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.

TOX介导T细胞耗竭的机制及其逆转研究

胸腺细胞选择相关的高迁移率族蛋白(TOX)是一种与DNA结合的转录因子,参与免疫细胞的发育过程。TOX是促进T细胞耗竭的关键转录因子,并与肿瘤的发生发展密切相关,提示TOX可能是潜在的免疫生物标志物和恶性肿瘤免疫治疗的靶点。本文就TOX介导T细胞耗竭的分子机制及其逆转研究的最新进展做一综述,为设计基于TOX更为精准的肿瘤免疫治疗策略提供依据。

Toxoplasma gondii adhesion and apoptosis of chicken erythrocytes

Toxoplasma gondi is thought to infect all nucleated cells in warm-blooded animals,including poultry,mammals,and humans.However,it is unclear whether T.gondi can infect chicken erythrocytes due to the nucleated nature of these cells.Due to the special role of chicken erythrocytes in innate immunity,we investigated the cell-cell interaction between T.gondi and erythrocytes to elucidate the role of chicken erythrocytes in T.gondi infection.Cellular apoptosis was analyzed by transwell assay and flow cytometry.An immunofluorescence method was used to examine the reorganization of vimentin during T.gondi infection in both Vero cells and chicken erythrocytes.The reorganization of actin was evaluated to further examine the invasion capacity of tachyzoites on chicken erythrocytes during infection.We discovered that T.gondi can adhere to but not invade chicken erythrocytes and eventually cause apoptosis in chicken erythrocytes.When tachyzoites were cocultured with chicken erythrocytes in vitro,the transcrip-tional levels of T.gondi MIC3,ROP16,and ROP18 were significantly decreased.In addition,the rearrangement of host cell vimentin,a type Il cytoskeleton protein regulated by T.gondii infection,was not observed.Similarly,the parasite-induced ring-shaped actin structure was not formed in the host-parasite junction.T.gondi(RH strain)tachyzoites pref-erentially invaded Vero cells and replicated in chicken blood monocytes,but they were not found in chicken erythro-cytes.These findings showed that although T.gondi could attach to the surface of chicken erythrocytes,but couldn't invade successfully.Interestingly,we found that the T.gondii secretome,lysates,and intact tachyzoites could cause apoptosis of chicken erythrocytes,which suggested a complex mechanism involved in the apoptosis of chicken erythrocytes induced by T.gondi.This study elucidated that T.gondi could not infect nucleated chicken erythrocytes and enriched our understanding of the transmission mechanism of T.gondii among avian species.

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.

Toxoplasma ROP16Ⅰ/Ⅲ ameliorated inflammatory bowel diseases via inducing M2 phenotype of macrophages

BACKGROUND Inflammatory bowel disease（IBD） is characterized by chronic and non-specific inflammation of the intestinal mucosa and mainly includes ulcerative colitis and Crohn’s disease.AIM To explore the beneficial effect of Toxo ROP16Ⅰ/Ⅲ-induced M2 phynotype macrophages in homeostasis of IBDs through downregulation of M1 inflammatory cells.METHODS RAW264.7 macrophages stimulated by lipopolysaccharide（LPS）（M1 cells） were co-cultured with Caco-2 cells as an inflammatory model of IBD in vitro.The expression of Toxo ROP16Ⅰ/Ⅲ was observed in RAW264.7 macrophages that were transfected with p EGFP-rop16Ⅰ/Ⅲ.The phenotypes of M2 and M1 macrophage cells were assessed by quantitative real-time reverse transcriptase polymerase chain reaction and the expression of tumor necrosis factor（TNF）-α,interleukin（IL）-1β,IL-6,transforming growth factor（TGF）-β1,IL-10,inducible nitric oxide synthase（i NOS）,and arginase-1（Arg-1） was detected.The expression of i NOS,Arg-1,signal transducer and activator of transcription 3（Stat3）,p-Stat3,Stat6,pStat6,programmed death ligand-2（PD-L2）,caspase-3,-8,and-9 was analyzed by Western blotting,and Griess assays were performed to detect nitric oxide（NO）.TNF-α,IL-1β,IL-6,TGF-β1,and IL-10 expression in the supernatants was detected by enzyme-linked immunosorbent assay,and Caco-2 cell apoptosis was determined by flow cytometry after mixing M1 cells with M2 cells in a Caco-2 cell co-culture system.RESULTS M1 cells exhibited significantly increased production of i NOS,NO,TNF-α,IL-1β,and IL-6,while Toxo ROP16Ⅰ/Ⅲ induced macrophage bias to M2 cells in vitro,showing increased expression of Arg-1,IL-10 and TGF-β1 and elevated production of p-Stat3 and p-Stat6.The mixed M1 and M2 cell culture induced by Toxo ROP16 Ⅰ/Ⅲ exhibited decreased production of NO and i NOS and upregulated expression of Arg-1 and PD-L2.Accordingly,Caco-2 cells became apoptotic,and apoptosis-associated proteins such as caspase-3,-8 and-9 were dampened during co-culture of M1 and M2 cells.Flow cytometry analysis showed that co-culture of M1 cells with Caco-2 cells facilitated the apoptosis of Caco-2 cells,but co-culture of M1 and M2 cells alleviated Caco-2 cell apoptosis.CONCLUSION Toxo ROP16 Ⅰ/Ⅲ-induced M2 macrophages inhibited apoptosis of Caco-2 cells caused by M1 macrophages.This finding may help gain a better understanding of the underlying mechanism and represent a promising therapeutic strategy for IBDs.

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.

Sero-prevalence and associated risk factors of Toxoplasma gondi infection among pregnant women attending antenatal care at Felege Hiwot Referral Hospital, northwest Ethiopia

Objective: To determine the prevalence of toxoplasmosis and to assess the possible risk factors associated with the infection among pregnant women attending antenatal care center at Felege Hiwot Referral Hospital, Bahir Dar town, northwest Ethiopia. Methods: A hospital based cross-sectional study was designed to determine the prevalence of toxoplasmosis among pregnant women. Three hundred eighty four serum samples were collected from November 2013 to January 2014. Data on socio-demographic and predisposing factors were collected from each study participant with simple random sampling technique. The serum samples were examined for anti- Toxoplasma gondii(T. gondii) antibodies using latex agglutination test. Results: The overall seroprevalence of T. gondii among the pregnant women was 18.5%. All of T. gondii positive cases found to be positive only for Ig G antibody. Significant association was observed between seroprevalence and presence of domestic cats [AOR=2.85, 95% CI: 1.66-4.90, P=0.000], consumption of raw or undercooked meat [AOR=1.98, 95% CI: 1.15-2.43, P=0.014] and history of abortion [AOR=2.47, 95% CI: 1.40-4.34, P=0.002]. No significant association was observed between seroprevalence and socio-demographic characters, gestational age, gravidity, consumption of raw vegetable, and blood transfusion. Conclusions: The seroprevalence of toxoplasmosis among pregnant women in Bahir Dar town was relatively high. Presence of domestic cats at home and consumption of raw or undercooked meat were identified as main risk factors for T. gondii infection. Therefore, health education towards avoiding eating raw or undercooked meat and avoiding contact with cats are recommended for prevention of miscarriage or defects during pregnancy.

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.

Toxoplasma encephalitis and AIDS in a patient with seizure and altered mental status:A case report

INTRODUCTIONIncreasing incidences of neurological complications are being encountered with the increase in the incidence of human immunodeficiency virus （HIV）. These can be due to the direct involvement of the central nervous system （CNS） by the virus or due to other opportunistic infections.

Seroprevalence of IgG and IgM anti-Toxoplasma antibodies in HIV/AIDS patients,northern Iran

ObjectiveTo determine the seroprevalence of anti-Toxoplasma gondii (T. gondii) IgG and IgM antibodies in HIV/AIDS patients and uninfected subjects.MethodsThis cross sectional survey was carried out on 78 healthy and 62 HIV+/AIDS individuals in northern Iran between September 2007 and October 2008. Five mL of blood samples were collected from each person in case and control groups. Determination of CD4+ counts was performed by flow cytometry. The serum separated from blood samples was evaluated by conventional ELISA technique to determine the presence of antibodies to T. gondii.ResultsForty eight out of 62 (77.4%) HIV/AIDS serum samples were found positive for anti-T. gondii IgG antibody, compared with 59 among 78 (75.6%) HIV negative samples from the same area (P > 0.05). Six out of 62 (9.7%) HIV+/AIDS patients showed anti-T. gondii IgM antibody in their serum samples, compared with 7 among 78 (9%) HIV negative samples (P > 0.05). The mean of CD4+ counts in HIV+/AIDS was (430.8±182.3) cells/μL and in control group was (871.0±243.3)% cells/μL (P<0.01). CD4+ estimation in 5 (11.1%) of HIV+/AIDS patients was <200 cells/μL (P < 0.0001).ConclusionsSeroprevalence of latent toxoplasmosis in HIV patients is high, therefore the prevention of toxoplasmic encephalitis, administration of primary prophylaxis with co-trimoxazole to all HIV+/AIDS patients are necessary.

Type I strain of Toxoplasma gondii from chicken induced different immune responses with that from human,cat and swine in chicken

In this study,four strains of Toxoplasma gondii with the same genetic type（Type I） originated from chicken,human,cat and swine were used to compare the immune responses in resistant chicken host to investigate the relationships between the parasite origins and the pathogenicity in certain host.A total of 300,10-day-old chickens were allocated randomly into five groups which named JS（from chicken）,CAT（from cat）,CN（from swine）,RH（from human） and a negative control group（—Ve） with 60 birds in each group.Tachyzoites of four different T.gondii strains（JS,CAT,CN and RH） were inoculated intraperitoneally with the dose of 1×10~7 in the four designed groups,respectively.The negative control（-Ve） group was mockly inoculated with phosphate-buffered saline（PBS） alone.Blood and spleen samples were obtained on the day of inoculation（day 0） and at days 4,11,25,39 and 53 post-infection to screen the immunopathological changes.The results demonstrated some different immune characters of T.gondii infected chickens with that of mice or swine previous reported.These differences included up-regulation of major histocompatibility complex class Ⅱ（MHC Ⅱ） molecules in the early stage of infection,early peak expressions of interleukin（IL）-12（IL-12） and-10（IL-10） and long keep of IL-17.These might partially contribute to the resistance of chicken to T.gondii infection.Comparisons to chickens infected with strains from human,cat and swine,chickens infected with strain from chicken showed significant high levels of CD4~＋ and CD8~＋ T cells,interferon gamma（IFN-γ）,IL-12 and IL-10.It suggested that the strain from chicken had different ability to stimulate cellular immunity in chicken.

Introduction of protocols for mass production of Toxoplasma gondii tachyzoites of the genotype II PRU strain

Background:Few investigations of genotype II of Toxoplasma gondii,the most preva-lent form of the Toxoplasma parasite in humans,have been carried out on due to the rapid conversion of tachyzoites to bradyzoites in its life cycle.The current study aimed to create animal and in vitro models for production of the tachyzoites of the Prugniaud(PRU)genotype II strain.Methods:To develop an immunocompromised model and obtain tachyzoites of the PRU strain,BALB/c mice were orally treated with dexamethasone(10 mg/kg),cyclo-phosphamide(36 mg/kg),and cyclosporine(18 mg/kg)from 5 days prior to inocula-tion.Then,10-15 tissue cysts of PRU strain were inoculated intraperitoneally into the mice.The tachyzoites obtained from mice were then cultivated in a HeLa cell culture.The resulting yield of tachyzoites was cryopreserved in 92%fetal calf serum,8%dimethyl sulfoxide.The infectivity of these tachyzoites was evaluated using in vivo and in vitro examinations.Results:Numerous tachyzoites were observed in the peritoneal fluid of the immuno-suppressed mice within 10-15 days after inoculation,and many tachyzoites were har-vested from the HeLa cell culture.Trypan Blue staining showed 80%viability of the tachyzoites recovered from cryopreservation and this was confirmed by HeLa cell culture.In addition,mice infected intraperitoneally with the recovered tachyzoites presented with cysts in the brain after 2 months.Conclusion:We have developed an animal model for mass production of T.gondii tachyzoites of the PRU strain.This method can provide fresh viable tachyzoites of Toxoplasma gondii for use as and when required in future investigations.