Jilong Shen

Genotypes and Mouse Virulence of Toxoplasma gondii Isolates from Animals and Humans in China

Background Recent population structure studies of T. gondii revealed that a few major clonal lineages predominated in different geographical regions. T. gondii in South America is genetically and biologically divergent, whereas this parasite is remarkably clonal in North America and Europe with a few major lineages including Types I, II and III. Information on genotypes and mouse virulence of T. gondii isolates from China is scarce and insufficient to investigate its population structure, evolution, and transmission. Methodology/Principal Findings Genotyping of 23 T. gondii isolates from different hosts using 10 markers for PCR-restriction fragment length polymorphism analyses (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) revealed five genotypes; among them three genotypes were atypical and two were archetypal. Fifteen strains belong to the Chinese 1 lineage, which has been previously reported as a widespread lineage from swine, cats, and humans in China. Two human isolates fall into the type I and II lineages and the remaining isolates belong to two new atypical genotypes (ToxoDB#204 and #205) which has never been reported in China. Our results show that these genotypes of T. gondii isolates are intermediately or highly virulent in mice except for the strain TgCtwh6, which maintained parasitemia in mice for 35 days post infection although it possesses the uniform genotype of Chinese 1. Additionally, phylogenetic network analyses of all isolates of genotype Chinese 1 are identical, and there is no variation based on the sequence data generated for four introns (EF1, HP2, UPRT1 and UPRT7) and two dense granule proteins (GRA6 and GRA7). Conclusion/Significance A limited genetic diversity was found and genotype Chinese 1 (ToxoDB#9) is dominantly circulating in mainland China. The results will provide a useful profile for deep insight to the population structure, epidemiology and biological characteristics of T. gondii in China.

Genotyping of Toxoplasma gondii isolates from cats in different geographic regions of China.

Fourteen isolates of Toxoplasma gondii were isolated from cats from 4 different geographic provinces (Anhui, Hubei, Shanxi and Guangdong) in China and their genetic diversity with 8 nuclear loci SAG2, SAG3, BTUB, GRA6, L358, PK1, c22-8, c29-2, and an apicoplast locus Apico, was analysed by restriction fragment length polymorphisms (RFLPs). Two genotypes from these 14 isolates were identified but none of them belongs to the typical genetic types (types I, II and III). It is unexpected that such high similarity was observed in these 14 isolates although their original regions are significantly distant. Our results strongly indicate that the three traditional clonal lineages of types I, II and III of this parasite may not be preponderant in China. In addition, our results show that the genotypes of T. gondii in China may be highly clonal with atypical genotypes and higher virulence.

Impact of Schistosoma japonicum Infection on Collagen-Induced Arthritis in DBA/1 Mice: A Murine Model of Human Rheumatoid Arthritis

Background The hygiene hypothesis suggests that helminth infections prevent a range of autoimmune diseases. Methodology/Principal Findings To investigate the effects of S. japonicum infection on collagen-induced arthritis (CIA), male DBA/1 mice were challenged with unisexual or bisexual S. japonicum cercariae two weeks prior to bovine type II collagen (CII) immunization or at the onset of CIA. S. japonicum infection prior to CII immunization significantly reduced the severity of CIA. ELISA (enzyme linked immunosorbent assay) showed that the levels of anti-CII IgG and IgG2a were reduced in prior schistosome-infected mice, while anti-CII IgG1 was elevated. Splenocyte proliferation against both polyclonal and antigen-specific stimuli was reduced by prior schistosome infection as measured by tritiated thymidine incorporation (3H-TdR). Cytokine profiles and CD4+ T cells subpopulation analysis by ELISA and flow cytometry (FCM) demonstrated that prior schistosome infection resulted in a significant down-regulation of pro-inflammatory cytokines (IFN-γ, TNF-α, IL-1β and IL-6) and Th1 cells, together with up-regulation of the anti-inflammatory cytokine IL-10 and Th2 cells. Interestingly, the expansion of Treg cells and the reduction of Th17 cells were only observed in bisexually infected mice. In addition, prior schistosome infection notably reduced the expression of pro-inflammatory cytokines and receptor activator of NF-κB ligand (RANKL) in the inflamed joint. However, the disease was exacerbated at one week after infection when established CIA mice were challenged with bisexual cercariae. Conclusion/Significance Our data provide direct evidence that the Th2 response evoked by prior S. japonicum infection can suppress the Th1 response and pro-inflammatory mediator and that bisexual infection with egg-laying up-regulates the Treg response and down-regulates the Th17 response, resulting in an amelioration of autoimmune arthritis. The beneficial effects might depend on the establishment of a Th2-dominant response rather than the presence of the eggs. Our results suggest that anti-inflammatory molecules from the parasite could treat autoimmune diseases.

Reactive Oxygen Species-Triggered Trophoblast Apoptosis Is Initiated by Endoplasmic Reticulum Stress via Activation of Caspase-12, CHOP, and the JNK Pathway in Toxoplasma gondii Infection in Mice

ABSTRACT Toxoplasma gondii infection in pregnant women may result in abortion or in fetal teratogenesis; however, the underlying mechanisms are still unclear. In this paper, based on a murine model, we showed that maternal infection with RH strain T. gondii tachyzoites induced elevated production of reactive oxygen species (ROS), local oxidative stress, and subsequent apoptosis of placental trophoblasts. PCR array analysis of 84 oxidative stress-related genes demonstrated that 27 genes were upregulated at least 2-fold and that 9 genes were downregulated at least 2-fold in the T. gondii infection group compared with levels in the control group. The expression of NADPH oxidase 1 (Nox1) and glutathione peroxidase 6 (Gpx6) increased significantly, about 25-fold. The levels of malondialdehyde (MDA) and 8-hydroxydeoxyguanosine (8-OHdG) increased significantly with T. gondii infection, and levels of glutathione (GSH) decreased rapidly. T. gondii infection increased the early expression of endoplasmic reticulum stress (ERS) markers, followed by cleavage of caspase-12, activation of ASK1/JNK, and increased apoptosis of trophoblasts, both in vivo and in vitro. The apoptosis of trophoblasts, the activation of caspase-12 and the ASK1/JNK pathway, and the production of peroxides were dramatically inhibited by pretreatment with N-acetylcysteine (NAC). The upregulation of Nox1 was contact dependent and preceded the increase in levels of ERS markers and the activation of the proapoptosis cascade. Thus, we concluded that apoptosis in placental trophoblasts was initiated predominantly by ROS-mediated ERS via activation of caspase-12, CHOP, and the JNK pathway in acute T. gondii infection. Elevated ROS production is the central event in T. gondii-induced apoptosis of placental trophoblasts.

Differences in iNOS and Arginase Expression and Activity in the Macrophages of Rats Are Responsible for the Resistance against T. gondii Infection

Toxoplasma gondii infects humans and warm blooded animals causing devastating disease worldwide. It has long been a mystery as to why the peritoneal macrophages of rats are naturally resistant to T. gondii infection while those of mice are not. Here, we report that high expression levels and activity of inducible nitric oxide synthase (iNOS) and low levels of arginase-1 (Arg 1) activity in the peritoneal macrophages of rats are responsible for their resistance against T. gondii infection, due to high nitric oxide and low polyamines within these cells. The opposite situation was observed in the peritoneal macrophages of mice. This discovery of the opposing functions of iNOS and Arg 1 in rodent peritoneal macrophages may lead to a better understanding of the resistance mechanisms of mammals, particularly humans and livestock, against T. gondii and other intracellular pathogens.

Toxoplasma gondii infection in pregnant women in China

Toxoplasmosis, caused by the protozoan parasite Toxoplasma gondii, is one of the most common parasitic infections in humans. Primary infection in pregnant women can be transmitted to the fetus leading to miscarriage or congenital toxoplasmosis. Carefully designed nationwide seroprevalence surveys and case-control studies of risk factors conducted primarily in Europe and America, have shaped our view of the global status of maternal and congenital infection, directing approaches to disease prevention. However, despite encompassing 1 in 5 of the worlds population, information is limited on the status of toxoplasmosis in China, partly due to the linguistic inaccessibility of the Chinese literature to the global scientific community. By selection and analysis of studies and data, reported within the last 2 decades in China, this review summarizes and renders accessible a large body of Chinese and other literature and aims to estimate the seroprevalence in Chinese pregnant women. It also reviews the prevalence trends, risk factors, and clinical manifestations. The key findings are (1) the majority of studies show that the overall seroprevalence in Chinese pregnant women is less than 10%, considerably lower than a recently published global analysis; and (2) the few available appropriate studies on maternal acute infection suggested an incidence of 0·3% which is broadly comparable to studies from other countries.

Toxoplasma gondii prevalence in food animals and rodents in different regions of China: isolation, genotyping and mouse pathogenicity

BackgroundRecent studies of Toxoplasma gondii isolates from animals in different regions of China have shown a limited genetic diversity and type China 1 was the dominant genotype of T. gondii prevalent in Chinese animals. However, little has been known concerning the isolation and genotyping of T. gondii circulating in chickens, pigs and rodents in China. The aim of the study was to characterize samples of T. gondii isolates obtained from naturally infected cats, pigs and free-range chickens slaughtered for human consumption in China.MethodsIn the present study, brain tissues of 77 animals collected from different areas of China, including 24 free-range chickens (Gallus domesticus) , 13 voles (Rattus flavipectus), 23 pigs and 17 cats, were bioassayed in mice and viable T. gondii were isolated from the brains of eleven. These eleven T. gondii isolates were maintained in Kunming (KM) outbred mice and DNA isolated from tissues of infected mice was characterized using 11 PCR-restriction fragment length polymorphism (PCR-RFLP) markers: SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico, and CS3. Moreover, to determine mouse virulence of China 1 lineage of parasites, a TgCtgy5 genotype isolate was selected randomly and assessed in KM mice with different inoculation doses.ResultsResults of genotyping revealed that ten isolates were type China 1 (ToxoDB PCR-RFLP genotype #9), and TgCksz1 was a new genotype that was reported for the first time designated here as ToxoDB PCR-RFLP #225. No clonal types I, II and III lineages were found. DNA sequencing of four introns (EF1, HP2, UPRT1 and UPRT7) and two genes (GRA6 and GRA7) from representative isolates confirmed the results of PCR-RFLP genotyping. The TgCtgy5 isolate was highly virulent in KM mice; all infected mice died of acute toxoplasmosis, irrespective of the inoculation dose. The results indicate that mouse virulent isolates of T. gondii are predominantly circulating in cats in China.ConclusionsT. gondii isolated from chickens, pigs, cats and rodents in different locations in China were genotyped and the results reconfirmed the limited diversity of T. gondii in China and showed that type China 1 lineage was dominant in this country.

Prevalence and genotypes of Toxoplasma gondii in pork from retail meat stores in Eastern China

Pork is known as one of the most important sources of Toxoplasma gondii infection in China. In the present study, 416 fresh pork samples were collected from different locations of Anhui province, Eastern China. Tissue fluid ELISA was conducted to detect the antibodies to T. gondii. Real-time PCR and bioassay were performed to identify the presence of T. gondii DNA and viable parasites, respectively. Seventy-five out of 416 samples (18.03%) demonstrated real-time PCR positive reaction and 42 out of 416 samples (10.1%) showed tissue fluid ELISA positive reaction. One isolate (Tgpkfx171) was obtained through bioassay in mice from 14 samples that demonstrated both PCR and ELISA positive reaction. The isolate and seven positive DNA samples were genotyped using 9 PCR-RFLP markers including SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico. Among these, only the isolate and two positive DNA samples were genotyped with complete data for all loci, belonging to ToxoDB#9 (Chinese 1) and ToxoDB#213, respectively. This is the first report of the prevalence and genetic typing of T. gondii from pork in retail meat stores in China. The present results provide an accurate picture of the risk of exposure to T. gondii in retail pork in China.

Serological proteome-oriented screening and application of antigens for the diagnosis of Schistosomiasis japonica.

Schistosomiasis remains a major parasitic disease, with 200 million people infected and 779 million people at risk worldwide. The lack of reliable diagnostic techniques makes this disease difficult to control. In an attempt to discover useful candidates for the diagnosis of schistosomiasis, proteomics in combination with western blotting were employed in this study. This serological proteome assay yielded more than 30 immunodominant spots. Ten of these spots were precisely matched with a homologous two-dimensional electrophoresis (2-DE) gel and successfully identified by LC/MS-MS as corresponding to four different proteins. Of these proteins, SjLAP and SjFBPA were successfully expressed, and their recombinant protein products were further applied in the diagnosis of human Schistosomiasis japonica using ELISA. The ELISA results revealed sensitivities of 98.1% and 87.8% for acute and chronic schistosomiasis with rSjLAP and 100% and 84.7% with rSjFBPA, whereas the assays showed a specificity of 96.7% with both recombinant proteins. After treatment with praziquantel, the titres of the antibodies against both antigens declined significantly (P<0.001). Our data therefore suggest that these antibody-oriented recombinant proteins had a high efficacy for the diagnosis of S. japonica, and 2-DE based screening followed by LC/MS-MS has promising potential in the screening of candidate antigens for the diagnosis of schistosomiasis.

Meta-analysis of prevention and treatment of toxoplasmic encephalitis in HIV-infected patients

Toxoplasmic encephalitis (TE) is one of the most common central nervous system (CNS) opportunistic infections in HIV-infected patients. It can be prevented and treated through drug regimen. However, drugs have serious adverse effects sometimes. The purpose of this review is to determine the most effective therapy for TE in HIV-infected patients. Different primary prophylaxis and treatment regimens have been compared with regard to episodes of TE, clinical response, morbidity, and serious adverse events. In September 2012, we searched PubMed, Google Scholar, EMBASE, and CENTRAL (the Cochrane Central Register of Controlled Trials) database for randomized and quasi-randomized controlled trials of any drug regimen for primary prophylaxis and treatment of TE in HIV-infected patients. We independently extracted data and assessed eligibility and risk of bias using a standardized data collection form, and resolved any disagreement through discussion. We combined dichotomous outcomes using odds ratio (OR), presenting with 95% confidence interval (CI). Eleven trials were found to meet the inclusion criteria. Six trials compared trimethoprim-sulfamethoxazole (TMP-SMX) with dapsone-pyrimethamine (D-P) were analyzed together for the outcome of episodes of TE, morbidity, and serious adverse events. The two treatment arms did not differ for episodes of TE (OR=0.98; 95% CI: 0.48-2.00). Compared with D-P, TMP-SMX showed a beneficial trend in terms of mortality despite a lack of statistical significance (OR=0.75; 95% CI: 0.53-1.06). However, TMP-SMX is still associated with substantial toxicity and intolerance (OR=1.47; 95% CI: 0.91-2.38). Three trials compared pyrimethamine-sulfadiazine (P-S) with pyrimethamine-clindamycin (P-C) were analyzed together for the outcome of clinical response, morbidity, and serious adverse events. Compared with P-C, P-S showed a beneficial trend in terms of clinical response (OR=1.63; 95% CI: 1.05-2.51); P-S also showed a beneficial trend in terms of mortality despite a lack of statistical significance (OR=0.66; 95% CI: 0.37-1.17). However, P-S is still associated with substantial toxicity and intolerance (OR=3.08; 95% CI: 1.82-5.24). Two trials compared P-S with TMP-SMX were analyzed together for the outcome of clinical response, morbidity, and serious adverse events. The two treatment arms did not differ for clinical response (OR=0.90; 95% CI: 0.39-2.06). Compared with TMP-SMX, P-S showed a beneficial trend in terms of mortality despite a lack of statistical significance (OR=0.12; 95% CI: 0.01-1.39). However, P-S is still associated with substantial toxicity and intolerance (OR=2.91; 95% CI: 0.99-8.55). The available evidence fails to identify any one superior regimen for the primary prophylaxis and treatment of TE. The choice of therapy will often be directed by available therapy. Although current evidence does not allow a definitive recommendation, administration of TMP-SMX for primary prophylaxis and treatment of TE in patients with HIV infection is consistent with the available data.