Intergenic spacer (IGS) polymorphism: a new genetic marker for differentiation of Toxoplasma gondii strains and Neospora caninum

The region between the 28S and 18S rRNA genes, including the intergenic spacer (IGS) region and the 5S rRNA gene, from 32 strains of Toxoplasma gondii and the NC1 strain of Neospora caninum was amplified and used for DNA sequencing and/or restriction fragment length polymorphism (RFLP) analysis. The 5S rDNA sequences from 20 strains of T. gondii were identical. The IGS region between the 5S and 18S rRNA genes (nontranscribed spacer 2 or NTS 2) showed 10 nucleotide variations. Six of the 10 variant positions correlated with the murine virulence of the strains. Intraspecific polymorphisms distinguished the virulent strains of zymodemes 5, 6, and 8 from other virulent strains (in zymodeme 1). RFLP methods (IGS-RFLP) were developed and used to characterize the virulent and avirulent patterns among 29 T. gondii strains. Sequence diversity of 19.8% was found between T. gondii and N. caninum when comparing a region of 919 bp at the 3' end of NTS 2. The sequence variation in ribosomal IGS could therefore be a useful marker for Toxoplasma strain identification and for distinguishing N. caninum from T. gondii.     

Attenuation of the virulent RH strain of Toxoplasma gondii by passages in mice immunized with Toxoplasma lysate antigens

The virulent RH strain of Toxoplasma gondii was attenuated after a few passages or just one long passage in mice immunized twice with a four-week interval between immunizations with an emulsion of Toxoplasma lysate antigens and complete Freund's adjuvant. Three avirulent strains, RH-cyst III, IV and VIII were established from the RH strain. The RH-cyst III strain was effective for vaccination against challenge with the original, virulent RH strain. The attenuation of T. gondii is an expression of the innate attributes of this parasite necessary to maintain its parasitic life cycle in nature.     

The relationship between nucleoside triphosphate hydrolase (NTPase) isoform and Toxoplasma strain virulence in rat and human toxoplasmosis

Avirulent strains of Toxoplasma gondii possess only the nucleoside triphosphate hydrolase II (NTPaseII) isoform, whilst virulent strains possess both NTPaseI and NTPaseII. To determine if it is possible to identify the infective strain type (virulent or avirulent) in T. gondii infections by serological methods, we developed isoform-specific peptide ELISAs from the NTPaseI and NTPaseII antigens of T. gondii. When rats were immunized with either recombinant NTPaseI or NTPaseII, the ELISA could differentially identify antibody reactivity to each NTPase isoform. This ELISA was then used to test six groups of rats that were infected with either one of three virulent (RH, P or Ent) or three avirulent (Me49, C or TPR) strains of T. gondii. No differential antibody reactivity was detected by either whole recNTPase ELISA or peptide ELISA in the sera of rats, whether infected by virulent or avirulent strains of T. gondii. We also studied a panel of human sera from patients infected with known laboratory strains of T. gondii or naturally infected patients where the parasite was isolated and its virulence determined in mice. Differential reactivity to whole recNTPase isoforms was detected in some human sera, but this reactivity was not detected by the isoform-specific peptide ELISAs. Although the NTPase peptides do exhibit differential antibody reactivity, this is not correlated with the virulence status of the infecting strain.     

Molecular typing of Toxoplasma gondii strains by GRA6 gene sequence analysis

The utility of sequence polymorphisms in the dense granule antigen GRA6 gene as typing markers for Toxoplasma gondii was investigated. The coding region of GRA6 was amplified, sequenced and compared for 30 Toxoplasma strains from eight different zymodemes (Z1-Z8). Sequence alignment identified nucleotide polymorphisms at 24 positions out of 690 bp, which correlated with murine-virulence. Types I, II, and III could be distinguished from each other on the basis of three, 10, and six variable positions, respectively. Two deletions of 15 bp and 3 bp existed in the avirulent (type II) strains. With one exception, all polymorphic positions resulted in amino acid substitutions, and the two gaps of 15 bp and 3 bp caused the deletion of six amino acids in type II strains. Intra-specific polymorphisms were also found in the virulent group. A high degree of sequence polymorphism correlating with the phenotypes of T. gondii strains points to the GRA6 gene being a good marker for strain characterisation and typing of the isolates of this apicomplexan. The large variety of amino acid changes supports the view that the GRA6 protein plays an important role in the antigenicity and pathogenicity of T. gondii. The existence of polymorphic restriction sites for endonuclease MseI was used to develop a PCR-RFLP method which could simply differentiate the three different groups (types I, II, III) of T. gondii.     

Role of Toxoplasma gondii HSP70 and Toxoplasma gondii HSP30/bag1 in antibody formation and prophylactic immunity in mice experimentally infected with Toxoplasma gondii.

Production of antibodies against Toxoplasma gondii (T. gondii)-derived stress proteins, T. gondii HSP70 (T.g.HSP70) and T.g.HSP30/bagl, in C57BL/6 and BALB/c mice perorally infected with cysts of the avirulent Fukaya strain of T. gondii was analyzed. Production of anti-T.g.HSP70 IgG antibodies was transient, whereas production of anti-T.g.HSP30/bag1 IgG antibodies persisted after infection in both C57BL/6 and BALB/c mice. C57BL/6 mice, a susceptible strain, predominantly produced IgG antibodies specific for T.g.HSP70, whereas BALB/c mice, a resistant strain, predominantly produced IgG antibodies specific for T.g.HSP30/bag1, after T. gondii infection. Immunization with rT.g.HSP30/bag1 enhanced, whereas immunization with rT.g.HSP70 reduced host protective immunity against T. gondii infection with a cyst-forming avirulent strain, Fukaya, and a virulent strain, RH.     

Role of L3T4+ and Lyt-2+ T cell subsets in protective immune responses of mice against infection with a low or high virulent strain of Toxoplasma gondii

In order to elucidate the role of T cell subsets in protective immunity against infection with high virulent and low virulent strains of Toxoplasma gondii, monoclonal antibodies specific for T cell subsets were injected into mice before immunization or challenge infection. Treatment of mice with monoclonal antibody to either L3T4+ or Lyt-2+ T cells before they were immunized with Toxoplasma cell homogenate prepared from high virulent RH strain tachyzoites markedly reduced survival after mice were challenged with low virulent bradyzoites of the Beverley strain. Thus, induction of protective immunity against bradyzoites of the Beverley strain requires the presence of both L3T4+ and Lyt-2+ T cells. In contrast, mice injected with living bradyzoites of the low virulent Beverley strain after immunization with Toxoplasma cell homogenate acquired protective immunity against high virulent tachyzoites of the RH strain. Lyt-2+ T cells alone appear to be final effector cells for protection against the challenge with high virulent RH strain tachyzoites, since treatment of the bradyzoite-immune mice with anti-Lyt-2 antibody, but not anti-L3T4 antibody, before challenge significantly increased mortality.     

Biological and genetic characterisation of Toxoplasma gondii isolates from chickens (Gallus domesticus) from S?o Paulo, Brazil: unexpected findings.

In spite of a wide host range and a world wide distribution, Toxoplasma gondii has a low genetic diversity. Most isolates of T. gondii can be grouped into two to three lineages. Type I strains are considered highly virulent in outbred laboratory mice, and have been isolated predominantly from clinical cases of human toxoplasmosis whereas types II and III strains are considered avirulent for mice. In the present study, 17 of 25 of the T. gondii isolates obtained from asymptomatic chickens from rural areas surrounding S?o Paulo, Brazil were type I. Antibodies to T. gondii were measured in 82 chicken sera by the modified agglutination test using whole formalin-preserved tachyzoites and mercaptoethanol and titres of 1:10 or more were found in 32 chickens. Twenty-two isolates of T. gondii were obtained by bioassay in mice inoculated with brains and hearts of 29 seropositive (> or =1:40) chickens and three isolates were obtained from the faeces of cats fed tissues from 52 chickens with no or low levels (<1:40) of antibodies. In total, 25 isolates of T. gondii were obtained by bioassay of 82 chicken tissues into mice and cats. All type I isolates killed all infected mice within 4 weeks whereas type III isolates were less virulent to mice. There were no type II strains. Tissue cysts were found in mice infected with all 25 isolates and all nine type I isolates produced oocysts. Infected chickens were from localities that were 18-200 km apart, indicating no common source for T. gondii isolates. This is the first report of isolation of predominantly type I strains of T. gondii from a food animal. Epidemiological implications of these findings are discussed.     

Experimental congenital toxoplasmosis in Wistar and Holtzman rats

Congenital toxoplasmosis was evaluated in Wistar and Holtzman rats using two strains of Toxoplasma gondii isolated in Brazil. Pregnant rats were inoculated by subcutaneous or intraperitoneal routes with 10(6) or 8 x 10(6) tachyzoites of N strain (virulent for mice) and by subcutaneous or oral routes with 10(2) or 1.2 x 10(3) cysts of P strain (avirulent for mice). The tissues of rat pups born from these rats were bioassayed for T. gondii infection. T. gondii was not observed in the pups born from rats inoculated with N strain. In the animals inoculated with P strain, congenital toxoplasmosis occurred in 22.8% (Wistar rats inoculated with 10(2) cysts by the subcutaneous route), 11.4% (Wistar rats inoculated with 10(2) cysts by the oral route), 21.2% (Wistar rats inoculated with 1.2 x 10(3) cysts by the oral route) and 2.9% of fetal infection (Holtzman rats inoculated with 10(2) cysts by the oral route). None of the pups born from chronically infected mother were infected with T. gondii.     

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.     

Speculation on possible life cycles for the clonal lineages in the genus toxoplasma

Recent evidence suggests that the strains currently classified in the genus Toxoplasma, ie. within the species Toxoplasma gondii, may actually comprise at least two clonal lineages correlated with their virulence in mice. Here, Alan Johnson reviews these data in the context of evolution and speciation within the genus, and raises hypotheses on how the virulent lineage may undergo an asexual life cycle in nature, similar to that found for the very closely related coccidian, Neospora camnum. The putative vertical transmission life cycle of this mouse virulent lineage of T. gondii could involve passage to the foetus late in pregnancy, or transmission in milk to the neonate after birth.     

Colonization of turbot tissues by virulent and avirulent Aeromonas salmonicida subsp. salmonicida strains during infection

Preventing disease outbreaks in cultured turbot Psetta maxima L. caused by Aeromonas salmonicida subsp. salmonicida (ASS) requires a better understanding of how this pathogen colonizes its host. Distribution of 1 virulent and 2 avirulent ASS strains in turbot tissues was investigated during early and late stages of infection following an immersion challenge. To track bacteria within the turbot, the ASS strains were tagged with green fluorescent protein (GFP). Both virulent and avirulent strains colonized the epidermal mucus, gills, and intestine within the first 12 h post challenge, suggesting that these sites may serve as points of entry into turbot. Although the avirulent strains colonized these initial sites in the turbot tissues, they were rarely found in the internal organs and were cleared from the host 4 d post challenge. In contrast, the virulent ASS strain was found in the liver and kidney as early as 12 h post challenge and was found in the muscle tissue at very late stages of infection. The virulent strain persisted in all tested host tissues until death occurred 7 d post challenge, suggesting that ASS must colonize and survive within the turbot tissues for an infection to result in death of the fish. Comparisons of the distribution profiles of both virulent and avirulent strains during early and late stages of an infection in turbot has provided important information on the route and persistence of an ASS infection in this host.     

Toxoplasma gondii: detection of MIC10 antigen in sera of experimentally infected mice

We developed a sandwich ELISA for the detection of circulating Toxoplasma gondii MIC10 antigens. In T. gondii culture supernatant, MIC10 was detected in a growth dependent manner. Mice were infected with a lethal dose of either a virulent RH strain, an avirulent Beverley strain or a sub-lethal dose of a PLK strain of T. gondii. MIC10 appeared 2 days after infection and increased gradually in the sera of RH-infected mice. A detectable but significantly lower amount of MIC10 was observed in the sera of mice infected intraperitoneally with Beverley tachyzoites. In contrast, the MIC10 antigen in mice sera following oral infection with Beverley cysts was below detectable levels during the course of the experiment. In sera of PLK-infected mice, MIC10 was predominantly observed between late acute and early chronic phase. Our data show that the kinetics of circulating MIC10 differs depending on the strain and route of infection.     

Electrophoretic comparison of endonuclease-digested plasmids from Neisseria gonorrhoeae.

In order to associate virulence in Neisseria gonorrhoeae with an alteration of the nucleotide sequence of its small covalently closed plasmid, plasmid deoxyribonucleic acid was isolated from both virulent (T1) and avirulent (T3) morphological types for two strains. Electrophoretic and contour length measurements of intact plasmids indicated a homogeneous population with a molecular weight of approximately 2.6 x 10(6). Digestion with two restriction endonucleases. Hinf I and Hpa II, generated distinct fragment patterns which in each case were identical for T1 and T3 plasmid molecules from the same strain. The analysis suggests no sequence differences between the plasmids from virulent and avirulent types. For both strains, however, a deletion or addition of about 1.5% of the total deoxyribonucleic acid appeared in the Hpa II C digestion fragment when patterns for gonococci serially passaged 300 times were compared to those for bacteria freshly established from frozen stocks. The significance of the plasmid instability remains undetermined.     

弓形虫特异DNA片段顺序分析及体外基因扩增

从弓形虫(ZS_2株)基因组文库中筛选出了一个弓形虫特异DNA片段的克隆,对克隆的片段进行了部分顺序分析。根据所得DNA顺序,自行设计并合成特异的寡核苷酸引物对,建立了体外扩增弓形虫特异DNA顺序的聚合酶链反应(PCR)方法。4种不同来源的弓形虫株DNA、人工感染弓形虫的三头幼猪白细胞和胸腺DNA经过PCR扩增,均出现特异的扩增条带;而正常人、正常幼猪外围血白细胞、正常小鼠脾脏、恶性疟原虫、卡氏肺孢子虫、溶组织内阿米巴和人巨细胞病毒DNA均不出现特异的扩增条带。对扩增产物进行了Southern印迹和限制性内切酶图谱分析,证明该PCR产物是弓形虫DNA特异的顺序。该方法可测出少至1pg的弓形虫DNA或1个弓形虫体的裂解液。本文分析的DNA顺序和设计合成的引物顺序数据,经电脑DNA数据库检索,证明无相同的顺序。本方法并具有简便、快速等优点,便于推广应用。     

First biologic and genetic characterization of Toxoplasma gondii isolates from chickens from Africa (Democratic Republic of Congo, Mali, Burkina Faso, and Kenya)

The prevalence of Toxoplasma gondii in free-ranging chickens (Gallus domesticus) is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. In the present study, prevalence of T. gondii in chickens from Democratic Republic of Congo, Mali, Burkina Faso, and Kenya is reported. The prevalence of T. gondii antibodies in sera of 50 free-range chickens from Congo was 50% based on the modified agglutination test (MAT); antibody titers were 1:5 in 7, 1:10 in 7, 1:20 in 6, 1:40 in 1, and 1:160 or more in 4 chickens. Hearts, pectoral muscles, and brains of 11 chickens with titers of 1:20 or more were bioassayed individually in mice; T. gondii was isolated from 9, from the hearts of 9, brains of 3, and muscles of 3 chickens. Tissues of each of the 14 chickens with titers of 1:5 or 1:10 were pooled and bioassayed in mice; T. gondii was isolated from 1 chicken with a titer of 1:10. Tissues from the remaining 25 seronegative chickens were pooled and fed to 1 T. gondii-free cat. Feces of the cat were examined for oocysts, but none was seen. The results indicate that T. gondii localizes in the hearts more often than in other tissues of naturally infected chickens. Genotyping of these 10 isolates using the SAG2 locus indicated that 8 were isolates were type III, 1 was type II, and 1 was type I. Two isolates (1 type I and 1 type III) were virulent for mice. Toxoplasma gondii was isolated by mouse bioassay from a pool of brains and hearts of 5 of 48 chickens from Mali and 1 of 40 chickens from Burkina Faso; all 6 isolates were avirulent for mice. Genetically, 4 isolates were type III and 2 were type II. Sera were not available from chickens from Mali and Burkina Faso. Toxoplasma gondii antibodies (MAT 100 or more) were found in 4 of 30 chickens from Kenya, and T. gondii was isolated from the brain of 1 of 4 seropositive chickens; this strain was avirulent for mice and was type II. This is the first report on isolation and genotyping of T. gondii from any source from these 4 countries in Africa.     

Isolation of viable Toxoplasma gondii from feral guinea fowl (Numida meleagris) and domestic rabbits (Oryctolagus cuniculus) from Brazil

Toxoplasma gondii was isolated from a feral guinea fowl (Numida meleagris) and domestic rabbits (Oryctologus cuniculus) from Brazil for the first time. Serum and brains from 10 guinea fowl and 21 rabbits from Brazil were examined for T. gondii infection. Antibodies to T. gondii were found in 2 of 10 fowl and 2 of 21 rabbits by the modified agglutination test (titer 1∶25 or higher). Viable T. gondii (designated TgNmBr1) was isolated from 1 of the 2 seropositive fowl by bioassay in mice but not from the 8 seronegative fowl by bioassay in cat. Viable T. gondii was isolated from both seropositive rabbits (designated TgRabbitBr1, TgRabbitBr2) by bioassay in mice from 1 and by bioassay in cat from the other. The TgRabbitBr1 strain was highly virulent for out-bred mice; mice fed 1 infective oocyst died of acute toxoplasmosis. The remaining 2 isolates were relatively avirulent for mice; lethal dose for mice was 10,000 oocysts. All 3 isolates were grown in cell culture, and tachyzoite-derived DNA were genotyped using 10 PCR-restriction fragment length polymorphism markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). The TgNmBr1 was found to be clonal Type II, a rare finding in Brazil in any host. The rabbit isolates were atypical, similar to isolates from cats from Brazil (TgRabbitBr1 was identical to TgCatBr5, and TgRabbitBr2 was identical to TgCatBr1, a common genotype in Brazil denoted type BrII). This is the first genetic characterization of T. gondii isolates from the rabbits and guinea fowl in Brazil and the first host record for T. gondii in the guinea fowl.     

Phosphorylation of mouse immunity-related GTPase (IRG) resistance proteins is an evasion strategy for virulent Toxoplasma gondii

Virulence of complex pathogens in mammals is generally determined by multiple components of the pathogen interacting with the functional complexity and multiple layering of the mammalian immune system. It is most unusual for the resistance of a mammalian host to be overcome by the defeat of a single defence mechanism. In this study we uncover and analyse just such a case at the molecular level, involving the widespread intracellular protozoan pathogen Toxoplasma gondii and one of its most important natural hosts, the house mouse (Mus musculus). Natural polymorphism in virulence of Eurasian T. gondii strains for mice has been correlated in genetic screens with the expression of polymorphic rhoptry kinases (ROP kinases) secreted into the host cell during infection. We show that the molecular targets of the virulent allelic form of ROP18 kinase are members of a family of cellular GTPases, the interferon-inducible IRG (immunity-related GTPase) proteins, known from earlier work to be essential resistance factors in mice against avirulent strains of T. gondii. Virulent T. gondii strain ROP18 kinase phosphorylates several mouse IRG proteins. We show that the parasite kinase phosphorylates host Irga6 at two threonines in the nucleotide-binding domain, biochemically inactivating the GTPase and inhibiting its accumulation and action at the T. gondii parasitophorous vacuole membrane. Our analysis identifies the conformationally active switch I region of the GTP-binding site as an Achilles' heel of the IRG protein pathogen-resistance mechanism. The polymorphism of ROP18 in natural T. gondii populations indicates the existence of a dynamic, rapidly evolving ecological relationship between parasite virulence factors and host resistance factors. This system should be unusually fruitful for analysis at both ecological and molecular levels since both T. gondii and the mouse are widespread and abundant in the wild and are well-established model species with excellent analytical tools available.