Colombia.     

Early hepatic immune response in rats infected with Fasciola hepatica

We investigated the phenotype of the T cells (CD4+ and CD8+) that produced Th1 (IFN-gamma) and Th2 cytokines (IL-4 and IL-10) during the firsttwo weeks of experimental fasciolosis in rats. We also followed the kinetics of the cytokine and proliferative responses of hepatic mononuclear cells (HMNC) over the same period. We found that HMNC were more numerous in the infected animals than in the controls. The percentage of CD4+ cells increased significantly after infection, whereas the percentage of CD8+ cells did not change. Moreover, the frequency of the cells producing (CP) cytokine changed after infection. The frequency of CP IFN-gamma on 7 days postinfection (pi) was similar to that in control animals. However, the frequency of CP IFN-gamma was clearly lower on day 14 pi, whereas the frequency of CP IL-4 and CP IL-10 had increased. The CP IL-10-were mostly CD4+. Mitogenic stimulation (phorbol myristate acetate/ionomycin) of HMNC led to an increase in the amounts of the Th2 cytokines in the supernatant on days 7 and 14 pi, with the increase more pronounced on day 14. In contrast, IFN-gamma levels also increased by day 7 pi but then decreased to below control levels by day 14. In addition, HMNC proliferation in response to mitogen followed a similar pattern to IFN-gamma production. These findings suggested that, during the first 2 weeks of infection, F hepatica induced a transient ThO cytokine profile followed by downregulation of the cellular response and the induction of a Th2 cytokine profile.     

Humoral and cell-mediated immune responses of d/d histocompatible pigs against classical swine fever (CSF) virus

A better understanding of cell-mediated immune responses to classical swine fever virus (CSFV) is essential for the future development of improved vaccines. We analyzed the generation of cell-mediated and humoral immune responses in d/d histocompatible pigs following CSFV infection or vaccination. Viral infection induced high T cell responses with high primary and secondary CTL activity correlated with high IFN-gamma production, whereas vaccination with a live vaccine followed by infection mainly induced neutralizing antibody but low cell-mediated responses. Moreover, high IgG1 response was associated with high IFN-gamma response following infection whereas a weak IFN-gamma response was related to a good IgG2 response but a low IgG1 production. These data could reflect Th1/Th2-like balance of immune responses depending upon immunization protocols, which has not yet been described in the pig. T-cell responses to CSFV were evidenced by CSFV-specific CD25 upregulation on CD4-CD8+, but not on CD4+CD8- cells, which further illustrated the importance of CTL responses after infection. Our results indicated that generation of cell-mediated immune responses was much higher following intranasal/oral CSFV infection than after intramuscular vaccination, which implies that the capacity of new CSFV vaccines to induce higher T-cell responses should be considered.     

Heligmosomoides polygyrus and Trypanosoma congolense infections in mice: effect of immunisation by abbreviated larval infection.

Concurrent African trypanosome and gastrointestinal helminth infections are prevalent in sub-humid savannah where they are endemic. However, acquired resistance in animals varies with their responder status and exposure. As a guide to study in the definitive hosts, the effects of Trypanosoma congolense infection on the development and maintenance of homologous Heligmosomoides polygyrus resistance were investigated in outbred TO mice. These mice were immunised by abbreviation of larval infection. Immune or naive mice were either infected with 500 infective larvae (L3) of H. polygyrus and/or 10(4) bloodstream forms of T. congolense or were not infected. The outcome of infection was monitored by routine parasitological and immunological techniques for 30 days after the day of the T. congolense infection. Significantly more immune mice concurrently infected with both parasites survived than did immune mice in which H. polygyrus was superimposed on a 10-day-old T. congolense infection. Although all the mice in this latter group died before the end of the experiment, larval immunisation prolonged their survival, relative to similarly treated naive mice. The antibody titres to H. polygyrus in the sera of immune mice challenged with H. polygyrus alone were significantly higher than those of immune mice concurrently infected with both parasites but the levels of protection obtained were comparable. It is concluded that T. congolense may not completely block the strong acquired resistance induced by abbreviated H. polygyrus larval infection in TO mice but is capable of interfering with protective responses, especially if the trypanosome infection occurs prior to H. polygyrus challenge infection.     

High tissue burden of Toxoplasma gondii is the hallmark of acute virulence in mice

Toxoplasma gondii virulence is commonly determined by mortality rate of infected mice. Limited data showed that virulent T. gondii strains had increased parasite growth in mice compared to that of less virulent strains. To determine if this is a common phenomenon for a variety of strains and to develop an alternative assay to test acute virulence in mice, we measured parasite burdens in experimentally infected outbred CD-1 mice for 19 T. gondii isolates, in which the virulence phenotypes had previously been determined by mortality assay. Our results showed that parasite concentrations in spleen tissues were two orders of magnitude higher in the virulent than the intermediately and non-virulent isolates at day 7 post infection. In competition assays, mice inoculated with mixed tachyzoites of virulent and intermediately virulent strains or virulent and non-virulent strains showed that the former always reached a higher concentration at day 7 post infection. In mixed infection of intermediate and non-virulent strains, both strains were detectable in mice at day 7 post infection. In conclusion, our data showed that the virulence of T. gondii can be predicted by parasite load in the spleen tissue of infected mice at 7 days post infection, providing an alternative method to determine virulence of Toxoplasma.     

Fatal toxoplasmosis and concurrent Calodium hepaticum infection in Korean squirrels (Tanias sibericus)

Four Korean squirrels (Tanias siberius) imported in Spain from People's Republic of China died 2 days after their arrival at a pet shop. They had neurological signs associated with generalized toxoplasmosis involving brain, lungs, liver, and the heart. Toxoplasma gondii-like tachyzoites and tissue cysts were found in organs of all four squirrels. The protozoa stained positively with T. gondii polyclonal antibodies and were ultrastructurally similar to T. gondii. Calodium (Capillaria) hepaticum infection was found in the liver of one squirrel.     

Expression of interleukin 4, interleukin 4 splice variants and interferon gamma mRNA in calves experimentally infected with Fasciola hepatica

Interleukin 4 (IL-4) is expected to play a dominant role in the development of T helper (Th) 2 cells. Th2 immune responses with expression of relatively large amounts of interleukin 4 (IL-4) but little interferon gamma (IFN-gamma) are characteristic for chronic helminth infections. But no information is available about IL4 expression during early Fasciola hepatica (F. hepatica) infections in cattle. Therefore, we investigated F. hepatica specific IL-4 and IFN-gamma mRNA expression in peripheral blood mononuclear cells (PBMCs) from calves experimentally infected with F. hepatica. Cells were collected prior to infection and on post-inoculation days (PIDs) 10, 28 and 70. Interestingly, PBMCs responded to stimulation with F. hepatica secretory-excretory products (FhSEP) already on PID 10 and expressed high amounts of IL-4 but not of IFN-gamma mRNA suggesting that F. hepatica induced a Th2 biased early immune response which was not restricted to the site of infection. Later in infection IL-4 mRNA expression decreased whereas IFN-gamma mRNA expression increased slightly. Isolated lymph node cells (LNCs) stimulated with FhSEP and, even more importantly, non-stimulated LN tissue samples indicated highly polarized Th2 type immune responses in the draining (hepatic) lymph node, but not in the retropharyngeal lymph node. During preliminary experiments, two splice variants of bovine IL-4 mRNA, boIL-4delta2 and boIL-4delta3, were detected. Since a human IL-4delta2 was assumed to act as competitive inhibitor of IL-4, it was important to know whether expression of these splice variants of bovine IL-4 have a regulatory function during an immune response to infection with F. hepatica. Indeed, IL-4 splice variants could be detected in a number of samples, but quantitative analysis did not yield any clue to their function. Therefore, the significance of bovine IL-4 splice variants remains to be determined.     

Induction of interferon-gamma (IFN-gamma) and T helper 1 (Th1) immune response by bitter gourd extract

Mice were inoculated intraperitoneally wih 34 different types of vegetable juices, and interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) were measured as markers for the induction of Th1 and Th2 cells, respectively. Serum IFN-gamma level was markedly increased in mice inoculated with bitter gourd (Momordica charantia) juice, but IL-4 levels were not increased with any of the 34 vegetable juices. Testing of the various components of bitter gourd, including peel, pulp, and seed, showed that the pulp induced the highest levels of IFN-gamma. Trial immunogen including the heat extract of the pulp induced specific IgG(2a) antibody of the mice serum inoculated with this immunogen. These results demonstrate that bitter gourd pulp induced IFN-gamma production and show its promise as a means of effective immunostimulatory therapy specific for Th1 cells and IFN-gamma production.     

Comparative infectivity of oocysts and bradyzoites of Toxoplasma gondii for intermediate (mice) and definitive (cats) hosts

Tachyzoites, bradyzoites (in tissue cysts), and sporozoites (in oocysts) are the three infectious stages of Toxoplasma gondii. The prepatent period (time to shedding of oocysts after primary infection) varies with the stage of T. gondii ingested by the cat. The prepatent period (pp) after ingesting bradyzoites is short (3-10 days) while it is long (18 days or longer) after ingesting oocysts or tachyzoites. The conversion of bradyzoites to tachyzoites and tachyzoites to bradyzoites is biologically important in the life cycle of T. gondii and it has been proposed that the pp can be used to study stage conversion. In the present study, infectivity of oocysts and bradyzoites released from tissue cysts of a recent isolate of T. gondii, TgCkAr23, to cats and mice was compared. Ten-fold dilutions of oocysts or bradyzoites were administered orally to cats, and orally and subcutaneously to mice. Of the 29 cats each fed 1-10 million oocysts only one cat shed oocysts and the pp was 23 days; all cats remained asymptomatic. In contrast, all mice administered the same 10-fold dilutions of oocysts either orally or subcutaneously died of toxoplasmosis. The results confirm that infectivity of the oocysts to cats is lower than for mice and that oocysts are non-pathogenic for cats. Of the 41 cats each fed 1-1,000 free bradyzoites, 15 shed oocysts with a short pp of 4-9 days, and all remained asymptomatic. The infectivity of bradyzoites to mice by the oral route was approximately 100 times lower than that by the subcutaneous route. The results confirm the hypothesis that the pp in cats is stage and not dose dependent, and that transmission of T. gondii is most efficient when cats consume tissue cysts (carnivory) or when intermediate hosts consume oocysts (fecal-oral transmission).     

Activation of bovine peripheral blood gammadelta T cells for cell division and IFN-gamma production

Bovine peripheral blood gammadelta T cells have been evaluated for effector function (IFN-gamma production) and clonal expansion in a variety of systems including following activation by mitogens, IL-12, and stimulation, through the T cell receptor (TCR) with anti-CD3 monoclonal antibody (mAb), a cell-bound molecule and a soluble antigenic extract. To evaluate cell division, carboxyfluorescein succinimidyl ester (CFSE) loading of cells and flow cytometric analysis were used, while IFN-gamma production was evaluated by intracytoplasmic staining. It was found that bovine gammadelta T cells produced IFN-gamma and clonally expanded when stimulated through the TCR/CD3 complex by a cell-associated autologous molecule on monocyte, by bacterial components following in vivo sensitization of gammadelta T cells with a leptospira vaccine or by anti-CD3 mAb. In addition, gammadelta T cells were activated efficiently for effector function but not clonal expansion by culturing with IL-12. In contrast, stimulation by Con A or PMA/ionomycin induced efficient replication but only low level IFN-gamma production which was not enhanced by the presence of IL-12. In several systems the amount of IFN-gamma produced per cell by gammadelta T cells was less than that produced by CD4 T cells in the same cultures.     

Relationship between type 1/type 2 immune responses and occurrence of vertical transmission in BALB/c mice infected with Neospora caninum

To examine the relationship between occurrence of vertical transmission and type 1/type 2 immune responses induced by Neospora caninum infection in BALB/c mice, pregnant (group 1 p) and non-pregnant mice (group 1 np) were inoculated with 2 x 10(6) of the N. caninum parasites and then we examined the vertical transmission rate and production of IFN-gamma and IL-4. We also studied chronically infected mice, which were bred at 4 weeks or more after infection (group 2), and mice inoculated during pregnancy and re-bred at 4 weeks or more after delivery (group 3). In groups 1p, 2 and 3, vertical transmission was observed in 27.4, 41.4, and 50% of the offspring, respectively. The serum IFN-gamma level increased on days 1 and 5 post-inoculation (p.i.) in groups 1 p and 1 np, while no increase level was observed in groups 2 and 3 during pregnancy or after delivery. When the mice in groups 2 and 3 were re-inoculated, all mice showed a transient increase in serum IFN-gamma on day 1 post-re-inoculation. The serum IL-4 level in both of groups 1p and np increased in a similar manner following infection. In group 3, the serum IL-4 level was somewhat higher than that in group 2 after re-inoculation. The anti-N. caninum antibody IgG1 titer in group 3 increased on day 10 post-re-inoculation. These results suggest that the mice infected during pregnancy may acquire a weaker immune response to the parasite than mice infected when they are not pregnant, and that mice infected during pregnancy may show an enhanced type 2 immune response in the recrudescence of the infection.