Brain Parasite Load Research Articles

Immunogenicity of a Virus-Like-Particle Vaccine Containing Multiple Antigenic Epitopes of Toxoplasma gondii Against Acute and Chronic Toxoplasmosis in Mice.

There is no effective protective vaccine against human toxoplasmosis, which is a potential threat to nearly a third of the world population. Vaccines based on virus-like particles (VLPs) have been highly successful in humans for many years, but have rarely been applied against Toxoplasma gondii infection. In this study, we inserted a B cell epitope (SAG182−102 or SAG1301−320), a CD8+ cell epitope (HF10 or ROP7), and a CD4+ cell epitope (AS15) of T. gondii into a truncated HBcΔ(amino acids1–149) particle to construct four chimeric VLP vaccine formulations, i.e., HBcΔH82, HBcΔH301, HBcΔ R82, and HBcΔ R301. When these chimeric HBc particles were expressed in Escherichia coli, they showed icosahedral morphology similar to that of the original VLPs and were evaluated as vaccine formulations against acute and chronic toxoplasmosis in a mouse model (BALB/c mice (H-2d). All these chimeric HBc VLPs induced strong humoral and cellular immune responses with high IgG antibody titers and interferon(IFN)-γ production. Only the mice immunized with HBcΔH82 showed prolonged survival time (15.6 ± 3.8 vs. 5.6 ± 0.8 days) against acute infection with RH tachyzoites and decrease in brain parasite load (1,454 ± 239 vs. 2,091 ± 263) against chronic infection with Prugniuad cysts, as compared to the findings for the control group. These findings suggest that HBc VLPs would act as an effective carrier for delivering effective multiple antigenic epitopes and would be beneficial for developing a safe and long-acting vaccine against toxoplasmosis.

The liver as an organ at risk for Toxoplasma transmission during transplantation: myth or reality?

AimToxoplasmosis following liver transplant with donor–recipient mismatch is rare, but is often life-threatening. However, there are no data on the frequency of cyst carriage in the liver, nor consensual chemoprophylaxis...

Survey on synergism effect of ketotifen in combination with pyrimethamine in treatment of acute murine toxoplasmosis

BackgroundStandard treatment of toxoplasmosis is accompanied by severe side effects and low tolerability; accordingly, alternative medicines are critically needed. Ketotifen (KET) as a cell membrane stabilizer could be an appropriate inhibitor of Toxoplasma gondii (T. gondii) parasite entrance into the host cells. Therefore, the focus of current study is characterization of the anti-Toxoplasma activity of KET in the acute phase of toxoplasmosis in murine model as pre-treatment and post-treatment (before and after infection with RH strain). KET was used intraperitoneally both individually (2 and 3 mg/kg/day) and in combination with pyrimethamine (PYR) (50 mg/kg/day). One week after the post infection, DNA was extracted from brain biopsies samples. Parasite load was calculated using Quantitative-PCR (Q-PCR) in a triplicate reaction for each DNA with the target for at RE (a 529 bp repeat element) gene.ResultsA significant difference between KET and control groups was observed (P < 0.001) in the pre-treatment and post-treatment groups. Both KET and the combination of KET and PYR showed a reduction in the parasite load in brain through the acute phase of the infection. 2 mg/kg/day dose of KET resulted in higher anti-Toxoplasma activity (15,698 parasites/ml) compared to 3 mg/kg/day dose of KET (72,898 parasites/ml) in brain in the pre-treatment group. In addition, KET combined with PYR significantly decreased the parasite load in the post-treatment group.ConclusionsOur results indicated that KET has both prophylactic and therapeutic effects on acute phases of the disease.

Histopathology of murine toxoplasmosis under treatment with dialyzable leukocyte extract.

BACKGROUNDDialyzable leukocyte extracts (DLEs) contain molecules smaller than 10 kDa with biological activity in receptor organisms. Primarily, they participate in the regulation of the Th1 immune response, which is essential for the control of several intracellular infections, such as toxoplasmosis. This disease is associated with congenital infection, encephalitis or systemic infections in immunocompromised individuals. The clinical course of this infection fundamentally depends on a well-regulated immune response and timely treatment with the appropriate drugs.OBJECTIVEThe aim of this study was to evaluate the effect of treatment with a leukocyte extract, derived from crocodile lymphoid tissue, on the histopathology and brain parasite load in NIH mice that had been infected with cysts of Toxoplasma gondii (ME-49 strain).METHODSThe treatment was applied during the acute and chronic stages of the infection. Histopathological changes were evaluated in the ileum, liver and spleen at one, four and eight weeks after infection and in the brain at week 8. The parasite load was evaluated by counting the cysts of T. gondii found in the brain.FINDINGSCompared to the control mouse group, the mice infected with T. gondii and under treatment with DLE showed less tissue damage, mainly at the intestinal, splenic and hepatic levels. In addition, a greater percentage of survival was observed, and there was a considerable reduction in the parasite load in the brain.CONCLUSIONSThe results suggest that DLE derived from crocodile is a potential adjunctive therapy in the conventional treatment of toxoplasmosis.

Induction of Protective Immunity against Toxoplasma gondii in Mice by Nucleoside Triphosphate Hydrolase-II (NTPase-II) Self-amplifying RNA Vaccine Encapsulated in Lipid Nanoparticle (LNP).

RNA-based vaccine represents an irresistible and safe immunization strategy with decreasing theoretical risks of genomic integration and malignant cell transformation. To our knowledge, however, there is no report about development of RNA vaccine against Toxoplasma gondii infection. We have previously demonstrated that the recombinant T. gondii nucleoside triphosphate hydrolase-II (NTPase-II) protein is able to provide protective Th1 cell-mediated immunity against T. gondii. Herein, we evaluated the immunogenic potential of a self-amplifying RNA vaccine-encoding T. gondii NTPase-II gene, RREP-NTPase-II, delivered by a synthetic lipid nanoparticle (LNP). Immunization of mice with naked RREP-NTPase-II induced a strong cellular and humoral immune response with high-IgG antibody titers and IFN-γ production. The immunized mice displayed significantly prolonged survival time and reduction in brain parasite load (46.4%) compared with control group. Furthermore, mice vaccinated with RREP-NTPase-II-encapsulated LNP displayed significantly enhanced protection against acute infection as well as chronic infection with PRU cyst, which shows 62.1% reduction in brain cyst burden in comparison to control group. These results suggest that the combination of self-amplifying RNA and LNP ion would be beneficial to the development of a safe and long-acting vaccine against toxoplasmosis.

Adoptive transfer of CD4+Foxp3+ regulatory T cells to C57BL/6J mice during acute infection with Toxoplasma gondii down modulates the exacerbated Th1 immune response

Infection of C57BL/6J mice with the parasite Toxoplasma gondii triggers a powerful Th1 immune response that is detrimental to the host. During acute infection, a reduction in CD4+Foxp3+ regulatory T cells (Treg) has been reported. We studied the role of Treg during T. gondii infection by adoptive transfer of cells purified from transgenic Foxp3EGFP mice to infected wild type animals. We found a less severe weight loss, a significant delayed mortality in infected Treg-transferred mice, and reduced pathology of the small intestine that were associated with lower IFN-γ and TNF-α levels. Nevertheless, higher cyst number and parasite load in brain were observed in these mice. Treg-transferred infected mice showed reduced levels of both IFN-γ and TNF-α in sera. A reduced number of CD4+ T cells producing IFN-γ was detected in these mice, while IL-2 producing CD4+ T cells were restored to levels nearly similar to uninfected mice. CD25 and CD69 expression of CD4+ T cells were also down modulated. Our data show that the low Treg cell number are insufficient to modulate the activation of CD4+ T cells and the production of high levels of IFN-γ. Thus, a delicate balance between an optimal immune response and its modulation by Treg cells must exist.

Critical role of IL-33 receptor ST2 in experimental cerebral malaria development.

Cerebral malaria, a severe complication of Plasmodium falciparum infection, can be modeled in murine Plasmodium berghei ANKA (PbA) infection. PbA-induced experimental cerebral malaria (ECM) is CD8(+) T-cell mediated, and influenced by TH 1/TH 2 balance. Here, we show that IL-33 expression is increased in brain undergoing ECM and we address the role of the IL-33/ST2 pathway in ECM development. ST2-deficient mice were resistant to PbA-induced neuropathology. They survived >20 days with no ECM neurological sign and a preserved cerebral microcirculation, while WT mice succumbed within 10 days with ECM, brain vascular leakage, distinct microvascular pathology obstruction, and hemorrhages. Parasitemia and brain parasite load were similar in ST2-deficient and WT mice. Protection was accompanied by reduced brain sequestration of activated CD4(+) T cells and perforin(+) CD8(+) T cells. While IFN-γ and T-cell-attracting chemokines CXCL9 and CXCL10 were not affected in the absence of functional ST2 pathway, the local expression of ICAM-1, CXCR3, and LT-α, crucial for ECM development, was strongly reduced, and this may explain the diminished pathogenic T-cell recruitment and resistance to ECM. Therefore, IL-33 is induced in PbA sporozoite infection, and the pathogenic T-cell responses with local microvascular pathology are dependent on IL-33/ST2 signaling, identifying IL-33 as a new actor in ECM development.

A 78 kDa host cell invasion protein of Neospora caninum as a potential vaccine candidate

Neosporosis is an intracellular protozoan disease caused by Neospora caninum. Until now, there is no effective vaccine to prevent neosporosis. The host cell binding protein has the potential as neosporosis vaccine. In the present study, a T7 phage display library was constructed and screened using Vero cells to obtain host cell binding protein of N. caninum. Two host cell binding proteins, a hypothetical protein of 78 kDa (named as NcP78) homologous to the acylglycerol lipase of Toxoplasma gondii ME49 (XP_002370319.1) and NcGRA7 (known as a dense granules protein that is involved in the invasion of N. caninum to the host cells), were identified. Immune responses induced by recombinant NcP78 and NcGRA7 proteins and their protective efficacies against homologous challenge in BALB/c mice were evaluated respectively. Results showed that recombinant NcP78 and NcGRA7 could elicit both Th1 and Th2 immune responses (with the elevated levels of IgG1 and IgG2a antibody), but predominately a Th2 immune response with a high level of IgG1. The ani-NcP78 and anti-NcGRA7 serum also had inhibitory effects on N. caninum invasion to Vero cells in vitro, which indicated that both NcP78 and NcGRA7 proteins were involved in host cell invasion. Recombinant NcP78 and NcGRA7 could not prolong the survival times and improve the survival rates of dams, but could prolong the survival times and improve the survival rates of offspring significantly. Moreover, the recombinant NcP78 and NcGRA7 could reduce the brain parasite load of dams and offspring. Though these protein vaccines could not effectively alleviate the symptom of abortion, they could increase the number of born offspring significantly, indicating that Nc78 and NcGRA7 recombinant proteins could provide a partial protection against N. caninum infection in mice.

Toxoplasma gondii parasite load elevation in diabetic rats as latent opportunistic character

Introduction: Toxoplasma gondii; is extreme zoonotic intracellular protozoan, exceeding human medical impact due congenital and cerebral toxoplasmosis. Diabetes is characterized by hyperglycemia resulting from perturbation in insulin secretion, insulin action or both, possible through its long depilating term to flare up the intrinsic (latent) stage of T. gondii. Objective: Is focused on studying the latent toxoplasmosis, based on identifying levels of both average brain parasite load and immunoglobulin titers in diabetic induced rat. Materials and Methods: A total number of 60 Wistar male rats (120-180 g) were divided into four groups, 15 each and experimented as follow; un-infected-normoglycaemic, un-infected-diabetic, infected-normoglycaemic and infected-diabetic groups. Blood samples were taken from rats for glucose determination and for serological assay, and at the same time brain tissue samples for parasite load estimation were collected from three sacrificed rats in each group at sequence days; 1, 15, 30, 45 and 60 cycle to days post diabetes, with the same compatible days at 45, 60, 75, 90 and 105 corresponding to days post infection. Results: Showed significance higher in both blood glucose levels and in cysts number in infected diabetic group than the uninfected-diabetic one. The elevated brain parasite load and IgM titer were always exceeding higher progression all over the course and parallel to the elevated glucose levels in infected diabetic group. Conclusion: Hyperglycemic factor was confirmed to opportunist higher T. gondii brain parasite load and elevate immunoglobulin titer in latent infected and diabetes induced rat, possible match with T. gondii sero-positive diabetic patient, and reflects the importance of serological monitoring in particular uncontrolled hyperglycemic ones.

Vaccination with a DNA vaccine coding for perforin-like protein 1 and MIC6 induces significant protective immunity against Toxoplasma gondii.

Host cell invasion by Toxoplasma gondii is tightly related to microneme protein 6 (MIC6) and T. gondii perforin-like protein 1 (TgPLP1). In this study, we constructed a DNA vaccine expressing a TgPLP1/MIC6 fusion protein using the pIRESneo vector, and we evaluated the immune response induced by this vaccine in Kunming mice. Levels of IgG antibody, gamma interferon (IFN-γ), interleukin 2 (IL-2), IL-12, IL-4, and IL-10 were examined. Five mice were chosen randomly from every group (vaccinated groups or the nonvaccinated control group) and were challenged intragastrically with 80 cysts of T. gondii strain PRU (genotype II) in order to observe mortality daily. To analyze protection against a less-virulent challenge, eight mice of each group were orally infected with 20 cysts of strain PRU at the 14th day after the last immunization. The brain parasite load was evaluated 6 weeks after infection. The results demonstrated that immunization with pIRESneo/MIC6/PLP1 resulted in the lowest brain cyst count and prolonged the survival time of immunized mice. The levels of Toxoplasma-specific IgG, IFN-γ, IL-2, and IL-12 increased significantly, and the numbers of cysts in brains decreased more obviously, in the group immunized with plasmid pIRESneo/MIC6/PLP1 than in the other groups (P < 0.05). Compared with pIRESneo/MIC6/PLP1, coimmunization with pIRESneo/MIC6/PLP1 and adjuvant murine IL-18 promoted cellular and humoral immune responses but did not contribute significantly to cyst reduction (65.43% versus 61.60%) or the survival of immunized mice (45.0 ± 2.9 days versus 42.8 ± 2.9 days) (P > 0.05). Furthermore, the study also showed that the immune efficacy induced by pIRESneo/MIC6/PLP1 was better than that induced by pVAX/PLP1 or pVAX/MIC6 alone.

Induction of protective Th1 immune responses in mice by vaccination with recombinant Toxoplasma gondii nucleoside triphosphate hydrolase-II

The Toxoplasma gondii nucleoside triphosphate hydrolase (TgNTPase) has apyrase activity, degrading ATP to the di- and mono-phosphate forms and may be used by the parasite to salvage purines from the host cell for survival and replication. To study the immune-protective value of TgNTPase-II, BALB/c mice were immunized with a recombinant form of the antigen rTgNTPase-II combined with alum. All immunized mice produced specific anti-rTgNTPase-II immunoglobulins, with high IgG antibody titers and a mixed IgG1/IgG2a response, with predominance of IgG2a production. The cellular immune response was associated with the production of IFN-γ and IL-2 cytokines and the increase of the percentage of CD8+ T cells. Vaccinated mice displayed significant protection against acute infection with the virulent RH strain ( P < 0.05 in survival rate) and also chronic infection with PRU cyst (62.9% and 57.6% reduction in brain parasite load for rTgNTPase-II + alum and rTgNTPase-II alone vaccinated groups) compared to the non-vaccinated control group. In conclusion, rTgNTPase-II elicits a strong specific Th1 immune response providing partial protection against both T. gondii acute and chronic infection.

Toxoplasma gondii protease inhibitor-1 (TgPI-1) is a novel vaccine candidate against toxoplasmosis

The Toxoplasma gondii serin protease inhibitor-1 (TgPI-1) is a dense granule antigen that showed to specifically inhibit trypsin, chymotrypsin and neutrophil elastase, suggesting a possible modulatory role during the parasite invasion process and on the development of the innate immune response. To study the immune-protective value of TgPI-1, C3H/HeN mice were immunized with a recombinant form of the antigen rTgPI-1 combined with alum. All immunized mice produced specific anti-rTgPI-1 immunoglobulins, with high IgG antibody titers and a mixed IgG 1/IgG 2a response, with predominance of IgG 1 production. The cellular immune response was associated with the production of IFN-γ and IL-10 cytokines. Vaccinated mice displayed significant protection against an oral challenge either after a lethal infection with Me49 cysts (90% survival vs. 50%) and also after a non-lethal infection (58% reduction in brain parasite load) compared to the non-vaccinated control group. In conclusion, rTgPI-1 elicits a strong specific immune response providing partial protection against both T. gondii acute and chronic infection, so it would be a good candidate in a vaccine against toxoplasmosis, which could be combined with other relevant parasite antigens.

Quantitative analysis of parasite DNA in the blood of immunized and naïve mice after infection with Neospora caninum

Real-time PCR was used to study the duration and level of parasitaemia in mice immunized with immune-stimulating complexes (iscoms) containing recombinant NcSRS2, one of the immunodominant surface antigens of Neospora caninum. After challenge infection, blood was collected daily for 9 days. During this period the amounts of parasite DNA detected in immunized mice were significantly lower (P<0.001), and the duration of parasitaemia appeared to be shorter, than in non-immunized controls. Furthermore, the degree of parasitaemia seemed to correlate well with the amount of N. caninum DNA in the brain 3 weeks post-inoculation and with disease severity measured as changes in body weight. These results indicate that the protective immunity induced by the NcSRS2-iscoms was sufficient to reduce the level of parasitaemia, which probably reduced the number of parasites reaching the brain, and could be the reason for the reduction in brain parasite load and clinical symptoms. Furthermore, real-time PCR was found to be a sensitive means for rapid assessment of N. caninum in blood.

Characterization of pathology and parasite load in outbred and inbred mouse models of chronic Neospora caninum infection.

Here, we analyzed histological findings and parasite burden in chronic Neospora caninum infection in BALB/c and ICR mice and studied the correlation between lesion severity and parasite load in brain. To obtain a better understanding of the infection, we examined the influence of various host pathogen factors. Groups of outbred (ICR) and inbred (BALB/c) mice were inoculated using several NC-1 parasite doses (4 x 10(5), 10(6), and 5 x 10(6) tachyzoites), inoculation routes (intraperitoneal and subcutaneous), and 3 immunosuppressive treatments (methylprednisolone, cyclophosphamide, and vinblastine). Lesion severity was analyzed in the liver, lung, heart, and brain tissues, and parasite load was measured by real-time polymerase chain reaction in brain tissue. The results indicated more severe cerebral lesions and higher brain parasite burdens in inbred than in outbred mice. Hepatic tissue was the primary lesion site in immunosuppressed ICR mice. We also observed that increased inoculum size was reflected in greater lesion severity and a higher cerebral parasite load. No difference was observed with respect to inoculation route. The study also showed an association between brain parasite burden and severity of cerebral lesions in BALB/c mice.

Recombinant feline herpesvirus type 1 expressing Toxoplasma gondi ROP2 antigen inducible protective immunity in cats.

In order to investigate whether a recombinant viral vaccine composed of a feline herpesvirus type 1 (FHVI) vector and an immunogenic antigen of Toxoplasma gondii can induce a protective immunity in cats, a recombinant FHV1 expressing ROP2 antigen of T. gondii was prepared. We introduced a DNA fragment encoding 1-538 amino acid residues of ROP2 precursor into FHV1 genome under the control of a cytomegalovirus promoter. The recombinant FHV1 (FHV/ROP2) successfully expressed a 59 kDa antigen that was recognized by anti-ROP2 antibodies. Vaccination of cats with FHV/ROP2 induced serum IgG recognizing the native antigen. Moreover the antibodies inhibited the in vitro invasion of tachyzoites. The vaccination also accelerated the IgG response after T. gondii infection and reduced brain parasite load in cats.

Interferon- γ and interleukin-12 mediate protection to acute Neospora caninum infection in BALB/c mice

The type of immune response required to protect mice against clinical disease during acute Neospora caninum challenge was investigated in BALB/c mice. Groups of female BALB/c mice were infected i.p. with N. caninum tachyzoites concomitant with either: (1) antibody to interferon- γ; (2) recombinant murine interleukin-12; or (3) recombinant murine interleukin-12 plus antibody to interferon- γ. Mice treated with anti-interferon- γ alone had increased morbidity/mortality, decreased body weight, increased foci of liver necrosis and increased numbers of N. caninum tachyzoites in the lung by 7 days p.i. compared with controls. Increased disease and parasite load in the anti-interferon- γ-treated mice was associated with antigen-specific antibody IgG1>IgG2a and a three-fold decreased ratio of antigen-specific interferon- γ : interleukin-4. Mice treated with recombinant murine interleukin-12 had decreased encephalitis and brain parasite load at 3 weeks p.i. compared with control mice treated with PBS. In recombinant murine interleukin-12-treated mice, decreased brain lesions and parasite load were associated with antigen-specific antibody IgG2a>IgG1 and a three-fold increased ratio of antigen-specific interferon- γ : interleukin-4 from splenocytes; the interleukin-12 effect was dependent upon interferon- γ, as indicated by concomitant in vivo interferon- γ neutralisation. By 6 weeks p.i. with N. caninum, there were no differences in brain lesions and parasite load between interleukin-12- and PBS-treated groups, indicating that the effects of interleukin-12 on driving a protective type 1 response were transient. These data indicate a role for interferon- γ, interleukin-12 and type 1 immune responses in control of acute neosporosis in mice.

Protective effect of vaccination with a combination of recombinant surface antigen 1 and interleukin-12 against toxoplasmosis in mice.

We studied the immune response induced in mice by recombinant Toxoplasma gondii surface antigen 1 (rSAG1) protein, alone or combined with interleukin-12 (IL-12) as an adjuvant, and the protective effect against toxoplasmosis. Immunization with rSAG1 alone induced a specific humoral type 2 immunity and did not protect the animals from infection. In contrast, immunization with rSAG1 plus IL-12 redirected humoral and cellular immunity toward a type 1 pattern and reduced the brain parasite load by 40%.