Effects of cyclosporin A on susceptibility and interferon-generating capacity in mice infected with Toxoplasma gondii

Cyclosporin A (Cs-A), a potent immunosuppressant, was administered to mice to evaluate the role of T lymphocytes for the development of a protective immunity to an obligate intracellular protozoan parasite, Toxoplasma gondii. Although daily administration of various amounts of Cs-A for 7 days enhanced the host susceptibility at all doses employed, a dose-dependent manner of Cs-A treatment was not observed as far as the dosage regimens applied here are concerned; mice died at the same rate (40%) among the groups receiving various amounts of Cs-A. Cs-A treatment had a differential effect on the course of disease depending on how it was given in relation to infection. All mice receiving 50 mg of Cs-A per kg per day for 10 days from the beginning of infection eventually died of toxoplasmosis. Cs-A did not suppress the production of intereferon(IFN)-alpha/beta that was induced shortly after the infection, whereas it reduced greatly the ability of Toxoplasma-infected mice to produce IFN-gamma induced by stimulation with Toxoplasma lysate antigen (TLA). Moreover, the decrease in IFN-gamma production correlated with an increase in the parasitic growth in the peritoneal cavities of Cs-A-treated mice. These results suggest that the immunosuppressive effect of Cs-A on the primary Toxoplasma infection in mice is expressed by inhibiting the development of effector T cells responsible for the production of IFN-gamma.     

Monoclonal antibodies to equine interferon-alpha (IFN-alpha): new tools to neutralize IFN-activity and to detect secreted IFN-alpha

Interferon-alpha (IFN-alpha) is a type I interferon that is secreted during the early stages of the innate immune response and is often induced upon infection with viral pathogens. IFN-alpha production affects multiple downstream events influencing both innate and adaptive immune responses. Here, we describe the expression of an equine rIFN-alpha/IgG4 fusion protein in mammalian cells. The anti-viral activity of rIFN-alpha/IgG4 was found to be 70-fold higher than that of a previously described IFN-gamma/IgG1 as tested by bioassay. The purified rIFN-alpha was subsequently used for the generation of six monoclonal antibodies (mAbs) to equine IFN-alpha. Four of these mAbs inhibited the protective anti-viral effect of equine leukocyte IFN in bioassays. One mAb (clone 240-2) showed a high-neutralizing capacity. An ELISA was established using two anti-equine IFN-alpha mAbs (clones 29B and 240-2) and its analytical sensitivity for was found to be around 800 pg/ml and 3 U/ml for rIFN-alpha and equine leukocyte IFN, respectively. When analyzing samples with a likely dominance of IFN-alpha among type I IFNs, such as supernatants from equine peripheral blood mononuclear cells stimulated with CpG-oligodeoxyribonucleotides, the results obtained by ELISA and IFN bioassay showed a high agreement (r(2)(sp)=0.98). When analyzing samples likely containing a mixture of type I IFNs, such as serum and nasal secretions from virally infected horses, the ELISA only detected some of the IFN-activity recorded in the bioassay. Overall, the data showed that the new anti-equine IFN-alpha mAbs are valuable tools to detect native IFN-alpha for further characterization of the early innate immune response and anti-viral immunity in horses.     

Immune production of interferon by cultured peripheral blood mononuclear cells from calves infected with BHV1 and PI3 viruses

Peripheral blood mononuclear cells (PBMC) from calves infected with bovine herpesvirus type 1 (BHV1) or parainfluenza 3 virus (PI3) were cultured in vitro in the presence of inactivated specific antigen presented on MDBK cells. In the presence of inactivated antigen, PBMC from both BHV1-infected and control calves produced interferon (IFN)-alpha in 24 hour cultures. Altering the culture conditions did not result in the detection of immune-specific IFN produced by mononuclear cells from BHV1-infected calves. However, spontaneous IFN was detected in the absence of antigen in 24 hour cultures from infected animals: this IFN was pH 2 labile and completely neutralised by antiserum to recombinant bovine IFN-gamma. Spontaneous IFN-gamma production was only seen in calves following a second BHV1 inoculation, given four to seven weeks after the primary dose. In contrast PBMC cultures from PI3 virus-infected calves did not produce IFN-gamma spontaneously, but did so in cultures which contained inactivated PI3 antigen. Mononuclear cells from control animals failed to produce either IFN-alpha or -gamma when cultured with inactivated PI3 virus. IFN-gamma was detected in PBMC cultures after the primary infection, with no increase in production occurring following subsequent PI3 virus inoculations. Immunospecific production of IFN-gamma provides a simple method for monitoring cell-mediated immunity in BHV1- and PI3 virus-infected calves and can be used for evaluating the efficacy of vaccines against these viruses.     

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.     

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.     

Comparison of the growth inhibitory effects of canine IFN-alpha, -beta and -gamma on canine cells infected with Neospora caninum tachyzoites

The growth inhibitory effects of recombinant canine interferon alpha (IFN-alpha), beta (IFN-beta) and gamma (IFN-gamma) were examined on Madin-Darby canine kidney cells infected with Neospora caninum tachyzoites. The parasite growth was inhibited by all IFNs in a dose-dependent manner. IFN-gamma inhibited the parasite growth with greater efficacy than IFN-alpha or IFN-beta. Moreover, the effect of IFNs on N. caninum growth associated with the suppression of the host cell viability. The present study indicates IFN-alpha and -beta, besides IFN-gamma, play a crucial role for N. caninum growth in host cells.     

The DNA vaccine vector pcDNA3 induces IFN-alpha production in pigs

The cytokine inducing capacity of the vaccine vector pcDNA3, a methylated form of the plasmid, and pcDNA3 encoding porcine interleukin (IL)-6 or granulocyte/macrophage colony-stimulating factor (GM-CSF) was studied in pigs, using a model with tissue chambers implanted subcutaneously. The production of interferon (IFN)-alpha, IFN-gamma, IL-6 and GM-CSF was studied at local (tissue chamber fluid (TCF)) and systemic (serum) levels during 3 days post-injection. All forms of the plasmid, except the methylated, induced a transient local production of IFN-alpha but no plasmid-induced production of IFN-gamma, GM-CSF or IL-6 could be detected after injection of the plasmids. The IFN-alpha response increased markedly at repeated injections of pcDNA3. This IFN-alpha inducing capacity of the plasmid is likely to affect immune responses at DNA vaccination of pigs.     

Immunostimulatory DNA activates production of type I interferons and interleukin-6 in equine peripheral blood mononuclear cells in vitro

This study aimed to evaluate different nucleic acid preparations as cytokine inducers in equine cells. To induce cytokine production, bacterial plasmid DNA or short synthetic oligodeoxyribonucleotides (ODN), with or without the transfection reagent lipofectin, were added to cultures of purified equine peripheral blood mononuclear cells (PBMC). Cytokine activity was detected with bioassays in cell culture supernatants after 24h of induction and cytokine mRNA expression was detected using RT-PCR at 6h post induction. For IFN-alpha/beta it was found that both plasmid DNA and phosphodiester ODN, containing an unmethylated CpG-motif, were able to induce IFN production in the presence of lipofectin but not without. The levels of IFN varied with individuals and were often quite low. Moreover, methylation or removal of the CpG sequence completely abolished IFN induction. CpG-containing ODN with poly-guanine (G) sequences in the 5' and 3' ends induced considerably higher levels of IFN, especially when the poly-G sequences had a phosphorothioate backbone. ODN with poly-G sequences also had the ability to induce IFN in the absence of lipofectin but the levels of IFN induced were radically reduced compared to those induced with lipofectin. In contrast to IFN, which was only detected upon induction, low spontaneous IL-6 production was observed in unstimulated control cultures. Nevertheless, plasmid DNA and CpG-containing ODN were able to increase the IL-6 production threefold. All the IFN inducing ODN also induced IL-6 production and the levels of IL-6 induced seemed influenced by addition of lipofectin and presence of poly-G sequences in the same way as was observed for the IFN-production. However, a complete phosphorothioate ODN with a central CpG-motif and poly-C sequences, that did not induce IFN, readily induced IL-6 both in the presence and absence of lipofectin. In addition, there was also evidence that some ODN induced increased expression of IL-12p40 mRNA. To conclude, equine PBMC were able to recognize CpG-DNA and respond with both IFN-alpha/beta and/or IL-6 production. The levels of cytokine induced, and sometimes which cytokine induced, varied with, e.g., CpG-motifs used, the presence of poly-G sequences, ODN backbone chemistry and presence of lipofectin.     

Epsilon-polylysine microparticle adjuvant drives cytokine production to Th1 profile

Epsilon-polylysine micro particles (SGEPL) and polyethyleneimine micro particles (SGPEI) were developed by the addition of a hydrophobic group and the immunological characterization of these micro particles and aluminum hydroxide (ALUM) was investigated. BALB/c mice were injected intraperitoneally with ovalbumin (OVA) as an antigen and SGEPL, SGPEI or ALUM as an adjuvant. The results showed that the mice injected with SGEPL produced a significant portion of anti-OVA antibody subclass IgG2a in the sera and suppressed interleukin (IL)-4 and IL-5, but enhanced IL-12 and interferon-gamma (IFN-gamma) from the spleen cells. Similar results relating to cytokines were also obtained, even without OVA. Direct stimulation with SGEPL to na?ve BALB/c mouse spleen cells induced IL-12 and IFN-gamma. Both spleen and purified B cells produced IgG1 and IgE after stimulation with IL-4 and the anti-CD40 monoclonal antibody. With the addition of SGEPL, the IgE production from the cells was suppressed as a result of enhanced IFN-gamma production. Furthermore, IgE production was also suppressed in the purified B cells without the influence of IFN-gamma or IL-12. Thus, we suggest SGEPL drives cytokine production to Th1 profile. It will be a novel promising adjuvant based on this viewpoint.     

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.     

Effects of an experimental infection with Actinobacillus pleuropneumoniae on the interferon-alpha and interleukin-6 producing capacity of porcine peripheral blood mononuclear cells stimulated with bacteria, virus or plasmid DNA

The effect of a bacterial infection on interferon-alpha (IFN-alpha) and interleukin-6 (IL-6) production by porcine cells was studied in specific pathogen-free (SPF) pigs, infected intranasally with Actinobacillus pleuropneumoniae serotype 2. Three experimental groups of five pigs were used: infected non-treated pigs, infected pigs that were treated with enrofloxacin at disease onset, and non-infected, non-treated control pigs. Blood samples were collected from all pigs on the day of infection and on days 1, 4, 7, 13 and 17 post-infection. Sera were analysed for presence of antibodies to A. pleuropneumoniae and for the cytokines IL-6 and IFN-alpha. Ability to produce these cytokines was tested in vitro using whole blood cultures stimulated with inactivated virus (Aujeszky's disease virus infected porcine kidney cells (ADV/PK-15)), inactivated bacteria (A. pleuropneumoniae) or bacterial plasmid (pcDNA3). All cytokine inducers were used neat or pre-incubated with the transfectious agent lipofectin. IL-6 appeared in the serum of all infected non-treated animals but no IFN-alpha was found in the serum of any of the experimental pigs. Accordingly, the bacteria induced a substantial IL-6 but hardly any IFN-alpha production when tested in vitro. However, following incubation with lipofectin, the inactivated bacteria as well as pcDNA3 became efficient inducers of IFN-alpha in whole blood cultures. The increased IFN-alpha production, previously recorded in vitro during the acute phase of infection with A. pleuropneumoniae, was confirmed using lipofected plasmid DNA and it was indicated that leukocytes obtained from infected but apparently cured animals also exhibited an increased production of IFN-alpha. Thus, even mild/sub-clinical bacterial infections may affect cytokine production in pigs.     

Augmentation of secreted and intracellular gamma interferon following johnin purified protein derivative sensitization of cows naturally infected with Mycobacterium avium subsp. paratuberculosis.

Measurement of secreted interferon (IFN)-gamma has proven to be a valuable tool for the detection of animals infected with mycobacterial pathogens, including Mycobacterium avium subsp. paratuberculosis. Previous reports have suggested that tuberculin skin testing can influence the performance of the IFN-gamma assay. In the present study, healthy noninfected cows, and cows subclinically and clinically infected with M. paratuberculosis were administered an intradermal injection of johnin purified protein derivative (JPPD) and effects on secreted and intracellular IFN-gamma were observed. Intradermal injection resulted in significant increases in secreted IFN-gamma for subclinically infected cows after stimulation of peripheral blood mononuclear cells (PBMC) with concanavalin A or M. paratuberculosis antigen preparations (whole-cell sonicate and JPPD) on days 7 and 10 postinjection. Intracellular IFN-gamma was increased after intradermal injection in total PBMC for all treatment groups and was higher within CD4+ and CD8+ subpopulations for infected cows compared to healthy controls throughout the study. When T-cell populations were further defined by CD45RO expression, intracellular IFN-gamma was higher within CD8+/CD45RO+ lymphocytes compared to CD4+/CD45RO+ cells for subclinically and clinically infected cows but similar within these subpopulations for healthy controls. These results indicate that intradermal sensitization of cows in the subclinical stage of infection will upregulate expression of IFN-gamma, enhancing the sensitivity of this assay. In addition, CD8+ lymphocytes appear to play an important role as a mediator of M. paratuberculosis infection in naturally exposed cattle.     

Characterization of Th1 activation by Bartonella henselae stimulation in BALB/c mice: Inhibitory activities of interleukin-10 for the production of interferon-gamma in spleen cells

This study was conducted to analyze cytokine production mechanisms in mice after Bartonella henselae stimulation. BALB/c mice were inoculated intraperitoneally with 3 x 10(6) colony forming units of B. henselae (Houston-1 strain) twice at 10-day interval. Spleen cells were harvested from the mice and stimulated with the organisms. Following the stimulation, interferon-gamma (IFN-gamma) and interleukin-4 (IL-4), IL-10, IL-12 and tumor necrosis factor-alpha (TNF-alpha) were measured in the culture supernatants of the spleen cells by ELISA. The spleen cells specifically secreted IFN-gamma, but not IL-4, indicating that T helper 1 (Th1) cells were activated following B. henselae stimulation. In addition, IL-10 and TNF-alpha productions were also detected in the culture supernatants of spleen cells. Neutralization of IL-10 in the culture supernatants significantly enhanced the production of IFN-gamma from the spleen cells stimulated with B. henselae. These results indicate that B. henselae predominantly stimulated Th1 cells and resulted in secreting IFN-gamma, however the production was partially inhibited by IL-10, which was produced simultaneously.     

Immunostimulation in mice infected with Sendai virus

The effect of Sendai virus infection on the splenic primary plaque-forming cell (PFC) response to sheep RBC in 2 strains of mice, with contrasting susceptibility to Sendai viral pneumonia, was examined. Mice were given single inoculations of sheep RBC, which varied relative to time of inoculation with Sendai virus, PFC were counted 6 days later, and were compared with PFC responses from noninfected mice. The IgM- and IgG-PFC responses were augmented in resistant C57BL/6J mice 7 and 9 days after inoculation with Sendai virus (sheep RBC given 1 and 3 days after inoculation with Sendai virus, respectively) and in susceptible DBA/2J mice 7, 9, 10, and 13 days after inoculation with Sendai virus. Augmentation was restricted mainly to IgM-, IgG3-, and IgG2b-PFC. The number of splenic background antitrinitrophenyl sheep RBC PFC in mice of both strains was examined during the course of Sendai virus infection. Only a marginal increase in background PFC was seen in C57BL/6J mice on or after viral inoculation day 11 and no change was seen in DBA/2J mice. Serum of infected mice also was examined sequentially for alpha/beta interferon (IFN). Despite vigorous lung IFN production, infected mice rarely had detectable circulating IFN. Seemingly, Sendai virus infection can induce transient hyperresponsiveness to a nonviral antigen.     

Changes of lymphocyte subpopulations and natural killer cells in mice sensitized with Toxoplasma lysate antigen before and after Babesia infection

When 8-week-old BALB/c mice were sensitized with two intramuscular injections of Toxoplasma lysate antigen (TLA) at 2 week interval, the numbers of sIg(+), Thy-1,2(+), Lyt-1,2(+) Lyt-2,2(+), and Asialo GM1(ASGM1)(+) cells in the spleen, liver and peripheral blood increased by 2 to 4 times over those found in unsensitized mice of the same age. When TLA-sensitized and unsensitized mice were infected with Babesia, 4 of 10 (40%) of the TLA-sensitized mice survived infection, while none of the unsensitized control mice lived longer than 14 days after Babesia infection. By contrast, sensitization of nude mice with TLA had no effect on survival, and mice did not live more than 12 days. The number of thymic Thy-1,2(+) cells decreased in TLA-sensitized and unsensitized BALB/c mice by almost 80% within 10 days after infection (AI). During the same time, the numbers of B cells, T cells, and NK cells increased in the spleen, liver and peripheral blood of both sensitized and unsensitized mice. Especially notable were increases in numbers of Lyt-2,2(+) cells in the spleen and blood and increases in numbers of NK cells in the spleen, liver and blood in both TLA-sensitized and unsensitized mice. When spleen cells from TLA-sensitized and unsensitized mice were cultured in the presence or absence of TLA for 6 days, assays for cytotoxicity using NK-insensitive P-815 target cells and NK-sensitive YAC-1 target cells demonstrated higher rates of cytotoxicity in cultures of TLA-sensitized spleen cells.     

Classical swine fever virus induces activation of plasmacytoid and conventional dendritic cells in tonsil, blood, and spleen of infected pigs

Classical swine fever virus (CSFV) compromises the host immune system, causing indirect leucopoenia and disruption of in vitro T cell stimulation capacity. In order to explore the potential role of dendritic cells (DC) in such phenomena, the activation of conventional DC (cDC) and plasmacytoid DC (pDC) in blood and secondary lymphoid organs of infected pigs was investigated in the early time course post-inoculation (pi), together with viral components dissemination and cytokine production in serum. Whereas CD11R1+CD172a+ cDC frequencies were markedly reduced in blood and spleen, analysis of CD4+CD172a+ pDC numbers revealed a rapid turn-over of this DC subset in tissues pi. Both subsets matured and were activated after infection, as demonstrated by down-regulation of CD1a, up-regulation of the co-stimulation molecule CD80/86 and expression of cytokines. cDC essentially expressed tumor necrosis factor alpha (TNF-alpha) and interleukin (IL)-10, whereas pDC produced alpha interferon (IFN-alpha) and IL-12. IFN-alpha and TNF-alpha productions revealed an enhancement of innate anti-viral immune responses. Detection of antigen activated B lymphocytes in tonsil T-cell areas at 72 h pi, subsequently to the transient translocation of the viral E2 protein within germinal centres at 48 h pi, indicates the initiation of humoral response. This response was also evidenced by an important IL-10 production in serum one week pi. IL-12 expression in organs, as well as transient detection of IL-18 and IFN-gamma in serum, reflected the initiation of cellular immune responses. However, the uncommonly high levels of TNF-alpha and IFN-alpha produced by DC and measured in serum early post-infection, together with IL-10 expression in spleen, could play a role in the disruption of immune system cells, either inducing apoptosis or impairing DC functionalities themselves.     

Murine model study of the practical implication of trypanosome-induced immunosuppression in vaccine-based disease control programmes

The relevance of trypanosome-induced immunosuppression in relation to the efficacy of vaccine-induced immunity was studied in mice. Mice were immunised with crude Trichinella spiralis muscle larvae homogenate vaccine and infected with T. spiralis and/or Trypanosoma brucei. Vaccination significantly decreased adult worm burden (p<0. 05) and accelerated worm expulsion in mice infected with T. spiralis only. T. brucei superinfection resulted in monocytosis, suppressed eosinophilia, significant decrease in PCV (p<0.001), higher numbers of adult worms (p<0.001) and failure to expel all adult worms by Day 12 post infection (p.i.). Regardless, they produced anti-Trichinella IgG(1) responses similar to those of the vaccinated non-T. brucei-infected group. T. brucei also suppressed the proliferative responses of spleen cells to stimulation with Con A and T. spiralis antigen, and induced strong production of interferon-gamma (IFN-gamma) in culture supernatants of antigen stimulated spleen and mesenteric lymph node cells. Interleukin-5 (IL-5) production was suppressed by T. brucei in supernatants of Con A- and antigen-stimulated spleen cells. It was concluded that trypanosome infections and the associated immunosuppression are of great practical significance in trypanosome endemic areas, especially with regards to disease control programmes involving vaccine-induced herd immunity.     

Effects of reticuloendotheliosis virus and Marek's disease virus infection and co-infection on IFN-gamma production in SPF chickens

Two experiments were used to examine the potential role of IFN-gamma in chickens infected with reticuloendotheliosis virus (REV) and Marek's disease virus (MDV). First, chickens were infected with REV and/or MDV at 5 days of age and examined from 3 to 50 days post-infection (dpi). In REV+MDV co-infection chickens, IFN-gamma ELISA demonstrated a 3-fold increase at 7 dpi compared to the controls, while REV alone caused a 5-fold increase, the IFN-gamma levels peaked, and then gradually decreased. IFN-gamma levels significantly decreased in MDV infection at 3 dpi and 15 dpi. Second, experiments were designed to determine the effects of different viruses and ConA on IFN-gamma production. For REV- or MDV-infected chickens, the IFN-gamma levels decreased slightly after adding ConA. This is the first report of IFN-gamma production in SPF chickens infected with REV and MDV measured by directly quantitative method.