Sequence Diversity in MIC6 Gene among Toxoplasma gondii Isolates from Different Hosts and Geographical Locations

Toxoplasma gondii is an opportunistic protozoan parasite that can infect almost all warm-blooded animals including humans with a worldwide distribution. Micronemes play an important role in invasion process of T. gondii, associated with the attachment, motility, and host cell recognition. In this research, sequence diversity in microneme protein 6 (MIC6) gene among 16 T. gondii isolates from different hosts and geographical regions and 1 reference strain was examined. The results showed that the sequence of all the examined T. gondii strains was 1,050 bp in length, and their A + T content was between 45.7% and 46.1%. Sequence analysis presented 33 nucleotide mutation positions (0-1.1%), resulting in 23 amino acid substitutions (0-2.3%) aligned with T. gondii RH strain. Moreover, T. gondii strains representing the 3 classical genotypes (Type I, II, and III) were separated into different clusters based on the locus of MIC6 using phylogenetic analyses by Bayesian inference (BI), maximum parsimony (MP), and maximum likelihood (ML), but T. gondii strains belonging to ToxoDB #9 were separated into different clusters. Our results suggested that MIC6 gene is not a suitable marker for T. gondii population genetic studies.     

Recent Advances in Toxoplasma gondii Immunotherapeutics

Toxoplasmosis is an opportunistic infection caused by the protozoan parasite Toxoplasma gondii. T. gondii is widespread globally and causes severe diseases in individuals with impaired immune defences as well as congenitally infected infants. The high prevalence rate in some parts of the world such as South America and Africa, coupled with the current drug treatments that trigger hypersensitivity reactions, makes the development of immunotherapeutics intervention a highly important research priority. Immunotherapeutics strategies could either be a vaccine which would confer a pre-emptive immunity to infection, or passive immunization in cases of disease recrudescence or recurrent clinical diseases. As the severity of clinical manifestations is often greater in developing nations, the development of well-tolerated and safe immunotherapeutics becomes not only a scientific pursuit, but a humanitarian enterprise. In the last few years, much progress has been made in vaccine research with new antigens, novel adjuvants, and innovative vaccine delivery such as nanoparticles and antigen encapsulations. A literature search over the past 5 years showed that most experimental studies were focused on DNA vaccination at 52%, followed by protein vaccination which formed 36% of the studies, live attenuated vaccinations at 9%, and heterologous vaccination at 3%; while there were few on passive immunization. Recent progress in studies on vaccination, passive immunization, as well as insights gained from these immunotherapeutics is highlighted in this review.     

Sterculic Acid and Its Analogues Are Potent Inhibitors of Toxoplasma gondii

Toxoplasmosis is a serious disease caused by Toxoplasma gondii, one of the most widespread parasites in the world. Lipid metabolism is important in the intracellular stage of T. gondii. Stearoyl-CoA desaturase (SCD), a key enzyme for the synthesis of unsaturated fatty acid is predicted to exist in T. gondii. Sterculic acid has been shown to specifically inhibit SCD activity. Here, we examined whether sterculic acid and its methyl ester analogues exhibit anti-T. gondii effects in vitro. T. gondii-infected Vero cells were disintegrated at 36 hr because of the propagation and egress of intracellular tachyzoites. All test compounds inhibited tachyzoite propagation and egress, reducing the number of ruptured Vero cells by the parasites. Sterculic acid and the methyl esters also inhibited replication of intracellular tachyzoites in HFF cells. Among the test compounds, sterculic acid showed the most potent activity against T. gondii, with an EC₅₀ value of 36.2 μM, compared with EC₅₀ values of 248-428 μM for the methyl esters. Our study demonstrated that sterculic acid and its analogues are effective in inhibition of T. gondii growth in vitro, suggesting that these compounds or analogues targeting SCD could be effective agents for the treatment of toxoplasmosis.     

Genotype of Toxoplasma gondii from Blood of Stray Cats in Gyeonggi-do, Korea

Genotyping of Toxoplasma gondii has been performed in 23 PCR positive blood samples from stray cats in Korea. We used 2 separate PCR-restriction fragment length polymorphism (RFLP) patterns of SAG2 gene, amplifying the 5''and 3''ends of the locus. The results revealed that all samples belonged to the type I clonal lineage. Although T. gondii organisms were not isolated from the samples, the results of the present study represent that stray cats with T. gondii infection should be seriously concerned in our environment. Adequate and continuous control programs of stray cats are needed to reduce the risk of transmission of T. gondii as a zoonotic infection threatening the public health.     

Immunosuppression with cyclophosphamide favors reinfection with recombinant Toxoplasma gondii strains

The aim of this study was to verify the effect of immunosuppression by cyclophosphamide (Cy) on susceptibility of BALB/c mice subjected to challenge with recombinant strains of Toxoplasma gondii. Animals were prime infected with the D8 (recombinant I/III) or the ME49 (type II) non-virulent strains, weekly immunosuppressed with Cy and challenged with the CH3 or EGS virulent strains (I/III). Parasites recovered from surviving mice were submitted to PCR-RFLP analysis to confirm co-infection. Prime-infection with the D8 strain conferred more protection against challenge with the CH3 and EGS strains when compared with ME49 prime infection. Cy treatment caused significant leukopenia in the infected mice, what probably favors reinfection after challenge. Reinfection was associated with increased levels of IgA. Otherwise, Cy-treated mice presented significantly lower IgA levels after challenge, suggesting involvement of this immunoglobulin on protection against reinfection. In conclusion, BALB/c mice susceptibility to reinfection by T. gondii is related to genetic differences among the strains used for primary and challenge infections. Alteration of the host's immune integrity by Cy probably compromises the protection previously established by primary infection.     

Sequence Variation in Superoxide Dismutase Gene of Toxoplasma gondii among Various Isolates from Different Hosts and Geographical Regions

Toxoplasma gondii, an obligate intracellular protozoan parasite of the phylum Apicomplexa, can infect all warm-blooded vertebrates, including humans, livestock, and marine mammals. The aim of this study was to investigate whether superoxide dismutase (SOD) of T. gondii can be used as a new marker for genetic study or a potential vaccine candidate. The partial genome region of the SOD gene was amplified and sequenced from 10 different T. gondii isolates from different parts of the world, and all the sequences were examined by PCR-RFLP, sequence analysis, and phylogenetic reconstruction. The results showed that partial SOD gene sequences ranged from 1,702 bp to 1,712 bp and A + T contents varied from 50.1% to 51.1% among all examined isolates. Sequence alignment analysis identified total 43 variable nucleotide positions, and these results showed that 97.5% sequence similarity of SOD gene among all examined isolates. Phylogenetic analysis revealed that these SOD sequences were not an effective molecular marker for differential identification of T. gondii strains. The research demonstrated existence of low sequence variation in the SOD gene among T. gondii strains of different genotypes from different hosts and geographical regions.     

High Toxoplasma gondii Seropositivity among Brain Tumor Patients in Korea

Toxoplasma gondii is an intracellular protozoan that can modulate the environment of the infected host. An unfavorable environment modulated by T. gondii in the brain includes tumor microenvironment. Literature has suggested that T. gondii infection is associated with development of brain tumors. However, in Korea, epidemiological data regarding this correlation have been scarce. In this study, in order to investigate the relationship between T. gondii infection and brain tumor development, we investigated the seroprevalence of T. gondii among 93 confirmed brain tumor patients (various histological types, including meningioma and astrocytoma) in Korea using ELISA. The results revealed that T. gondii seropositivity among brain tumor patients (18.3%) was significantly (P<0.05) higher compared with that of healthy controls (8.6%). The seropositivity of brain tumor patients showed a significant age-tendency, i.e., higher in younger age group, compared with age-matched healthy controls (P<0.05). In conclusion, this study supports the close relationship between T. gondii infection and incidence of brain tumors.     

Molecular cloning and characterization of peroxiredoxin from Toxoplasma gondii.

A cDNA of 1.1 kb comprising the gene encoding the peroxiredoxin of Toxoplasma gondii (TgPrx) has been cloned. The open reading frame of 591 bp was translated into a protein of 196 amino acids with a molecular mass of 25 kDa. Conserved 2 cysteine domains of Phe-Val-Cys-Pro and Glu-Val-Cys-Pro indicated TgPrx belonged to 2-Cys Prx families. TgPrx showed the highest homology with that of Arabidopsis thaliana by 53.9% followed by Entamoeba histolytica with 39.5% by the amino acid sequence alignment. Polyclonal antibody against recombinant TgPrx detected 25 kDa band in T. gondii without binding to host cell proteins. TgPrx was located in the cytoplasm of T. gondii extracellularly or intracellularly by immunofluorescence assay. The expression of TgPrx was increased as early as 30 min after the treatment with artemisinin in the intracellular stage, while no changes in those of host Prx I and TgSOD. This result implies that TgPrx may function as an antioxidant protecting the cell from the attack of reactive oxygen intermediates. It is also suggested that TgPrx is a possible target of chemotherapy.     

Toxoplasma gondii in wild and domestic animals from New Caledonia

Samples (serum or meat juice) collected from 205 animals in New Caledonia in April 2009 were tested for antibodies against Toxoplasma gondii by ELISA using the multi-species ID Screen^® Toxoplasmosis Indirect kit (IDVET, Montpellier). Antibodies to T. gondii were detected in 2% (1/49) of the pigs, in 3.3% (1/30) of the cattle, in 13.8% (4/29) of Rusa deers, in 16% (4/25) of the horses, in 32.8% (21/64) of the dogs, and in 50% (4/8) of cats. Statistically, no significant difference was observed between T. gondii seroprevalence and age or sex. No survey on the prevalence of T. gondii in animals has ever been conducted in New Caledonia and this is the first serological evidence of T. gondii in Rusa deer (Cervus timorensis russa). These results indicate an important circulation of T. gondii exists in the animal populations of New Caledonia. In view of humans being exposed, it is advisable to insist on sanitary education and on respect for good hygienic and food practice.     

Seropositivity and Serointensity of Toxoplasma gondii Antibodies and DNA among Patients with Schizophrenia

The aim of this cross sectional case control study was to examine the serofrequency and serointensity of Toxoplasma gondii (Tg) IgG, IgM, and DNA among patients with schizophrenia. A total of 101 patients with schizophrenia and 55 healthy controls from Sungai Buloh Hospital, Selangor, Malaysia and University Malaya Medical Center (UMMC) were included in this study. The diagnosis of schizophrenia was made based on the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV). The presence of Tg infection was examined using both indirect (ELISA) and direct (quantitative real-time PCR) detection methods by measuring Tg IgG and IgM and DNA, respectively. The serofrequency of Tg IgG antibodies (51.5%, 52/101) and DNA (32.67%, 33/101) among patients with schizophrenia was significantly higher than IgG (18.2%, 10/55) and DNA (3.64%, 2/55) of the controls (IgG, P=0.000, OD=4.8, CI=2.2-10.5; DNA, P=0.000, OD=12.9, CI=2.17-10.51). However, the Tg IgM antibody between patients with schizophrenia and controls was not significant (P>0.005). There was no significant difference (P>0.005) in both serointensity of Tg IgG and DNA between patients with schizophrenia and controls. These findings have further demonstrated the strong association between the active Tg infection and schizophrenia.     

Genotyping of a Korean isolate of Toxoplasma gondii by multilocus PCR-RFLP and microsatellite analysis

Although the Korean isolate KI-1 of Toxoplasma gondii has been considered to be a virulent type I lineage because of its virulent clinical manifestations, its genotype is unclear. In the present study, genotyping of the KI-1 was performed by multilocus PCR-RFLP and microsatellite sequencing. For 9 genetic markers (c22-8, c29-2, L358, PK1, SAG2, SAG3, GRA6, BTUB, and Apico), the KI-1 and RH strains exhibited typical PCR-RFLP patterns identical to the type I strains. DNA sequencing of tandem repeats in 5 microsatellite markers (B17, B18, TUB2, W35, and TgM-A) of the KI-1 also revealed patterns characteristic of the type I. These results provide strong genetic evidence that KI-1 is a type I lineage of T. gondii.     

Proliferation of Toxoplasma gondii Suppresses Host Cell Autophagy

Autophagy is a process of cytoplasmic degradation of endogenous proteins and organelles. Although its primary role is protective, it can also contribute to cell death. Recently, autophagy was found to play a role in the activation of host defense against intracellular pathogens. The aims of our study was to investigate whether host cell autophagy influences Toxoplasma gondii proliferation and whether autophagy inhibitors modulate cell survival. HeLa cells were infected with T. gondii with and without rapamycin treatment to induce autophagy. Lactate dehydrogenase assays showed that cell death was extensive at 36-48 hr after infection in cells treated with T. gondii with or without rapamycin. The autophagic markers, LC3 II and Beclin 1, were strongly expressed at 18-24 hr after exposure as shown by Western blotting and RT-PCR. However, the subsequent T. gondii proliferation suppressed autophagy at 36 hr post-infection. Pre-treatment with the autophagy inhibitor, 3-methyladenine (3-MA), down-regulated LC3 II and Beclin 1. The latter was also down-regulated by calpeptin, a calpain inhibitor. Monodansyl cadaverine (MDC) staining detected numerous autophagic vacuoles (AVs) at 18 hr post-infection. Ultrastructural observations showed T. gondii proliferation in parasitophorous vacuoles (PVs) coinciding with a decline in the numbers of AVs by 18 hr. FACS analysis failed to confirm the presence of cell apoptosis after exposure to T. gondii and rapamycin. We concluded that T. gondii proliferation may inhibit host cell autophagy and has an impact on cell survival.     

Toxoplasma gondii: Ultrastructure study of the entry of tachyzoites into mammalian cells

Toxoplama gondii (Apicomplexa: Coccidia), an obligatory intracellular parasite with a unique capacity to invade virtually all nucleated cell type from warm-blooded vertebrate hosts. Despite the efficiency with which Toxoplasma enters its host cell, it remains unresolved if invasion occurs by direct penetration of the parasite or through phagocytosis. In the present work, electron microscopic study was designed to examine the entry process of Toxoplasma (RH strain) into macrophages and non phagocytic-host cells (Hela cells) and to observe the ultrastructure changes associated with intracellular parasitism. The results showed that both active invasion and phagocytosis were occurred and revealed that invasion is an ordered process that initiates with binding of the parasite at its apical end followed by tight-fitting invagination of the host cell membrane and a prominent constriction in the parasite at the site of penetration. The process ended by the professional parasitophorous vacuole that is distinct at the outset from those formed by phagocytosis in which once Toxoplasma triggered, phagocytic uptake can proceed by capture of the parasite within a loose fitting vacuole formed by localized membrane ruffling. The cytopathic effects of the parasite on macrophages and Hela cells were demonstrated within 5–15 h post-inoculation in the form of degenerative mitochondria, swelling Golgi apparatus and widening of endoplasmic reticulum indicating intracellular oedema. These changes were exaggerated and several cells were found dead after 48–72 h.     

Malondialdehyde, Glutathione, and Nitric Oxide Levels in Toxoplasma gondii Seropositive Patients

The aim of this study was to investigate the difference in the serum malondialdehyde (MDA), glutathione (GSH), and nitric oxide (NO) levels between normal and T. gondii-infected patients. To this end, MDA, GSH, and NO levels in the sera of 37 seropositive patients and 40 participants in the control group were evaluated. In Toxoplasma ELISA, IgG results of the patient group were 1,013.0 ± 543.8 in optical density (mean ± SD). A statistically significant difference was found between patients and the control group in terms of MDA, GSH, and NO levels. A decrease in GSH activity was detected, while MDA and NO levels increased significantly. Consequently, it is suggested that the use of antioxidant vitamins in addition to a parasite treatment shall prove useful. The high infection vs control ratio of MDA and NO levels probably suggests the occurrence as a mechanism of tissue damage in cases of chronic toxoplasmosis. Moreover, it is recommended that the patient levels of MDA, GSH, and NO should be evaluated in toxoplasmosis.     

Protease activity of 80 kDa protein secreted from the apicomplexan parasite Toxoplasma gondii

This study describes the characterization of 80 kDa protease showing gelationlytic property among three proteases in the excretory/secretory proteins (ESP) from Toxoplasma gondii. The protease activity was detected in the ESP but not in the somatic extract of RH tachyzoites. This protease was active only in the presence of calcium ion but not other divalent cationic ions such as Cu(2+), Zn(2+), Mg(2+), and Mn(2+), implying that Ca(2+) is critical factor for the activation of the protease. The 80 kDa protease was optimally active at pH 7.5. Its gelatinolytic activity was maximal at 37 degrees C, and significant level of enzyme activity of the protease remained after heat treatment at 56 degrees C for 30 min or 100 degrees C for 10 min. This thermostable enzyme was strongly inhibited by metal chelators, i.e., EDTA, EGTA, and 1,10- phenanthroline. Thus, the 80 kDa protease in the ESP secreted by T. gondii was classified as a calcium dependent neutral metalloprotease.     

Prevalence of Toxoplasma gondii Infection in Stray and Household Cats in Regions of Seoul, Korea

The principal objective of this study was to investigate the prevalence of toxoplasmosis in household and stray cats in Seoul, Republic of Korea. We collected blood samples from 72 stray and 80 household cats, and all samples were examined by ELISA and nested PCR. The overall positive rates of Toxoplasma gondii in stray cats were 38.9% (28/72), with 15.3% (11/72) in ELISA and 30.6% (22/72) in PCR. The positive rate in male stray cats was slightly higher than that of female stray cats. The highest positive rate of T. gondii infection was noted in Gangnam and Songpa populations in ELISA and in Gwangjin population in PCR. In household cats, however, we could not detect any specific antibodies or DNA for T. gondii. In conclusion, we recognized that the infection rate of toxoplasmosis in stray cats in Seoul was considerably high but household cats were free from infection.     

Prevalence and Genetic Characterization of Toxoplasma gondii in Pet Dogs in Central China

The prevalence and genotype of Toxoplasma gondii infection in dogs in Henan Province, Central China was investigated. A total of 125 blood samples were collected from pet dogs during April to June 2013, and all samples were examined by indirect hemagglutination antibody test (IHA) and nested PCR. The overall T. gondii prevalence in pet dogs was 24.0% (30/125), with 20.8% (26/125) in IHA and 10.4% (13/125) in PCR, respectively. No statistical associations were found between animal gender and age and the prevalence of T. gondii infection. Thirteen positive DNA samples were genotyped using 11 PCR-RFLP markers, including SAG1, (3’+5’) SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico. Of these, only 2 samples were genotyped with complete data for all loci, and a novel genotype (type III at SAG3 and GRA6 loci, and type I at other loci) was identified. This is the first report of genetic characterization of T. gondii infection in dogs in China.     

Toxoplasma gondii and mucosal immunity

Toxoplasma gondii, an intracellular parasite infects the host through the oral route. Infection induces a cascade of immunological events that involve both the components of the innate and adaptative immune responses. Alteration of the homeostatic balance of infected intestine results in an acute inflammatory ileitis in certain strains of inbred mice. Both the infected enterocytes as well as the CD4 T cells from the lamina propria produce chemokines and cytokines that are necessary to clear the parasite whereas CD8 intraepithelial lymphocytes secrete transforming growth factor beta that reduces the inflammation. In this review, we describe the salient features of this complex network of interactions among the different components of the gut-associated lymphoid tissue cell population that are induced after oral infection with T. gondii.     

Production and Evaluation of Toxoplasma gondii Recombinant GRA7 for Serodiagnosis of Human Infections

The precise diagnosis of the acute toxoplasmosis in pregnant women and immunocompromsied patients has critical importance. Most of the commercially available assays use the whole Toxoplasma soluble extract as the antigen. However, the assays currently available for the detection of specific anti-Toxoplasma antibodies may vary in their abilities to detect serum immunoglobulins, due to the lack of a purified standardized antigen. The aim of this study was production and evaluation of the usefulness of the recombinant Toxoplasma gondii GRA7 antigen for the serodiagnosis of Toxoplasma gondii IgM and IgG by ELISA. A total of 70 T. gondii IgM positive sera, 74 T. gondii IgG positive sera, and 60 sera from subjects who were not infected with T. gondii were examined. These sera were shown different absorbance values in ELISA test. To control the specificity of the rGRA7 other parasitic diseases, for example, echinococcosis, malaria, leishmaniasis, fascioliasis, and strongyloidiasis were tested of which none showed positive results. Sensitivity and specificity of the generated recombinant IgG ELISA in comparison with commercial ELISA (com ELISA) were 89% and 90%, and the sensitivity and specificity of the generated recombinant IgM ELISA were 96% and 90%, respectively. The results obtained here show that this antigen is useful for diagnostic purposes.