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.

弓形虫(Toxoplasma gondii)的PCR检测方法在笼养猕猴群中的应用

目的建立快速、灵敏、特异及检测结果易判断的PCR方法,并应用于大规模猕猴种群的弓形虫常规检测中。同时比较巢式PCR和单一PCR的一致性。方法根据弓形虫保守基因p30(SAG1)设计了内、外两对进行巢式PCR扩增以及B1基因设计一对引物进行单一PCR扩增,将DNA样本进行10倍倍比稀释,以检测巢式PCR反应的灵敏度;并对医学生物学研究所自繁猕猴共150只进行了弓形虫检测。结果巢式PCR检测法检测限度可达10-3ng/uL,而且方法特异。两种PCR法检测结果基本一致,其中巢式PCR检测阳性率(10%)稍高于单一PCR检测阳性率(8.67%)。结论巢式PCR和一次PCR方法都可应用于猕猴弓形虫的常规检测中,并提示巢式PCR比单一PCR更敏感、检出率更高。

刚地弓形虫(Toxoplasma gondii)不同地理株对钴^(60)丙线敏感性的比较研究

对中国NT株弓形虫和美国的ME—49株及TS—2株弓形虫的包囊进行了抗丙线耐受性的比较，试验采用上述三株弓形虫包囊为材料，分设0．4、0．5、0．6、0．7、1．OKGy丙线剂量组和不经照射处理的对照组。通过感染小鼠和幼猫的生物学检测，求得使包囊丧失感染性的丙线剂量，得出不同株包囊对丙线的敏感性。结果表明：丙线控制中国NT株的最小有效剂量为0．55KGy，控制ME—49株和TS—2株的感染性的最小有效剂量为0．60KGy。两者接近，提示这三个地理株的弓形虫包囊对钴60丙线的敏感性虽略有差异，但其差异并不明显。

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Research Progress in NTPase of Toxoplasma gondii and Other Protozoa

Nucleoside triphosphate hydrolase （NTPase） is a multifunctional enzymatic family widely existing in vivo. They can hydrolyze NTP to NMP or dNTP to dNMP to produce energy. In this article, the structure of Toxoplasma gondii NTPase is analyzed. The research progress in NT- Pase of Toxoplasrna gondii, Trypanosoma cruzi, Sarcocystis neurona and Neospora caninum was briefly reviewed.

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