Selection at a single locus leads to widespread expansion of toxoplasma gondii lineages that are virulent in mice

The determinants of virulence are rarely defined for eukaryotic parasites such as T. gondii, a widespread parasite of mammals that also infects humans, sometimes with serious consequences. Recent laboratory studies have established that variation in a single secreted protein, a serine/threonine kinase known as ROPO18, controls whether or not mice survive infection. Here, we establish the extent and nature of variation in ROP18among a collection of parasite strains from geographically diverse regions. Compared to other genes, ROP18 showed extremely high levels of diversification and changes in expression level, which correlated with severity of infection in mice. Comparison with an out-group demonstrated that changes in the upstream region that regulates expression of ROP18 led to an historical increase in the expression and exposed the protein to diversifying selective pressure. Surprisingly, only three atypically distinct protein variants exist despite marked genetic divergence elsewhere in the genome. These three forms of ROP18 are likely adaptations for different niches in nature, and they confer markedly different virulence to mice. The widespread distribution of a single mouse-virulent allele among geographically and genetically disparate parasites may have consequences for transmission and disease in other hosts, including humans

Additional Haplogroups of Toxoplasma gondii out of Africa: Population Structure and Mouse-Virulence of Strains from Gabon

Prevalence of human toxoplasmosis in tropical African countries usually exceeds 50%. Its role as a major opportunistic infection of AIDS patients is regularly described. Due to the lack of investigation, congenital infection is certainly underestimated in Africa. Incidence of Toxoplasma ocular disease is higher in Africa and South America than in Europe. Severe cases in immunocompetent patients were described after infection acquired in Amazonia, but nothing is known about such cases in Africa. Several studies argued for a role of genotypes in the clinical expression of human toxoplasmosis, and for a geographical structuration of Toxoplasma across continents. Genetic data concerning isolates from Africa are scarce. Here, apart from the worldwide Type III, we described two main haplogroups, Africa 1 and 3. We detected genetic exchanges between urban centers favored by trade exchange and transportation. It shows how important human influence is, even in shaping the genetic structure of a zoonotic disease agent. Finding of identical haplogroups in South America suggested that these African and American strains share a common ancestor. As a higher pathogenicity in human of South American genotypes has been described, this similarity of genotypes should encourage further clinical studies with genotype analysis in Africa

Comparison of two DNA targets for the diagnosis of Toxoplasmosis by real-time PCR using fluorescence resonance energy transfer hybridization probes

BACKGROUND: Toxoplasmosis is an infectious disease caused by the parasitic protozoan Toxoplasma gondii. It is endemic worldwide and, depending on the geographic location, 15 to 85% of the human population are asymptomatically infected. Routine diagnosis is based on serology. The parasite has emerged as a major opportunistic pathogen for immunocompromised patients, in whom it can cause life-threatening disease. Moreover, when a pregnant woman develops a primary Toxoplasma gondii infection, the parasite may be transmitted to the fetus and cause serious damnage. For these two subpopulations, a rapid and accurate diagnosis is required to initiate treatment. Serological diagnosis of active infection is unreliable because reactivation is not always accompanied by changes in antibody levels, and the presence of IgM does not necessarily indicate recent infection. Application of quantitative PCR has evolved as a sensitive, specific, and rapid method for the detection of Toxoplasma gondii DNA in amniotic fluid, blood, tissue samples, and cerebrospinal fluid. METHODS: Two separate, real-time fluorescence PCR assays were designed and evaluated with clinical samples. The first, targeting the 35-fold repeated B1 gene, and a second, targeting a newly described multicopy genomic fragment of Toxoplasma gondii. Amplicons of different intragenic copies were analyzed for sequence heterogeneity. RESULTS: Comparative LightCycler experiments were conducted with a dilution series of Toxoplasma gondii genomic DNA, 5 reference strains, and 51 Toxoplasma gondii-positive amniotic fluid samples revealing a 10 to 100-fold higher sensitivity for the PCR assay targeting the newly described 529-bp repeat element of Toxoplasma gondii. CONCLUSION: We have developed a quantitative LightCycler PCR protocol which offer rapid cycling with real-time, sequence-specific detection of amplicons. Results of quantitative PCR demonstrate that the 529-bp repeat element is repeated more than 300-fold in the genome of Toxoplasma gondii. Since individual intragenic copies of the target are conserved on sequence level, the high copy number leads to an ultimate level of analytical sensitivity in routine practice. This newly described 529-bp repeat element should be preferred to less repeated or more divergent target sequences in order to improve the sensitivity of PCR tests for the diagnosis of toxoplasmosis

Transcriptional Analysis of Murine Macrophages Infected with Different Toxoplasma Strains Identifies Novel Regulation of Host Signaling Pathways

Most isolates of Toxoplasma from Europe and North America fall into one of three genetically distinct clonal lineages, the type I, II and III lineages. However, in South America these strains are rarely isolated and instead a great variety of other strains are found. T. gondii strains differ widely in a number of phenotypes in mice, such as virulence, persistence, oral infectivity, migratory capacity, induction of cytokine expression and modulation of host gene expression. The outcome of toxoplasmosis in patients is also variable and we hypothesize that, besides host and environmental factors, the genotype of the parasite strain plays a major role. The molecular basis for these differences in pathogenesis, especially in strains other than the clonal lineages, remains largely unexplored. Macrophages play an essential role in the early immune response against T. gondii and are also the cell type preferentially infected in vivo. To determine if non-canonical Toxoplasma strains have unique interactions with the host cell, we infected murine macrophages with 29 different Toxoplasma strains, representing global diversity, and used RNA-sequencing to determine host and parasite transcriptomes. We identified large differences between strains in the expression level of known parasite effectors and large chromosomal structural variation in some strains. We also identified novel strain-specifically regulated host pathways, including the regulation of the type I interferon response by some atypical strains. IFNβ production by infected cells was associated with parasite killing, independent of interferon gamma activation, and dependent on endosomal Toll-like receptors in macrophages and the cytoplasmic receptor retinoic acid-inducible gene 1 (RIG-I) in fibroblasts.National Institutes of Health (U.S.) (R01-AI080621)New England Regional Center of Excellence for Biodefense and Emerging Infectious Diseases (Developmental Grant AIO57159)Pew Charitable Trusts (Biomedical Scholars Program)Robert A. Swanson Career Development awardThe Knights Templar Eye Foundation, Inc.Pre-Doctoral Grant in the Biological Sciences (5-T32-GM007287-33)Cleo and Paul Schimmel Foundatio

Clinical pattern of ocular toxoplasmosis treated in a referral centre in Serbia

Purpose To analyze the clinical pattern of ocular toxoplasmosis (OT) in a referral centre in Serbia. Patients and methods The medical records of consecutive patients admitted for OT to the single referral centre for uveitis in Serbia between 2006 and 2010 were retrospectively analyzed. OT was diagnosed on the basis of typical fundus lesions and positive serology for Toxoplasma. Results In a total of 457 uveitis patients, OT was the third leading cause, with 59 patients (12.9%). Most OT cases (73%) were monocular. An active primary retinal lesion was observed in 36% and recurrent OT in 64% patients. Localization of lesions was central/paracentral (44%), juxtapapillar (27%), peripheral (19%), and multifocal (10%). Other ocular manifestations of inflammation included vitritis (44%), anterior uveitis (19%), and retinal vasculitis (10%). Complications included choroidal neovascularization in two and exudative retinal detachment with cataract, glaucoma, and cystoid macular oedema in one patient each. The detection of Toxoplasma-specific IgM antibodies in a single patient indicates a low rate of OT concomitant with acute infection. After treatment, the mean best-corrected visual acuity (BCVA) increased significantly. However, 14 (24%) patients ended up legally blind in the affected eye, of which 2 (3%) with bilateral blindness, all with a very poor BCVA (0.047 +/- 0.055) at presentation. Visual impairment and treatment outcome were both associated with central localization of lesions (P lt 0.0001 and P = 0.006, respectively). Conclusion OT is a significant cause of posterior uveitis in Serbia. Patients should be aware of the recurring nature of OT and react immediately if symptoms occur. Eye (2012) 26, 723-728; doi: 10.1038/eye.2012.20; published online 24 February 201

Factors associated to infection by Toxoplasma gondii in pregnant women attended in Basic Health Units in the city of Rolândia, Paraná, Brazil

The aim of the present work was to determine the prevalence of IgG and IgM anti-Toxoplasma gondii antibodies and the factors associated to the infection in pregnant women attended in Basic Health Units in Rolândia, Paraná, Brazil. The sample was divided in two groups: group I (320 pregnant women who were analyzed from July 2007 to February 2008) and group II (287 pregnant women who were analyzed from March to October 2008). In group I, it was found 53.1% of pregnant women with IgG reactive and IgM non-reactive, 1.9% with IgG and IgM reactive, 0.3% with IgG non-reactive and IgM reactive and 44.7% with IgG and IgM non-reactive. In group II, it was found 55.1% with IgG reactive and IgM non-reactive and 44.9% with IgG and IgM non-reactive. The variables associated to the presence of IgG antibodies were: residence in rural areas, pregnant women between 35-40 years old, low educational level, low family income, more than one pregnancy, drinking water which does not originate from the public water supply system and the habit of handling soil or sand. Guidance on primary prevention measures and the quarterly serological monitoring of the pregnant women in the risk group are important measures to prevent congenital toxoplasmosis

Are eukaryotic microorganisms clonal or sexual? A population genetics vantage.

We argue that the mode of reproduction of microorganisms in nature can only be decided by population genetic information. The evidence available indicates that many parasitic protozoa and unicellular fungi have clonal rather than sexual population structures, which has major consequences for medical research and practice. Plasmodium falciparum, the agent of malaria, is a special case: the scarce evidence available is contradictory, some suggesting that uniparental lineages may exist in nature. This is puzzling (because P. falciparum is known to have a sexual stage) and poses a challenge that can be readily settled by ascertaining the frequency distribution of genotypes in natural populations