Gondii Strains Research Articles

A rare case of acute toxoplasmosis in a stray dog due to infection of T. gondii clonal type I: public health concern in urban settings with stray animals?

BackgroundTyping of Toxoplasma gondii strains is important in epidemiological surveys, to understand the distribution and virulence of different clones of the parasite among human and animal populations. Stray dogs can be consider sentinel animals for contaminated environments playing an important but probably under- evaluated role in the epidemiology of T. gondii. We reported a rare case of acute toxoplasmosis in a stray dog due to clonal type I infection. The clonal type I, sporadic in Europe, is frequently associated with severe toxoplasmosis in humans and the control of its circulation is particularly relevant for public health. The symptomatology suggested a potential infection with the high similar parasite Neospora caninum but differential diagnosis showed that only T. gondii was involved highlighting the importance of multiple diagnostic methods beyond the clinical signs.Case presentationA female stray dog approximately six-month of age presented muscular atrophy of the femoral region and hyperextension of hind limbs. Body condition score (BCS) was 20% below ideal weight, ribs had almost no fat and the sensor state was depressed. Haematological values were normal and the dog did not show any neurological abnormalities. Serological analysis showed a positive response for T. gondii immunoglobulin G (IgG) antibodies and exclude N. caninum infection.To confirm T. gondii infection, a muscle biopsy was performed and genomic DNA was extracted. PCR analysis resulted positive to T. gondii and strain genotyping reveals clonal type I infection. The dog recovered after 4 weeks of treatment with clindamycin hydrochloride and aquatic physiotherapy.ConclusionsOur study reports a rare and severe case of T. gondii clonal type I infection in a stray dog feeding in garbage containers. The data confirm the importance of an in vivo early diagnosis for toxoplasmosis in dog. Clinical signs are often related to specific T. gondii genotype and parasite genotyping is important in the epidemiological survey of toxoplasmosis in public health. The detection of parasitic DNA in the tissue could be an useful diagnostic method in facilitating early treatment of the disease, which is important for a timely clinical recovery.

Enrofloxacin and Toltrazuril Are Able to Reduce Toxoplasma gondii Growth in Human BeWo Trophoblastic Cells and Villous Explants from Human Third Trimester Pregnancy.

Classical treatment for congenital toxoplasmosis is based on combination of sulfadiazine and pyrimethamine plus folinic acid. Due to teratogenic effects and bone marrow suppression caused by pyrimethamine, the establishment of new therapeutic strategies is indispensable to minimize the side effects and improve the control of infection. Previous studies demonstrated that enrofloxacin and toltrazuril reduced the incidence of Neospora caninum and Toxoplasma gondii infection. The aim of the present study was to evaluate the efficacy of enrofloxacin and toltrazuril in the control of T. gondii infection in human trophoblast cells (BeWo line) and in human villous explants from the third trimester. BeWo cells and villous were treated with several concentrations of enrofloxacin, toltrazuril, sulfadiazine, pyrimethamine, or combination of sulfadiazine+pyrimethamine, and the cellular or tissue viability was verified. Next, BeWo cells were infected by T. gondii (2F1 clone or the ME49 strain), whereas villous samples were only infected by the 2F1 clone. Then, infected cells and villous were treated with all antibiotics and the T. gondii intracellular proliferation as well as the cytokine production were analyzed. Finally, we evaluated the direct effect of enrofloxacin and toltrazuril in tachyzoites to verify possible changes in parasite structure. Enrofloxacin and toltrazuril did not decrease the viability of cells and villous in lower concentrations. Both drugs were able to significantly reduce the parasite intracellular proliferation in BeWo cells and villous explants when compared to untreated conditions. Regardless of the T. gondii strain, BeWo cells infected and treated with enrofloxacin or toltrazuril induced high levels of IL-6 and MIF. In villous explants, enrofloxacin induced high MIF production. Finally, the drugs increased the number of unviable parasites and triggered damage to tachyzoite structure. Taken together, it can be concluded that enrofloxacin and toltrazuril are able to control T. gondii infection in BeWo cells and villous explants, probably by a direct action on the host cells and parasites, which leads to modifications of cytokine release and tachyzoite structure.

Antibody Detection, Isolation, Genotyping, and Virulence of Toxoplasma gondii in Captive Felids from China.

The felids are the only definitive hosts of Toxoplasma gondii, which could excrete oocysts into the environment and provide an infection source for toxoplasmosis in various warm-blooded animal species, particularly the captive felids that live close to human communities. The infection rate of the captive felids is a perfect standard in detecting the presence of Toxoplasma gondii oocysts in the environment. In this study, sera or tissue samples from zoo (1 young tiger, 2 adult tigers, 6 young lions), farm (10 masked palm civets), and pet hospital (28 cats) from Henan Province (China) were collected. The sera (n = 47) were tested for immunoglobulin G (IgG) antibodies against T. gondii by using modified agglutination test (MAT), whereas the hearts tissue (n = 40) were bioassayed in mice to isolate T. gondii strains. The genotype was distinguished by using PCR-RFLP of 10 loci (SAG1, SAG2, SAG3, GRA6, BTUB, L358, c22-8, PK1, c29-2, and Apico). The detection rate for the T. gondii antibody in captive felids was 21.3% (10/47). One viable T. gondii strain (TgCatCHn4) was obtained from a cat heart tissue, and its genotype was ToxoDB#9. The oocysts of ToxoDB#9 were collected from a T. gondii-free cat. The virulence of TgCatCHn4 was low and no cysts were detected in the brain of mice at 60 days post-inoculation. The finding of the present study suggested a widespread exposure of T. gondii for felids in Henan Province of central China, particularly those from the zoological gardens and homes. ToxoDB#9 was the predominant strain in China. Preventive measures against T. gondii oocyst contamination of various components of the environment should thus be implemented, including providing pre-frozen meat, well-cooked cat food, cleaned fruits and vegetables, monitoring birds and rodents, inactive T. gondii oocysts in felids feces, and proper hygiene.

Toxoplasmosis treatment with diphenyl diselenide in infected mice modulates the activity of purinergic enzymes and reduces inflammation in spleen

Toxoplasma gondii, an intracellular protozoan, may cause chronic infection in the brain tissue of the host inducing a systemic pro-inflammatory profile. Chronic infections can induce numerous physiological changes, such as alterations in the immune and oxidative profiles. Diphenyl diselenide (PhSe)2, an organoselenium compound, has shown antioxidant and immunomodulatory activities in recent studies. So, the aim of this study was to investigate the activity of purinergic enzymes and reactive oxygen species (ROS) in serum and spleen of mice chronically infected by T. gondii, untreated and treated with (PhSe)2. For this experiment, were divided into four groups: Group A (healthy mice), Group B (healthy mice treated with (PhSe)2), Group C (infected mice) and Group D (infected mice treated with (PhSe)2). Group C and group D were infected via oral route with ME49 Toxoplasma gondii strain. Groups B and D were treated subcutaneously with 5 μmol kg−1 of (PhSe)2. Chronic T. gondii infection induced splenomegaly and physiological changes in the spleen and raised histologic inflammatory markers, ROS levels and the activity of purinergic enzymes activity such as NTPDase, 5´nucleotidase and ADA. In serum, the infection increased 5´nucleotidase and ADA activities. (PhSe)2per se has managed to decrease ROS levels and ADA activity and increase NTPDase and 5´nucleotidase in spleen. In infected mice, treatment with (PhSe)2 reversed splenomegaly, reduced histological inflammatory markers, ROS levels and ADA activity in the spleen. Our results prove that chronic toxoplasmosis can induce splenomegaly, heightens ROS levels and purinergic enzyme activity in mice. These results suggest that (PhSe)2 is a potential therapy for the alterations found in the spleen in chronic T. gondii infection.

A more sensitive, efficient and ISO 17025 validated Magnetic Capture real time PCR method for the detection of archetypal Toxoplasma gondii strains in meat

Toxoplasma gondii is a globally prevalent, zoonotic parasite of major importance to public health. Various indirect and direct methods can be used for the diagnosis of toxoplasmosis. Whereas serological tests are useful to prove contact with the parasite has occurred, the actual presence of the parasite in the tissues of a seropositive animal is not demonstrated. For this, a bioassay is still the reference method. As an alternative, various PCR methods have been developed, but due to the limited amount of sample that can be tested, combined with a low tissue cyst density, those have proved to be insufficiently sensitive. A major improvement of the sensitivity was achieved with magnetic capture-based DNA extraction. By combining the hybridization of specific, biotinylated probes with the capture of those probes with streptavidin-coated paramagnetic beads, T. gondii DNA can selectively be “fished out” from a large volume of meat lysate. Still, several studies showed an insufficient sensitivity compared with the mouse bioassay. Here we present a method that is more sensitive (99% limit of detection: 65.4 tachyzoites per 100g of meat), economical and reliable (ISO 17025 validated) by adding a non-competitive PCR inhibition control (co-capture of cellular r18S) and making the release of the target DNA from the streptavidin-coated paramagnetic beads UV-dependent. The presented results demonstrate the potential of the modified Magnetic Capture real time PCR as a full alternative to the mouse bioassay for the screening of various types of tissues and meat, with the additional advantage of being quantitative.

Comparative proteomic analysis of virulent and avirulent strains of Toxoplasma gondii reveals strain-specific patterns.

Research exploring the proteome of Toxoplasma gondii oocysts has gained momentum over the past few years. However, little is known about the oocyst's protein repertoires that contribute to differential virulence among T. gondii strains. Here, we used isobaric tag for relative and absolute quantitation-based proteomic analysis of oocysts of two T. gondii strains exhibiting the virulent PYS (ToxoDB#9) phenotype versus the less virulent PRU (Type II, ToxoDB#1) phenotype. Our aim was to determine protein expression patterns that contribute to the virulence of a particular phenotype. A total of 2,551 proteins were identified, of which 374 were differentially expressed proteins (DEPs) (|log2 fold change| ≥ 0.58 and P < 0.05). DEPs included 192 increased and 182 decreased proteins. Gene Ontology and KEGG pathway analyses revealed a large number of DEPs enriched in various metabolic processes. Protein interaction network analysis using STRING identified inosine monophosphate dehydrogenase (IMPDH), Bifunctional GMP synthase/glutamine amidotransferase protein, Glucose-6-phosphate 1-dehydrogenase, and Citrate synthase as the top four hubs. Of the 22 virulence proteins commonly expressed in the oocysts of the two strains, 13 and 2 proteins were increased in PYS strain and PRU strain, respectively. Also, 10 and 3 of the 22 identified oocyst wall proteins showed higher expression in oocysts of PRU strain and PYS strain, respectively. These findings revealed new proteomic differences in the oocysts of T. gondii strains of different genotypic backgrounds.

Immunization with Toxoplasma gondii GRA17 Deletion Mutant Induces Partial Protection and Survival in Challenged Mice.

Toxoplasmosis remains a world-threatening disease largely because of the lack of a fully effective vaccine. Here, we created a ΔGRA17 mutant by disrupting the virulence factor GRA17 using CRISPR-Cas9 method. Then, we tested whether ΔGRA17 tachyzoites can be used as a live-attenuated vaccine against acute, chronic, and congenital Toxoplasma gondii infection in mice. Immune response evoked by ΔGRA17 immunization suggested a sequential Th1 and Th2 T cell response, indicated by high levels of Th1 and a mixed Th1/Th2 cytokines at 28 and 70 days after immunization, respectively. ΔGRA17-mediated immunity fully protected mice against lethal infection with wild-type (wt) RH strain, heterologous challenge with PYS, and TgC7 strains of the Chinese ToxoDB#9 genotype, and T. gondii Pru strain. Although parasite cysts were detected in 8 out of 10 immunized mice, cyst burden in the brain was significantly reduced (P < 0.05) in immunized mice (53 ± 15 cysts/brain) compared to non-immunized mice (4,296 ± 687 cysts/brain). In respect to congenital infection, the litter size, survival rate, and body weight (BW) of pups born to ΔGRA17-immunized dams were not different compared to pups born to naïve control dams (P = 0.24). However, a marked reduction in the litter size (P < 0.001), survival rate, and BW (P < 0.01) of pups born to non-immunized and infected dams was detected. Also, immunized dams infected with type II Pru strain had significantly (P < 0.001) less cyst burden in the brain compared with non-immunized and infected dams. These findings show that immunization with ΔGRA17 strain evokes cell-mediated and neutralizing antibody responses and confers some degree of protection against challenge with homologous and heterologous virulent T. gondii strains.

Strain- and Dose-Dependent Reduction of Toxoplasma gondii Burden in Pigs Is Associated with Interferon-Gamma Production by CD8+ Lymphocytes in a Heterologous Challenge Model.

Toxoplasma gondii is a worldwide prevalent parasite of humans and animals. The global infection burden exceeds yearly one million disability-adjusted life years (DALY's) in infected individuals. Therefore, effective preventive measures should be taken to decrease the risk of infection in humans. Although human toxoplasmosis is predominantly foodborne by ingestion of tissue cysts in meat from domestic animals such as pigs, the incidence risk is difficult to estimate due to the lack of screening of animals for infection and insights in location and persistence of the parasite in the tissues. Hence, experimental infections in pigs can provide more information on the risk for zoonosis based on the parasite burden in meat products intended for human consumption and on the immune responses induced by infection. In the present study, homo- and heterologous infection experiments with two distinct T. gondii strains (IPB-LR and IPB-Gangji) were performed. The humoral and cellular immune responses, the presence of viable parasites and the parasite load in edible meat samples were evaluated. In homologous infection experiments the parasite persistence was clearly strain-dependent and inversely correlated with the infection dose. The results strongly indicate a change in the amount of parasite DNA and viable cysts in porcine tissues over time. Heterologous challenge infections demonstrated that IPB-G strain could considerably reduce the parasite burden in the subsequent IPB-LR infection. A strong, however, not protective humoral response was observed against GRA7 and TLA antigens upon inoculation with both strains. The in vitro IFN-γ production by TLA-stimulated PBMCs was correlated with the infection dose and predominantly brought about by CD3+CD4−CD8αbright T-lymphocytes. The described adaptive cellular and humoral immune responses in pigs are in line with the induced or natural infections in mice and humans. Previous studies underscored the heterogeneity of T. gondii strains and the corresponding virulence factors. These findings suggest the potential of the IPB-G strain to elicit a partially protective immune response and to reduce the parasite burden upon a challenge infection. The IPB-G strain could be used as a promising tool in limiting the number of viable parasites in edible tissues and, hence, in lowering the risk for human toxoplasmosis.

Genetic analyses of Chinese isolates of Toxoplasma gondii reveal a new genotype with high virulence to murine hosts

A great deal of evidence demonstrates that a strongly clonal population structure of Toxoplasma gondii strains exists in humans and animals in North America and Europe, while the strains from South America are genetically separate and more diverse. Potential differences in virulence between different strains mean that an understanding of strain diversity is important to human and animal health. However, to date, only one predominant genotype, ToxoDB#9 (Chinese I), and a few other genotypes, including ToxoDB#205, have been identified in China. By using DNA sequence-based phylogenetic analyses, we have re-evaluated the population structure of T. gondii strains collected from China and compared them with other global strains. Based on phylogenetic analysis of restriction fragment length polymorphisms, multilocus sequence typing and intron sequences from T. gondii, we propose that the Chinese isolates described as Chinese I are divided into two groups called Chinese I and Chinese III. Our results demonstrate that significant differences were found in mouse mortality caused by some Chinese strains, and also the archetypal I, II, III strains in mice. Furthermore, a comparison of cyst loading in the brains of infected rats showed some Chinese strains to be capable of a high degree of cyst formation. Furthermore we show that genotyping using neutral genetic markers may not be a useful predictor of pathogenic phenotypes.

Intestinal, extra-intestinal and systemic sequelae of Toxoplasma gondii induced acute ileitis in mice harboring a human gut microbiota.

BackgroundWithin seven days following peroral high dose infection with Toxoplasma gondii susceptible conventionally colonized mice develop acute ileitis due to an underlying T helper cell (Th) -1 type immunopathology. We here addressed whether mice harboring a human intestinal microbiota developed intestinal, extra-intestinal and systemic sequelae upon ileitis induction.Methodology/Principal findingsSecondary abiotic mice were generated by broad-spectrum antibiotic treatment and associated with a complex human intestinal microbiota following peroral fecal microbiota transplantation. Within three weeks the human microbiota had stably established in the murine intestinal tract as assessed by quantitative cultural and culture-independent (i.e. molecular 16S rRNA based) methods. At day 7 post infection (p.i.) with 50 cysts of T. gondii strain ME49 by gavage human microbiota associated (hma) mice displayed severe clinical, macroscopic and microscopic sequelae indicating acute ileitis. In diseased hma mice increased numbers of innate and adaptive immune cells within the ileal mucosa and lamina propria and elevated intestinal secretion of pro-inflammatory mediators including IFN-γ, IL-12 and nitric oxide could be observed at day 7 p.i. Ileitis development was accompanied by substantial shifts in intestinal microbiota composition of hma mice characterized by elevated total bacterial loads and increased numbers of intestinal Gram-negative commensals such as enterobacteria and Bacteroides / Prevotella species overgrowing the small and large intestinal lumen. Furthermore, viable bacteria translocated from the inflamed ileum to extra-intestinal including systemic compartments. Notably, pro-inflammatory immune responses were not restricted to the intestinal tract as indicated by increased pro-inflammatory cytokine secretion in extra-intestinal (i.e. liver and kidney) and systemic compartments including spleen and serum.Conclusion/SignificanceWith respect to the intestinal microbiota composition “humanized” mice display acute ileitis following peroral high dose T. gondii infection. Thus, hma mice constitute a suitable model to further dissect the interactions between pathogens, human microbiota and vertebrate host immunity during acute intestinal inflammation.

The first isolation and molecular characterization of Toxoplasma gondii from horses in Serbia

BackgroundConsumption of undercooked or insufficiently cured meat is a major risk factor for human infection with Toxoplasma gondii. Although horsemeat is typically consumed rare or undercooked, information on the risk of T. gondii from infected horse meat to humans is scarce. Here, we present the results of a study to determine the presence of T. gondii infection in slaughter horses in Serbia, and to attempt to isolate viable parasites.MethodsThe study included horses from all regions of Serbia slaughtered at two abattoirs between June 2013 and June 2015. Blood sera were tested for the presence of specific IgG T. gondii antibodies by the modified agglutination test (MAT), and samples of trypsin-digested heart tissue were bioassayed in mice. Cyst-positive mouse brain homogenates were subjected to DNA extraction and T. gondii strains were genotyped using 15 microsatellite markers (MS).ResultsA total of 105 slaughter horses were sampled. At the 1:6 cut-off 48.6% of the examined horses were seropositive, with the highest titre being 1:400. Viable parasites were isolated from two grade type mares; both parasite isolates (RS-Eq39 and RS-Eq40) were T. gondii type III, and both displayed an increased lethality for mice with successive passages. These are the first cases of isolation of T. gondii from horses in Serbia. When compared with a worldwide collection of 61 type III and type III-like strains, isolate RS-Eq39 showed a combination of MS lengths similar to a strain isolated from a duck in Iran, and isolate RS-Eq40 was identical in all markers to three strains isolated from a goat from Gabon, a sheep from France and a pig from Portugal. Interestingly, the source horses were one seronegative and one weakly seropositive.ConclusionsThe isolation of viable T. gondii parasites from slaughter horses points to horsemeat as a potential source of human infection, but the fact that viable parasites were isolated from horses with only a serological trace of T. gondii infection presents further evidence that serology may not be adequate to assess the risk of toxoplasmosis from horsemeat consumption. Presence of T. gondii type III in Serbia sheds more light into the potential origin of this archetypal lineage in Europe.

Seroprevalence, isolation and co-infection of multiple Toxoplasma gondii strains in individual bobcats (Lynx rufus) from Mississippi, USA

Toxoplasma gondii causes lifelong chronic infection in both feline definitive hosts and intermediate hosts. Multiple exposures to the parasite are likely to occur in nature due to high environmental contamination. Here, we present data of high seroprevalence and multiple T. gondii strain co-infections in individual bobcats (Lynx rufus). Unfrozen samples (blood, heart, tongue and faeces) were collected from 35 free ranging wild bobcats from Mississippi, USA. Toxoplasma gondii antibodies were detected in serum by the modified agglutination test (1:≥200) in all 35 bobcats. Hearts from all bobcats were bioassayed in mice and viable T. gondii was isolated from 21; these strains were further propagated in cell culture. Additionally, DNA was extracted from digests of tongues and hearts of all 35 bobcats; T. gondii DNA was detected in tissues of all 35 bobcats. Genetic characterisation of DNA from cell culture-derived isolates was performed by multiplex PCR using 10 PCR-RFLP markers. Results showed that ToxoDB genotype #5 predominated (in 18 isolates) with a few other types (#24 in two isolates, and #2 in one isolate). PCR-DNA sequencing at two polymorphic markers, GRA6 and GRA7, detected multiple recombinant strains co-infecting the tissues of bobcats; most possessing Type II alleles at GRA7 versus Type X (HG-12) alleles at GRA6. Our results suggest that individual bobcats have been exposed to more than one parasite strain during their life time.

Seroprevalence, Isolation, Genotyping, and Pathogenicity of Toxoplasma gondii Strains from Sheep in China.

Toxoplasma gondii is an important cause of reproductive failure in small ruminants that also poses a risk to consumers who consume undercooked meat. However, little is known about sheep toxoplasmosis in China for the world. Therefore, this study was conducted to assess the prevalence of T. gondii infection in sheep from China, to isolate T. gondii via bioassay in mice and to evaluate the virulence of the isolated T. gondii based on vero cell invasion and mice. A total of 840 samples (304 unfrozen hearts and 536 sera) from sheep in China were collected from 2014 to 2016. Heart samples (n = 36) of T. gondii seropositive sheep (MAT, ≥25) were bioassayed in mice individually. DNA derived from cell cultured tachyzoites of the isolated T. gondii was characterized by PCR-RFLP of 10 loci (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). The virulence of the T. gondii was evaluated based on the mortality and encystation in mice, as well as their growth characteristics in cell culture. Antibodies to T. gondii were found in 174 of 840 (20.71%, 304 hearts juice and 536 sera) sheep by the modified agglutination test (cut-off 1:25). Viable T. gondii was isolated from the hearts of two of 36 seropositive sheep hearts. Both genotypes of the sheep heart isolates were ToxoDB#9. The virulence of the two ToxoDB#9 isolations varied significantly. To the best of our knowledge, this is the first report of isolation of ToxoDB#9 strain of T. gondii from sheep in China.

Molecular and virulence characterization of Toxoplasma gondii strains isolated from humans in Portugal.

Toxoplasma gondii is an apicomplexan parasite responsible for toxoplasmosis which infects all warm-blooded vertebrates, including mammals and birds. The majority of studies conducted in Europe have revealed that more than 80% of strains isolated from human infections belong to genotype II, whereas genotypes I and III are responsible for a small number of cases. Atypical and recombinant strains are generally associated with more severe infections. In Portugal, there is a lack of data concerning genetic diversity as the classical typing studies in humans have never been performed. We aimed to determine the Sag2 and microsatellite-based (TUB2, TgM-A, W35, B17, B18) genotypes of T. gondii isolated from humans in Portugal, as well as to study their virulence in mice. We analyzed 48 strains from congenital and acquired toxoplasmosis collected during the last two decades. Sag2-based genotyping of T. gondii was achieved in all 48 strains where 35 (73%) were classified as type II and 13 (27%) were type I. The multilocus PCR of five microsatellites allowed the classification of 10 strains (21%) as recombinant strains that had been previously identified as type II or I by Sag2 typing. Seven out of the 48 strains, including three type I, three recombinant, and one type I, were virulent in mice. This study constitutes the first evidence of recombinant strains circulating in Portugal in humans from congenital infection, highlighting the need for a better evaluation of these strains as their phenotype is still barely understood.

Host Cell Vimentin Restrains Toxoplasma gondii Invasion and Phosphorylation of Vimentin is Partially Regulated by Interaction with TgROP18.

The obligate intracellular parasite, Toxoplasma gondii, manipulates the cytoskeleton of its host cells to facilitate infection. A significant rearrangement of host cell vimentin around Toxoplasma parasitophorous vacuoles is observed during the course of infection. ROP18 (TgROP18) is a serine-threonine kinase secreted by T. gondii rhoptry and a major virulence factor; however, the mechanisms by which this kinase modulates host factors remain poorly understood. Different and dynamic patterns of vimentin solubility, phosphorylation, and expression levels were observed in host cells infected with T. gondii strain RH and RH Δrop18 strains, suggesting that TgROP18 contributes to the regulation of these dynamic patterns. Additionally, host cell vimentin was demonstrated to interact with and be phosphorylated by TgROP18. A significant increase in T. gondii infection rate was observed in vimentin knockout human brain microvessel endothelial cells (HBMEC), while vimentin knockout or knock down in host cells had no impact on parasite proliferation and egress. These results indicate that host cell vimentin can inhibit T. gondii invasion. Interestingly, western blotting of different mouse tissues indicated that the lowest vimentin expression level was present in the brain, which may explain the mechanism underlying the nervous system tropism of T. gondii, and the phenomenon of huge cyst burdens developing in the mouse brain during chronic infection.

In contrast to Toxoplasma gondii, Neospora caninum tachyzoites did not sustain multiplication in vitro at increased incubation temperatures

Neospora caninum and Toxoplasma gondii are coccidian parasites that infect a wide range of mammalian and avian species. While viable T. gondii has been in vitro isolated in natural infections from wild and domestic birds, attempts to isolate N. caninum from naturally-infected birds were unsuccessful. We speculate that body temperatures of birds, which are usually higher than those of mammals, may impair the multiplication of N. caninum. In contrast to N. caninum, T. gondii can grow in vitro at temperatures higher than 37°C. To test the hypothesis that N. caninum tachyzoites are impaired to grow in vitro at high temperatures, three strains of N. caninum (NC-1, NC-Liverpool, and NC-Bahia) and three of T. gondii (RH, ME-49 and NED) were cultivated at gradually increasing temperatures starting at 37°C up to 41.5°C. A permanent chicken cell line was chosen for the study. Parasites were observed microscopically and their presence in culture was evaluated by species-specific conventional PCRs. In a second experiment, growth rates of T. gondii (RH strain) and N. caninum (NC-1 strain) were evaluated after direct passage of tachyzoites from 37°C to 41.5°C, and quantified by real-time PCR. In addition to comparisons between N. caninum and T. gondii, growth rates of three T. gondii strains were compared at high temperatures. Neospora caninum tachyzoites could not sustain multiplication at temperatures between 39°C and 41.5°C. Toxoplasma gondii tachyzoites continued to multiply at the same experimental conditions. Direct passage of N. caninum tachyzoites from 37°C to 41.5°C caused a significant decrease in the number of parasites during 96h of observation, while T. gondii had a significant increase in the number of stages after the same period of time. T. gondii RH strain (clonal type I) presented a different growth rate at 41.5°C when compared with type II and type III strains. In conclusion, multiplication of N. caninum tachyzoites in vitro was inhibited at temperatures similar to those of chickens, what may be one of the reasons that isolation of the parasite is difficult in avian species. In contrast to N. caninum, T. gondii continued to grow at 41.5°C.

Congenital Toxoplasmosis in Chronically Infected and Subsequently Challenged Ewes.

This experiment studied congenital transmission in sheep experimentally infected with oocysts of Toxoplasma gondii and reinfected at one of three stages of pregnancy. Twenty ewes were experimentally infected with T. gondii strain ME49 (day 0). After the T. gondii infection became chronic (IFAT≤512), the ewes were allocated with rams for coverage. After the diagnosis of pregnancy, these ewes were allocated into four experimental groups (n = 5): I-reinfected with T. gondii on the 40th day of gestation (DG); II-reinfected on DG 80; III-reinfected on DG 120; and IV-saline solution on DG 120 (not reinfected). Five ewes (IFAT<64) were kept as negative controls (uninfected, group V), therefore in groups I-III were infected prior to pregnancy and re-infected during pregnancy, group IV was only infected prior to pregnancy, and group V was not infected. Parasitism by T. gondii was investigated (histopathology, immunohistochemistry, mouse bioassay and PCR) in mothers and lambs tissue. All ewes produced lambs serologically positive for T. gondii. The results of the mouse bioassay, immunohistochemistry and PCR assays revealed the presence of T. gondii in all 20 sheep and their lambs. The congenital transmission of T. gondii was associated with fetal loss and abnormalities in persistently infected sheep and in ewes infected and subsequently reinfected by this protozoan. Therefore, congenital T. gondii infection was common when ewes were chronically infected prior to pregnancy, with or without reinfection during at various stages of gestation.

Comparative Analysis of Conventional Natural Killer Cell Responses to Acute Infection with Toxoplasma gondii Strains of Different Virulence.

Conventional natural killer (cNK) cells, members of group 1 innate lymphoid cells, are a diverse cell subpopulation based on surface receptor expression, maturation, and functional potential. cNK cells are critical for early immunity to Toxoplasma gondii via IFNγ production. Acute cNK cell responses to infection with different strains of T. gondii have not yet been characterized in detail. Here, we comprehensively performed this analysis with Type I virulent RH, Type II avirulent ME49, and fully attenuated Type I cps1-1 strains. In response to these three parasite strains, murine cNK cells produce IFNγ and become cytotoxic and polyfunctional (IFNγ+CD107a+) at the site of infection. In contrast to virulent RH and avirulent ME49 T. gondii strains, attenuated cps1-1 induced only local cNK cell responses. Infections with RH and ME49 parasites significantly decreased cNK cell frequency and numbers in spleen 5 days post infection compared with cps1-1 parasites. cNK cell subsets expressing activating receptors Ly49H, Ly49D, and NKG2D and inhibitory receptors Ly49I and CD94/NKG2A were similar when compared between the strains and at 5 days post infection. cNK cells were not proliferating (Ki67−) 5 days post infection with any of the strains. cNK cell maturation as measured by CD27, CD11b, and KLRG1 was affected after infection with different parasite strains. RH and ME49 infection significantly reduced mature cNK cell frequency and increased immature cNK cell populations compared with cps1-1 infection. Interestingly, KLRG1 was highly expressed on immature cNK cells after RH infection. After RH and ME49 infections, CD69+ cNK cells in spleen were present at higher frequency than after cps1-1 infection, which may correlate with loss of the mature cNK cell population. Cytokine multiplex analysis indicated cNK cell responses correlated with peritoneal exudate cell, spleen, and serum proinflammatory cytokine levels, including IL-12. qPCR analysis of parasite-specific B1 gene revealed that parasite burdens may affect cNK cell responses. This study demonstrates infection with RH and ME49 parasites impacts cNK cell maturation during acute T. gondii infection. Different cNK cell responses could impact early immunity and susceptibility to these strains.

Identification and characterization of an immunogenic antigen, enolase 2, among excretory/secretory antigens (ESA) of Toxoplasma gondii

An immunogenic protein, enolase 2, was identified among the secreted excretory/secretory antigens (ESAs) from Toxoplasma gondii strain RH using immunoproteomics based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Enolase 2 was cloned, sequenced, and heterologously expressed. BLAST analysis revealed 75–96% similarity with enolases from other parasites. Immunoblotting demonstrated good immunoreactivity of recombinant T. gondii enolase (Tg-enolase 2) to T. gondii-infected animal serum. Purified Tg-enolase 2 was found to catalyze dehydration of 2-phospho-d-glycerate to phosphoenolpyruvate. In vitro studies revealed maximal activity at pH 7.5 and 37 °C, and activity was inhibited by K+, Ni2+, Al3+, Na+, Cu2+ and Cr3+. A monoclonal antibody against Tg-enolase 2 was prepared, 1D6, with the isotype IgG2a/κ. Western blotting revealed that 1D6 reacts with Tg-enolase 2 and native enolase 2, present among T. gondii ESAs. The indirect immunofluorescence assays showed that enolase 2 could be specifically detected on the growing T. gondii tachyzoites. Immunoelectron microscopy revealed the surface and intracellular locations of enolase 2 on T. gondii cells. In conclusion, our results clearly show that the enzymatic activity of T. gondii enolase 2 is ion dependent and that it could be influenced by environmental factors. We also provide evidence that enolase 2 is an important immunogenic protein of ESAs from T. gondii and that it is a surface-exposed protein with strong antigenicity and immunogenicity. Our findings indicate that enolase 2 could play important roles in metabolism, immunogenicity and pathogenicity and that it may serve as a novel drug target and candidate vaccine against T. gondii infection.

MIC16 gene represents a potential novel genetic marker for population genetic studies of Toxoplasma gondii.

BackgroundThe zoonotic agent Toxoplasma gondii is distributed world-wide, and can infect a broad range of hosts including humans. Microneme protein 16 of T. gondii (TgMIC16) is responsible for binding to aldolase, and is associated with rhomboid cleavage and presence of trafficking signals during invasion. However, little is known of the TgMIC16 sequence diversity among T. gondii isolates from different hosts and geographical locations.ResultsIn this study, we examined sequence variation in MIC16 gene among T. gondii isolates from different hosts and geographical regions. The entire genomic region of the MIC16 gene was amplified and sequenced, and phylogenetic relationship was reconstructed using Bayesian inference (BI) and maximum parsimony (MP) based on the MIC16 gene sequences. The results of sequence alignments showed two lengths of the sequence of MIC16 gene among all the examined 12 T. gondii strains: 4391 bp for strains TgCatBr5 and MAS, and 4394 bp for strains RH, TgPLH, GT1, PRU, QHO, PTG, PYS, GJS, CTG and TgToucan. Their A+T content ranged from 50.30 to 50.59 %. A total of 107 variable nucleotide positions (0.1–0.9 %) were identified, including 29 variations in 10 exons and 78 variations in 9 introns. Phylogenetic analysis of MIC16 sequences showed that typical genotypes (Type I, II and III) were able to be grouped into their respective genotypes. Moreover, the three major clonal lineages (Type I, II and III) can be differentiated by PCR-RFLP using restriction enzyme Pst I.ConclusionsPhylogenetic analysis and PCR-RFLP of the MIC16 locus among T. gondii isolates from different hosts and geographical regions allowed the differentiation of three major clonal lineages (Type I, II and III) into their respective genotypes, suggesting that MIC16 gene may provide a novel potential genetic marker for population genetic studies of T. gondii isolates.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0726-3) contains supplementary material, which is available to authorized users.