Screening Of Drugs For Treating Toxoplasma Gondii Infection In Chicken And Identification And Functional Study Of Proteins Binding To Mouse Macrophages By Shotgun In Excretory/Secretory Antigens Of T.Gondii

Toxoplasma gondii(T.gondii)is an obligate intracellular parasite that is capable of infecting a variety of mammals and birds and causing toxoplasmosis.Almost one-third of people throughout the world are infected by this parasite.Human toxoplasmosis can be life-threatening in immunocompromised individuals and has been associated with psychiatric disorders,beside it could cause severe congenital pathologies,spontaneous abortion,or stillbirth.Economically,toxoplasmosis is also important,since the infection during pregnancy,especially in sheep and pigs,often results in abortion,representing considerable economic loss.Chicken is considered as one resistant host of T.gondii.Toxoplasmosis in chickens has been not received adequate attention because of the inapparent infection.However,epidemiological investigations worldwide indicated that the seropositive rate of T.gondii infection was very high in free-range chickens.As chickens are one of the most important meat resources for humans,the high infection and long persistence of T.gondii indicated that ingestion of contaminated chicken meat might be a risk factor for T.gondii infections in humans and other animals.Currently,studies on toxoplasmosis in chickens mainly were focused on epidemiological investigations and genotypic analysis.However,the relationships between the age of the chicken and the infection of T.gondii were rarely reported.Meanwhile,the drug treatment for T.gondii infections in chickens was rarely reported.To find the most suitable age for the establishment the model of T.gondii infections in chickens,and on this basis,then to screen the drugs for treating T.gondii infection in chicken,which will be beneficial to the development of model and control the T.gondii infections in chickens.This research mainly focuses on the following:1.Comparision on pathogenicity of two different sources of Toxoplasma gondii isolates to different ages' chickensThis experiment was conducted to investigate the pathogenicity of Toxoplasma gondii on broiler chickens with different age.Tachyzoites of RH and JS strain were injected intraperitoneally with dose of 1×108 to 7,14,21 and 28-day-old chickens,respectively.The negative control group was mockly inoculated with physiological saline alone.After that,the clinical signs of each animal were checked everyday post inoculation and the death number of chickens were recorded.Serum samples were obtained on the day of inoculation(day 0)and at days 4,11,18,25,32,39,46 and 53 post-infection to screen the T.gondii circulating antigens(TCA)and antibodies(TCAb)using enzyme linked immunosorbent assay(ELISA)technique.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 day 53 by PCR method.Histopathological sections were used to observe the pathological changes in the died chickens during acute phage and the living animals at day 53.The results indicated that T.gondii infection at 7-day-old chickens mostly could cause acute death,the mortality(100%)of JS strain was significantly higher than the RH strain(70%).14-day-old chickens only showed several slight clinical symptoms,but no death.Neither clinical symptoms nor death were found at 21 and 28-day-old chickens.In conclusion,the inoculated age has an important effect on the pathogenicity of T.gondii,and the smaller age,the stronger the pathogenicity of T.gondii to chickens.Chicks aged from 7 to 14-day-old can be used to establish the model of acute toxoplasmosis of chicken,while 14 to 28-day-old chicks can be used to establish a model of chronic toxoplasmosis of chicken.2.Screening of drugs for treating Toxoplasma gondii infection in chickenFor the purpose of screening the effective drugs on chicken toxoplasmosis,preliminary therapeutic experiments were conducted on the ICR mice infected with Toxoplasma gondii.Based on these results,three drugs or compounds of drugs including high,middle and poor therapeutic ones on mouse toxoplasmosis were choosed to evaluate their effectivenesses on chicken toxoplasmosis.In this study,1×104 tachyzoites of RH and JS strain of T.gondii were intraperitoneally inoculated in mice,while 1×108 tachyzoites of JS strain were infected i.p with chickens.Four hours later,all the target animals were administrated orally with different drugs.All these drugs were given once a day and treated for 5 days consecutively.After that,the clinical signs of each animal were checked everyday and the survival time was recorded.The experiment was ended 20 days post the infection for mice and 10 days for chickens.According to the survival rate and the average survival time,all these drugs were evaluated for their therapeutic effects.The results shown that the most effective therapy was the combination of Sulfaclozine Sodium and Trimethoprim on the mice,and Azithromycin was also effective.However,the other drugs had no siginificantly effectivenesses.When compared the effects of anti-T.gondii infection on chickens,both Azithromycin and the combination of Sulfaclozine Sodium and Trimethoprim could notablely improve the survival rate of infected chickens.The survival rate(44.4%)of former was slighly higher than that of the latter(33.3%),but no significantly differences were found between these two groups.In conclusion,Azithromycin and the combination of Sulfaclozine Sodium and Trimethoprim were the effective drugs for the treatments of chicken toxoplasmosis.Toxoplasma gondii excretory/secretory antigens secretion began in the early stage of invasion and play key roles in the host cell invasion.It has been widely used for the diagnosis of toxoplasmosis because of its good immunogenicity.Previous studies confirmed that excretory/secretory antigens from T.gondii could triggered strong humoral and cellular responses,and induced effective protection in mice against acute T.gondii infection.Furthermore,ESA also plays an important role in antitumor.From now on,the research on regulation effects of T.gondii ESA on the host cells,in particular for macrophages was rarely reported.To identify new proteins binding to the host cells and to participate in the invasion of the cell and to modulate the cell functions,which will help to illuminate the molecular mechanisms of T.gondii pathogenesis.So,the research work was sequentially carried as follows:3.Effects of excretory/secretory antigens from T.gondii on the celluar functions of murine macrophages in vitroIn this study,we evaluated the modulating effects of excretory/secretory antigens produced by Toxoplasma gondii on murine macrophage cell line Ana-1.Mouse macrophage Ana-1 cells were incubated with different concentrations of ESA.The cell viability was determined by an CCK-8 assay.The phagocytotic assay,cell apoptosis and the expression of toll-like receptor 2 and 4 were analyzed by flow cytometric analysis.The ELISA assay was used to examine the expression of interleukin-1?(IL-1?),interleukin-10(IL-10),tumor necrosis factor-a(TNF-a),transforming growth factor-?1(TGF-?1)of macrophages Ana-1 after incubation with ESA.The results showed that ESA decreased the viability of Ana-1 cells and induced apoptosis in a dose dependent manner.Phagocytotic assay by FITC-dextran internalization showed that ESA inhibited the phagocytosis of macrophages.Moreover,incubation with ESA downregulated the expression of pro-inflammatory cytokines tumor necrosis factor-?(TNF-?)and interleukin-1?(IL-1?),and upregulated the expression of anti-inflammatory cytokines interleukin-10(IL-10)and transforming growth factor-?1(TGF-?1)in the culture supernatant of Ana-1 cells.Incubation with ESA also inhibited the activation of Toll-like receptor 2 and 4,and the production of Nitric oxide(NO).ESA from T.gondii inhibited nuclear translocation of NF-?B in LPS-induced Ana-1 macrophages.Taken together,these results suggested that ESA produced by T.gondii regulate the celluar functions of murine macrophage cell line Ana-1.4.Identification of proteins binding to mouse macrophages by shotgun in Excretory/secretory antigens of Toxoplasma gondiiThe purpose of this study was to screen the Toxoplasma gondii invasion related or immunomodulatory proteins binding to mouse macrophages(Ana-1 cell)after incubation with Excretory/secretory antigens(ESA).Ana-1 cells were incubated with ESA and the binding was investigated by an immunofluorescence assay.The ESA which binded to Ana-1 cells were collected by Co-Immunoprecipitation assay and identified employing shotgun LC-MS/MS.Results showed that ESA could bind to the surfaces of Ana-1 cells.Total 109 proteins of T.gondii were identified from T,gondii protein database by using SEQUEST searches.Fifty-seven proteins were annotated according to Gene Ontology Annotation in terms of molecular function,biological process,and cellular localization.Out of 57 annotated proteins,40 proteins(70.2%)had binding activity and 31 proteins(54.4%)had catalytic activity,which might be related to the process of host cell invasion or modulation of the host celluar functions by T.gondii.The findings in this investigation provided an insight into the interactive relationship between T.gondii and the host macrophages,and will shed new lights on the understanding of molecular mechanisms of T.gondii pathogenesis.5.Molecular cloning,expression and immunoprotection of elongation factor-1 alpha and 10 kDa excretory-secretory antigen from Toxoplasma gondiiIn this study,we cloned full-length cDNA encoding Toxoplasma gondii elongation factor-1 alpha(TgEF-1?)and 10 kDa excretory-secretory antigen(TgESA10)from the cDNA of RH tachyzoites,respectively.The open reading frame(ORF)of the TgEF-1?gene was 1347 base pairs(bp)encoding a protein of 448 amino acids,the ORF of the TgESA10 gene was 390 base pairs(bp)encoding a protein of 129 amino acids.TgEF-la and TgESA10 were subcloned into pET-32a(+)and expressed in Escherichia coli(E.coli)BL21(DE3).Passive immunization of mice with anti-rTgEF-la or anti-rTgESA10 polyclonal antibody following challenge with a lethal dose of tachyzoites significantly increased survival time compared with PBS control group.The survival time of mice challenged with tachyzoites pretreated with anti-rTgEF-la or anti-rTgESA10 PcAb were significantly increased.Invasion of tachyzoites into macrophages was significantly inhibited in the anti-rTgEF-la and anti-rTgESA10 PcAb pretreated group.Mice vaccinated with rTgEF-la or rTgESA10 induced a high level of specific anti-T.gondii antibodies and production of IFN-gamma,interleukin-4.The expression levels of MHC-I and MHC-II molecules as well as the percentages of CD4+ and CD8+ T cells in mice vaccinated with rTgEF-la or rTgESA10 was significantly increased respectively(P<0.05),compared with all the controls.Immunization with rTgEF-la significantly(P<0.05)prolonged survival time(14.53±1.72 days)while immunization with rTgESA10 significantly(P<0.05)prolonged survival time(14.13±1.63 days)after challenge infection with the virulent T.gondii RH strain.These results indicate that T.gondii EF-1?and ESA 10 play essential roles in mediating host cell invasion by the parasite and,as such,could be candidate vaccine antigens against toxoplasmosis.6.Protective immunity against acute toxoplasmosis in BALB/c mice induced by a DNA vaccine encoding Toxoplasma gondii elongation factor-1 alpha(TgEF-la)or 10 kDa excretory-secretory antigen(TgESA10)In the present study,DNA vaccine(pVAX-EF-1?)encoding T.gondii EF-1?(TgEF-1?)gene and DNA vaccine(pVAX-ESA10)encoding T.gondii 10 kDa excretory-secretory antigen(TgESA10)were constructed and the immune response and protective efficacy of this vaccine against lethal challenge in BALB/c mice were evaluated,respectively.Both mice inoculated with pVAX-EF-1?or pVAX-ESA10 vaccine induced a high level of specific anti-T.gondii antibodies and production of IFN-gamma,interleukin-4 and interleukin-17.The expression levels of MHC-?and MHC-?molecules as well as the percentages of CD4+ and CD8+ T cells in mice vaccinated with pVAX-EF-1?and pVAX-ESA 10 were significantly increased respectively(P<0.05),compared with all the controls(blank control,PBS,and pVAX).Immunization with pVAX-EF-1?and pVAX-ESA10 significantly(P<0.05)prolonged survival time(14.1±1.7 days)and(14.3±1.7 days)after challenge infection with the virulent T.gondii RH strain,compared with the control groups which died within 8 days.These results suggested that DNA vaccines pVAX-EF-1?and pVAX-ESA10 triggered strong humoral and cellular responses,and induced effective protection in mice against acute T.gondii infection,indicating that TgEF-la and TgESA10 are promising vaccine candidates against acute toxoplasmosis.7.Effects of four recombinant proteins of T.gondii on the celluar functions of murine macrophages in vitroIn this study,we cloned the full-length cDNA encoding T.gondii calcium-dependent protein kinase 1(TgCDPK1)and 14-3-3 protein(Tg14-3-3)and purified the recombinant proteins TgCDPKl and Tg14-3-3,and evaluated the modulating effects of four recombinant proteins of T.gondii on murine macrophage cell line Ana-1.Mouse macrophage Ana-1 cells were incubated with recombinant proteins of different concentrations.The cell viability was determined by an CCK-8 assay.The phagocytotic assay,cell apoptosis and the expression of toll-like receptor 2 and 4 were analyzed by flow cytometric analysis.The ELISA assay was used to examine the expression of interleukin-1?(IL-1?),interleukin-10(IL-10),tumor necrosis factor-?(TNF-?),transforming growth factor-?1(TGF-?1)of macrophages Ana-1 after incubation with recombinant proteins.The results showed that rTgl4-3-3,rTgCDPK1 and rTgESA10 of high doses decreased the viability of Ana-1 cells and induced apoptosis,while rTgEF-la had no significant effect on them.Phagocytotic assay by FITC-dextran internalization showed that rTg 14-3-3 and rTgCDPK1 inhibited the phagocytosis of macrophages,rTgESA10 increased the phagocytosis of macrophages,while rTgEF-1?had no significant effect on phagocytosis.Moreover,rTgEF-1?,rTg14-3-3,rTgCDPK1 and rTgESA10 of high doses upregulated the expression of pro-inflammatory cytokines tumor necrosis factor-?(TNF-?)and interleukin-1?(IL-1?),and anti-inflammatory cytokines interleukin-10(IL-10)and transforming growth factor-?1(TGF-?1)in the culture supernatant of Ana-1 cells.All of the recombinant proteins could activate the Toll-like receptor 2(TLR2)except individual dose group,while rTgESA10 and rTgEF-la activated the TLR4.Taken together,these results suggested that four recombinant proteins of T.gondii regulate the celluar functions of murine macrophage cell line Ana-1 in different patterns.