A Toxoplasma gondii Oxopurine Transporter Binds Nucleobases and Nucleosides Using Different Binding Modes.

Toxoplasma gondii is unable to synthesize purines de novo, instead salvages them from its environment, inside the host cell, for which they need high affinity carriers. Here, we report the expression of a T. gondii Equilibrative Nucleoside Transporter, Tg244440, in a Trypanosoma brucei strain from which nucleobase transporters have been deleted. Tg244440 transported hypoxanthine and guanine with similar affinity (Km ~1 µM), while inosine and guanosine displayed Ki values of 4.05 and 3.30 µM, respectively. Low affinity was observed for adenosine, adenine, and pyrimidines, classifying Tg244440 as a high affinity oxopurine transporter. Purine analogues were used to probe the substrate-transporter binding interactions, culminating in quantitative models showing different binding modes for oxopurine bases, oxopurine nucleosides, and adenosine. Hypoxanthine and guanine interacted through protonated N1 and N9, and through unprotonated N3 and N7 of the purine ring, whereas inosine and guanosine mostly employed the ribose hydroxy groups for binding, in addition to N1H of the nucleobase. Conversely, the ribose moiety of adenosine barely made any contribution to binding. Tg244440 is the first gene identified to encode a high affinity oxopurine transporter in T. gondii and, to the best of our knowledge, the first purine transporter to employ different binding modes for nucleosides and nucleobases.

Role of amylopectin synthesis in Toxoplasma gondii and its implication in vaccine development against toxoplasmosis.

Toxoplasma gondii is a ubiquitous pathogen infecting one-third of the global population. A significant fraction of toxoplasmosis cases is caused by reactivation of existing chronic infections. The encysted bradyzoites during chronic infection accumulate high levels of amylopectin that is barely present in fast-replicating tachyzoites. However, the physiological significance of amylopectin is not fully understood. Here, we identified a starch synthase (SS) that is required for amylopectin synthesis in T. gondii. Genetic ablation of SS abolished amylopectin production, reduced tachyzoite proliferation, and impaired the recrudescence of bradyzoites to tachyzoites. Disruption of the parasite Ca2+-dependent protein kinase 2 (CDPK2) was previously shown to cause massive amylopectin accumulation and bradyzoite death. Therefore, the Δcdpk2 mutant is thought to be a vaccine candidate. Notably, deleting SS in a Δcdpk2 mutant completely abolished starch accrual and restored cyst formation as well as virulence in mice. Together these results suggest that regulated amylopectin production is critical for the optimal growth, development and virulence of Toxoplasma. Not least, our data underscore a potential drawback of the Δcdpk2 mutant as a vaccine candidate as it may regain full virulence by mutating amylopectin synthesis genes like SS.

Outbreaks of clinical toxoplasmosis in humans: five decades of personal experience, perspectives and lessons learned.

BACKGROUND: The protozoan parasite Toxoplasma gondii has a worldwide distribution and a very wide host range, infecting most warm-blooded hosts. Approximately 30% of humanity is infected with T. gondii, but clinical toxoplasmosis is relatively infrequent. Toxoplasmosis has a wide range of clinical symptoms involving almost all organ systems. In most persons that acquire infection postnatally, symptoms (when present) are mild and mimic other diseases such as flu, Lyme disease, Q fever, hematological alterations, or mumps. It is likely that clinical disease is more common than reported. The ingestion of infected meat or food and water contaminated with oocysts are the two main modes of postnatal transmission of Toxoplasma gondii. The infective dose and the incubation period of T. gondii infection are unknown because there are no human volunteer experiments. METHODS: Here, I have critically reviewed outbreaks of clinical toxoplasmosis in humans for the past 55 years, 1966-2020. Information from oocyst-acquired versus meat-acquired infections was assessed separately. RESULTS: Most outbreaks were from Brazil. There were no apparent differences in types or severity of symptoms in meat- versus oocyst-acquired infections. Fever, cervical lymphadenopathy, myalgia, and fatigue were the most important symptoms, and these symptoms were not age-dependent. The incubation period was 7-30 days. A genetic predisposition to cause eye disease is suspected in the parasites responsible for three outbreaks (in Brazil, Canada, and India). Only a few T. gondii tissue cysts might suffice to cause infection, as indicated by outbreaks affecting some (but not all) individuals sharing a meal of infected meat. CONCLUSIONS: Whether the high frequency of outbreaks of toxoplasmosis in humans in Brazil is related to environmental contamination, poor hygiene, socioeconomic conditions, or to genotypes of T. gondii needs investigation.

Epidemiologic and Public Health Significance of Toxoplasma gondii Infections in Venison: 2009-2020.

Toxoplasma gondii infections are common in humans and animals worldwide. The ingestion of food or water contaminated with oocysts excreted by infected cats or ingestion of uncooked or undercooked meat containing tissue cysts of T. gondii are the 2 major modes of transmission of T. gondii. Deer are a popular game. Recently, outbreaks of clinical toxoplasmosis were reported in humans in North America linked to ingestion of undercooked venison. Here, we review prevalence, persistence of infection, clinical disease, epidemiology, and public health risks of T. gondii infections in deer and other cervids for the past decade. Estimates of worldwide serological prevalence are summarized individually for each species of deer, elk, moose, and caribou. Genetic diversity of 112 viable isolates of T. gondii from cervids is discussed, including its public health significance. Prevalence of T. gondii in deer is very high. Any part of a deer, including liver, spleen, and muscles, should be cooked thoroughly before human consumption.

Early immune responses and parasite tissue distribution in mice experimentally infected with oocysts of either archetypal or non-archetypal genotypes of Toxoplasma gondii.

In most of the world Toxoplasma gondii is comprised of archetypal types (types I, II and III); however, South America displays several non-archetypal strains. This study used an experimental mouse model to characterize the immune response and parasite kinetics following infection with different parasite genotypes. An oral inoculation of 50 oocysts per mouse from T. gondii M4 type II (archetypal, avirulent), BrI or BrIII (non-archetypal, virulent and intermediate virulent, respectively) for groups (G)2, G3 and G4, respectively was used. The levels of mRNA expression of cytokines, immune compounds, cell surface markers and receptor adapters [interferon gamma (IFNγ), interleukin (IL)-12, CD8, CD4, CD25, CXCR3 and MyD88] were quantified by SYBR green reverse transcription-quantitative polymerase chain reaction. Lesions were characterized by histology and detection by immunohistochemistry established distribution of parasites. Infection in G2 mice was mild and characterized by an early MyD88-dependent pathway. In G3, there were high levels of expression of pro-inflammatory cytokines IFNγ and IL-12 in the mice showing severe clinical symptoms at 8­11 days post infection (dpi), combined with the upregulation of CD25, abundant tachyzoites and tissue lesions in livers, lungs and intestines. Significant longer expression of IFNγ and IL-12 genes, with other Th1-balanced immune responses, such as increased levels of CXCR3 and MyD88 in G4, resulted in survival of mice and chronic toxoplasmosis, with the occurrence of tissue cysts in brain and lungs, at 14 and 21 dpi. Different immune responses and kinetics of gene expression appear to be elicited by the different strains and non-archetypal parasites demonstrated higher virulence.

Public Health Significance of Toxoplasma gondii Infections in Cattle: 2009-2020.

Toxoplasma gondii infections are common in humans and animals worldwide. The ingestion of food or water contaminated with oocysts excreted by infected cats or ingesting uncooked or undercooked meat containing tissue cysts of T. gondii are the 2 major modes of transmission of T. gondii. Viable T. gondii is more prevalent in pork and lamb than in beef. In the past decade, there have been many articles on the high seroprevalence in cattle, particularly from China. There is a report of an outbreak of acute toxoplasmosis in humans suspected to be linked to the ingestion of Artisan fresh cheese from cow's milk. There are conflicting reports concerning the rate of congenital transmission of T. gondii in cattle, especially from Brazil. In a report from Brazil, viable T. gondii was isolated from the blood of 1 of 60 pregnant cows slaughtered at an abattoir and from 1 fetus. The role of beef in the epidemiology of T. gondii infections is still not clear. Here, we review prevalence, persistence of infection, clinical disease, epidemiology, and public health risks of T. gondii infections in cattle from beef and cow's milk worldwide for the past decade.

Toxoplasma gondii Chinese I genotype Wh6 strain infection induces tau phosphorylation via activating GSK3ß and causes hippocampal neuron apoptosis.

Toxoplasma gondii (T. gondii) is a neurophilic and intracellular parasite that can affect plenty of vertebrate animals, including humans. Recent researches indicate that T. gondii infection is associated with neurodegenerative diseases such as Alzheimer's disease(AD). In addition, tau hyper-phosphorylation is a crucial event leading to the formation of nerve fiber tangles in AD. Despite the efforts to understand the interactions between T. gondii and AD, there are no clear results available so far. Here, we infected mice with the T. gondii of the Chinese 1 genotype Wh6 strain (TgCtwh6) for 60 days. Then we observed the formation of tissue cysts in the brain, the damage of neuron and the increased expression of phosphorylated tau (p-tau) in the hippocampal tissue of the mice. Similarly, we also found that p-tau, glycogen synthase kinase 3 beta (GSK3ß), and phosphorylated GSK3ß (p-GSK3ß) were upregulated in vitro in TgCtwh6 challenged hippocampal neuron cell strain, HT22 cells. We noted a down-regulated expression of GSK3ß,p-GSK3ß, and p-tau in HT22 cells, which were pretreated with LiCl, an inhibitor of GSK3ß. These data suggested that p-GSK3ß may mediate tau phosphorylation after TgCtwh6 infection. Furthermore, TgCtwh6 infection also caused the increased expression of Bax and Caspase3, the decreased expression of Bcl-XL in HT22 cells, which had both early and late apoptosis. In all, our results indicated that TgCtwh6 infection not only led to phosphorylation of tau via activating GSK3ß but also promoted hippocampal neuron apoptosis. Our research may partially reveal the mechanism with which TgCtwh6 induce neurofibrillary pathology.

Application of Toxoplasma gondii GRA15 peptides in diagnosis and serotyping.

Previous studies showed that three clonal strain types (I, II, and III) of Toxoplasma gondii can be distinguished using serotyping based on a series of polymorphic proteins. However, to establish a systematic serotyping method with higher resolution even being equal to that of genotyping, more specific peptide markers are needed. The objective of the present study was to determine the possibility of the polymorphic dense granule protein 15 (GRA15) for diagnosis and serotyping of T. gondii infection. Three different T. gondii GT1 strain GRA15 gene fragments encoding a 584-residue peptide, a 199-residue peptide and a 84-residue peptide were amplified, expressed and purified, respectively. Anti-T. gondii GT1 strain antibodies, anti-T. gondii RH strain antibodies and anti-T. gondii PRU strain antibodies were used for immunoblotting analysis of the three peptides. Western blotting analysis showed that the 584-residue peptide of GT1 strain GRA15 was a potential candidate for serological diagnosis of T. gondii infection. RH strain from GT1 strain could be distinguished by serotyping based on the GRA15199 or GRA1584, and T. gondii GT1 strain could be distinguished from PRU strain by using serotyping based on the GRA1584. These findings reveal, for the first time, a novel potential role of GRA15-derived peptides in diagnosis and serological differentiation of T. gondii infection.

In vivo characterization of a Toxoplasma gondii strain TgCatJpTy1/k-3 isolated from a stray cat in Japan.

The Toxoplasma gondii strain TgCatJpTy1/k-3 (K-3), isolated from a stray cat in Tokyo, Japan, is categorized as a type II genotype. Since the K-3 strain is empirically known to form relatively larger cysts and exhibit weak pathogenesis in a mouse, it could serve as a useful model organism to study chronic T. gondii infection in the host. However, a detailed biological characterization of this strain had not been performed. In this study, we thoroughly assessed the K-3 strain in vivo using a mouse model. Tests indicated that pathogenicity of the K-3 strain was lower than that of the PLK strain, a clonal laboratory strain with a moderately pathogenic type II genotype. Further, cyst sizes of the K-3 strain were significantly larger than those of the PLK strain. Interestingly, K-3 cyst sizes in T. gondii-resistant ICR mice were larger than those in T. gondii-susceptible C57BL/6N mice. Our study suggests that the K-3 strain is suitable to study T. gondii cystogenesis and chronic infection, which are currently difficult to analyze using cell-adopted T. gondii strains.

A review of toxoplasmosis in humans and animals in Turkey.

Infections by the protozoan parasite Toxoplasma gondii are widely prevalent in humans and animals in Turkey but little is known of the burden of their clinical toxoplasmosis. Many early papers on toxoplasmosis in Turkey were published in Turkish and often not available widely. Here, we review prevalence, clinical spectrum, epidemiology and diagnosis of T. gondii in humans and animals in Turkey. This knowledge should be useful to biologists, public health workers, veterinarians and physicians. Although one-third of the human population in Turkey is seropositive, the rate of congenital toxoplasmosis is unknown and no information is available in children 12 years old or younger. One large outbreak of acute toxoplasmosis has been reported in 14-18-year old school children in Turkey. An alarming rate (36%) of T. gondii tissue cysts were reported in tissues of sheep and water buffalo meats destined for human consumption; these reports require verification. Genetically, T. gondii strains from domestic cats and wild birds in Turkey were generally classical type II and III, like those prevalent in Europe. A separate genotype, Type 1 Africa, was isolated from two congenitally infected children and a domestic cat in Turkey.

Serotyping of Toxoplasma gondii Infection Using Peptide Membrane Arrays.

The intracellular parasite Toxoplasma gondii can cause chronic infections in most warm-blooded animals, including humans. In the USA, strains belonging to four different Toxoplasma clonal lineages (types 1, 2, 3, and 12) are commonly isolated, whereas strains not belonging to these lineages are predominant in other continents such as South America. Strain type plays a pivotal role in determining the severity of Toxoplasma infection. Therefore, it is epidemiologically relevant to develop a non-invasive and inexpensive method for determining the strain type in Toxoplasma infections and to correlate the genotype with disease outcome. Serological typing is based on the fact that many host antibodies are raised against immunodominant parasite proteins that are highly polymorphic between strains. However, current serological assays can only reliably distinguish type 2 from non-type 2 infections. To improve these assays, mouse, rabbit, and human infection serum were reacted against 950 peptides from 62 different polymorphic Toxoplasma proteins by using cellulose membrane peptide arrays. This allowed us to identify the most antigenic peptides and to pinpoint the most relevant polymorphisms that determine strain specificity. Our results confirm the utility of previously described peptides and identify novel peptides that improve and increase the specificity of the assay. In addition, a large number of novel proteins showed potential to be used for Toxoplasma diagnosis. Among these, peptides derived from several rhoptry, dense granule, and surface proteins represented promising candidates that may be used in future experiments to improve Toxoplasma serotyping. Moreover, a redesigned version of the published GRA7 typing peptide performed better and specifically distinguished type 3 from non-type 3 infections in sera from mice, rabbits, and humans.

Testing New Peptides From Toxoplasma gondii SAG1, GRA6, and GRA7 for Serotyping: Better Definition Using GRA6 in Mother/Newborns Pairs With Risk of Congenital Transmission in Mexico.

Toxoplasma gondii variant influences clinical profile in human congenital and ocular toxoplasmosis. Parasite genotyping represents a challenge due to insufficient amount of genetic material of the protozoan in the host samples, and isolates are hard to obtain, especially from pediatric patients. An alternative is serotyping, which is based on the presence of specific antibodies against polymorphic proteins related to virulence; the more widely used are GRA6 and GRA7, but most works report cross reactions among the classical strains (I, II, and III). We designed new peptides of GRA6, GRA7, and SAG1 proteins, with more SNPs among the three clonal strains than those previously designed. This was done by identifying BcR and polymorphic epitopes by means of bioinformatics; then we designed peptides with linkers joining the specific regions and predicted their 3D structure. With the commercial molecules synthesized on the basis of these designs, we tested 86 serum samples from 42 mother/newborn pairs and two congenitally infected newborns, by indirect ELISA. We implemented a strategy to determine the serotype based on scatter plots and a mathematical formula, using ratios among reactivity indexes to peptides. We found low frequency of samples reactive to GRA7 and SAG1, and cross reactions between GRA6 serotypes I and III; we modified these later peptides and largely improved distinction among the three clonal strains. The chronicity of the infection negatively affected the reactivity index against the peptides. Serotyping both members of the mother/child pair improves the test, i.e., among 26% of them only one member was positive. Serotype I was the most frequent (38%), which was congruent with previous genotyping results in animals and humans of the same area. This serotype was significantly more frequent among mothers who transmitted the infection to their offspring than among those who did not (53 vs. 8%, p = 0.04) and related to disease dissemination in congenitally infected children, although non-significantly. In conclusion, serotyping using the improved GRA6 peptide triad is useful to serotype T. gondii in humans and could be implemented for clinical management and epidemiological studies, to provide information on the parasite type in specific areas.

The ROP16III-dependent early immune response determines the subacute CNS immune response and type III Toxoplasma gondii survival.

Toxoplasma gondii is an intracellular parasite that persistently infects the CNS and that has genetically distinct strains which provoke different acute immune responses. How differences in the acute immune response affect the CNS immune response is unknown. To address this question, we used two persistent Toxoplasma strains (type II and type III) and examined the CNS immune response at 21 days post infection (dpi). Contrary to acute infection studies, type III-infected mice had higher numbers of total CNS T cells and macrophages/microglia but fewer alternatively activated macrophages (M2s) and regulatory T cells (Tregs) than type II-infected mice. By profiling splenocytes at 5, 10, and 21 dpi, we determined that at 5 dpi type III-infected mice had more M2s while type II-infected mice had more pro-inflammatory macrophages and that these responses flipped over time. To test how these early differences influence the CNS immune response, we engineered the type III strain to lack ROP16 (IIIΔrop16), the polymorphic effector protein that drives the early type III-associated M2 response. IIIΔrop16-infected mice showed a type II-like neuroinflammatory response with fewer infiltrating T cells and macrophages/microglia and more M2s and an unexpectedly low CNS parasite burden. At 5 dpi, IIIΔrop16-infected mice showed a mixed inflammatory response with more pro-inflammatory macrophages, M2s, T effector cells, and Tregs, and decreased rates of infection of peritoneal exudative cells (PECs). These data suggested that type III parasites need the early ROP16-associated M2 response to avoid clearance, possibly by the Immunity-Related GTPases (IRGs), which are IFN-γ- dependent proteins essential for murine defenses against Toxoplasma. To test this possibility, we infected IRG-deficient mice and found that IIIΔrop16 parasites now maintained parental levels of PECs infection. Collectively, these studies suggest that, for the type III strain, rop16III plays a key role in parasite persistence and influences the subacute CNS immune response.

Inter- and intra-genotype differences in induced cystogenesis of recombinant strains of Toxoplasma gondii isolated from chicken and pigs.

The ability to differentiate from the proliferative (tachyzoite) to the latent (bradyzoite) stage of isolates of Toxoplasma gondii recombinant genotypes (I/II/III and I/III) and reference strains from a clonal line (RH and ME49) was investigated in this study. Two isolates from chicken (#114 and #277; ToxoDB) and 3 from pigs (#114; ToxoDB) were the subjects for evaluation. The isolates were grown in cell culture under 2 different conditions: culture medium at pH 7.0 (neutral, without stress induction) or pH 8.0 (alkaline, stress inducing). After 4 days, the cultures were fixed and the events resulting from infection and induction were labeled. T. gondii cysts were labeled using Dolichos biflorus-FITC lectin (DBL-cysts) and free tachyzoites or vacuolar were labeled using an anti-T. gondii polyclonal antibody followed by an Alexa 594-conjugated secondary antibody (DBL-negative structures compatible with parasite structures - lysis plaques or vacuole). Differences in DBL-cysts formation in vitro in response to exogenous stress were observed between recombinant genotype isolates and the typical genotypes. The differences in conversion rates and the patterns of lysis plate production between genotype I/III isolates (#114) indicate that care should be taken when extrapolating the in vitro phenotypic characteristics of parasites from the same genotype.

Toxoplasma gondii in water buffaloes (Bubalus bubalis) from Romania: what is the importance for public health?

The purpose of our study was to evaluate the prevalence of Toxoplasma gondii infection in autochthonous Carpathian buffaloes from northwestern Romania by serology, PCR techniques, and mouse bioassay. Agreement between MAT and ELISA, correlation between indirect and direct detection methods, and risk factors were evaluated. The apparent overall seroprevalence of T. gondii was 8.1% by MAT and 6.6% by ELISA. The agreement between ELISA and MAT was fair. The apparent seroprevalence was significantly higher in adult buffaloes (12.5%) compared to calves (0.0%) and juveniles (1.9%) by MAT. Most of the positive adult buffaloes detected by MAT had antibodies at a low sera dilution and the highest dilution was 1:768 in a juvenile female (30 months). No viable T. gondii was detected by mouse bioassay, as no T. gondii cyst or DNA was found in the brain of mice and they did not seroconvert. However, T. gondii DNA was detected in two buffaloes: in a 30-month-old male buffalo by qPCR on the diaphragm digest and in a 252-month-old female buffalo by RE nPCR on the mesenteric lymph node. Both animals were negative in MAT and ELISA. The total prevalence of T. gondii by direct detection methods was 2.7%. There was no correlation between indirect and direct detection methods. Since no viable T. gondii was detected in buffaloes, the risk of human infection from buffalo meat is minimal. Buffaloes' biological response to a T. gondii infection appears to be very similar to the response of cattle.

An unusual and vital protein with guanylate cyclase and P4-ATPase domains in a pathogenic protist.

cGMP signaling is one of the master regulators of diverse functions in eukaryotes; however, its architecture and functioning in protozoans remain poorly understood. Herein, we report an exclusive guanylate cyclase coupled with N-terminal P4-ATPase in a common parasitic protist, Toxoplasma gondii This bulky protein (477-kD), termed TgATPaseP-GC to fairly reflect its envisaged multifunctionality, localizes in the plasma membrane at the apical pole of the parasite, whereas the corresponding cGMP-dependent protein kinase (TgPKG) is distributed in the cytomembranes. TgATPaseP-GC is refractory to genetic deletion, and its CRISPR/Cas9-assisted disruption aborts the lytic cycle of T. gondii Besides, Cre/loxP-mediated knockdown of TgATPaseP-GC reduced the synthesis of cGMP and inhibited the parasite growth due to impairments in the motility-dependent egress and invasion events. Equally, repression of TgPKG by a similar strategy recapitulated phenotypes of the TgATPaseP-GC-depleted mutant. Notably, despite a temporally restricted function, TgATPaseP-GC is expressed constitutively throughout the lytic cycle, entailing a post-translational regulation of cGMP signaling. Not least, the occurrence of TgATPaseP-GC orthologs in several other alveolates implies a divergent functional repurposing of cGMP signaling in protozoans, and offers an excellent drug target against the parasitic protists.

Efficacy of sulfadiazine and pyrimetamine for treatment of experimental toxoplasmosis with strains obtained from human cases of congenital disease in Brazil.

Toxoplasmosis in South America presents great health impacts and is a topic of research interest not only because of the severity of native cases but also due to the predominant atypical genotypes of the parasite circulating in this continent. Typically, symptomatic toxoplasmosis is treated with a combination of sulfadiazine (SDZ) and pyrimethamine (PYR). However, some clinical cases present treatment failures due to an inability of the drugs to control the infection or their significant adverse effects, which can lead to treatment interruption. Although resistance/susceptibility to the aforementioned drugs has been well described for clonal strains of Toxoplasma gondii spread to the Northern Hemisphere, less is known about the South American atypical strains. In this study, the effectiveness of SDZ and PYR for the treatment of mice during acute infection with different atypical T. gondii strains was evaluated. Swiss mice were infected with seven T. gondii strains obtained from newborn patients with congenital toxoplasmosis in Brazil. The infected mice were treated with 10-640 mg/kg per day of SDZ, 3-200 mg/kg per day of PYR, or a combination of both drugs with a lower dosage. The mice were evaluated for parameters including mortality, anti-T. gondii IgG production by ELISA and the presence of brain cysts. In addition, the presence of polymorphisms in the dhps gene was verified by gene sequencing. A descriptive analysis was used to assess the association between susceptibility to SDZ and/or PYR and the genotype. The TgCTBr4 and TgCTBr17 strains (genotype 108) presented lower susceptibility to SDZ or PYR treatment. The TgCTBr1 and TgCTBr25 strains (genotype 206) presented similar susceptibility to PYR but not SDZ treatment. The TgCTBr9 strain (genotype 11) was the only strain with high susceptibility to treatment with both drugs. The TgCTBr13 strain (genotype 208) was not susceptible to treatment with the lower PYR or SDZ doses. The TgCTBR23 strain (genotype 41) was more susceptible to PYR than to SDZ treatment. However, the association of low SDZ and PYR doses showed good efficacy for the treatment of experimental toxoplasmosis with T. gondii atypical strains obtained from newborns in Brazil. A new mutation in the T. gondii dhps gene (I347M) was identified that might be associated with the SDZ low sensitivity profile observed for the TgCTBr4 and TgCTBr17 isolates.

The expressive identification and localization of bicoid-interacting protein 3 in the toxoplasma gondii Chinese I genotype Wh3 strain.

Toxoplasma gondii is an important intracellular parasite that is distributed worldwide and can infect almost all warm-blooded animals. The Chinese I genotype Wh3 strain is the most common in China and has unique pathogenicity compared with other strains of T. gondii. Bicoid-interacting protein (Bin3) is predicted to be involved in the development and polarity of T. gondii, and the localization of this protein is necessary for studying the biological characteristics of the Chinese I genotypeWh3 strain of T. gondii. In this study, we established an in vitro the method of transforming the tachyzoites into bradyzoites to lay the foundations for further experimental studies. Parasites were induced by culturing in alkaline conditions, then the changes in parasites morphology were evaluated. SAG2C, BAG1 and SAG1 were used to identify parasites. The results show that the Chinese I genotype Wh3 strain exhibited pseudocysts and cysts in the alkaline conditions after being induced, and the bradyzoite stage expressed specific proteins at the same time. Bradyzoites, induced using an alkaline medium (pH = 8.2), had higher expression levels of the Bin3 protein than tachyzoites. The results of indirect immunofluorescence, using a Bin3 monoclonal antibody showed that the Bin3 protein is expressed in both free-state and pseudocysts tachyzoites, and in the cysts of the Chinese I genotype Wh3 strain. The Bin3 protein is located in the cytoplasm of free-state tachyzoites, secreted between the parasite and the pseudocyst membrane in pseudocysts, and distributed inside the cyst wall of cysts. These findings provide a basis for further study on the biological characteristics of the Chinese I genotype Wh3 strain.

Systematic review and meta-analysis of variation in Toxoplasma gondii cyst burden in the murine model.

Toxoplasma gondii is an obligate intracellular protozoan parasite that infects approximately 30% of the population of the United States, with worldwide distribution. The chronic (latent) infection, mediated by the bradyzoite parasite life stage, has attracted attention due to possible links to host behavioral alteration and psychomotor effects. Mice are a common model organism for studying the chronic stage, as they are natural hosts of infection. Notably, published studies demonstrate vast ranges of measured cyst burden within the murine brain tissue. The inconsistency of measured cyst burden within and between experiments makes interpretation of statistical significance difficult, potentially confounding studies of experimental anti-parasitic approaches. This review analyzes variation in measured cyst burden in a wide array of experimental mouse infections across published literature. Factors such as parasite infection strain, mouse strain, mode of infection, and infectious dose were all examined. The lowest variation in measured cyst burden occurred with the commonly available Balb/c and CBA mice undergoing infection by the ME49 strain of T. gondii. A summary of cyst variation and average cyst counts in T. gondii mouse models is presented, which may be useful for designing future experiments.

A review of Cystoisospora felis and C. rivolta-induced coccidiosis in cats.

Until the discovery of Toxoplasma gondii oocysts in cat feces in 1970, little was known of coccidiosis in cats. Until 1970, three coccidian parasites based on different sized oocysts were recognized, the parasite with large oocysts (∼40 µm long and called Isospora felis), medium sized oocysts (∼25 µm long, called Isospora rivolta), and small sized oocysts (14 µm or less, called Isospora bigemina) were known and they were considered not host-specific. Later, it was demonstrated that these parasites were host-specific and had also extra-intestinal stages. The Isospora bigemina turned out to be more than 25 organisms belonging to T. gondii, Hammondia spp., Sarcocystis spp., Besnoitia spp., and Neospora spp.; these subjects have been reviewed previously in detail. The present paper summarizes biology of Isospora felis, and I. rivolta (now transferred to genus Cystoisospora), including taxonomy, life cycle, diagnosis, and treatment. Re-excretion of T. gondii oocysts from chronically infected cats after superinfection with Cystoisospora felis oocysts is discussed. There are only two species of Cystoisospora species in cats, C. felis and C. rivolta; Isospora novocati and Cystoisospora frenkeli named for I. rivolta-like parasites of cats are considered synonym of C. rivolta. Clinical coccidiosis occurs more commonly in recently weaned kittens and C. felis infections are more prevalent than C. rivolta.