Functional analyses of Toxoplasma gondii dihydroorotase reveal a promising anti-parasitic target.

Toxoplasma gondii relies heavily on the de novo pyrimidine biosynthesis pathway for fueling the high uridine-5'-monophosphate (UMP) demand during parasite growth. The third step of de novo pyrimidine biosynthesis is catalyzed by dihydroorotase (DHO), a metalloenzyme that catalyzes the reversible condensation of carbamoyl aspartate to dihydroorotate. Here, functional analyses of TgDHO reveal that tachyzoites lacking DHO are impaired in overall growth due to decreased levels of UMP, and the noticeably growth restriction could be partially rescued after supplementation with uracil or high concentrations of L-dihydroorotate in vitro. When pyrimidine salvage pathway is disrupted, both DHOH35A and DHOD284E mutant strains proliferated much slower than DHO-expressing parasites, suggesting an essential role of both TgDHO His35 and Asp284 residues in parasite growth. Additionally, DHO deletion causes the limitation of bradyzoite growth under the condition of uracil supplementation or uracil deprivation. During the infection in mice, the DHO-deficient parasites are avirulent, despite the generation of smaller tissue cysts. The results reveal that TgDHO contributes to parasite growth both in vitro and in vivo. The significantly differences between TgDHO and mammalian DHO reflect that DHO can be exploited to produce specific inhibitors targeting apicomplexan parasites. Moreover, potential DHO inhibitors exert beneficial effects on enzymatic activity of TgDHO and T. gondii growth in vitro. In conclusion, these data highlight the important role of TgDHO in parasite growth and reveal that it is a promising anti-parasitic target for future control of toxoplasmosis.

A global phosphoproteomics analysis of adult Fasciola gigantica by LC-MS/MS.

Protein phosphorylation plays key roles in a variety of essential cellular processes. Fasciola gigantica is a tropical liver fluke causing hepatobiliary disease fascioliasis, leading to human health threats and heavy economic losses. Although the genome and protein kinases of F. gigantica provided new insights to understand the molecular biology and etiology of this parasite, there is scant knowledge of protein phosphorylation events in F. gigantica. In this study, we characterized the global phosphoproteomics of adult F. gigantica by phosphopeptide enrichment-based LC-MS/MS, a high-throughput analysis to maximize the detection of a large repertoire of phosphoproteins and phosphosites. A total of 1030 phosphopeptides with 1244 phosphosites representing 635 F. gigantica phosphoproteins were identified. The phosphoproteins were involved in a wide variety of biological processes including cellular, metabolic, and single-organism processes. Meanwhile, these proteins were found predominantly in cellular components like membranes and organelles with molecular functions of binding (51.3%) and catalytic activity (40.6%). The KEGG annotation inferred that the most enriched pathways of the phosphoproteins included tight junction, spliceosome, and RNA transport (each one contains 15 identified proteins). Combining the reports in other protozoa and helminths, the phosphoproteins identified in this work play roles in metabolic regulation and signal transduction. To our knowledge, this work performed the first global phosphoproteomics analysis of adult F. gigantica, which provides valuable information for development of intervention strategies for fascioliasis.

Seroprevalence of Toxoplasma gondii infection in the endangered Père David's deer (Elaphurus davidianus) in China.

Père David's deer (Elaphurus davidianus) is an emblematic endangered species and regarded as a national treasure, toxoplasmosis is a serious zoonotic parasitic disease for wild animals. Little is known about the prevalence of antibodies to this parasite in Père David's deer. In this study, sera from 43 wild Père David's deer, from Dafeng nature reserve China were tested for antibodies to Toxoplasma gondii by MAT. The investigation showed that antibodies to toxoplasma were detected in 8 of 43 (18.60%, 95% CI 6.97-30.24) samples. Seroprevalence ranged from 15.00% to 21.74% between the different genders, but the difference was not significant according to SPSS analysis (P > 0.05). This report of seroprevalence of antibodies to T. gondii in Père David's deer provides basic data of T. gondii infection data, which is important for controlling and preventing toxoplasmosis in Père David's deer.

First report on the prevalence of Fasciola hepatica in the endangered Père David's deer (Elaphurus davidianus) in China.

BACKGROUND: Fasciola hepatica is an important zoonotic parasite that causes fasciolosis in a broad range of animals. No information is available about the prevalence of F. hepatica in Père David's deer (Elaphurus davidianus), an endangered species in the world. Therefore, the purpose of the study was to evaluate the prevalence of fasciolosis in Père David's deer in the Dafeng Elk National Natural Reserve, Jiangsu province, China. RESULTS: In this study, 142 fecal samples from Père David's deer were analyzed for F. hepatica by microscopy and nest-PCR. Only one sample was positive for F. hepatica according to microscopy examination, while 18 of 142 (12.68, 95%CI: 2.841-22.45%) samples were positive for F. hepatica according to nest-PCR results. CONCLUSIONS: This is the first report of prevalence of F. hepatica in Père David's deer. The prevalence data indicated that F. hepatica was also present in this endangered animal, which may cause a potential threat to this precious species.

Evaluation of protective immunity induced by recombinant calcium-dependent protein kinase 1 (TgCDPK1) protein against acute toxoplasmosis in mice.

Toxoplasma gondii is an intracellular zoonotic parasite that causes toxoplasmosis, which can cause economic losses and serious public health problems worldwide. A member of the T. gondii calcium-dependent protein kinases family, TgCDPK1 was recently identified as an essential regulator of exocytosis in T. gondii, and participated in direct parasite motility, host-cell invasion and egress. In the present study, the protective immunity of recombinant TgCDPK1 protein (rTgCDPK1) was evaluated against acute toxoplasmosis in mice. rTgCDPK1 were expressed and purified, BABL/c mice were intraperitoneally immunized with rTgCDPK1 and challenged with the highly virulent RH strain of T. gondii. The specific immune responses were analyzed by measuring the cytokine and serum antibody, and lymphocyte proliferation assays, flow cytometry of lymphocytes and the survival curve were employed to evaluate the protective efficacy. From the results we found that special humoral and cellular responses could be elicited in vaccine mice, and higher level of IgG antibody, and the significant increased levels of Th1-type cytokines IFN-γ, IL-12 (p70), IL10 and CD3+CD4+CD8- and CD3+CD8+CD4- T cells could also be detected comparing to control mice (P < 0.05). All vaccinated mice prolonged survival time (14.90 ±â€¯2.89 days) challenge with 1000 tachyzoites of RH, while the control mice died within 8 days. These results indicated that TgCDPK1 protein was a potential vaccine candidate against acute toxoplasmosis.

Seroprevalence and risk assessment of Toxoplasma gondii in Java sparrows (Lonchura oryzivora) in China.

BACKGROUND: Toxoplasma gondii, an intracellular zoonotic parasite, infects all mammalian and birds. Understanding the prevalence of Toxoplasma in bird is important for evaluating the transmission of this parasite. No information about the seroprevalence of T. gondii in Java sparrows (Lonchura oryzivora) is available. RESULTS: In this study, from 2014 to 2015, 350 serum samples from Java sparrows were collected in Beijing and Shangqiu, Henan province, and the antibodies against T. gondii were evaluated with MAT. The seroprevalence in Java sparrows was 34.29% (CI95% 29.31-39.26). A phenomenon of seropositivity tended to increase with age were observed, but the difference is not significant. The prevalence was significant different in gender and color, which could be risk factors. CONCLUSIONS: This study firstly reported T. gondii seroprevalence in Java sparrows, which extended the host range of T. gondii. Java sparrows may pose significant transmission medium, accelerating the spread of T. gondii diffusion.

Proteomic analysis of Fasciola gigantica excretory and secretory products (FgESPs) interacting with buffalo serum of different infection periods by shotgun LC-MS/MS.

Fasciolosis, caused by Fasciola hepatica and Fasciola gigantica, is an important zoonotic disease in the world. It affects livestock, especially for sheep and cattle, causing major economic loss due to morbidity and mortality. Although the excretory and secretory products (ESPs) of F. hepatica have been relatively well studied, little is known about the interaction between the ESP and host, and the mechanism of the key proteins involved in interaction. In this study, buffaloes were infected by Fasciola gigantica, and infection serum was collected at three different periods (42dpi, 70dpi, and 98dpi). The interaction proteins were pulled down with three different period serum by Co-IP assay, respectively, and then identified by LC-MS/MS analysis. A number of proteins were identified; some of them related to the biological function of the parasite, while most of them the functions were unknown. For the annotated proteins, 13, 5, and 7 proteins were pulled down by the infected serum in 42dpi, 70dpi, and 98dpi, respectively, and 18 proteins could be detected in all three periods. Among them, 13 belong to the cathepsin family, 4 proteins related to glutathione S-transferase, and 3 proteins are calcium-binding protein; other proteins related to catalytic activity and cellular process. This study could provide new insights into the central role played by ESPs in the protection of F. gigantica from the host immune response. At the same time, our research provided material for further studies about the interaction between F. gigantica and host.

Live Attenuated Pru:Δcdpk2 Strain of Toxoplasma gondii Protects Against Acute, Chronic, and Congenital Toxoplasmosis.

Background: The threat of Toxoplasma gondii infection in immunocompromised individuals and pregnant women necessitates the development of a safe and effective vaccine. Here, we examined the immune protection conferred by a live attenuated strain of T. gondii. Methods: We tested the efficacy of intraperitoneal vaccination using 500 Ca2+-dependent protein kinase 2 (cdpk2)-deficient tachyzoites of T. gondii Pru strain against acute, chronic, and congenital toxoplasmosis in mice. The kinetics of antibody response, cytokines, and other quantifiable correlates of protection against T. gondii infection were determined. Results: Vaccination with Pru:Δcdpk2 induced a high level of anti-T. gondii immunoglobulin G titer, type 1 T-helper (Th1) response at 28 days postvaccination, and a mixed Th1/type 2 T-helper response at 70 days postvaccination. All vaccinated mice survived a heterologous challenge with 1000 tachyzoites of RH or ToxoDB#9 (PYS or TgC7) strains. Also, vaccination protected against homologous infection with 20 T. gondii Pru cysts, and improved pregnancy outcome by reducing parasite cyst load in the brain, maintaining litter size and body weight of pups born to vaccinated dams challenged with 10 Pru cysts compared to pups born to unvaccinated dams. Conclusions: The use of T. gondii Pru:Δcdpk2 mutant strain represents a promising approach to protection against acute, chronic, and congenital toxoplasmosis in mice.

Evaluation of protective immunity induced by DNA vaccination with genes encoding Toxoplasma gondii GRA17 and GRA23 against acute toxoplasmosis in mice.

Toxoplasma gondii, an obligatory intracellular protozoan, can cause serious public health problems and economic losses worldwide. Two novel dense granule proteins (GRA17 and GRA23) were recently identified as T. gondii-secreted proteins which are localized to the parasitophorous vacuole membrane (PVM) and can mediate the movement of small molecules between the host cell and parasitophorous vacuole (PV). In the present study, we evaluated the protective immunity induced by DNA vaccination with genes encoding GRA17 and GRA23 against acute toxoplasmosis in mice. Eukaryotic expressing plasmids pVAX-TgGRA17 and pVAX-TgGRA23 were constructed. Then, BALB/c mice were intramuscularly immunized with pVAX-TgGRA17, pVAX-TgGRA23, or pVAX-TgGRA17 + pVAX-TgGRA23 followed by challenge infection with the highly virulent RH strain of T. gondii. The specific immune responses and protective efficacy against T. gondii were examined by cytokine and serum antibody measurements, lymphocyte proliferation assays, flow cytometry of lymphocytes and the survival time after challenge. Our results showed that mice immunized with pVAX-TgGRA17, pVAX-TgGRA23, or pVAX-TgGRA17 + pVAX-TgGRA23 induced specific humoral and cellular responses, with higher level of IgG antibody, increased levels of Th1-type cytokines IFN-γ and IL-12 (p70), and CD3+CD4+CD8- and CD3+CD8+CD4- T cells, as well as prolonged survival time (9.1 ± 0.32 days for pVAX-TgGRA17, 10.8 ± 0.79 days for pVAX-TgGRA23, and 12.6 ± 2.55 days for pVAX-TgGRA17 + pVAX-TgGRA23) compared to the blank control (7.11 ± 0.33 days), PBS control (7.22 ± 0.44 days), and pVAX I control (7.11 ± 0.33 days). These results demonstrated that both TgGRA17 and TgGRA23 are potential vaccine candidates, TgGRA23 has a better immunogenicity, and co-immunization of pVAX-TgGRA17 and pVAX-TgGRA23 induces better protective efficacy.

Proteomic analysis of Fasciola hepatica excretory and secretory products (FhESPs) involved in interacting with host PBMCs and cytokines by shotgun LC-MS/MS.

Fasciola hepatica is a helminth parasite with a worldwide distribution, which can cause chronic liver disease, fasciolosis, leading to economic losses in the livestock and public health in many countries. Control is mostly reliant on the use of drugs, and as a result, drug resistance has now emerged. The identification of F. hepatica genes involved in interaction between the parasite and host immune system is utmost important to elucidate the evasion mechanisms of the parasite and develop more effective strategies against fasciolosis. In this study, we aimed to identify molecules in F. hepatica excretory and secretory products (FhESPs) interacting with the host peripheral blood mononuclear cells (PBMCs), Th1-like cytokines (IL2 and IFN-γ), and Th17-like cytokines (IL17) by Co-IP combined with tandem mass spectrometry. The results showed that 14, 16, and 9 proteins in FhESPs could bind with IL2, IL17, and IFN-γ, respectively, which indicated that adult F. hepatica may evade the host immune responses through directly interplaying with cytokines. In addition, nine proteins in FhESPs could adhere to PBMCs. Our findings provided potential targets as immuno-regulators, and will be helpful to elucidate the molecular basis of host-parasite interactions and search for new potential proteins as vaccine and drug target candidates.

Vaccination with Toxoplasma gondii calcium-dependent protein kinase 6 and rhoptry protein 18 encapsulated in poly(lactide-co-glycolide) microspheres induces long-term protective immunity in mice.

BACKGROUND: Toxoplasmosis is a worldwide zoonosis caused by the intracellular parasite Toxoplasma gondii. However, no effective vaccine is yet available. Poly(lactide-co-glycolide) polymers can reduce protein degradation and sustain the release of antigens over a long period, which could generate a long-lasting immune response in vivo. Using a mouse model of toxoplasmosis, we evaluated the protective efficacy of vaccination with two recombinant proteins, which are formulated in biodegradable polymers. METHODS: Two recombinant proteins, rCDPK6 and rROP18, were encapsulated in poly(D,L-lactide-co-glycolide) (PLG), and then injected subcutaneously into Kunming mice. The mice immune responses were evaluated in terms of lympho-proliferation, cytokine expression, and antibodies. The survival of infected mice and brain cyst formation were also evaluated at 6 weeks after challenge with T. gondii RH strain (genotype I) or PRU strain (genotype II). RESULTS: Both protein vaccines induced Th1-biased immune responses, with increased specific antibodies and T cells, high levels of interferon-γ and interleukin 2, and strong lymphocyte proliferative responses. The mice immunized with the various protein vaccines survived slightly longer time than the control groups (P > 0.05) after injection with T. gondii RH strain. There were fewer brain cysts in the mice in all the immunized groups than that in the control groups, and the brain cysts were significantly reduced in mice immunized with proteins + 206, rCDPK6 + PLG and rCDPK6 + rROP18 + PLG (P < 0.05) compared controls. Further comparison of the immune responses to the proteins adjuvanted with PLG or Montanide™ ISA 206 VG 6 weeks after the last immunization revealed that antigens encapsulated in PLG conferred greater protective immunity against challenge. CONCLUSIONS: These findings suggest that the two recombinant T. gondii proteins encapsulated in PLG conferred immunity to T. gondii for an extended period, providing the foundation for the further development of a commercial vaccine against toxoplasmosis.

Evaluation of the basic functions of six calcium-dependent protein kinases in Toxoplasma gondii using CRISPR-Cas9 system.

Toxoplasma gondii, an important protozoan parasite, infects almost all warm-blooded animals and humans. Although treatments in T. gondii are limited by the lack of effective drugs, some calcium-dependent kinases were demonstrated as the promising drug targets to chemotherapy against T. gondii due to their essential roles in T. gondii and absence from their hosts. The objectives of the present study were to investigate the functions of six calcium-dependent protein kinases (CDPK4, CDPK4A, CDPK5, CDPK6, CDPK8, and CDPK9) in T. gondii to assess whether they are suitable for designing as drug targets. We used the CRISPR-Cas9 system to disrupt six CDPK genes successfully by insertion of DHFR* at the guide RNA-targeted region in the six endogenous CDPK loci and successfully obtained the six knockout (KO)-CDPK strains. The biological characteristics of the six strains were evaluated by plaque assays, invasion, egress, replication, and virulence assays, respectively. The results indicated that there was no significant difference between the six KO-CDPK strains and wild-type strain in virulence and the lytic cycle including invasion, egress, and replication. The conclusion was the six CDPKs are not essential for T. gondii lytic cycle and also not virulence factors for mice, suggesting that the six CDPKs may participate in other functions in T. gondii.

TgERK7 is involved in the intracellular proliferation of Toxoplasma gondii.

Toxoplasma gondii uses a unique mechanism to fulfill its asexual life cycles by which the parasite can infect all the warm-blooded animals including humans. Mitogen-activated protein kinase (MAPK) or extracellular signal-regulated kinase (ERK) pathway widely existed in eukaryotic cells mediates the conversion of environmental stimuli to intracellular events such as proliferation and differentiation. Their counterparts have been identified in Apicomplexan parasites such as ERK7 in T. gondii. To confirm whether the unique mechanism of T. gondii is relevant to MAPK/ERK member, we created a mutant (ΔTgERK7) in GT1 tachyzoites using double homologous recombination method. Our results of virulence evaluation showed 100 % survival of all the ΔTgERK7-infected mice until 35 days post-challenge compared to no survival in wild-type GT1-infected group (10.6 ± 0.34 days). Furthermore, lower parasite loads were detected in the peritoneal fluid of ΔTgERK7-infected mice (P < 0.05). To ensure whether or not ERK7 gene knockout leads to the growth deficiency of T. gondii, the intracellular proliferation of ΔTgERK7 was also examined in vitro. Our data indicated that the proliferation of ΔTgERK7 parasites was significantly prolonged in comparison with wild-type GT1 tachyzoites (P < 0.05). Therefore, we concluded that TgERK7 is important for the intracellular proliferation of T. gondii, which further emphasized that MAPK/ERK derived from T. gondii participates in the regulation of the asexual life cycles to ensure the survival and reinfections of this parasite.

Transcriptomic analysis of global changes in cytokine expression in mouse spleens following acute Toxoplasma gondii infection.

Toxoplasma gondii is a global pathogen that infects a wide range of animals and humans. During T. gondii infection, the spleen plays an important role in coordinating the adaptive and innate immune responses. However, there is little information regarding the changes in global gene expression within the spleen following T. gondii infection. To address this gap in knowledge, we examined the transcriptome of the mouse spleen following T. gondii infection. We observed differential expression of 2310 transcripts under these conditions. Analysis of KEGG and GO enrichment indicated that T. gondii alters multiple immune signaling cascades. Most of differentially expressed GO terms and pathways were downregulated, while immune-related GO terms and pathways were upregulated with response to T. gondii infection in mouse spleen. Most cytokines were upregulated in infected spleens, and all differentially expressed chemokines were upregulated which enhanced the immune cells chemotaxis to promote recruitment of immune cells, such as neutrophils, eosinophils, monocytes, dendritic cells, macrophages, NK cells, basophils, B cells, and T cells. Although IFN-γ-induced IDO (Ido1) was upregulated in the present study, it may not contribute a lot to the control of T. gondii because most differentially expressed genes involved in tryptophan metabolism pathway were downregulated. Innate immunity pathways, including cytosolic nucleic acid sensing pathway and C-type lectins-Syk-Card9 signaling pathways, were upregulated. We believe our study is the first comprehensive attempt to define the host transcriptional response to T. gondii infection in the mouse spleen.

Genetic Characterization of Toxoplasma gondii Isolates from Pigs in Jilin Province, Northeastern China.

Toxoplasma gondii is prevalent in humans and animals worldwide. The present study aimed to determine the genetic diversity of T. gondii isolates from pigs in Jilin province, northeastern China. A total of 100 DNA samples were extracted from the hilar lymph nodes of slaughtered pigs, and 9 (9.0%, 95% confidence interval: 3.4-14.6%) were detected positive for T. gondii B1 gene by a nested polymerase chain reaction (PCR). The positive DNA samples were typed at 11 genetic markers, including 10 nuclear loci (SAG1, 5'-SAG2, and 3'-SAG2, alternative SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, and PK1) and an apicoplast locus (Apico) using the multilocus PCR-restriction fragment length polymorphism technology. Only three isolates were completely typed at all loci, showing that they all belonged to the clonal type I. One isolate was typed at five loci, including 5' +3'-SAG2, SAG2, SAG3, GRA6, and L358, revealing the possible clonal type I. This is the first report of the genetic characterization of T. gondii isolates in pigs in Jilin province, northeastern China, which has implications for better understanding the population structure of T. gondii infection in China.

Seroprevalence and risk factors of Toxoplasma gondii in Tibetan Sheep in Gansu province, Northwestern China.

BACKGROUND: Toxoplasma gondii, a protozoan parasite, infects almost all warm-blooded animals and humans. Limited information is available about T. gondii infection in Tibetan Sheep in Gansu province, northwestern China. In the present study, we estimated the seroprevalence and risk factors of T. gondii infection in this region of China. RESULTS: A total of 1732 Tibetan Sheep were included from Tianzhu and Maqu in Gansu province. Antibodies to T. gondii were examined by modified agglutination test (MAT), and 352 (20.3%) out of 1732 Tibetan sheep were found positive. Multivariate logistic regression analysis was used to analyze the risk factors associated with seroprevalence, the results showed that age, gender, and numbers of past pregnancies were not the significant risk factors. However, Tibetan sheep in Maqu had a 1.64 times (odds ratio [OR] =1.637, 95% CI =1.291-2.075, P < 0.001) higher seroprevalence compared to Tianzhu, and the seropositivity in summer were 1.61 times (OR =1.608, 95% CI =1.122-2.303, P = 0.010) higher compared to Tibetan sheep in winter, followed by 1.42 times (OR =1.419, 95% CI =1.002-2.011, P = 0.049) in spring. Thus, season and location were considered as risk factors associated with T. gondii infection in this study. CONCLUSIONS: This is the first report of T. gondii seroprevalence in Tibetan sheep in Gansu province, which enriches the epidemiological data of T. gondii infection in Tibetan sheep in China. The results of this study indicate that Tibetan sheep in Gansu province are frequently exposed to T. gondii, posing a direct threat to the public health as well as to local sheep industry. These data is useful to strengthen future prevention and control of T. gondii infection in Tibetan sheep in this region.

Seroprevalence and risk factors of Chlamydia abortus infection in free-ranging white yaks in China.

BACKGROUND: Chlamydia is gram-negative obligate bacteria which causes a wide variety of diseases in humans and animals. To date, there are a few reports about the seroprevalence of Chlamydia and the risk factors associated with Chlamydia infection in yaks in the world. In this study, 974 blood samples were collected from white yaks (Bos grunniens) in Tianzhu Tibetan Autonomous County, Gansu province, northwest China from June 2013 to April 2014. RESULTS: Antibodies against Chlamydia abortus were examined by the indirect hemagglutination (IHA) test, and 158 of 974 (16.22%) white yaks were seropositive for C. abortus antibodies at the cut-off of 1:16. The risk factors associated with seroprevalence were evaluated by a multivariate logistic regression analysis. Region, gender and age of white yak were left out of the final model, due to its insignificance in the logistic regression analysis (P > 0.05). However, season was considered as a major risk factor associated with C. abortus infection in white yaks. CONCLUSIONS: To our knowledge, this is the first survey of C. abortus seroprevalence in white yaks in China, which extends the host range for C. abortus and has important implications for public health and the local Tibetan economy.

Rhoptry protein 47 gene sequence: A potential novel genetic marker for population genetic studies of Toxoplasma gondii.

Toxoplasma gondii, an obligate intracellular parasite, is able to infect many animal species and humans, and can cause toxoplasmosis of the host. In this study, we examined sequence variation in rhoptry protein 47 (ROP47) gene among T. gondii isolates originating from different hosts and geographical regions. The entire genome region of the ROP47 gene was amplified and sequenced, and phylogenetic relationship was reconstructed using maximum parsimony (MP), maximum likelihood (ML) and neighbor-joining (NJ), based on the ROP47 gene sequences. The results of sequence alignments showed that all ROP47 gene sequences were 396 bp in length. There were 19 variable nucleotide positions in the coding region, resulted in 16 amino acid substitutions (12.21%) among all examined T. gondii strains and the existence of polymorphic restriction sites for endonucleases SacI and AflIII, allowing the differentiation of the three major clonal lineage types I, II and III by PCR-RFLP. Phylogenetic analysis of ROP47 gene sequences showed that three major clonal lineage types I, II and III were clustered differently, consistent with PCR-RFLP results. These results suggest that ROP47 gene sequence may represent a potential novel genetic marker for population genetic studies of T. gondii isolates.

Genetic Diversity of Toxoplasma gondii Strains from Different Hosts and Geographical Regions by Sequence Analysis of GRA20 Gene.

Toxoplasma gondii is a eukaryotic parasite of the phylum Apicomplexa, which infects all warm-blood animals, including humans. In the present study, we examined sequence variation in dense granule 20 (GRA20) genes among T. gondii isolates collected from different hosts and geographical regions worldwide. The complete GRA20 genes were amplified from 16 T. gondii isolates using PCR, sequence were analyzed, and phylogenetic reconstruction was analyzed by maximum parsimony (MP) and maximum likelihood (ML) methods. The results showed that the complete GRA20 gene sequence was 1,586 bp in length among all the isolates used in this study, and the sequence variations in nucleotides were 0-7.9% among all strains. However, removing the type III strains (CTG, VEG), the sequence variations became very low, only 0-0.7%. These results indicated that the GRA20 sequence in type III was more divergence. Phylogenetic analysis of GRA20 sequences using MP and ML methods can differentiate 2 major clonal lineage types (type I and type III) into their respective clusters, indicating the GRA20 gene may represent a novel genetic marker for intraspecific phylogenetic analyses of T. gondii.

Comparative proteomic analysis of different Toxoplasma gondii genotypes by two-dimensional fluorescence difference gel electrophoresis combined with mass spectrometry.

Toxoplasma gondii is a protozoan parasite infecting almost all warm-blooded animals and humans. There are three infective stages of T. gondii: the tachyzoites, the bradyzoites, and the oocysts. The tachyzoite is a rapidly multiplying stage and the main pathogenic factor. In North America and Europe, T. gondii is consisted of four major clonal lineages (namely Types I, II, III, and Type 12). In this study, we explored the proteomic profiles of different genotypes (Type I-RH strain, Type II-PRU strain, Type II-TgQHO strain, and ToxoDB 9-TgC7 strain) of T. gondii tachyzoites by using 2D DIGE combined with MALDI-TOF MS. Totally, 110 differentially abundant protein spots were selected. Of these, 98 spots corresponding to 56 proteins from T. gondii were successfully identified. These included surface antigen (SAG1), heat shock protein 70 (Hsp 70), disulfide isomerase, coronin, heat shock protein 60 (Hsp 60), pyruvate kinase, receptor for activated C kinase 1, and peroxiredoxin. Gene ontology enrichment analysis revealed that most of the differentially abundant proteins were involved in biological regulation, metabolic process, response to stress, binding, antioxidant activity, and transporter activity. According to the KEGG metabolic pathway maps of T. gondii, some identified proteins were involved in the glycolytic/gluconeogenesis pathway. The present study identified differentially abundant proteins among different genotypes of T. gondii and these findings have implications for the better understanding of the phenotypic differences among the examined T. gondii genotypes, which in turn may contribute to the better control of toxoplasmosis.