Comparisons Of Immunopathological Changes Of Four Isolates Of Toxoplasma Gondii From Different Animals In Mice And Chickens

Toxoplasmosis is a globally spread zoonosis which is caused by Toxoplasma gondii. It is estimated that around25%to50%of the world human population is carrying the parasite. T. gondii is remarkably known to have a wide range of hosts. Domestic cats and other felids are the definitive hosts, and virtually all warm blooded animals, including humans, are the intermediate hosts. The pathogenicity of T. gondii infection to most animals varies widely, ranging from inapparent infection to fatal disease conditions. It is well-known that mice are most susceptible to T. gondii and always act as an animal model of acute infection. While chickens were found to display the chronic type of infection without apparent clinical signs to toxoplasmosis, and considered to be a quite resistant intermediate host to T. gondii. To study the immunopathological changes of these two hosts trying to find why chickens are resistant to T. gondii, this research mainly focuses on the following:1. Isolation of Toxoplasma gondii from cats in Jiangsu Province and identification of the genotype for the different original isolates by PCR-RFLP.In this research,11cats from Nanjing Jiangsu Province were tested for IgG against Toxoplasma gondii. Positive cats were killed humanlly and hearts, brains and tongues of each cat were bioassayed for Toxoplasma gondii individually. Tissues of each cat were mixed with five volumes of acidic pepsin. After that, the samples were centrifuged, neutralized with1.2%sodium bicarbonate (pH8.3), mixed with antibiotics (ampicillin and gentamycin sulfate). And then lml of tissue homogenate was inoculated intraperitoneally into each of six mice. At the third blind passages of the survived mice, two strains of T. gondii were isolated from cats No.2and No.3and named CAT2and CAT3. These two isolates and other three chicken (named JS), swine (named CN) and human (named RH) original Toxoplasma gondii isolates were identified by PCR-RFLP method. Seven genetic markers (5’-SAG2,3’-SAG2, SAG3, cB21-4, L358, BTUB and GRA6) of the five isolates were obtained by PCR. Digestion of these seven loci with appropriate endonucleases revealed that these isolates had the common haplotype to the type I lineage. 2. The pathogenicity of four isolates of T. gondii from different animals in chickens and miceThe pathogenicity of T. gondii infection to mice and chickens varies widely. Mice are susceptible while chickens are not. In this study, five isolates (RH, CN, JS, CAT2and CAT3) of T. gondii which well conserved in liquid nitrogen in Laboratory of Veterinary Molecular and Immunological Parasitology, Nanjing Agricultural University, China were used to study the pathogenicity on chickens and mice, respectively, and to construct the animal model which infected on the broiler chickens. Two-hundred and ten chickens aging10-day-old were allocated into21groups of10chickens each. The chickens were infected intraperitoneally with these five isolates with different dosages (5×108,1×108,1×107and1×106). The negative control (-Ve) group was mockly inoculated with physiological saline alone. After that, clinical symptoms and the rectal temperatures of the chickens were checked every day. The died cases during acute phage were checked for T.gondii tachyzoites by making wet smears after necropsy. And the living cases were detected for the infection situation of T. gondii at day53by PCR method. Histopathological sections were used to observe the pathological changes in the died chickens during acute phage and the living animals at day53. For the study of pathogenicity in mice, two routes of infection were used. For one route,260ICR mice were allocated into26groups of10mice each. The mice were infected intraperitoneally and intragastrically with the tachyzoites of these five isolates, respectively. The infection doses were1×105,1×104,1×103,1×102and1×101, respectively. The survival time of each mouse was recorded post inoculation. Results show that no significant differences of pathogenicity were found in chickens and mice among the RH, CN, CAT2and CAT3isolates while the pathogenicity of JS isolate was weak to the mice but comparatively strong to the chicken. These findings suggested that the differences might be related to the sources of the isolate origin. However, the exact mechanisms of them need to be further investigated. Meanwhile, inoculation intraperitoneally at the dosage of1×107tachyzoites on the ten days old chickens can effectively infect the chickens, making them display moderate clinical signs and at the same time stimulate a proper immunopathological changes beside avoidance of losing them during the experiment suggesting that it is a suitable model to study the in vivo infection of T. gondii on chickens. Considering the infection rate, survival time and the causal factors during the infection of mice, it was found that1×102intraperitoneally injected and1×104intragastrically delievered were the suitable dosages to experimentally infect the mice model in this study. 3. Comparisons of immunopathological changes of four isolates of Toxoplasma gondii from different animals intraperitoneally infected in miceA total of200,(18-22g) mice were allocated randomly into5groups which named JS, CAT, CN, RH and a negative control group (-Ve) with40mice in each group. Tachyzoites of four different T. gondii isolates JS, CAT, CN and RH were inoculated intraperitoneally with the dose of1×102in the four designed groups, respectively. The negative control (-Ve) group was mockly inoculated with PBS alone. Blood and spleen samples were obtained on the day of inoculation (day0) and at days3,6and8post-infection to screen the immune responses. Toxoplasma circulating antigens (TCA) and T. gondii specific antibodies (IgA, IgG, IgE, IgM and subsets of IgG) in the serum were detected by enzyme-linked immunosorbent assay (ELISA) method. Subsets of T-lymphocytes (CD4+and CD8+), NK cells and major histocompatibility complex (MHC) molecules (MHC-I and MHC-II) from spleen cells were analyzed by flow cytometric method. The kinetics of cytokines (IFN-y, IL-12, IL-4, IL-10and IL-17) were evaluated by ELISA method. Serum iron, serum nitric oxide (NO) and inducible nitric oxide synthase (iNOS) activity changes were detected by colorimetric technique. IgM specific antibody to T. gondii was first to be found at day6and this level was kept untill the end of the experiment, meanwhile IgA was first detected at day8. However, no specific IgG, subsets of IgG and IgE antibodies were found during the experiment. Flow cytometric analysis indicated that increases of CD4+, CD8+T lymphocytes and NK cells in the four infected groups at days6,3and3, respectively. Thereafter, all these three factors were dropped at day8. For CD4+T lymphocytes, only CN group was showed a significant increase compared to the negative control group, while RH, CN and CAT groups were significantly higher for NK cells.However, all these four infected groups were significantly higher than the negative control group for CD8+T lymphocytes at this timepoint. MHC class I increased in the four infected groups at day6and decreased at day8while MHC class II was down-regulated from day3to8in the four infected groups but no significant differences were shown among the four infected groups. Increases in expression of IFN-y and IL-12were detected in the four infected groups at day6and decreased at day8. However, the levels of IFN-y in the four infected groups were still higher than the negative control group but only RH, CN and CAT groups showed higher levels of IL-12. Notable increases of IL-10and IL-4in the infected groups were found at day3, but dropped at day6. At this time, the levels of IL-10were still higher than the negative control group but IL-4was not. Levels of IL-17were increased markedly at day6 and kept the high levels to the day8of the experiment. A significant increase in the concentration of NO, serum iron and the activity of iNOS were observed in the infected groups at day3, and reached the peaks at day6. After that, concentrations of serum iron in the four targeted groups and the concentration of NO in CAT group were significantly higher than the negative control group at day8. However, no significant differences regarding the activity of iNOS were found in these four infected groups. These findings might partially be explain the immunopathological changes through which T. gondii infect mice. Differences of the detected markers among the four isolates suggested that the bionomic characteristics of varied origin isolates might be present variance when infected the certain host.4. Comparisons of immunopathological changes of four isolates of Toxoplasma gondii from different animals intragastrically infected in miceA total of250,(18-22g) mice were allocated randomly into5groups which named JS, CAT, CN, RH and a negative control group (-Ve) with50mice in each group. Tachyzoites of four different T. gondii isolates JS, CAT, CN and RH were inoculated intragastrically with the dose of1×104in the four designed groups, respectively. The negative control (-Ve) group was mockly inoculated with PBS alone. Blood and spleen samples were obtained on the day of inoculation (day0) and at days3,6,8and10post-infection to screen the immune responses. Toxoplasma circulating antigens (TCA) and T. gondii specific antibodies (IgA, IgG, IgE, IgM and subsets of IgG) in the serum were detected by ELISA method. Subsets of T-lymphocytes (CD4+and CD8+), NK cells and major histocompatibility complex (MHC) molecules (MHC-Ⅰ and MHC-Ⅱ) from spleen cells were analyzed by flow cytometric method. The kinetics of cytokines (IFN-y, IL-12, IL-4, IL-10and IL-17) were evaluated by ELISA method. Serum iron, serum nitric oxide (NO) and inducible nitric oxide synthase (iNOS) activity changes were detected by colorimetric technique. All the animals in the four infected groups were positive in TCA at day6and the high levels were kept untill the end of the experiment. IgG specific antibody to T. gondii was first to be detected at day10and the subsets IgG2a and IgG2b were both found while IgGl and IgG3were not. IgA and IgM antibodies were first to be found at day6and kept the high levels till day10without any significant differences among the four infected groups. Flow cytometric analysis indicated that increases of CD4+and CD8+T lymphocytes in the four infected groups at day6and they reached the peaks at day8. At day10, the levels of CD4+in the four infected groups were decreased without any significant differences compared to the negative control group. However, the levels of CD8+in CN, CAT and JS groups were still higher than the control negative. At day3, only JS and CAT groups were showed a markedly increase in NK cells while all the four infected groups were significantly higher than the negative control group from day8to10. Similar with NK cells, MHC class I molecules of CAT group were increased notablely at day3while all the four infected groups got the higher levels at day8. At day10, all the targeted groups except JS were still higher than the control negative. However, MHC class II molecules were down-regulated from day3to10without any significant differences among the four infected groups. Increases in expression of IFN-y were detected in the different infected groups at days6and8, respectively, and dropped to the level of negative control group at day10. For IL-12, levels of RH and CAT groups were started to increase at day3. However, at day6all the infected groups showed a significant increase. Except of CAT group, at day10, the levels of IL-12were dropped and showed no significance compared with the negative control group. For IL-4, only RH and CAT groups showed the significant increase at day6. IL-17of CN, JS and CAT groups was increased at day8and level declined at day10except JS group. A noteably increase in the concentrations of NO, serum iron and the activity of iNOS were observed in the infected groups at day3and kept the levels to day8. While no significant differences were found at day10when compared to the negative control group. These findings might partially explain the immunopathological changes through which T. gondii infect mice. When compared with the results of intraperitoneally infected mice, positive response of IgG antibody, higher titres and early expressed of IgA and delayed TCA might be present variance when using different routes to establish an infection with T. gondii.5. Comparisons of the immunopathological changes with four different isolates of Toxoplasma gondii in chickensIn this study,4isolates (JS, CAT, CN and RH) were used to compare the immunopathological changes in chickens and to investigate whether the origins of T. gondii could play some roles in the pathogenicities of T. gondii and the susceptibilities of different animals. A total of300,10-day-old chickens were allocated randomly into five groups which named JS, CAT, CN, RH and a negative control group (-Ve) with sixty birds in each group. Tachyzoites of JS, CAT, CN and RH were inoculated intraperitoneally with the dose of1×107in the four designed groups, respectively. The negative control (-Ve) group was mockly inoculated with PBS alone. Blood and spleen samples were obtained on the day of inoculation (day O) and at days4,11,25,39and53post-infection to screen the immunopathological changes. Toxoplasma gondii circulating antigens (TCA) and T. gondii specific antibodies (IgA, IgG and IgM) in the serum were detected by ELISA method. Subsets of T-lymphocytes (CD4+and CD8+) and major histocompatibility complex (MHC) molecules (MHC-Ⅰ and MHC-Ⅱ) from spleen cells were analyzed by flow cytometric method. The kinetics of cytokines (IFN-y, IL-12, IL-4, IL-10and IL-17), serum iron, serum nitric oxide (NO) and inducible nitric oxide synthase (iNOS) activity changes were evaluated by ELISA method. Differential cell counts including neutrophils, lymphocytes and monocytes in the peripheral blood were carried out with an automated electronic cell Coulter counter. Higher levels of specifc IgA, IgG and IgM antibodies were detected in the targated groups. Increased CD4+and CD8+T cells, highly expressed MHC I molecules and up-regulated MHC Ⅱ molecules were noticed during the early stages of infection. However, MHC Ⅱ molecules was down-regulated after the the early stages. High concentrations of IFN-y, IL-12, IL-10and IL-17were also observed. Higher concentrations of NO, iron and high activity of iNOS were also noticed in the infected groups. These findings might partially explain the resistance to T. gondii infection in chickens. Compared to the other3isolates, JS group showed significant high levels of CD4+and CD8+T cells, high levels of IFN-y, IL-12, IL-10, high concentrations of NO and high activity of iNOS. These might be related to the sources of the isolates origins. When compared with the results of mice, positive response of IgG antibody, up-regulated MHC II molecules at the early stage of infection and early expressed of IFN-y and IL-12might be the reasons for the sensitive differences of chicken and mouse when infected with T. gondii.