ROP18 and ROP5 alleles combinations are related with virulence of T. gondii isolates from Argentina

The allelic combination of ROP18/ROP5 genes of Toxoplasma gondii has been shown to be highly predictive of mouse virulence in canonical isolates and strains. The aims of this study were to analyze the alleles present in the ROP18/ROP5 genes from T. gondii isolates obtained in Argentina, to associate the results with the virulence registered in mouse model, and to compare with other isolates and reference strains using a phylogenetic network. Fourteen T. gondii isolates from Argentina were analyzed by nPCR-RFLP for ROP18/ROP5. Phylogenetic network analysis was inferred using the ToxoDB genotypes and the ROPs molecular markers. All isolates and reference strains were categorized as lethal or non-lethal. As results, combinations 2/2, 3/3 and 4/3 for ROP18/ROP5 were detected in 12 isolates, whereas only alleles 1 and 2 of ROP5 were detected in 2 isolates. The majority of the isolates had a mouse virulence matching to that predicted by the ROP18/ROP5 allele combination. The 3 isolates that differed from the expected virulence presented non-clonal genotypes. ROPs incorporation increased the accuracy of the phylogenetic network relations among the T. gondii samples, prevailing the clustering according to regions. Our results indicate a predominance of type 3 allele in both ROP18 and ROP5 markers and an association of allelic profiles 3/3 and 4/3 of non-clonal genotypes from Argentina, both with virulent and avirulent profiles in mice.     

Virulence and molecular characterization of Toxoplasma gondii isolated from goats in Ceará, Brazil

One hundred and sixty-nine fragments of heart muscle collected from goats in the State of Ceará, Brazil, were analyzed by mouse bioassay with the aim of isolating Toxoplasma gondii. Two T. gondii isolates, named G1 and G2, were obtained and were characterized by PCR-RFLP. In addition, their virulence was evaluated by experimental inoculation into BALB/c mice. The G1 isolate presented high virulence leading to 100% mortality of mice after inoculations with 10¹, 10², and 10³ tachyzoites. The G2 isolate presented low virulence and none of the doses tested lead to mortality of mice. The PCR-RFLP analysis showed that the two isolates are recombinants of the types I/III. However, they differ in the haplotype combination for the genotypes analyzed.     

Detection of Toxoplasma gondii Infections using Virus-Like Particles Displaying T. gondii ROP4 Antigen

Toxoplasma gondii ME49 infections are typically diagnosed by serological tests. However, serological diagnosis of RH strain-induced toxoplasmosis remains unknown. In order to develop seradiagnosis of above 2 kinds of infections, we generated recombinant virus-like particles (VLPs) displaying the T. gondii rhoptry protein 4 (ROP4) and evaluated their potential in T. gondii ME49 or RH strain infection diagnostics. Mice were orally infected with either the tachyzoites of T. gondii (RH) or cysts of T. gondii (ME49) at various dosages, and sera were collected at regular intervals. ELISA-based serological tests were performed to assess IgG, IgM, and IgA antibody responses against ROP4 VLP antigen and tissue lysate antigen (TLA). Compared to TLA, IgG, IgM, and IgA levels to ROP4 VLP antigen were significantly higher in the sera of T. gondii RH-infected mice 1 and 2 week post-infection (PI). T. gondii-specific IgG antibody was detected at 1, 2, 4, and 8 week PI in the T. gondii ME49-infected mice with infection dose-dependent manner. These results indicated that the ROP4 VLP antigen was highly sensitive antigens detecting T. gondii RH and ME49 antibodies at an early stage.     

Western Australian Marsupials Are Multiply Infected with Genetically Diverse Strains of Toxoplasma gondii

Five different organs from 16 asymptomatic free-ranging marsupial macropods (Macropus rufus, M. fuliginosus, and M. robustus) from inland Western Australia were tested for infection with Toxoplasma gondii by multi-locus PCR-DNA sequencing. All macropods were infected with T. gondii, and 13 had parasite DNA in at least 2 organs. In total, 45 distinct T. gondii genotypes were detected. Fourteen of the 16 macropods were multiply infected with genetically distinct T. gondii genotypes that often partitioned between different organs. The presence of multiple T. gondii infections in macropods suggests that native mammals have the potential to promote regular cycles of sexual reproduction in the definitive felid host in this environment.     

Guanylate-binding Protein 1 (Gbp1) Contributes to Cell-autonomous Immunity against Toxoplasma gondii

IFN-γ activates cells to restrict intracellular pathogens by upregulating cellular effectors including the p65 family of guanylate-binding proteins (GBPs). Here we test the role of Gbp1 in the IFN-γ-dependent control of T. gondii in the mouse model. Virulent strains of T. gondii avoided recruitment of Gbp1 to the parasitophorous vacuole in a strain-dependent manner that was mediated by the parasite virulence factors ROP18, an active serine/threonine kinase, and the pseudokinase ROP5. Increased recruitment of Gbp1 to Δrop18 or Δrop5 parasites was associated with clearance in IFN-γ-activated macrophages in vitro, a process dependent on the autophagy protein Atg5. The increased susceptibility of Δrop18 mutants in IFN-γ-activated macrophages was reverted in Gbp1^−/− cells, and decreased virulence of this mutant was compensated in Gbp1^−/− mice, which were also more susceptible to challenge with type II strain parasites of intermediate virulence. These findings demonstrate that Gbp1 plays an important role in the IFN-γ-dependent, cell-autonomous control of toxoplasmosis and predict a broader role for this protein in host defense.     

Isolation and Genetic Characterization of Toxoplasma gondii from Black Bears (Ursus americanus), Bobcats (Lynx rufus), and Feral Cats (Felis catus) from Pennsylvania

Toxoplasma gondii infects virtually all warm‐blooded hosts worldwide. Recently, attention has been focused on the genetic diversity of the parasite to explain its pathogenicity in different hosts. It has been hypothesized that interaction between feral and domestic cycles of T. gondii may increase unusual genotypes in domestic cats and facilitate transmission of potentially more pathogenic genotypes to humans, domestic animals, and wildlife. In the present study, we tested black bear (Ursus americanus), bobcat (Lynx rufus), and feral cat (Felis catus) from the state of Pennsylvania for T. gondii infection. Antibodies to T. gondii were found in 32 (84.2%) of 38 bears, both bobcats, and 2 of 3 feral cats tested by the modified agglutination test (cut off titer 1:25). Hearts from seropositive animals were bioassayed in mice, and viable T. gondii was isolated from 3 of 32 bears, 2 of 2 bobcats, and 2 of 3 feral cats. DNA isolated from culture‐derived tachyzoites of these isolates was characterized using multilocus PCR‐RFLP markers. Three genotypes were revealed, including ToxoDB PCR‐RFLP genotype #1 or #3 (Type II, 1 isolate), #5 (Type 12, 3 isolates), and #216 (3 isolates), adding to the evidence of genetic diversity of T. gondii in wildlife in Pennsylvania. Pathogenicity of 3 T. gondii isolates (all #216, 1 from bear, and 2 from feral cat) was determined in outbred Swiss Webster mice; all three were virulent causing 100% mortality. Results indicated that highly mouse pathogenic strains of T. gondii are circulating in wildlife, and these strains may pose risk to infect human through consuming of game meat.     

Characterization of a spontaneous cyst-forming strain of Toxoplasma gondii isolated from Tokachi subprefecture in Japan

Apicomplexan parasite Toxoplasma gondii has three distinct clonal lineages: high, medium and low virulent strains, type I, II and III, respectively. T. gondii avoids the immune response by transforming from fast multiplying tachyzoite to slow multiplying bradyzoite, and establishing a chronic infection. In the present study, we isolated a new strain of T. gondii from cat feces in the Tokachi subprefecture, Hokkaido, Japan and named it as TgCatJpObi1 (Obi1) strain. Genotyping analysis of 12 loci revealed atypical characters close to type II, genotype 4 according to ToxoDB classification. Phenotypically, Obi1 strain shows slow growth rate and the ability of spontaneous cyst formation in both human foreskin fibroblast (HFFs) and mouse peritoneal macrophages in vitro without bradyzoite induction. Parasite virulence was assessed by means of mouse survival upon infection with either Obi1 or ME49 strains. Obi1 strain displayed no mortalities in comparison to type II clonal lineage, ME49 at LD₅₀ to LD₁₀₀ range (1 × 10³–10⁶ tachyzoites). Although virulence of Obi1 strain is significantly lower than that of ME49, nucleotide sequences analyses revealed that genes of virulence factors such as Gra15, Rop5, 16, 17, and 18 in Obi1 strain were 100% identical to those in the type II strain. Thus, characterization of a newly isolated strain, Obi1, is crucial to clarify the development of toxoplasmosis in both humans and animals.     

Toxoplasma gondii Protein Disulfide Isomerase (TgPDI) Is a Novel Vaccine Candidate against Toxoplasmosis

Toxoplasma gondii is a ubiquitous protozoan parasite that can infect all warm-blooded animals, including both mammals and birds. Protein disulfide isomerase (PDI) localises to the surface of T. gondii tachyzoites and modulates the interactions between parasite and host cells. In this study, the protective efficacy of recombinant T. gondii PDI (rTgPDI) as a vaccine candidate against T. gondii infection in BALB/c mice was evaluated. rTgPDI was expressed and purified from Escherichia coli. Five groups of animals (10 animals/group) were immunised with 10, 20, 30, 40 μg of rTgPDI per mouse or with PBS as a control group. All immunisations were performed via the nasal route at 1, 14 and 21 days. Two weeks after the last immunisation, the immune responses were evaluated by lymphoproliferative assays and by cytokine and antibody measurements. The immunised mice were challenged with tachyzoites of the virulent T. gondii RH strain on the 14th day after the last immunisation. Following the challenge, the tachyzoite loads in tissues were assessed, and animal survival time was recorded. Our results showed that the group immunised with 30 μg rTgPDI showed significantly higher levels of specific antibodies against the recombinant protein, a strong lymphoproliferative response and significantly higher levels of IgG2a, IFN-gamma (IFN-γ), IL-2 and IL-4 production compared with other doses and control groups. While no changes in IL-10 levels were detected. After being challenged with T. gondii tachyzoites, the numbers of tachyzoites in brain and liver tissues from the rTgPDI group were significantly reduced compared with those of the control group, and the survival time of the mice in the rTgPDI group was longer than that of mice in the control group. Our results showed that immunisation with rTgPDI elicited a protective immune reaction and suggested that rTgPDI might represent a promising vaccine candidate for combating toxoplasmosis.     

Pathogenicity of Five Strains of Toxoplasma gondii from Different Animals to Chickens

Toxoplasma gondii is a protozoan parasite with a broad range of intermediate hosts. Chickens as important food-producing animals can also serve as intermediate hosts. To date, experimental studies on the pathogenicity of T. gondii in broiler chickens were rarely reported. The objective of the present study was to compare the pathogenicity of 5 different T. gondii strains (RH, CN, JS, CAT2, and CAT3) from various host species origin in 10-day-old chickens. Each group of chickens was infected intraperitoneally with 5×10⁸, 1×10⁸, 1×10⁷, and 1×10⁶ tachyzoites of the 5 strains, respectively. The negative control group was mockly inoculated with PBS alone. After infection, clinical symptoms and rectal temperatures of all the chickens were checked daily. Dead chickens during acute phage of the infection were checked for T. gondii tachyzoites by microscope, while living cases were checked for T. gondii infection at day 53 post-inoculation (PI) by PCR method. Histopathological sections were used to observe the pathological changes in the dead chickens and the living animals at day 53 PI. No significant differences were found in survival periods, histopathological findings, and clinical symptoms among the chickens infected with the RH, CN, CAT2, and CAT3 strains. Histopathological findings and clinical symptoms of the JS (chicken origin) group were similar to the others. However, average survival times of infected chickens of the JS group inoculated with 5×10⁸ and 1×10⁸ tachyzoites were 30.0 and 188.4 hr, respectively, significantly shorter than those of the other 4 mammalian isolates. Chickens exposed to 10⁸ of T. gondii tachyzoites and higher showed acute signs of toxoplasmosis, and the lesions were relatively more severe than those exposed to lower doses. The results indicated that the pathogenicity of JS strain was comparatively stronger to the chicken, and the pathogenicity was dose-dependent.     

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.     

The Polymorphic Pseudokinase ROP5 Controls Virulence in Toxoplasma gondii by Regulating the Active Kinase ROP18

Secretory polymorphic serine/threonine kinases control pathogenesis of Toxoplasma gondii in the mouse. Genetic studies show that the pseudokinase ROP5 is essential for acute virulence, but do not reveal its mechanism of action. Here we demonstrate that ROP5 controls virulence by blocking IFN-γ mediated clearance in activated macrophages. ROP5 was required for the catalytic activity of the active S/T kinase ROP18, which phosphorylates host immunity related GTPases (IRGs) and protects the parasite from clearance. ROP5 directly regulated activity of ROP18 in vitro, and both proteins were necessary to avoid IRG recruitment and clearance in macrophages. Clearance of both the Δrop5 and Δrop18 mutants was reversed in macrophages lacking Irgm3, which is required for IRG function, and the virulence defect was fully restored in Irgm3^−/− mice. Our findings establish that the pseudokinase ROP5 controls the activity of ROP18, thereby blocking IRG mediated clearance in macrophages. Additionally, ROP5 has other functions that are also Irgm3 and IFN-γ dependent, indicting it plays a general role in governing virulence factors that block immunity.     

Analysis of Toxoplasma gondii clonal type-specific antibody reactions in experimentally infected turkeys and chickens

Due to their ground-feeding behaviour, free-ranging chickens and turkeys are exposed to oocysts and are good indicators of the presence of Toxoplasma gondii in the environment. In addition, poultry may become infected by ingestion of tissues of infected intermediate hosts such as small rodents. Free-ranging poultry are considered an important source of T. gondii infection in humans, especially in developing countries. Knowledge on T. gondii genotypes in infected animals and humans is important for understanding the epidemiology of T. gondii infections. The aim of the present study was to analyse the ability of experimentally infected turkeys and chickens to develop a T. gondii clonal type-specific antibody response (IgY) after i.v. inoculation with tachyzoites of three T. gondii clonal lineages, types I, II and III. A peptide microarray displaying a panel of 101 different synthetic peptides was used for serotyping. Peptide sequences were derived from polymorphic regions of 16?T. gondii proteins (GRA1, GRA3-7, SAG1, SAG2A, SAG3, SAG4, SRS1, SRS2, ROP1, NTPase I and NTPase III and BSR4). The array was probed with 120 sera from experimentally infected chickens and turkeys inoculated with different doses of T. gondii tachyzoites (10⁴, 10³ and 10²) collected from isolates representative for T. gondii clonal types I (RH), II (ME49) or III (NED) and uninfected controls. After screening of the peptides with reference sera from chickens and turkeys, and evaluation of data by Receiver Operating Characteristics analysis, 41 and 40 peptides were identified that appeared suitable to detect type-specific reactions with sera collected at 2, 5, 7 and 9?weeks p.i. Selected peptides allowed the identification of T. gondii clonal types, until 9?week p.i., which the chickens or turkeys had been inoculated with. At 9?weeks p.i., a high proportion of the experimentally infected chickens (67% (12/18)) and turkeys (61% (11/18)) no longer reacted with the selected peptides. Serotyping of the infection in individual chickens or turkeys was only possible when the whole peptide panel was applied. Clonal type-specific antibody responses were dynamic in both poultry species and depended on the individual animal and the time after infection.     

Imidazo[1,2-b]pyridazines targeting Toxoplasma gondii calcium-dependent protein kinase 1 decrease the parasite burden in mice with acute toxoplasmosis

The current therapeutic arsenal for toxoplasmosis is restricted to drugs non-specific to the parasite which cause important side effects. Development of more efficient and specific anti-Toxoplasma compounds is urgently needed. Imidazo[1,2-b]pyridazines designed to inhibit the calcium-dependent protein kinase 1 of Toxoplasma gondii (TgCDPK1) and effective against tachyzoite growth in vitro at submicromolar ranges were modified into hydrochloride salts to be administered in vivo in a mouse model of acute toxoplasmosis. All protonated imidazo[1,2-b]pyridazine salts (SP230, SP231 and SP232) maintained their activity on TgCDPK1 and T. gondii tachyzoites. Rat and mouse liver microsomes were used to predict half-life and intrinsic clearance, and the pharmacokinetic profile of the most rapidly degraded imidazo[1,2b]pyridazine salt (SP230) was determined in serum, brain and lungs of mice after a single administration of 50?mg/kg. Compounds were then tested in vivo in a murine model of acute toxoplasmosis. Mice infected with tachyzoites of the ME49 strain of T. gondii were treated for 4, 7 or 8?days with 25 or 50?mg/kg/day of SP230, SP231 or SP232. The parasite burdens were strongly diminished (>90% reduction under some conditions) in the spleen and the lungs of mice treated with imidazo[1,2-b]pyridazine salts compared with untreated mice, without the need for pre-treatment. Moreover, no increases in the levels of hepatic and renal toxicity markers were observed, demonstrating no significant signs of short-term toxicity. To conclude, imidazo[1,2-b]pyridazine salts have strong efficacy in vivo on acute toxoplasmosis and should be further tested in a model of mouse congenital toxoplasmosis.     

Effects of Toxoplasma gondii (Gleadle strain) on the host-parasite relationship in trichinosis.

The effects of concurrent infection with Toxoplasma gondii on the host-parasite relationship in trichinosis were studied. Infected mice showed a delay in expulsion of Trichinella spiralis adults from the gut. Persisting adult female worms were fecund but the numbers of larvae recovered from the muscles were not increased. Increased resistance to the systemic phase of trichinosis was shown by reduced numbers of muscle larvae after intravenous injection of newborn larvae in animals with toxoplasmosis as compared with control mice. There were no differences in small bowel pathology of trichinous mice with and without toxoplasmosis but inflammation around muscle cysts of T. spiralis was reduced in mice with toxoplasmosis. The eosinophilia which normally develops in mice with trichinosis was suppressed by concurrent toxoplasmosis. Trichinella infection did not alter the numbers of T. gondii cysts recovered from the brain 4 weeks after infection. It is suggested that the delay in expulsion of adult worms, decrease in muscle inflammation around T. spiralis cysts, and inhibition of eosinophilia result from immune suppression, while the reduction in numbers of muscle larvae after intravenous injection of newborn larvae reflects enhanced nonspecific resistance to infection in toxoplasmosis.     

The Toxoplasma gondii rhoptry protein ROP18 is an Irga6‐specific kinase and regulated by the dense granule protein GRA7

In mice, avirulent strains (e.g. types II and III) of the protozoan parasite Toxoplasma gondii are restricted by the immunity‐related GTPase (IRG) resistance system. Loading of IRG proteins onto the parasitophorous vacuolar membrane (PVM) is required for vacuolar rupture resulting in parasite clearance. In virulent strain (e.g. type I) infections, polymorphic effector proteins ROP5 and ROP18 cooperate to phosphorylate and thereby inactivate mouse IRG proteins to preserve PVM integrity. In this study, we confirmed the dense granule protein GRA7 as an additional component of the ROP5/ROP18 kinase complex and identified GRA7 association with the PVM by direct binding to ROP5. The absence of GRA7 results in reduced phosphorylation of Irga6 correlated with increased vacuolar IRG protein amounts and attenuated virulence. Earlier work identified additional IRG proteins as targets of T. gondii ROP18 kinase. We show that the only specific target of ROP18 among IRG proteins is in fact Irga6. Similarly, we demonstrate that GRA7 is strictly an Irga6‐specific virulence effector. This identifies T. gondii GRA7 as a regulator for ROP18‐specific inactivation of Irga6. The structural diversity of the IRG proteins implies that certain family members constitute additional specific targets for other yet unknown T. gondii virulence effectors.     

Evaluation of immunization with tachyzoite excreted-secreted proteins in a novel susceptible mouse model (A/Sn) for Toxoplasma gondii

Toxoplasma gondii is an important food-borne parasite transmitted primarily from animals to humans through meat consumption, mainly pork and lamb, as well as through oocysts shed by cats. Infection in humans can cause severe neonatal malformations, ocular complications or encephalitis. Toxoplasmosis infection during pregnancy, especially in sheep, often results in abortion, representing considerable economic loss. The aim of this study was to investigate whether Toxoplasma gondii pooled excreted-secreted antigens (ESA), recovered from infected culture supernatants with tachyzoites used as immunogen, can protect experimental mice against T. gondii infection. For immunization experiments, we evaluated A/Sn inbred mice, a novel susceptible mouse model for T. gondii and a virulent strain (RH) for challenge experiments. The antigen selection was based on those produced by tachyzoites since they are responsible for disseminating the infection as well as stimulating the humoral and cellular immune responses. ESA were recovered from VERO cell-culture supernatants infected with virulent RH strain tachyzoites harvested after 48 h. Groups of 5 female mice were intraperitoneally (i.p.) immunized with 4 doses at 2 week intervals with 20 μg of ESA adsorbed to 0.5 mg of alum. The control group received only the adjuvant in PBS on the same dates. Pooled serum collected from chronically infected mice was used as positive control. Blood samples were collected from tail veins 14 days after each immunization. Antibody was detected using ELISA, indirect immunofluorescence and immunoblotting. Anti-ESA antibodies were also evaluated by agglutination, complement-mediated lysis and antibody-mediated cellular toxicity. Fifteen days after the last immunization, both groups were challenged (i.p.) with 1 × 10³ RH strain tachyzoites. The parasitemia was evaluated by PCR, and survival was followed daily. The results showed an increase of antibody levels after each immunization. Anti-ESA antibodies also reacted with a crude tachyzoite antigen and bonded on the parasite surface, with particularly high intensity at the apical region. Anti-ESA antibodies were also able to agglutinate and kill tachyzoites in vitro through interactions with complement and cellular pathways. Even though the tachyzoite challenge was lethal to the mice, PCR results suggested that immunized mice had lower parasitemia as well as longer survival (72 h) than mice from the control group.     

Antibody Detection and Molecular Characterization of Toxoplasma gondii from Bobcats (Lynx rufus), Domestic Cats (Felis catus), and Wildlife from Minnesota,USA

Little is known of the epidemiology of toxoplasmosis in Minnesota. Here, we evaluated Toxoplasma gondii infection in 50 wild bobcats (Lynx rufus) and 75 other animals on/near 10 cattle farms. Antibodies to T. gondii were assayed in serum samples or tissue fluids by the modified agglutination test (MAT, cut‐off 1:25). Twenty nine of 50 bobcats and 15 of 41 wildlife trapped on the vicinity of 10 farms and nine of 16 adult domestic cats (Felis catus) and six of 14 domestic dogs resident on farms were seropositive. Toxoplasma gondii oocysts were not found in feces of any felid. Tissues of all seropositive wild animals trapped on the farm were bioassayed in mice and viable T. gondii was isolated from two badgers (Taxidea taxus), two raccoons (Procyon lotor), one coyote (Canis latrans), and one opossum (Didelphis virginiana). All six T. gondii isolates were further propagated in cell culture. Multi‐locus PCR‐RFLP genotyping using 10 markers (SAG1, SAG2 (5′‐3′SAG2, and alt.SAG2), SAG3, BTUB, GRA6, c22‐8, c29‐2, L358, PK1, and Apico), and DNA from cell culture derived tachyzoites revealed three genotypes; #5 ToxoDataBase (1 coyote, 1 raccoon), #1 (1 badger, 1 raccoon, 1 opossum), and #2 (1 badger). This is the first report of T. gondii prevalence in domestic cats and in bobcats from Minnesota, and the first isolation of viable T. gondii from badger.     

Toxoplasma gondii α-amylase deletion mutant is a promising vaccine against acute and chronic toxoplasmosis

Individuals with inhibited immunity may develop lethal toxoplasmosis; thus, a safe and effective vaccine is urged to be developed. Toxoplasma gondii (T. gondii) α-amylase (α-AMY) is one of the enzymes responsible for starch digestion. In the present study, we first generated a ME49Δα-amy mutant and discovered that loss of α-AMY robustly grew in vitro but contributed to significant virulence attenuation in vivo. Therefore, we established a mouse model to explore the protective immunity of Δα-amy mutant against acute and chronic toxoplasmosis. The results indicated that the survival rates of short-term or long-term immunized mice re-infected with the tachyzoites of multiple T. gondii strains were nearly 100%. ME49Δα-amy not only could provide protective immunity against tachyzoites infection but also could resist the infection of tissue cysts. Furthermore, we detected that ME49Δα-amy vaccination could effectively eliminate the proliferation of parasites in mice and prevent the formation of cysts. The significant increases of Th1-type cytokines, Th2-type cytokines and specific total IgG and IgG subclasses (IgG2a and IgG1) confirmed efficiency of a combination of cellular and humoral immunity against infection. In conclusion, ME49Δα-amy attenuated strain can produce strong immune responses to provide efficient protection against toxoplasmosis, which signifies that ME49Δα-amy mutant may be a potential vaccine candidate.     

Monoclonal antibodies against nucleoside triphosphate hydrolase-II can reduce the replication of Toxoplasma gondii

Nucleoside triphosphate hydrolase (NTPase) is an abundant protein secreted by the obligate protozoan parasite Toxoplasma gondii, which has a wide specificity toward NTP. In the present study, two monoclonal antibodies (mAbs, MNT1 and MNT2) against recombinant T. gondii NTPase-II (rTgNTPase-II) were developed. Western blot analysis displayed that these two mAbs can recognize specifically rTgNTPase-II as well as a 63 kDa molecule in tachyzoites soluble antigens that corresponded to native NTPase-II. T. gondii tachyzoites pretreated with two mAbs were observed under Confocal Laser Microscope and a specific reaction was displayed on tachyzoites after indirect fluorescence antibody test (IFAT). When COS-7 cells were co-cultured with tachyzoites pretreated with two mAbs, the number of intracellular parasites per infected cell was significantly decreased compared with the control. Furthermore, incubation of T. gondii tachyzoites with two mAbs can inhibit NTPase activity in the presence of dithiothreitol, which hinted that the reduction of tachyzoite replication might be owing to the inhibition of NTPase-II by the mAbs. The passive immunization test indicated that the transferred mAbs can significantly prolong the survival time of challenge infected mice. Taken together, we concluded that the mAbs against NTPase-II can reduce the replication of T. gondii and have a crucial effect on the protection of host from T. gondii infection.