Research Progress on the Association between Schizophrenia and Toxoplasma gondii Infection

Toxoplasma gondii(T.gondii or Tg),is an obligatory intracellular parasite with humans as its intermediate hosts.In recent years,significant correlations between T.gondii infection and schizophrenia have been reported,including the possible mediating mechanisms.Currently,mechanisms and hypotheses focus on central neurotransmitters,immunity,neuroinflammation,and epigenetics;however,the exact underlying mechanisms remain unclear.In this article,we review the studies related to T.gondii infection and schizophrenia,particularly the latest research progress.Research on dopamine(DA)and other neurotransmitters,the blood-brain barrier,inflammatory factors,disease heterogeneity,and other confounders is also discussed.In addition,we also summarized the results of some new epidemiological investigations.

Association of Host Interferon-γ Gene Polymorphism with Toxoplasma gondii Infection in Pregnant Women of Bangladesh

Human toxoplasmosis is caused by the intracellular protozoan parasite Toxoplasma gondii. Although T. gondii infection is generally asymptomatic for most of the immunocompetent adults, severe complications may occur particularly in pregnant women and immunocompromised individual. Host cell immunity plays a critical role in parasite differentiation and persistence in the host. Therefore, genetic polymorphism in the host immune genes, for instance interferon-γ gene could be linked with possibility of T. gondii infection. The objective of the study was to verify the link between the single nucleotide polymorphisms (SNPs) in the IFN-γ gene of pregnant women and T. gondii infection through correlating with anthropometric and sociodemographic parameters. In this study, ninety-two (N = 92) pregnant women (16 - 40 years) and healthy controls (N = 95) with similar age ranges were included. Among them, 25% (n = 23) pregnant women were seropositive for T. gondii IgG antibodies by Rapid Test Assay. Allelic and genotypic frequencies of IFN-γ +874T/A (rs2430561) SNPs were evaluated by using ARMS-PCR. The distribution of the A and T alleles in the specific position of the IFN-γ gene in the T. gondii-infected pregnant women and the control groups did not differ significantly, according to the data. However, we found a higher frequency (13.04%) of A/A genotype in T. gondii infected pregnant women as compared to non-infected individuals (8.70%), demonstrating that T. gondii infection susceptibility may be increased by homozygosity for the A allele. Further studies are to be needed to find out the link between host gene polymorphism and T. gondii infection in Bangladesh.

Seroprevalence of Toxoplasma gondii among AIDS Patients in Saudi Arabia

Background: Toxoplasma gondii (T. gondii) is an intracellular parasite mainly found in the central nervous system CNS, however, it can persist in multiple tissues in the body. Moreover, T. gondii is the commonest protozoans causing infections among individuals with acquired immunodeficiency syndrome (AIDS). Thus, the aim of this study was to investigate the frequency of T. gondii infection among AIDS patients in Makkah at Saudi Arabia. Methods: Fifty patients with AIDS proved to be positive by ULTRA HIV Ag-Ab Enzyme Immunoassay, and thirty healthy volunteers negative for AIDS by ULTRA HIV Ag-Ab Enzyme Immunoassay were subjected to determination of anti T. gondii immunoglobulin M (IgM) antibody seropositivity and anti T. gondii immunoglobulin G (IgG) antibody seropositivity using commercially available enzyme-linked immunosorbent assay kits. Results: The results showed that the seropositivity rate of anti T. gondii IgM antibodies among AIDS patients (18%) was significantly higher than in the healthy volunteers group (3.33%). Regarding the serum level of anti T. gondii IgG antibodies among AIDS patients, it was 30% significantly higher compared with those of the seropositive healthy volunteers (6.67%). Conclusions: These statistically significant results support the association between T. gondii infection and AIDS and suggest the usefulness of providing data for an educational program that will be designed to prevent T. gondii infection among AIDS patients.

Analysis Concentration of <i>Toxoplasma gondii</i>on Anti-Toxoplasma IgG-IgM Antibody Levels, and the Outcomes of Pregnancy in Mice Balb/c

Introduction: Toxoplasma gondii is an obligate intracellular protozoan parasite that can infect any warm blood vertebrae, and if first trimester pregnant woman infected, it may cause abortion. The objective is to prove the effect of the Toxoplasma gondii concentration in anti-toxoplasma IgG-IgM antibody levels, and the outcomes of Balb/c mice pregnancies. Materials and Methods: The study was conducted in Balb/c mice with inclusion criteria, and was conditioned pregnant. The pathogen strains of Toxoplasma gondii tachyzoite injected intraperitoneally. The blood samples were taken serially to be tested for anti-toxoplasma IgG-IgM antibody levels. After the mice were injected with tachyzoite, they are assessed every day to observe their body weight, vaginal bleeding, and labor. Anti-toxoplasma IgG-IgM antibody levels examined using qualitative mouse IgG-IgM antibody ELISA KIT. Results: Anti-toxoplasma IgM antibody levels increased significantly after 24 hours of injection tachyzoites in all dose groups, and remained high through day 21. Anti-toxoplasma antibody IgG levels increased significantly after 72 hours post injection and remained elevated until day 21. The incidence of abortion is 100% in mice which injected tachyzoite levels 1 × 103 and 1 × 104, and the incidence of abortion approximately 2 - 4 days post injection. 100% of mice that were injected with tachyzoites 1 × 101 and 1 × 102 have labor at term. Physical anomaly was found in baby mice from mice that were injected with tachyzoite 1 × 102. Conclusion: There is a significant correlation between the concentrations of Toxoplasma gondii tachyzoite with anti-toxoplasma IgG-IgM antibody levels, and there is a significant relationship between the concentrations of tachyzoite with abortion.

Challenging loop-mediated isothermal amplification(LAMP) technique for molecular detection of Toxoplasma gondii

Objective:To compare analytical sensitivity and specificity of a newly described DNA amplification technique.LAMP and nested PCR assay targeting the RE and Bl genes for the detection of Toxoplasma gondii(T.gondii) DNA.Methods:The analytical sensitivity of LAMP and ncstcd-PCR was obtained against 10-fold serial dilutions of T.gondii DNA ranging from 1 ng to 0.01 fg.DNA samples of other parasites and human chromosomal DNA were used to determine the specificity of molecular assays.Results:After testing LAMP and nesled-PCR in duplicate,the detection limit of RE-LAMP.B1-LAMP,RE-nested PCR and B1-nested PCR assays was one fg.100 fg,1 pg and 10 pg of T.gondii DNA respectively.All the LAMP assays and nested PCRs were 100% specific.The RE-LAMP assay revealed the most sensitivity for the detection of T.gondii DNA.Conclusions:The obtained results demonstrate that the LAMP technique has a greater sensitivity for detection of T.gondii.Furthermore,these findings indicate that primers based on the RE are more suitable than those based on the B1 gene.However,the B1-LAMP assay has potential as a diagnostic tool for detection of T.gondii.

Diagnosis of Toxoplasma gondii infection in pregnant women using automated chemiluminescence and quantitative real time PCR

Objective: To identify serodiagnosis and quantification of Toxoplasma gondii(T. gondii) infection among pregnant women in Salmas, northwest of Iran. Methods: In this crosssectional study, 276 blood samples were collected from pregnant women referred to the health care centers in Salmas city. The demographic variables were also recorded. Titers of antiToxoplasma IgM and IgG antibodies(Ab) were determined using the chemiluminescence immunoassay. Quantitative real-time PCR targeting the T. gondii repeated element gene was also performed on the blood sample. Results: Out of all, 19.92%(55/276) and 2.17%(6/276) patients were seropositive for anti-Toxoplasma IgG and IgM Ab, respectively. Moreover, the presence of T. gondii DNA was observed in 12.31%(34/276) blood samples. A significant relationship was observed between the IgG Ab seropositivity and contact with the cat as a risk factor(P=0.022). Conclusions: The seroprevalence rate of T. gondii infection in pregnant women is relatively low. Consequently, the seronegative pregnant women are at risk, and a considerable rate of positive blood samples for the presence of parasite's DNA should not be ignored. Besides, quantitative real-time PCR could be considered as an accurate method for diagnosis of acute toxoplasmosis especially when the precise results are of the most importance in pregnancy. Limiting contact with cats is also suggested for pregnant women.

Tissue tropism and parasite burden of Toxoplasma gondii RH strain in experimentally infected mice

Objective:To evaluate parasite distribution and tissue tropism of Toxoplasma gondii tachyzoites in experimentally infected mice using real time QPCR.Methods:In this survey 16 Balb/c mice were inoculated with 1×10~4 alive tachyzoites of Toxoplasma gondii RH strain.After 1,2,3 days post infection and the last day(before death),different tissues of mice including blood,brain,eye,liver,spleen,kidney,heart and muscle were harvested.Following tissues DNA extraction,the parasite burden was quantified using real time QPCR targeting the B1 gene(451 bp).Results:It showed that Toxoplasma after intraperitoneal injection was able to movement to various tissues in24 hours.Parasite burden was high in all tissues but the most number of parasites were observed in kidney,heart and liver,respectively.Conclusions:These data provide significant baseline information about Toxoplasma pathogenesis,vaccine monitoring and drug efficiency.

Investigation on the co-infections of Toxoplasma gondii with PRRSV, CSFV or PCV-2 in swine in part of China

The objective of the present investigation was to estimate the prevalence of Toxoplasma gondii infection and co-infection with porcine reproductive and respiratory syndrome virus(PRRSV), classical swine fever virus(CSFV) and porcine circovirus type 2(PCV-2) in pigs in China. A total of 372 tissues or serum samples collected from pigs distributed in 9 provinces/municipalities of China during the period from February 2011 to November 2012 were assayed for T. gondii antigens and antibodies using enzyme linked immunosorbent assay(ELISA) technique, while the PCR was designed for the detection of the PRRSV, CSFV and PCV-2, respectively. The total positive rate of T. gondii, PRSSV, CSFV and PCV-2 was 9.14%(34/372), 50.00%(186/372), 37.10%(138/372) and 3.23%(12/372), respectively. Among the 34 T. gondii positive samples, 26 samples were simultaneously infected with T. gondii and viruses, while the remaining eight samples were infected with T. gondii alone. In addition, the co-infection rate of T. gondii with PRSSV, T. gondii with PRSSV and CSFV, T. gondii with PRSSV and PCV-2, T. gondii with CSFV and PCV-2, T. gondii with PRSSV, CSFV and PCV-2 was 1.61%(6/372), 4.03%(15/372), 0.27%(1/372), 0.27%(1/372) and 0.81%(3/372), respectively. The results of the present survey revealed that PRRSV and CSFV were the common pathogens co-existing with porcine toxoplasmosis in China, and both of them could increase the chances of T. gondii infection in pig. This is the first report of T. gondii co-infections with viruses in pigs. It is very important to understand the interactions of parasite and virus, and can be used as reference data for the control and prevention of co-infections of T. gondii and viruses in pigs.

Isolation and Molecular Characterization of Toxoplasma gondii from Chickens in China

One strain of Toxoplasma gondii was successfully isolated from chickens in China by bioassay in mice. Antibodies and circulating antigens of T. gondii were assayed by the ELISA kits in 100 free range chickens from a rural area surrounding Funing, China. Fifty-three chickens were antibody-positive and 21 chickens were antigen positive. Hearts, brains, spleens, lungs, livers, and kidneys of 21 antibody or antigen-positive chickens were bioassayed in mice. One strain of T. gondii was isolated from 1 of 21 （4.76%） chickens. The isolated T. gondii killed all of the inoculated mice. Genotyping of this isolate using polymorphisms at the loci 5′-SAG2, 3′-SAG2, SAG3, cB21-4, L358, BTUB, and GRA6 revealed that it was Type I. These indicated that it was virulent for mice. This is the first report of isolation of T. gondii from chickens in China.

Biological role of surface Toxoplasma gondii antigen in development of vaccine

AIM： To analyze the biological role of the surface antigen of Toxoplasma gondii（Tgondii） in development of vaccine. METHODS： The surface antigen of Tgondii （SAG1） was expressed in vitro. The immune response of the host to the antigen was investigated by detection of specific antibody reaction to SAG1 and production of cytokines. Mice were immunized with recombinant SAG1 and challenged with lethal strain of Tgondii RH. The monoclonal antibody to r-SAG1 was prepared and used to study the effects of SAG1 on Tgondii tachyzoites under electromicroscope. RESULTS： The mice immunized with recombinant SAG1 delayed death for 60 h compared to the control group. The recombinant SAG1 induced specific high titer of IgG and IgM antibodies as well as IFN-y, IL-2 and IL-4 cytokines in mice. In contrast, IL-12, IL-6 and TNF-α were undetectable. When T gondii tachyzoites were treated with the monoclonal antibody to r-SAG1, the parasites were gathered together, destroyed, deformed, swollen, and holes and gaps formed on the surface. CONCLUSION： SAG1 may be an excellent vaccine candidate against T gondii. The immune protection induced by SAG1 against Tgondii may be regulated by both hormone- and cell-mediated immune response.

Toxoplasma gondii infection among chronic hepatitis C patients:A casecontrol study

Objective:To determine the detection rate of anti-Toxoplasma gondii(T.gondii) IgG and IgM in chronic HCV patients attending the Department of Tropical Medicine Mansoura University hospital in Egypt.Methods:This study included 120 adult chronic HCV patients.81 decompensate cirrhosis(late-stage)and 39 chronic HCV non cirrhotic patients(early-stage) and40 healthy blood donors as controls.Serum samples uere examined for anti-Toxoplasma IgM and anti-Toxoplasma IgG antibodies by ELISA.Real-time RT-polymerase chain reaction assay was done for quantitation of hepatitis C virus.Results:Anti-T.gondii IgG antibodies were detected in 75(92.6%) of 81 late-stage cirrhotic patients.30(76.9%) of the 39 chronic HCV non cirrhotic patients(early-Stage) and in 6(15ft) of 40 controls with statistically significant difference(P<0.001).Anti-T.gondii IgM antibodies were found in 11(13.6%) in late stage patients,5(12.8%)in early stage and in 3(7.5%) of controls with no statistical significant difference(P=0.610).There was no correlation between stage of fibrosis and IgM or IgG antibodies positivity in our studied groups(P=0.526).High IgG levels significantly correlated with high viral load(P=0.026).Conclusions:Our findings suggest that the serious opportunistic T.gondii infection represent a potential significant risk for chronic HCV patients.So.toxoplasmosis should be considered in their investigations and follow-up.

THE ULTRASTRUCTURE OF TOXOPLASMA GONDII TACHYZOITES AND THE EFFECT OF USNIC ACID ON IT

The normal fine structures of toxoplasma gondii tachyzoite and the erfect of usnic acid on the ultrastructure of the tachyzoites were observed by transmission electron microscope (TEM).The studies indicate that the pathological changes or the ultrastructure of the parasite took place under the effect of the 10 mg/L usnic acid for 30 min.These changes can be illustrated as folio'vs; 1)The posterior portion of the rhopties were destroyed.2)The membrane of the daughter cell fractured. 3)The membranate organellae or the organisms were demaged.

Infection of <i>Toxoplasma gondii</i>in Humans and Livestock Animals: An Emerging Silent Threat for Bangladesh

Toxoplasma gondii is an intracellular, zoonotic protozoan parasite that causes toxoplasmosis. It can potentially infect almost all mammalian and avian hosts including one-third of the human population world-wide. The major target group of the parasite includes immunocompromised patients (e.g. AIDS, cancer, organ transplantation) and fetus bearing pregnant women where it develops toxoplasmic encephalitis, myocarditis, chorioretinitis and abnormal fetal brain development or stillbirths respectively. In this review, we have presented the current status of T. gondii infection in livestock animals and human population in Bangladesh to assess the country-wide relative risk. Although exact prevalence is difficult to predict due to the scarcity of data, nevertheless existing literature suggests that 16% - 39% humans and 8% - 70% domestic animals are infected with T. gondii, which implies Bangladeshi population is at high risk of toxoplasmosis. Furthermore, we have proposed a potential area of research to decipher the genetic diversity and transmission routes of T. gondii infection into Bangladeshi population.

Influence of Toxoplasma gondii on in vitro proliferation and apoptosis of hepatoma carcinoma H7402 cell

Objective:To discuss the influence of tachyzoite of Toxoplasma gondii（T.gondii） RH strain on proliferation and apoptosis of hepatoma carcinoma（HCC） H7402 cell.Methods:The HCC H7402 cell in logarithmic phase and tachyzoite of T.gondii RH strain in different concentrations（1×107/mL,2×107/mL.4×107/mL,8×107mL and 16×107/mL） were co-cultured.CCK-8was utilized to determine the inhibition rate of T.gondii tachyzoite on H7402 cell growth.Flow cytometry was used to detect the change of cell cycle.RT-PCR method was used to detect the expression of cyclinB1 and cdc2-two genes related to cell cycle.Western blot method was used to detect the expression of apoptosis-related proteins Caspase-3 and Bcl-2.Results:The tachyzoite of T.gondii RH strain can inhibit the proliferation of HCC H7402 cells.The inhibition rate of tumor cell growth increased with the increase of concentration of T.gondii tachyzoite.With the increase of concentration of T.gondii tachyzoite,the proportion of G0/G1 phase of H7402 cell increased,the proportion of S phase decreased,and PI value decreased accordingly.The expression of cyclinB1 and cdc2 genes decreased with the increase of the concentration of T.gondii tachyzoite.With the increase of the concentration of tachyzoite of T.gondii RH strain,the expression quantity of Caspase-3 in H7402 cell increased,but the expression quantity of Bcl-2protein decreased.Conclusions:T.gondii can inhibit the in vitro proliferation of HCC H7402 cell,and induce its apoptosis.This effect shows a trend of concentration-dependent increase.Moreover,it is related to the down-regulation of cyclinB1 and cdc2（cell cycle-related genes）,the increase of apoptosis-related protein Caspase-3.and the decreasc of Bcl-2 expression.

Seroprevalence of Toxoplasma gondii infection in dogs and cats in Zhenjiang City,Eastern China

Objective:To determine the seroprevalence of Toxoplasma gondii(T.gondii) infection in dogs and cats in Zhenjiang City,Jiangsu Province.Eastern China,and to evaluate the main associated risk factors relating to exposure to 71 gondii in this region.Methods:Sera from 160 clogs and 116 cats from Zhenjiang City were tested for anti-T.gondii antibodies using EUSA.The seropositivity by area of activity,sex and age was analyzed.Results:Overall.21 dogs(13.l%) and 24 cats(20.7%) had antibodies to T.gondii.The infection rate in stray dogs(38.7%) and cats(28.6%! was significantly higher(P<0.05) than in household dogs(6.9%) and cats(18.2%).The seroprevalence in male clogs(14.8%) and cats(21.05%) were slighlly higher than their female counterparts(11.4%in dogs and 20.0%in cats),but were not significantly differenent(P>0.05).A high proportion of dogs at 3 to 6 years of age were positive to T.gondii(20.0%)while cats with relatively high seropositivity rates were at 0 to 1 year of age(33.3%).Conclusions:The prevalence of T.gondii infection in dogs and cats in Zhenjiang City was high,which is probably the main source of T.gondii infection in this area.

The level of chemerin and adipocyte fatty acid binding protein in Toxoplasma gondii seropositive obese individuals

Objective: To know the difference between chemerin and adipocyte fatty acid-binding protein(AFABP) levels in obese individuals with positive Toxoplasma gondii(T. gondii)immunoglobulin G(IgG) compared with negative T. gondii IgG.Methods: This study is a cross-sectional study by using consecutive sampling methods conducted from January to April 2013. The subjects were 57 obese individuals who were divided into obese group of positive and negative T. gondii IgG. The level of chemerin,AFABP and T. gondii IgG was done by ELISA. The data were analyzed by independent t test.Results: The results showed that the level of chemerin of positive T. gondii IgG group was significantly higher than the negative T. gondii IgG group [(70.0 ± 16.5) vs.(64.4 ± 16.1) pg/mL; P = 0.003], but there was not significant AFABP difference between seropositive and negative IgG groups [(83.6 ± 41.9) vs.(74.2 ± 36.7) pg/mL; P = 0.598].Conclusions: It can be concluded that the level of chemerin of seropositive T. gondii IgG was higher than that in the negative T. gondii IgG group.

Toxoplasma gondii infection can induce retinal DNA damage: an experimental study

AIM:To detect whether Toxoplasma gondii(T.gondii)infection of mice can induce retinal DNA damage.METHODS:A total of 20 laboratory-bred male Swiss albino mice were used and divided into four groups:control group(non-infected animals);T.gondii infected group;immunosuppressed infected group;and infected group treated with sulfadiazine and pyrimethamine.Mice eyes were collected 6wk post infection and retinas were obtained.Each retina was immediately processed for comet assay and the frequency of tailed nuclei(DNA damage)was calculated.In addition,retinal DNA damage was revealed by various comet assay parameters that were provided by the image analysis software including tail length,percentage of DNA in the tail,percentage of tailed cells and tail moment.RESULTS:The obtained results showed that T.gondii infection induced a statistically significant increase in the frequency of tailed nuclei,tail length,percentage of DNA in the tail,and tail moment in mice retinal cells compared to the control group(which showed some degree of DNA damage).In immunosuppressed infected group,retinal DNA damage was severing and there was significant increase in various comet assay parameters compared to both control and infected groups.After treatment with sulfadiazine and pyrimethamine,retinal DNA damage decreased and all comet assay parameters showed a statistical significant decrease compared to infected groups.CONCLUSION:T.gondii infection can induce DNA damage in mice retinal cells.

Expression of p30, the major surface antigen of Toxoplasma gondii, in baculovirus-insect cell system and the evaluation of immune response induced by p30

Objective: To produce the major surface antigen (p30) of Toxoplasma gondii from the Baculovirus Expression System. Methods: The p30 coding sequence was cloned into a transfer vector, then the recombinant baculovirus containing p30 gene was cloned and purified by the co-transfection and plaque assay. The expression and immunoactivity of the recombinant p30 were analyzed by SDS-PAGE and Western blot. The immune responses in mice for being immunized with recombinant p30 were tested. Results: About 750μg of purified (95% purity) p30 was obtained from a culture of 108 in- sect Sf21 cells. Mice in injected with the recombinant protein produced specific humoral and cellular immune responses. Immunization with p30 also prolonged the period of mice survival infected by Toxoplasma gondii. Conclusion: It is indicated that the recombinant p30 from baculovirus expression system can stimulate mice to produce effective protection from Toxo- plasma gondii infection.

Amplification,cloning,sequencing and expression of the gene encoding the major surface antigen of Toxoplasma gondii isolated in China

According to the published gene sequence of the major surface antigen (P30) of Toxoplasma gondii, a pair of primers were designed and synthesized. Using polymerase chain reaction (PCR), the coding sequences of P30 gene were amplified from a Chinese strain of T. gondii, The amplified gene fragment and plasmid pB220 were digested with EcoRI and BamHI and then ligated. The inserted gene fragment was sequenced by the chain termination method, the reading reveals that nucleotide sequence determined was the same as the P30 sequecne of RH strain pubilished by Burg (1988), except that one base was changed. The recombinant plasmid containing P30 gene was transformed to E. coli DH5α.After temperature inducing culture, the total cellular proteins were analysed by SDS-PAGE and Western blot. The results show that the p30 gene cloned into the plasmid could express in E. coli, and the expression product had immunogenicity.

Expression of P30 gene of Toxoplasma gondii in Spodoptera frugiperda cell and Argyrogramma agnata larva

The gene encoding the major surface antigen(P30) of Toxoplasrna gondii was cloned into a transfer plasmid vector pSXIVVI￣+X3,then the recombinant plasmid pSXIVVI￣+X3-P30 DNA and the parent virus TnNPV DNA were used to cotransfect the cultured Spodoptera f