Experimental infection with Toxoplasma gondii in broiler chickens (Gallus domesticus): seroconversion, tissue cyst distribution, and prophylaxis.

Toxoplasma gondii is a widespread zoonotic protozoan that infects most species of mammals and birds, including poultry. This study aimed to investigate the course of T. gondii infection and the efficacy of diclazuril and Artemisia annua in preventing infection in experimentally infected chickens. Seventy-five 1-month-old chickens, female and male, were randomly divided into five groups (n = 15 each) as follows: (1) uninfected untreated (negative control, NC); (2) infected with T. gondii genotype II/III isolated from a wild cat (group WC); (3) infected with T. gondii genotype II isolated from a domestic cat (group DC); (4) infected with T. gondii domestic cat strain and treated with the anticoccidial diclazuril (group DC-D); and (5) infected with T. gondii domestic cat strain and treated with the medicinal plant Artemisia annua (group DC-A). Clinical signs, body temperature, mortality rate, weight gain, feed conversion ratio, hematological parameters, and the presence of T. gondii-specific IgY antibodies were recorded in all groups. Five chickens per group were euthanized 28 days post-infection (p.i.) and their brains, hearts, and breast muscle tested for T. gondii by mouse bioassay and polymerase chain reaction (PCR). No clinical signs related to the experimental infection were observed throughout the study period. T. gondii-specific antibodies were detected by day 28 p.i., but not in all infected chickens. Overall, T. gondii DNA was detected (bioassay or tissue digests) in all infected and untreated chickens (10/10), while viable parasite (bioassay) was isolated from 7 out of 10 chickens. The parasite was most frequently identified in the brain (7/10). There were no differences in the T. gondii strains regarding clinical infection and the rate of T. gondii detection in tissues. However, higher antibody titers were obtained in chickens infected with T. gondii WC strain (1:192) comparing with T. gondii DC strain (1:48). A. annua reduced replication of the parasite in 3 out of 5 chickens, while diclazuril did not. In conclusion, broiler chickens were resistant to clinical toxoplasmosis, irrespective of the strain (domestic or wild cat strain). The herb A. annua presented prophylactic efficacy by reduced parasite replication. However, further studies are required aiming at the efficacy of diclazuril and A. annua for the prevention of T. gondii infection in chickens using quantitative analysis methods.

Anti-Sarcocystis Antibodies in Lambs Deprived of Colostrum.

INTRODUCTION: The objective of this study was to evaluate the presence of anti-Sarcocystis spp. specific IgG antibodies in serum samples from precolostral lambs to determine the occurrence of transplacental transmission of Sarcocystis spp. in sheep. METHODS: Blood samples were collected from 80 ewes and their respective lambs, immediately after lambing and before colostrum ingestion, respectively. The presence of anti-Sarcocystis spp. IgG was evaluated in serum samples using the indirect fluorescent antibody test (IFAT). Positive samples of the lambs were submitted to titration and IFAT to detect anti-T. gondii and anti-N. caninum specific IgG. RESULTS: Anti-Sarcocystis spp. IgG was detected in 62.5% of the ewes (50/80) and in 4% of the lambs of the seropositive ewes (2/50). None of the lambs from seronegative ewes were positive. The final titers of the positive lambs were 80. No cross reaction was detected among the positive samples to anti-Sarcocystis spp., anti-N. caninum, and anti-T. gondii IgG. The detection of anti-Sarcocystis spp. antibodies in serum samples of lambs deprived of colostrum suggests transplacental transmission of infection. Thus, the vertical transmission may be an alternative route of infection of Sarcocystis spp. also in sheep. Further studies are warranted to confirm transplacental transmission in sheep and to explain the importance of this infection pathway.

Protective efficacy induced by DNA prime and recombinant protein boost vaccination with Toxoplasma gondii GRA14 in mice.

Toxoplasma gondii, the etiological agent of toxoplasmosis, can cause severe or lethal damages in both animals and man. So, tends to develop a more effective vaccine to prevent this disease is extremely needed and would be so prominent. The novel dense granule antigen 14 (GRA14) has been identified as a potential vaccine candidate against T. gondii infection. The aim of this study was evaluation of protective immunity induced by prime/boost vaccination strategy of GRA14 antigen with calcium phosphate (CaPNs) or Aluminum hydroxide (Alum) nano-adjuvants in BALB/c mice. The finding showed that immunization with the prime-boost strategy using plasmid DNA (pcGRA14) and recombinant protein (rGRA14) with nano-adjuvants significantly elicited levels of specific IgG antibodies and cytokines against T. gondii infection. Given that, there were the high levels of total IgG, IgG2a, IFN-γ in mice of rGRA14-CaPNs and pcGRA14 + rGRA14-CaPNs groups, which indicating a Th-1 type response. While immunization of mice with Alum based rGRA14 and pcGRA14 + rGRA14 elicited specific IgG1 and IL-4 levels, which was confirmed a Th-2 type response. Mice immunized with DNA prime-protein boost vaccine with nano-adjuvants produce more vigorous specific lymphoproliferative responses than mice immunized with other antigen formulations. In addition, the CaPNs-based prime-boost vaccine of pcGRA14 + rGRA14 showed the longest survival time in mice and the lowest parasitic load in their brain tissue compared to the other groups. The results obtained in this study show that the use of GRA14 based DNA prime-protein boost vaccination regime with CaPNs can dramatically enhanced both humoral and cellular immune responses. Therefore, this strategy can provide a promising approach to the development of an effective vaccine against T. gondii infection in the future.

Antigenic Epitope Analysis and Efficacy Evaluation of GRA41 DNA Vaccine Against T. gondii Infection.

BACKGROUND: Toxoplasma gondii has a comprehensive impact on a great range of warm-blood mammals, in which one-third of the population all over the world is involved. Dense granular proteins, regarded as GRA family, mediating substantial interface between host cell cytoplasm and parasite, are widely studied for preventing the infection of T. gondii. PURPOSE: As is handled in our study, the effect of intramuscularly injecting the genetic vaccine pEGFP-C1/GRA41 encoding a novel dense granule protein, GRA41, was evaluated. METHODS: At the beginning, bioinformatics analysis was used to evaluate epitopes of both B cells and T cells on the GRA41 protein of T. gondii. Afterwards, recombinant plasmids (pEGFP-C1/GRA41) were injected into BALB/c mice and the quantity of IgG and its subclass IgG2a remarkably increased. IFN-γ, distinctive from the other cytokines (IL-4, and IL-10), was significant in growth. Afterwards, the intraperitoneal challenge was executed for recording survival time with tachyzoites with high virulence (in RH strain) and counting the number of brain cysts was carried out after the infection of PRU strain (low virulence). RESULTS: In pEGFP-C1/GRA41 group, the survival period was significantly longer (13.3 ± 3.37 days) after tachyzoites attack with the RH strain in high virulence, compared with the other groups (less than 8 days). Additionally, the cyst quantity is remarkably lower and the rate of reduction could reach 59.34%. CONCLUSION: All the results indicated effective protection of DNA vaccine encoding GRA41 against T. gondii.

Spatial and simultaneous representative seroprevalence of anti-Toxoplasma gondii antibodies in owners and their domiciled dogs in a major city of southern Brazil.

Toxoplasmosis, caused by Toxoplasma gondii, has traditionally been considered an important water and foodborne protozoonosis with important public health considerations. Although felids play a well-established role as definitive hosts, canine epidemiological involvement in the parasite's life cycle remains questionable and controversial. The increasing closeness of the human-dog bond, particularly seen in urban settings, has been recognized as a historically unprecedented worldwide movement. Sharing daily lives in the same households, dogs may be exposed to similar associated risks of T. gondii infection as their owners. Thus, epidemiological assessment of the intra-domiciled environment, especially among socio-economically different human populations, may provide novel information regarding the actual role of dogs in animal and human toxoplasmosis. Despite spatial approaches being recently used for other water and foodborne diseases, no study has been conducted on the simultaneous spatial seroprevalence of both human and animal IgG anti-T. gondii antibodies in urban areas of major cities. Accordingly, the aim of the present study was to assess the seroprevalence and associated variables of Toxoplasma infection in owners and their domiciled dogs in Londrina, southern Brazil. Human and canine seroprevalence rates and variables associated with seroprevalence were investigated through representative random sampling among 564 households, which included 597 owners and 729 dogs. Overall, statistically significant differences between the seroprevalence of human and dog anti-T. gondii antibodies were found by Immunofluorescence Antibody Testing in 248/597 (41.54%) owners and 119/729 (16.32%) dogs. Through multiple analysis, significant concomitant variables for seropositivity of household individuals (people and dogs) were determined, including public sewer service, yard cleaning frequency, and having a dirty yard. Although no statistically significant multiple logistic model was observed among owners, univariate analysis detected associations with monthly income, soil contact, and occupation. Among dogs, the absence of other dogs and the absence of a dirty yard were concomitant significantly protective associated factors. Age differences between seropositive and seronegative individuals was significant only for human beings, with the median age of negative individuals significantly higher than positive individuals. Although no spatial clusters were identified for humans or residences, a significant cluster was identified for dogs. In conclusion, characteristics of urban toxoplasmosis may include significantly higher owner seroprevalence than their owned dogs, with canine seroprevalence directly associated with having more dogs and a dirty backyard, and spatial differences in both human and dog exposures. Although not a good indicator for human foodborne diseases, dogs may be a reliable sentinel for environmental infection. Moreover, such a holistic approach may provide crucial information for more focused prevention and monitoring programs, particularly in households with multiple pets and trash-filled backyards.

Enhancing immune responses to a DNA vaccine encoding Toxoplasma gondii GRA14 by calcium phosphate nanoparticles as an adjuvant.

Several approaches have been used to improve the immunogenicity of DNA vaccines. In the current study, we constructed the plasmid encoding T. gondii dense granule 14 (GRA14) and investigated the immunological properties of calcium phosphate nanoparticles (CaPNs) as nano-adjuvant to enhance the protective efficacy of pcGRA14. BALB/c mice intramuscularly injected three times at two-week intervals and the immune responses were evaluated using lymphocyte proliferation assay, cytokine and antibody measurements, survival times, and parasite load of mice challenged with the virulent T. gondii RH strain. The results showed that the immune responses were induced in mice receiving pcGRA14 DNA vaccine. Interestingly, pcGRA14 coated with nanoparticles led to statistically significant enhancements of cellular and humoral immune responses against Toxoplasma infection (P<0.05). After challenge with RH strain of T. gondii, immunized mice with pcGRA14 showed prolong survival time compared to control groups (P<0.05). In addition, pcGRA14 coated with nano-adjuvant exhibited the lowest parasitic load in the infected mice tissues. For the first time, our data indicate that the pcGRA14 coated with CaPN was more effective for stimulation of immune responses and should be considered as an adjuvant in the design of vaccines against toxoplasmosis.

Unconventional Peptide Presentation by Major Histocompatibility Complex (MHC) Class I Allele HLA-A*02:01: BREAKING CONFINEMENT.

Peptide antigen presentation by major histocompatibility complex (MHC) class I proteins initiates CD8+ T cell-mediated immunity against pathogens and cancers. MHC I molecules typically bind peptides with 9 amino acids in length with both ends tucked inside the major A and F binding pockets. It has been known for a while that longer peptides can also bind by either bulging out of the groove in the middle of the peptide or by binding in a zigzag fashion inside the groove. In a recent study, we identified an alternative binding conformation of naturally occurring peptides from Toxoplasma gondii bound by HLA-A*02:01. These peptides were extended at the C terminus (PΩ) and contained charged amino acids not more than 3 residues after the anchor amino acid at PΩ, which enabled them to open the F pocket and expose their C-terminal extension into the solvent. Here, we show that the mechanism of F pocket opening is dictated by the charge of the first charged amino acid found within the extension. Although positively charged amino acids result in the Tyr-84 swing, amino acids that are negatively charged induce a not previously described Lys-146 lift. Furthermore, we demonstrate that the peptides with alternative binding modes have properties that fit very poorly to the conventional MHC class I pathway and suggest they are presented via alternative means, potentially including cross-presentation via the MHC class II pathway.

Immunological evaluation of a DNA cocktail vaccine with co-delivery of calcium phosphate nanoparticles (CaPNs) against the Toxoplasma gondii RH strain in BALB/c mice.

Many recent studies have been conducted to evaluate protective immunity mediated by DNA vaccines against toxoplasmosis. Cocktail DNA vaccines showed better immune responses compared to single vaccines. The objective of the current study was to evaluate the protective efficacy of rhomboid 4 (ROM4) and cocktail DNA vaccines (ROM4 + GRA14) of the Toxoplasma gondii RH strain with or without coated calcium phosphate nanoparticles (CaPNs) as the adjuvant to improve the immunogenicity against the T. gondii RH strain in BALB/c mice. Cocktail DNA vaccines of pcROM4 + pcGRA14 of the T. gondii RH strain were constructed. CaPNs were synthesized and the cocktail DNA vaccine was coated with the adjuvant of CaPNs. Immunogenicity and the protective effects of cocktail DNA vaccines with or without CaPNs against lethal challenge were evaluated in BALB/c mice. pcROM4 and cocktail DNA vaccine coated with CaPNs significantly enhanced cellular and humoral immune responses against Toxoplasma compared to pcROM4 and cocktail DNA vaccine without CaPNs (p < 0.05). These findings indicate that the survival time of immunized mice after challenge with the RH strain of T. gondii was increased compared to that of controls and the DNA vaccine provided significant protection in mice (p < 0.05). The CaPN-based cocktail DNA vaccine of pcROM4 + pcGRA14 showed the longest survival time compared to the other groups. Co-immunization with CaPN-based cocktail DNA vaccine (pcROM4 + pcGRA14) boosted immune responses and increased the protective efficacy against acute toxoplasmosis in BALB/c mice compared to both single gene and bivalent DNA vaccine without nano-adjuvants.

Relationship between Toxoplasma gondii seropositivity and acoustic startle response in an inner-city population.

Toxoplasma gondii (TOXO) is a neuroinvasive protozoan parasite that induces the formation of persistent cysts in mammalian brains. It infects approximately 1.1million people in the United States annually. Latent TOXO infection is implicated in the etiology of psychiatric disorders, especially schizophrenia (SCZ), and has been correlated with modestly impaired cognition. The acoustic startle response (ASR) is a reflex seen in all mammals. It is mediated by a simple subcortical circuit, and provides an indicator of neural function. We previously reported the association of TOXO with slowed acoustic startle latency, an index of neural processing speed, in a sample of schizophrenia and healthy control subjects. The alterations in neurobiology with TOXO latent infection may not be specific to schizophrenia. Therefore we examined TOXO in relation to acoustic startle in an urban, predominately African American, population with mixed psychiatric diagnoses, and healthy controls. Physiological and diagnostic data along with blood samples were collected from 364 outpatients treated at an inner-city hospital. TOXO status was determined with an ELISA assay for TOXO-specific IgG. A discrete titer was calculated based on standard cut-points as an indicator of seropositivity, and the TOXO-specific IgG concentration served as serointensity. A series of linear regression models were used to assess the association of TOXO seropositivity and serointensity with ASR magnitude and latency in models adjusting for demographics and psychiatric diagnoses (PTSD, major depression, schizophrenia, psychosis, substance abuse). ASR magnitude was 11.5% higher in TOXO seropositive subjects compared to seronegative individuals (p=0.01). This effect was more pronounced in models with TOXO serointensity that adjusted for sociodemographic covariates (F=7.41, p=0.0068; F=10.05, p=0.0017), and remained significant when psychiatric diagnoses were stepped into the models. TOXO showed no association with startle latency (t=0.49, p=0.63) in an unadjusted model, nor was TOXO associated with latency in models that included demographic factors. After stepping in individual psychiatric disorders, we found a significant association of latency with a diagnosis of PTSD (F=5.15, p=0.024), but no other psychiatric diagnoses, such that subjects with PTSD had longer startle latency. The mechanism by which TOXO infection is associated with high startle magnitude is not known, but possible mechanisms include TOXO cyst burden in the brain, parasite recrudescence, or molecular mimicry of a host epitope by TOXO. Future studies will focus on the neurobiology underlying the effects of latent TOXO infection as a potential inroad to the development of novel treatment targets for psychiatric disease.

Lectins from Synadenium carinatum (ScLL) and Artocarpus heterophyllus (ArtinM) Are Able to Induce Beneficial Immunomodulatory Effects in a Murine Model for Treatment of Toxoplasma gondii Infection.

Infection by Toxoplasma gondii affects around one-third of world population and the treatment for patients presenting toxoplasmosis clinically manifested disease is mainly based by a combination of sulfadiazine, pyrimethamine, and folinic acid. However, this therapeutic protocol is significantly toxic, causing relevant dose-related bone marrow damage. Thus, it is necessary to improve new approaches to investigate the usefulness of more effective and non-toxic agents for treatment of patients with toxoplasmosis. It has been described that lectins from plants can control parasite infections, when used as immunological adjuvants in vaccination procedures. This type of lectins, such as ArtinM and ScLL is able to induce immunostimulatory activities, including efficient immune response against parasites. The present study aimed to evaluate the potential immunostimulatory effect of ScLL and ArtinM for treatment of T. gondii infection during acute phase, considering that there is no study in the literature accomplishing this issue. For this purpose, bone marrow-derived macrophages (BMDMs) were treated with different concentrations from each lectin to determine the maximum concentration without or with lowest cytotoxic effect. After, it was also measured the cytokine levels produced by these cells when stimulated by the selected concentrations of lectins. We found that ScLL showed high capacity to induce of pro-inflammatory cytokine production, while ArtinM was able to induce especially an anti-inflammatory cytokines production. Furthermore, both lectins were able to increase NO levels. Next, we evaluated the treatment effect of ScLL and ArtinM in C57BL/6 mice infected by ME49 strain from T. gondii. The animals were infected and treated with ScLL, ArtinM, ArtinM plus ScLL, or sulfadiazine, and the following parameters analyzed: Cytokines production, brain parasite burden and survival rates. Our results demonstrated that the ScLL or ScLL plus ArtinM treatment induced production of pro-inflammatory and anti-inflammatory cytokines, showing differential but complementary profiles. Moreover, when compared with non-treated mice, the parasite burden was significantly lower and survival rates higher in mice treated with ScLL or ScLL plus ArtinM, similarly with sulfadiazine treatment. In conclusion, the results demonstrated the suitable potential immunotherapeutic effect of ScLL and ArtinM lectins to control acute toxoplasmosis in this experimental murine model.

Oocyst-Derived Extract of Toxoplasma Gondii Serves as Potent Immunomodulator in a Mouse Model of Birch Pollen Allergy.

INTRODUCTION: Previously, we have shown that oral infection with Toxoplasma gondii oocysts prevented type I allergy in mice. Here we investigated whether the application of a T. gondii oocyst lysate antigen (OLA) could also reduce allergy development. BALB/c mice were immunised twice with OLA followed by sensitisation with the major birch pollen (BP) allergen Bet v 1 and an aerosol challenge with BP extract. METHODS: First, we tested OLA in vitro. Stimulation of splenocytes and bone marrow-derived dendritic cells (BMDC) with OLA led to the production of pro-inflammatory and regulatory cytokines such as IL-6, IFN-γ and IL-10. Moreover, BMDC exposed to OLA upregulated the maturation markers CD40, CD80, CD86, and MHCII. Furthermore, OLA was recognised by TLR2-transfected human embryonic kidney cells. RESULTS: Immunisation of mice with OLA induced high levels of Toxoplasma-specific IgG antibodies in sera along with increased production of IFN-γ and IL-10 in Toxoplasma-antigen restimulated splenocytes. OLA reduced allergic airway inflammation as manifested by significant reduction of eosinophils in bronchoalveolar fluids, decreased cellular infiltrates and mucus production in the lungs. Accordingly, Bet v 1-specific IgE was decreased in OLA-pretreated mice. The reduced allergic immune responses were accompanied by increased numbers of CD4+CD25highFoxp3+ regulatory T cells in spleens as well as by increased numbers of granulocytic myeloid-derived suppressor cells in lungs when compared to sensitised controls suggesting that these two cell populations might be involved in the suppression of the allergic immune responses. CONCLUSION: Our data demonstrate that pretreatment with the oocyst extract can exert anti-allergic effects comparable to T. gondii infection. Thus, the immunomodulatory properties of the parasite extract indicate that this extract and in the future defined molecules thereof might serve as immunomodulatory adjuvants in allergy treatment and prophylaxis.

Vaccination with Toxoplasma gondii calcium-dependent protein kinase 6 and rhoptry protein 18 encapsulated in poly(lactide-co-glycolide) microspheres induces long-term protective immunity in mice.

BACKGROUND: Toxoplasmosis is a worldwide zoonosis caused by the intracellular parasite Toxoplasma gondii. However, no effective vaccine is yet available. Poly(lactide-co-glycolide) polymers can reduce protein degradation and sustain the release of antigens over a long period, which could generate a long-lasting immune response in vivo. Using a mouse model of toxoplasmosis, we evaluated the protective efficacy of vaccination with two recombinant proteins, which are formulated in biodegradable polymers. METHODS: Two recombinant proteins, rCDPK6 and rROP18, were encapsulated in poly(D,L-lactide-co-glycolide) (PLG), and then injected subcutaneously into Kunming mice. The mice immune responses were evaluated in terms of lympho-proliferation, cytokine expression, and antibodies. The survival of infected mice and brain cyst formation were also evaluated at 6 weeks after challenge with T. gondii RH strain (genotype I) or PRU strain (genotype II). RESULTS: Both protein vaccines induced Th1-biased immune responses, with increased specific antibodies and T cells, high levels of interferon-γ and interleukin 2, and strong lymphocyte proliferative responses. The mice immunized with the various protein vaccines survived slightly longer time than the control groups (P > 0.05) after injection with T. gondii RH strain. There were fewer brain cysts in the mice in all the immunized groups than that in the control groups, and the brain cysts were significantly reduced in mice immunized with proteins + 206, rCDPK6 + PLG and rCDPK6 + rROP18 + PLG (P < 0.05) compared controls. Further comparison of the immune responses to the proteins adjuvanted with PLG or Montanide™ ISA 206 VG 6 weeks after the last immunization revealed that antigens encapsulated in PLG conferred greater protective immunity against challenge. CONCLUSIONS: These findings suggest that the two recombinant T. gondii proteins encapsulated in PLG conferred immunity to T. gondii for an extended period, providing the foundation for the further development of a commercial vaccine against toxoplasmosis.

Co-administration of interleukins 7 and 15 with DNA vaccine improves protective immunity against Toxoplasma gondii.

Toxoplasma gondii is an obligatory intracellular parasite, which can infect all warm-blooded animals including humans. Cytokines, including IL-15 and IL-7, play a critical role in the regulation of the homeostasis of naive and memory T cells. Co-administration the DNA vaccine with cytokines may improve its efficacy. IL-7 and IL-15 from splenic tissues of Kunming mice were cloned, and eukaryotic plasmid pVAX-IL-7-IL-15 was constructed. Kunming mice were administrated with DNA vaccine expressing T. gondii calcium-dependent protein kinase 1 (TgCDPK1), pVAX-CDPK1, in the presence or absence of IL-7 and IL-15 plasmids (pVAX-IL-7-IL-15), immune responses were analyzed including lymphoproliferative assay, cytokine and serum antibody measurements, flow cytometric surface markers on lymphocytes, and thus protective immunity against acute and chronic T. gondii infection was estimated. Mice injected with pVAX-CDPK1 supplemented with pVAX-IL-7-IL-15 showed higher Toxoplasma-specific IgG2a titers, Th1 responses associated with the production of IFN-γ, IL-2 as well as cell-mediated cytotoxic activity where stronger frequencies of IFN-γ secreting CD8+ and CD4+ T cells (CD8+/CD4+ IFN-γ+ T cells) compared to controls. Co-administration of pVAX-IL-7-IL-15 and pVAX-CDPK1 significantly (P < 0.05) increased survival time (18.07 ± 5.43 days) compared with pVAX-CDPK1 (14.13 ± 3.85 days) or pVAX-IL-7-IL-15 (11.73 ± 1.83 days) alone, and pVAX-IL-7-IL-15 + pVAX-CDPK1 significantly reduced the number of brain cysts (73.5%) in contrast to pVAX-CDPK1 (46.0%) or pVAX-IL-7-IL-15 alone (45.0%). Our results indicate that supplementation of DNA vaccine with IL-7 and IL-15 would facilitate specific humoral and cellular immune responses elicited by DNA vaccine against acute and chronic T. gondii infection in mice.

Evaluation of colostrum as an alternative biological sample for the diagnosis of human congenital toxoplasmosis.

BACKGROUND: Toxoplasmosis is a zoonosis caused by Toxoplasma gondii, an intracellular protozoan parasite able to infect a wide range of hosts, including humans. Congenital infection can cause severe damage to the fetus. Thus, it is important to detect antibodies against the parasite to confirm clinical manifestations. Considering that all immunoglobulin isotypes may be present in biological samples from newborns and their mothers, this study aimed to evaluate the ability to diagnose recent toxoplasmosis by using colostrum, as an alternative noninvasive way to obtain biological samples, as well as to determine correlation rates between antibodies from serum samples to detect IgG, IgM and IgA isotypes against T. gondii. METHODS: A total of 289 puerperal women from Clinical Hospital of Federal University of Uberlândia (mean age: 24.8 years, range: 14 - 43 years) took part in this study. Serum and colostrum samples from these patients were analyzed using ELISA and immunoblotting assays for soluble antigens from T. gondii. RESULTS: ELISA immunoassays with serum samples showed reactivity in 47.0, 6.9 and 2.8 % of samples to anti-T. gondii IgG, IgM and IgA, respectively, in comparison with colostrum samples, which showed reactivity in 46.0, 7.9 and 2.8 % of samples to the same isotypes. Also, significant correlation rates of anti-T. gondii antibody levels between serum and colostrum samples were observed. Interestingly, reactivity to IgM and/or IgA in colostrum and/or serum confirmed clinical manifestations of congenital toxoplasmosis in three newborns. Immunoblotting assays showed that it is possible to detect IgG, IgM and IgA antibodies against various antigens of T. gondii in serum and colostrum samples. IgG antibodies in serum and colostrum samples recognized more antigenic fractions than IgM and IgA antibodies. Serum IgG detected more antigenic fractions than IgG antibodies present in the colostrum of the same patient. In contrast, specific IgA present in colostrum recognized a higher number of antigens than IgA present in serum samples of the same patient. CONCLUSIONS: Overall, the results show that it is important to investigate the occurrence of congenital toxoplasmosis, even at puerperal period. Furthermore, this study demonstrates that T. gondii-specific IgG, IgM and IgA antibodies in serum and colostrum samples from puerperal women may be detected with a significant correlation, suggesting that colostrum may also be used as an alternative biological sample to efficiently diagnose recent human toxoplasmosis.

Immunoglobulin and cytokine changes induced following immunization with a DNA vaccine encoding Toxoplasma gondii selenium-dependent glutathione reductase protein.

Toxoplasma gondii Glutathione Reductase (TgGR) plays important role during the survival of the parasite. In this investigation, immunological changes and protection efficiency of this protein delivered as a DNA vaccine (pTgGR) have been evaluated. Mice were immunized with pTgGR, followed by challenge with virulent T. gondii RH strain, 2 weeks after the booster immunization. Compared to the control groups pVAX1, PBS and Blank groups, the results showed that pTgGR stimulated specific humoral response defined by significant titers of total IgG, subclasses IgG1 and IgG2a, classes IgA and IgM, but not IgE. Analysis of IFN-γ, IL-4, IL-17 and TGF-ß1 cytokines after immunization and compared with the control groups showed significant increments in pTgGR group. Additionally, T lymphocytes subpopulation CD4(+) T was positively recruited with significant percentage detected, while subset CD8(+) appeared not to be involved in response to this antigen. Vaccinated mice showed a significantly longer survival time, 15 days, in contrast with control groups which died within 8-10 days after challenge. These results demonstrated that TgGR could induce significant humoral and cell mediated responses leading to a considerable level of resistance against toxoplasmosis infection.

Evaluation of the adjuvant effect of pidotimod on the immune protection induced by UV-attenuated Toxoplasma gondii in mouse models.

The current anti-Toxoplasma gondii drugs have many shortcomings and effective vaccines against T. gondii may contribute to the control of this pathogen. Pidotimod is a synthetic substance capable of stimulating both cellular and humoral immunity. To investigate the possible adjuvant effect of pidotimod on the immune response to T. gondii in Kunming mice induced by ultraviolet-attenuated T. gondii (UV-T.g), in this study, mice were immunized intraperitoneal (i.p.) with UV-T.g or UV-T.g co-administered with pidotimod (UV-T.g + PT). After infection or challenge by i.p. injection of 10(2) RH tachyzoites, the animal survival rate, parasite burden in peritoneal lavage fluids, liver histopathology, the level of serum anti-toxoplasma IgG antibody, and the mRNA expressions of IL-2, IFN-γ, and TNF-α from spleen analyzed using real-time PCR, were compared among different groups. The results showed that, compared with infected controls, infected mice treated with pidotimod had significantly increased survival rate and extended survival time, decreased parasite burden, improved liver histopathology, increased level of anti-toxoplasma IgG antibody, and increased mRNA expressions of Th1-type cytokine (IL-2, IFN-γ, and TNF-α) (P < 0.01), while mice vaccinated with UV-T.g and then challenged had even significantly increased survival rate and extended survival time, decreased parasite burden, improved liver histopathology, and increased mRNA expressions of Th1-type cytokines (IL-2, IFN-γ, and TNF-α) (P < 0.01); furthermore, vaccinated mice co-administered with pidotimod had even more lower parasite burden, milder liver histopathology, and higher levels of Th1-type cytokine and anti-toxoplasma IgG antibody (P < 0.01). Our data demonstrated that pidotimod in vivo could promote strong and specific humoral and cellular immune response to T. gondii challenge infection when co-administered with UV-attenuated T. gondii. It suggests that pidotimod may have the potential to be used as an effective vaccine adjuvant.

Seroprevalence and risk factors of toxoplasmosis in cattle from extensive and semi-intensive rearing systems at Zona da Mata, Minas Gerais state, Southern Brazil.

BACKGROUND: Concerning the infection of humans by T. gondii, limited efforts have been directed to the elucidation of the role of horizontal transmission between hosts. One of the main routes of transmission from animals to humans occurs through the ingestion of raw or insufficiently cooked meat. However, even though the detection of T. gondii in meat constitutes an important short-term measure, control strategies can only be accomplished by a deeper understanding of the epidemiology of toxoplasmosis. The present study aimed to investigate the seroprevalence of toxoplasmosis in cattle from Zona da Mata, Minas Gerais, Brazil, and to identify associated risk factors, through an epidemiological investigation. METHODS: The animals studied (Bos indicus, breed Nelore or Gir) were reared in the Zona da Mata micro-region and killed at a commercial slaughterhouse at Juiz de Fora, Minas Gerais state. The animals came from 53 cattle farms with extensive (predominantly pasture feeding management) or semi-intensive (food management based on grazing, salt mineral and feed supplementation) rearing systems. Blood samples were collected from 1200 animals, and assigned to Indirect Fluorescent Antibody Test. RESULTS: When analyzing IgG anti-T.gondii we found an overall seroprevalence of 2.68%. In Brazil prevalences vary from 1.03% to 60%. Although in the present study, the seroprevalence per animal is considered low compared to those observed in other studies, we found out that of the 53 farms analyzed, 17 (34.69%) had one or more positive cattle. It is a considerable percentage, suggesting that the infection is well distributed through the Zona da Mata region. The results of the epidemiological investigation showed that the main risk factors of Toxoplasma gondii infection are related to animal management and to the definive host. There was a relationship between the number of seropositive cattle and the presence and number of resident cats, presence and number of stray cats, presence of cats walking freely, rat control by using cats and feed storage. CONCLUSION: These results may contribute to the development of preventive strategies in Brazil and other developing countries were extensive and semi-intensive cattle rearing systems are very widespread and the efforts to control this important zoonotic disease have attained little success.

Increased survival time in mice vaccinated with a branched lysine multiple antigenic peptide containing B- and T-cell epitopes from T. gondii antigens.

To develop a multiple antigenic peptide (MAP) vaccine against toxoplasmosis, tri-epitope MAP constructs were made in dimeric fashion. The constructs included one B-cell and two T-cell epitopes derived from Toxoplasma gondii antigens (SAG1, GRA4 and GRA1) situated in tandem through the GGG spacer sequence, with the latter positioned adjacent to a polylysine core. Immunization of BALB/c and Kunming mice with the MAP construct in Freund's adjuvant induced not only humoral immunes response but cellular responses. These responses were accompanied by significant levels of splenocyte proliferation and interferon gamma (IFN-γ) in vitro. After lethal challenge, vaccinated mice had increased survival time in comparison to unvaccinated controls. Our data demonstrate that a MAP construct could trigger strong humoral and cellular responses against T. gondii, and that this MAP is a vaccine candidate worth further development.

Congenital toxoplasmosis--a report on the Danish neonatal screening programme 1999-2007.

OBJECTIVES: This paper reports on the national neonatal screening programme for congenital toxoplasmosis (CT) in Denmark conducted from 1999 to 2007, including background, basis for initiation of screening, methods, results, and finally reasons for the discontinuation of the screening. METHODS: A nationwide screening was conducted at Statens Serum Institut, including >98% newborns, and using filter paper eluates (Guthrie card, PKU card) obtained from newborns 5-10 days old. These were analysed for Toxoplasma gondii-specific antibodies (IgM), and if positive, then IgM (ISAGA). Confirmatory serology was performed on children and their mothers (IgM, IgG, IgA, dye test) where infection was suspected, and children with suspected or confirmed CT initiated a 3-month treatment regimen with pyrimethamine, sulfadiazine and folinic acid supplements. Selective cohorts were followed with regard to developmental and clinical outcome. RESULTS: A total of 100 children were diagnosed with CT in the screening period, and only 2 cases were detected outside of the screening programme. CT prevalence was 1.6 per 10,000 live-born infants. Follow-up studies showed new retinochoroidal lesions in affected children despite treatment. CONCLUSION: Screening was terminated August 2007, after it became apparent that no benefit of treatment could be shown. CT was evaluated using a Danish adaptation of the Uniform Screening Panel (ACMG), showing CT as an unlikely candidate for screening today. Whereas results might be comparable with other low-endemic countries with similar strains of T. gondii, neonatal screening and treatment might offer different results in regions with either high prevalence or different strains of T. gondii.

Evaluation of the adjuvant properties of Astragalus membranaceus and Scutellaria baicalensis GEORGI in the immune protection induced by UV-attenuated Toxoplasma gondii in mouse models.

Human vaccines are not available and current anti-toxoplasma treatment is disappointing. To investigate the possible adjuvant effect of aqueous extracts obtained from medicinal herbs of Astragalus membranaceus (Am) and Scutellaria baicalensis GEORGI (Sb) on the immune response to Toxoplasma gondii in the mouse models induced by ultraviolet (UV)-attenuated T. gondii, this paper studies the possible vaccination strategies to help combat infections with Toxoplasma and looking towards developing new vaccine and approaches. We used UV-attenuated T. gondii (UV-T.g) of RH strain as a vaccine and the extracts of Am (AmE) and Sb (SbE) as adjuvant. Mice were infected by intraperitoneal (i.p.) injection of 10(2) RH tachyzoites alone (infected controls), infected and treated with AmE (T.g+AmE) and SbE (T.g+SbE), respectively; and mice immunized i.p. with UV-T.g alone, UV-T.g co-administrated with AmE (UV-T.g+AmE) or SbE (UV-T.g+SbE), and then challenged with T.g, respectively. The animal survival time, parasite burden in peritoneal lavage fluids, liver histopathological analysis, and levels of serum antibodies among the groups were compared after either infection or challenge. The results showed that, compared to infected controls, infected mice treated with AmE or SbE, or vaccinated mice and then challenged, had significantly prolonged survival time, decreased parasite burden, improved liver histopathological score, and increased Th1-type cellular immune response; furthermore, vaccinated mice co-administrated with AmE or SbE had even longer survival, lower parasite burden, lower liver histopathological score, and higher Th1 response after challenge. Our data demonstrated that the protective immunity of UV-attenuated T. gondii could be markedly enhanced by AmE or SbE co-administration, which suggests that both AmE and SbE may have the potential to be used as effective vaccine adjuvant.