Prevalence and Genetic Characterization of Toxoplasma gondii in House Sparrows (Passer domesticus) in Lanzhou,China

The prevalence of Toxoplasma gondii infection in birds has epidemiological significance because birds are indeed considered as a good indicator of environmental contamination by T. gondii oocysts. In this study, the prevalence of T. gondii in 313 house sparrows in Lanzhou, northwestern China was assayed by the modified agglutination test (MAT). Antibodies to T. gondii were positive in 39 (12.46%) of 313 samples (MAT titer ≥ 1:5). Tissues of heart, brain, and lung from the 39 seropositive house sparrows were tested for T. gondii DNA, 11 of which were found to be positive for the T. gondii B1 gene by PCR amplification. These positive DNA samples were typed at 9 genetic markers, including 8 nuclear loci, i.e., SAG1, 5''- and 3''-SAG2, alternative SAG2, SAG3, GRA6, L358, PK1, c22-8 and an apicoplast locus Apico. Of them, 4 isolates were genotyped with complete data for all loci, and 2 genotypes (Type II variants; ToxoDB #3 and a new genotype) were identified. These results showed that there is a potential risk for human infection with T. gondii in this region. To our knowledge, this is the first report of T. gondii seroprevalence in house sparrows in China.     

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.     

Detection and genetic characterisation of Toxoplasma gondii circulating in free-range chickens,pigs and seropositive pregnant women in Benue state,Nigeria

Toxoplasma gondii parasites present strong but geographically varied signatures of population structure. Populations sampled from Europe and North America have commonly been defined by over-representation of a small number of clonal types, in contrast to greater diversity in South America. The occurrence and extent of genetic diversity in African T. gondii populations remains understudied, undermining assessments of risk and transmission. The present study was designed to establish the occurrence, genotype and phylogeny of T. gondii in meat samples collected from livestock produced for human consumption (free-range chickens, n = 173; pigs, n = 211), comparing with T. gondii detected in blood samples collected from seropositive pregnant women (n = 91) in Benue state, Nigeria. The presence of T. gondii DNA was determined using a published nested polymerase chain reaction, targeting the 529 bp multicopy gene element. Samples with the highest parasite load (assessed using quantitative PCR) were selected for PCR-restriction fragment length polymorphism (PCR-RFLP) targeting the surface antigen 3 (SAG3), SAG2 (5’ and 3’), beta-tubulin (BTUB) and dense granule protein 6 (GRA6) loci, and the apicoplast genome (Apico). Toxoplasma gondii DNA was detected in all three of the populations sampled, presenting 30.6, 31.3 and 25.3% occurrence in free-range chickens, pigs and seropositive pregnant women, respectively. Quantitative-PCR indicated low parasite occurrence in most positive samples, limiting some further molecular analyses. PCR-RFLP results suggested that T. gondii circulating in the sampled populations presented with a type II genetic background, although all included a hybrid type I/II or II/III haplotype. Concatenation of aligned RFLP amplicon sequences revealed limited diversity with nine haplotypes and little indication of host species-specific or spatially distributed sub-populations. Samples collected from humans shared haplotypes with free-range chickens and/or pigs. Africa remains under-explored for T. gondii genetic diversity and this study provides the first detailed definition of haplotypes circulating in human and animal populations in Nigeria.     

Toxoplasma gondii isolates: multilocus RFLP-PCR genotyping from human patients in Sao Paulo State, Brazil identified distinct genotypes

This study investigated the genetic characteristics of Toxoplasma gondii samples collected from 62 patients with toxoplasmosis in Sao Paulo State, Brazil. DNA samples were isolated from blood, cerebrospinal fluid and amniotic fluids of 25 patients with cerebral toxoplasmosis and AIDS, two patients with acute toxoplasmosis, 12 patients with ocular toxoplasmosis, six newborns with congenital toxoplasmosis and 17 pregnant women with acute infection. Diagnosis of toxoplasmosis was based in clinical, radiological and laboratory features. Genotyping was performed using multilocus PCR–RFLP genetic markers including SAG1, SAG2, 5′- and 3′-SAG2, alt.SAG2, SAG3, BTUB, GRA6, C22-8, c29-2, L358, PK1 and Apico. Among the 62 clinical samples, 20 (32%) were successfully genotyped at eight or more genetic loci and were grouped to three distinct genotypes. Eighteen samples belonged to ToxoDB Genotype #65 and the other two samples were identified as ToxoDB Genotypes #6 and #71, respectively (http://toxodb.org/toxo/). Patients presenting Genotypes #6 and #71 had severe and atypical cerebral toxoplasmosis, characterized by diffuse encephalitis without extensive brain lesions. These results indicate that T. gondii Genotype #65 may have a high frequency in causing human toxoplasmosis in Sao Paulo State, Brazil. This unusual finding highlights the need to investigate the possible association of parasite genotypes with human toxoplasmosis.     

Identification and genetic characterization of a new Brazilian genotype of Toxoplasma gondii from sheep intended for human consumption

Recent studies have demonstrated that strains of Toxoplasma gondii in Brazil are frequently different from those detected in other countries, thus making an accurate phylogenetic analysis difficult. The aim of this study was to genetically characterize T. gondii samples from sheep raised in southern Bahia and intended for human consumption, by means of PCR–RFLP and sequencing techniques. Experimental samples were obtained from 200 sheep brains purchased at butcher's shops in Itabuna, Bahia, Brazil. In total, three samples (#54, #124 and #127) were T. gondii-positive. The application of multilocus PCR–RFLP using ten molecular markers (SAG1, SAG2, SAG3, BTUB, c22-8, PK1, GRA6, L358, c-29-2 and Apico) revealed a single genotype common to all samples of this study, which differed from any other published T. gondii genotypes. An atypical allele was detected in the L358 genetic marker; this has not previously been shown in any other South American T. gondii isolates. Phylogenetic analysis on the sequences from multilocus PCR sequencing revealed that these three samples were classified into the same lineage. Extensive indel regions were detected in the Apico genetic marker. Together, our findings revealed a new Brazilian T. gondii genotype. Further research should be conducted to enrich the database of Brazilian T. gondii genotypes from different regions. This will make it possible to understand the phylogenetic relationship between isolates.     

Prevalence of Toxoplasma gondii in Dogs in Zhanjiang,Southern China

Toxoplasmosis, caused by Toxoplasma gondii, is a parasitic zoonosis with worldwide distribution. The present study investigated the prevalence of T. gondii in dogs in Zhanjiang city, southern China, using both serological and molecular detection. A total of 364 serum samples and 432 liver tissue samples were collected from the slaughter house between December 2012 and January 2013 and were examined for T. gondii IgG antibody by ELISA and T. gondii DNA by semi-nested PCR based on B1 gene, respectively. The overall seroprevalence of T. gondii IgG antibody was 51.9%, and T. gondii DNA was detected in 37 of 432 (8.6%) liver tissue samples. These positive DNA samples were analyzed by PCR-RFLP at 3''- and 5''-SAG2. Only 8 samples gave the PCR-RFLP data, and they were all classified as type I, which may suggest that the T. gondii isolates from dogs in Zhanjiang city may represent type I or type I variant. This study revealed the high prevalence of T. gondii infection in dogs in Zhanjiang city, southern China. Integrated measures should be taken to prevent and control toxoplasmosis in dogs in this area for public health concern.     

Toxoplasmosis in a Pet Peach-Faced Lovebird (Agapornis roseicollis)

Toxoplasma gondii atypical type II genotype was diagnosed in a pet peach-faced lovebird (Agapornis roseicollis) based on histopathology, immunohistochemistry, and multilocus DNA typing. The bird presented with severe neurological signs, and hematology was suggestive of chronic granulomatous disease. Gross post-mortem examination revealed cerebral hemorrhage, splenomegaly, hepatitis, and thickening of the right ventricular free wall. Histologic sections of the most significant lesions in the brain revealed intralesional protozoan organisms associated with malacia, spongiform changes, and a mild histiocytic response, indicative of diffuse, non-suppurative encephalitis. Immunohistochemistry confirmed the causative organisms to be T. gondii. DNA isolated from the brain was used to confirm the presence of T. gondii DNA. Multilocus genotyping based on SAG1, altSAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico markers demonstrated the presence of ToxoDB PCR-RFLP genotype #3 and B1 gene as atypical T. gondii type II. The atypical type II strain has been previously documented in Australian wildlife, indicating an environmental transmission route.     

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.     

Clonal types of Toxoplasma gondii among immune compromised and immune competent individuals in Accra,Ghana

There are three major clonal lineages, types I, II, and III, of Toxoplasma gondii known to cause human toxoplasmosis worldwide. Toxoplasma gondii infections have, however, not been genotyped in Ghana. This study detected the clonal types infecting immune compromised and immune competent individuals in Accra, Ghana. Blood samples were obtained from 148 HIV seropositive pre-antiretroviral therapy individuals (0 ≤ CD4⁺ T-cell count/μl blood ≤ 200) at the Fevers Unit and 149 HIV seronegative apparently healthy blood donors at the blood bank, all of the Korle-Bu Teaching Hospital. Genomic DNA was extracted and multilocus genotyping conducted by nested PCR-RFLP analysis using GRA6, SAG3, and BTUB gene markers. Among the HIV seropositive participants, 54.7% (81/148) were T. gondii DNA positive for any of the markers. Out of the 81, 42.0% (34) were positive for SAG3 only, 30.9% (25) for GRA6 only, 24.7% (20) for both SAG3 and GRA6, and 2.5% (2) for SAG3, GRA6, and BTUB. Overall, 93.8% of the positives were of clonal type II, 1.2% type I, while 4.9% (4) were atypical or mixed types (I and II). In the healthy blood donors, prevalence of T. gondii DNA positivity was 3.4% (5/149) by SAG3 and/or GRA6; among them, 60.0% (3/5) were type I, and the remaining 40.0%, type II. This study showed a relatively high prevalence of active T. gondii infections in immune compromised patients and low prevalence in immune competent individuals in Accra. Type II was highly prevalent. Detection of T. gondii in blood donors raises public health concerns and screening for T. gondii should be considered.     

Two viable Toxoplasma gondii isolates from red-necked wallaby (Macropus rufogriseus) and red kangaroo (M. rufus)

Wallabies and kangaroos are susceptible to Toxoplasma gondii. However, little information concerning T. gondii infection in captive macropods is available. Three dead macropods collected from a zoo exhibited no clinical symptoms associated with toxoplasmosis. Heart fluids were tested for T. gondii antibodies using a modified agglutination test. T. gondii DNA samples derived from macropod tissues were tested by Polymerase Chain Reaction. Viable T. gondii were isolated from myocardium of macropods via mouse bioassay. Tissues (brain, lungs, or mesenteric lymph nodes) from T. gondii-positive mice were seeded into Vero cell culture flasks. The virulence of the isolated T. gondii strains was evaluated in Swiss mice. The DNA from T. gondii tachyzoites obtained from cell cultures was characterized by 10 PCR-RFLP markers and the virulence genes, ROP18 and ROP5. T. gondii antibodies were identified in two of the three macropods (Macropod^#5 and ^#7). T. gondii DNA was obtained from the heart and lungs of Macropod^#7. Two viable T. gondii strains were isolated from the myocardium of Macropus rufogriseus (Macropod^#5) and M. rufus (Macropod^#7) via mouse bioassay and designated as TgRooCHn2 and TgRooCHn3, respectively. TgRooCHn2 was ToxoDB genotype^#3, and TgRooCHn3 was ToxoDB genotyp^#2. Both 10⁴ TgRooCHn2 and TgRooCHn3 tachyzoites had intermediate virulence in mice. M. rufogriseus (Macropod^#5) and M. rufus (Macropod^#7) may have been in the initial stages of toxoplasmosis, due to a recent T. gondii infection with oocysts. This study is the first to document the T. gondii ToxoDB^#3 isolate in macropods. T. gondii infection in captive macropods indicates the urgent need to control the transmission of this parasite in the environment, food and water of zoo animals.     

Detection of toxoplasmic encephalitis in HIV positive patients in urine with hydrogel nanoparticles

BackgroundDiagnosis of toxoplasmic encephalitis (TE) is challenging under the best clinical circumstances. The poor clinical sensitivity of quantitative polymerase chain reaction (qPCR) for Toxoplasma in blood and CSF and the limited availability of molecular diagnostics and imaging technology leaves clinicians in resource-limited settings with few options other than empiric treatment.Methology/principle findingsHere we describe proof of concept for a novel urine diagnostics for TE using Poly-N-Isopropylacrylamide nanoparticles dyed with Reactive Blue-221 to concentrate antigens, substantially increasing the limit of detection. After nanoparticle-concentration, a standard western blotting technique with a monoclonal antibody was used for antigen detection. Limit of detection was 7.8pg/ml and 31.3pg/ml of T. gondii antigens GRA1 and SAG1, respectively. To characterize this diagnostic approach, 164 hospitalized HIV-infected patients with neurological symptoms compatible with TE were tested for 1) T. gondii serology (121/147, positive samples/total samples tested), 2) qPCR in cerebrospinal fluid (11/41), 3) qPCR in blood (10/112), and 4) urinary GRA1 (30/164) and SAG1 (12/164). GRA1 appears to be superior to SAG1 for detection of TE antigens in urine. Fifty-one HIV-infected, T. gondii seropositive but asymptomatic persons all tested negative by nanoparticle western blot and blood qPCR, suggesting the test has good specificity for TE for both GRA1 and SAG1. In a subgroup of 44 patients, urine samples were assayed with mass spectrometry parallel-reaction-monitoring (PRM) for the presence of T. gondii antigens. PRM identified antigens in 8 samples, 6 of which were concordant with the urine diagnostic.Conclusion/significancesOur results demonstrate nanoparticle technology’s potential for a noninvasive diagnostic test for TE. Moving forward, GRA1 is a promising target for antigen based diagnostics for TE.     

Genotyping of Toxoplasma gondii from Rats (Rattus rattus) in Riyadh,Saudi Arabia

Toxoplasma 3 main clonal lineages are designated as type I, II, and III; however, atypical and mixed genotypes were also reported. This study was conducted for detection of Toxoplasma gondii genotypes in rats (Rattus rattus) in Riyadh region, Saudi Arabia. PCR test on T. gondii B1 gene was conducted on ELISA IgM positive samples for confirmation of the infection. However, genetic analysis of the SAG2 locus was performed to determine T. gondii genotypes using PCR-RFLP technique. PCR test on T. gondii B1gene showed that 22 (81.5%) out of the 27 ELISA IgM positive samples have T. gondii DNA. Genotypic analysis shows that, of the total 22 PCR positive samples, only 13 (59.1%) were of type II, 7 (31.8%) were of type III, and 2 (9.1%) were of an unknown genotype. It is obvious that the prevalence of both type II and III is high in rats. No reports have been available on T. gondii genotypes among rats in Riyadh region, and only little is known about its seroprevalence in rats. Future studies on T. gondii genotypes in rats using multi-locus markers is needed in Riyadh region, Saudi Arabia for better understanding of T. gondii pathogenesis and treatment in humans and animals.     

Genetic characterization of Toxoplasma gondii isolates from China

Toxoplasma gondii infections are prevalent in humans and animals worldwide. In North America and Europe, T. gondii is highly clonal, consisting of three distinct lineages (Types I, II and III), whereas in South America, T. gondii is highly diverse with a few lineages expanded in the population. However, there is limited data on the diversity of T. gondii in Asia. Here we report the genetic characterization of T. gondii isolates from different hosts and geographical locations in China using the multilocus PCR–RFLP. A total of 17 T. gondii isolates from humans (3 strains), sheep (1 strain), pigs (5 strains) and cats (8 strains) were typed at 10 genetic markers including 9 nuclear loci SAG1, SAG2, SAG3, BTUB, GRA6, L358, PK1, c22-8, c29-2 and an apicoplast locus Apico. Four genotypes were revealed, including three previously reported and one new genotype. Three isolates belong to the clonal Type I lineage, one isolate belongs to the clonal Type II lineage, and the rest 13 isolates are grouped into two genotypes. This is the first report of genetic typing of T. gondii isolates from different hosts and geographical locations in China using a number of genetic markers, which has implications for the studies of population genetic structures of T. gondii, as well as for the prevention and control of T. gondii infections in humans and animals in China.     

Toxoplasma gondii: serological characterization and immunogenicity of recombinant surface antigen 2 (SAG2) expressed in the yeast Pichia pastoris

The full length surface antigen 2 (SAG2) gene of the protozoan parasite Toxoplasma gondii was cloned and intracellularly expressed in the Pichia pastoris expression system. The molecular weight of the expressed recombinant SAG2 (36 kDa) was much larger than the native SAG2 (22 kDa). This discrepancy in size was due to hyperglycosylation, as deglycosylation assay reduced the size of the recombinant SAG2 to 22 kDa. Despite being hyperglycosylated, the recombinant SAG2 reacted strongly with pooled anti-Toxoplasma human serum, pooled anti-Toxoplasma mouse serum and a SAG2-specific monoclonal antibody. The glycosylated recombinant SAG2 was further evaluated in Western blot and in-house enzyme-linked immunosorbent assay (ELISA) using 80 human serum samples, including confirmed early acute (IgM positive, IgG negative; n = 20), acute (IgM positive, IgG positive; n = 20) and chronic (IgM negative, IgG positive; n = 20) toxoplasmosis patients, and toxoplasmosis negative control patients (n = 20). Results of the Western blot showed that the recombinant SAG2 reacted with all 60 samples of the toxoplasmosis cases but not with the Toxoplasma-negative samples. The sensitivity of in-house ELISA was 80%, 95% and 100% for early acute, acute and chronic patients’ serum samples, respectively. Vaccination study showed that serum from mice immunised with the glycosylated recombinant SAG2 reacted specifically with the native SAG2 of T. gondii. The mice were significantly protected against lethal challenge with live T. gondii RH strain tachyzoites (P < 0.01) and their survival time was increased compared to controls. Therefore, the present study shows that the P. pastoris-derived recombinant SAG2 was specific and suitable for use as antigen for detecting anti-Toxoplasma IgG and IgM antibodies. The vaccination study showed that recombinant SAG2 protein was immunoprotective in mice against lethal challenge.     

Analysis of Toxoplasma gondii surface antigen 2 gene (SAG2). Relevance of genotype I in clinical toxoplasmosis

Toxoplasma gondii is one of the most successful protozoan parasites given its ability to manipulate the immune system and establish a chronic infection. It is a parasite with a significant impact on human health, mainly in immunocompromised patients. In Europe and North America, only a few clonal genotypes (I, II and III) seem to be responsible for the vast majority of Toxoplasma infections. Surface antigen 2 gene (SAG2) has been extensively used for genotyping T. gondii isolates. The analysis of this locus reveals that in Northern hemisphere, human disease causing isolates are mainly type II, whereas T. gondii isolated from different animals are both type II and III. Since the immune response depends on parasite genotype, it seems relevant to characterize parasites producing human toxoplasmosis in different geographical areas. The growing information about the prevalent T. gondii genotypes in South America mostly refers to domestic animals. This is the first report of genetic characterization of T. gondii isolates from clinical samples in Chile, South America. All the samples analyzed corresponded to SAG2 type I isolates, and they differ from classic SAG2 type I by genetic polymorphisms. This study contributes to the scarce available information on T. gondii at South America, and reinforces an emerging concept suggesting that SAG2 type I, rather than II, parasites are a frequent cause of clinical toxoplasmosis in this continent.     

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.     

Characterization of Toxoplasma gondii isolates from herds of sheep in southern Brazil reveals the archetypal type II genotype and new non-archetypal genotypes

Recent studies have demonstrated that, in Brazil and South America, strains of Toxoplasma gondii are often genotypically and biologically different from those found in countries on other continents. The objective of this study was to genotypically characterize T. gondii isolates from naturally infected sheep in herds in the southern region of the state of Rio Grande do Sul, Brazil, by means of the polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP). Five T. gondii isolates obtained from sheep in five municipalities in the state of Rio Grande do Sul were used. Application of multilocus PCR-RFLP multilocus using 12 genetic markers (SAG1, 5′3′ SAG2, alt. SAG2, SAG3, BTUB, c22-8, c29-2, GRA6, L358, PK1, APICO and CS3) revealed four different genotypes in the five isolates studied: clonal type II (TgOvBrRS4), type BrIV (TgOvBrRS2 and TgOvBrRS3) and two new non-archetypal genotypes, ToxoDB-RFLP#270 and #271 (TgOvBrRS1 and TgOvBrRS5, respectively). The genotype structure found in the T. gondii isolates from naturally infected sheep in the southern region of Brazil was revealed to have high diversity. This study confirms the presence of rare circulation of the clonal type II genotype in Brazil.     

Genetic characterization of Toxoplasma gondii isolates from pigs in southwestern China

The genetic diversity of Toxoplasma gondii varies in different geographical regions. Isolates of T. gondii in South America, for example, are genetically and biologically divergent from those in North America and Europe, where the population structure is highly clonal and composed mainly of 3 distinct lineages, i.e., Types I, II, and III. However, little is known of the T. gondii genotypes in the People's Republic of China. Toxoplasma gondii infection in pigs causes significant economic loss and presents a risk for human infection. We conducted a survey to determine the genetic diversity of this parasite in slaughtered pigs from Yunnan Province, southwestern China. In total, 412 DNA samples were extracted from hilar lymph nodes and livers of pigs from slaughterhouses in Yunnan Province in southwest China, 56 of which were found to be positive for the T. gondii SAG3 gene. These positive DNA samples were typed at 10 genetic markers, including 9 nuclear loci, i.e., SAG1, SAG2, SAG3, BTUB, GRA6, L358, PK1, c22-8, c29-2, and an apicoplast locus Apico. Of these, 5 isolates were genotyped with complete data for all loci. Only 1 genotype (ToxoDB 9) was identified, previously reported as a widespread lineage from pigs, cats, and human patients in China. The results indicate that this genotype may be the major T. gondii lineage in China and possibly all of eastern Asia. This is the first report of genetic typing of T. gondii isolates from pigs in China's southwestern Yunnan Province, the results of which have implications for the prevention and control of T. gondii infections in humans and other animals.     

Chronic Toxoplasma gondii in Nurr1-Null Heterozygous Mice Exacerbates Elevated Open Field Activity

Latent infection with Toxoplasma gondii is common in humans (approximately 30% of the global population) and is a significant risk factor for schizophrenia. Since prevalence of T. gondii infection is far greater than prevalence of schizophrenia (0.5-1%), genetic risk factors are likely also necessary to contribute to schizophrenia. To test this concept in an animal model, Nurr1-null heterozygous (+/-) mice and wild-type (+/+) mice were evaluate using an emergence test, activity in an open field and with a novel object, response to bobcat urine and prepulse inhibition of the acoustic startle response (PPI) prior to and 6 weeks after infection with T. gondii. In the emergence test, T. gondii infection significantly decreased the amount of time spent in the cylinder. Toxoplasma gondii infection significantly elevated open field activity in both +/+ and +/- mice but this increase was significantly exacerbated in +/- mice. T. gondii infection reduced PPI in male +/- mice but this was not statistically significant. Aversion to bobcat urine was abolished by T. gondii infection in +/+ mice. In female +/- mice, aversion to bobcat urine remained after T. gondii infection while the male +/- mice showed no aversion to bobcat urine. Antibody titers of infected mice were a critical variable associated with changes in open field activity, such that an inverted U shaped relationship existed between antibody titers and the percent change in open field activity with a significant increase in activity at low and medium antibody titers but no effect at high antibody titers. These data demonstrate that the Nurr1 +/- genotype predisposes mice to T. gondii-induced alterations in behaviors that involve dopamine neurotransmission and are associated with symptoms of schizophrenia. We propose that these alterations in murine behavior were due to further exacerbation of the altered dopamine neurotransmission in Nurr1 +/- mice.     

Prime-Boost Vaccination with Toxoplasma Lysate Antigen,but Not with a Mixture of Recombinant Protein Antigens,Leads to Reduction of Brain Cyst Formation in BALB/c Mice

IntroductionInfection with the ubiquitous parasite Toxoplasma gondii is a threat for immunocompromised patients and pregnant women and effective immune-prophylaxis is still lacking.MethodsHere we tested a mixture of recombinant T. gondii antigens expressed in different developmental stages, i.e., SAG1, MAG1 and GRA7 (SMG), and a lysate derived from T. gondii tachyzoites (TLA) for prophylactic vaccination against cyst formation. Both vaccine formulations were applied systemically followed by an oral TLA-booster in BALB/c mice.ResultsSystemic priming with SMG and oral TLA-booster did not show significant induction of protective immune responses. In contrast, systemic priming and oral booster with TLA induced higher levels of Toxoplasma-specific IgG, IgG1 and IgG2a in sera as well as high levels of Toxoplasma-specific IgG1 in small intestines. Furthermore, high levels of Toxoplasma-specific Th1-, Th17- and Th2-associated cytokines were only detected in restimulated splenocytes of TLA-vaccinated mice. Importantly, in mice orally infected with T. gondii oocysts, only TLA-vaccination and booster reduced brain cysts. Furthermore, sera from these mice reduced tachyzoites invasion of Vero cells in vitro, indicating that antibodies may play a critical role for protection against Toxoplasma infection. Additionally, supernatants from splenocyte cultures of TLA-vaccinated mice containing high levels of IFN-γ lead to substantial production of nitric oxide (NO) after incubation with macrophages in vitro. Since NO is involved in the control of parasite growth, the high levels of IFN-γ induced by vaccination with TLA may contribute to the protection against T. gondii.ConclusionIn conclusion, our data indicate that prime-boost approach with TLA, but not with the mixture of recombinant antigens SMG, induces effective humoral and cellular Toxoplasma-specific responses and leads to significant reduction of cerebral cysts, thereby presenting a viable strategy for further vaccine development against T. gondii infection.