Vaccination with Recombinant Microneme Proteins Confers Protection against Experimental Toxoplasmosis in Mice

Toxoplasmosis, a zoonotic disease caused by Toxoplasma gondii, is an important public health problem and veterinary concern. Although there is no vaccine for human toxoplasmosis, many attempts have been made to develop one. Promising vaccine candidates utilize proteins, or their genes, from microneme organelle of T. gondii that are involved in the initial stages of host cell invasion by the parasite. In the present study, we used different recombinant microneme proteins (TgMIC1, TgMIC4, or TgMIC6) or combinations of these proteins (TgMIC1-4 and TgMIC1-4-6) to evaluate the immune response and protection against experimental toxoplasmosis in C57BL/6 mice. Vaccination with recombinant TgMIC1, TgMIC4, or TgMIC6 alone conferred partial protection, as demonstrated by reduced brain cyst burden and mortality rates after challenge. Immunization with TgMIC1-4 or TgMIC1-4-6 vaccines provided the most effective protection, since 70% and 80% of mice, respectively, survived to the acute phase of infection. In addition, these vaccinated mice, in comparison to non-vaccinated ones, showed reduced parasite burden by 59% and 68%, respectively. The protective effect was related to the cellular and humoral immune responses induced by vaccination and included the release of Th1 cytokines IFN-γ and IL-12, antigen-stimulated spleen cell proliferation, and production of antigen-specific serum antibodies. Our results demonstrate that microneme proteins are potential vaccines against T. gondii, since their inoculation prevents or decreases the deleterious effects of the infection

Terrestrial behavior in titi monkeys (Callicebus, Cheracebus, and Plecturocebus) : potential correlates, patterns, and differences between genera

For arboreal primates, ground use may increase dispersal opportunities, tolerance to habitat change, access to ground-based resources, and resilience to human disturbances, and so has conservation implications. We collated published and unpublished data from 86 studies across 65 localities to assess titi monkey (Callicebinae) terrestriality. We examined whether the frequency of terrestrial activity correlated with study duration (a proxy for sampling effort), rainfall level (a proxy for food availability seasonality), and forest height (a proxy for vertical niche dimension). Terrestrial activity was recorded frequently for Callicebus and Plecturocebus spp., but rarely for Cheracebus spp. Terrestrial resting, anti-predator behavior, geophagy, and playing frequencies in Callicebus and Plecturocebus spp., but feeding and moving differed. Callicebus spp. often ate or searched for new leaves terrestrially. Plecturocebus spp. descended primarily to ingest terrestrial invertebrates and soil. Study duration correlated positively and rainfall level negatively with terrestrial activity. Though differences in sampling effort and methods limited comparisons and interpretation, overall, titi monkeys commonly engaged in a variety of terrestrial activities. Terrestrial behavior in Callicebus and Plecturocebus capacities may bolster resistance to habitat fragmentation. However, it is uncertain if the low frequency of terrestriality recorded for Cheracebus spp. is a genus-specific trait associated with a more basal phylogenetic position, or because studies of this genus occurred in pristine habitats. Observations of terrestrial behavior increased with increasing sampling effort and decreasing food availability. Overall, we found a high frequency of terrestrial behavior in titi monkeys, unlike that observed in other pitheciids

Azitromicina inibe a infecção por Toxoplasma gondii em oculares de fetos e reduz o parasitismo na infecção congênita e adquirida em modelo experimental de Calomys callosus

Toxoplasma gondii is an obligatory intracelullar parasite of large geographical distribution that causes severe sequelae to fetuses of mothers infected for the first time during pregnancy and in immunossuppressed adults. The drugs most frequently used for the treatment of toxoplasmosis are a combination of sulfadiazine and pirimethamine plus folinic acid. However, these drugs present severe side effects and currently their efficacy is under study. Other medicines that are considered as alternatives for the treatment are clindamycin, atovaquone and azithromycin. The latter has demonstrated to be effective againts both tachyzoites and bradyzoites in vitro with lesser side effects in clinical practice. In this study we tested the efficacy of azithromycin on the reduction of vertical transmission of Toxoplasma, by the analyses of fetal eyes of Calomys callosus, as well as the acquired adult infection analyzing brains of the mothers and adult males. For the analysis of the treatment on the vertical transmission, females of C. callosus were inoculated perorally with 20 cysts of the ME49 strain of T. gondii on the first day of pregnancy and then received the different treatments. Fetuses and mothers brains were collected on days 15, 17 e 19 of gestation/infection. Females were sorted into three treatment groups: control (vehicle), azithromycin (300mg/kg/dia) and the association of the drugs sulfadiazine (100 or 75 mg/Kg/day), pyrimethamine (100 or 50mg/Kg/day) and folinic acid (15mg/Kg/day). The drugs were administered orally starting at different days post-infection and animals were bled on the first day of pregnancy and on the day of sacrifice to perform ELISA tests for the detection of anti-T. gondii antibodies. For the analyses of acquired infection, adult males were infected and treated using the same drugs as the females, for either the same or longer periods. After treatment, animals were sacrificed and the brains were collected from the adults and eyes from the fetuses. All samples were processed for immunohistochemistry, using a policlonal antibody against T.gondii , while one of the eyes of fetuses from day 19 were also processed for real time PCR detection of parasite DNA. Parasite load was significantly reduced in brain tissues of females treated with azithromycin when compared to SPAf treatment. In fetuses of azithromycin-treated mothers no parasites were detected in the eyes analyzed. No difference was seen in parasite load when the SPAf-treated group was compared to the control group. In brain tissues of adult males, there was significant reduction in parasite numbers in the azithromycin-treated animals. Our study demonstrated a good efficacy of azithromycin for the treatment of pregnant females, diminishing fetal ocular infection, as well as for the treatment of adult animals, representing an alternative choice for the treatment of toxoplasmosis.Universidade Federal de UberlândiaMestre em Imunologia e Parasitologia AplicadasToxoplasma gondii é um parasita intracelular obrigatório, amplamente distribuído e que causa danos graves aos fetos quando são transmitidos durante a gestação e em adultos imunossuprimidos. As drogas mais utilizadas para o tratamento da toxoplasmose são a combinação de medicamentos: sulfadiazina, pirimetamina e ácido folínico. Entretanto, essas drogas demonstram graves efeitos colaterais e, atualmente, a sua eficácia é bastante discutida na literatura. Outros medicamentos são tidos como alternativos ao tratamento da toxoplasmose adquirida, dentre eles a clindamicina, a atovaquona e a azitromicina. Essa última demonstra-se eficaz no combate as formas taquizoítas e bradizoítas, in vitro , do parasita e apresenta-se com poucos efeitos colaterais na prática clínica. Neste estudo testamos a eficácia da azitromicina na redução da transmissão congênita de Toxoplasma analisando olhos de fetos de Calomys callosus, bem como da infecção adquirida em adultos através da análise do tecido cerebral de fêmeas prenhes e de machos imunocompetentes. Para a avaliação da infecção vertical, fêmeas de C. callosus foram inoculadas, oralmente, com 20 cistos da cepa ME49 de T. gondii no 1º dia de gestação e tratadas com os differentes protocolos. Os fetos foram coletados nos dias 15, 17 e 19 de gestação-infecção materna. As fêmeas foram divididas em três grupos de tratamento: PBS, azitromicina (300mg/Kg/dia) e a combinação (SPAf) de sulfadiazina (100 ou 75 mg/Kg/dia), pirimetamina (100 ou 50mg/Kg/dia) e ácido folínico (15mg/Kg/dia). Os medicamentos foram administrados oralmente em diferentes dias após a infecção. As fêmeas foram sangradas no primeiro dia de gestação e no dia do sacrifício para a realização de teste ELISA para confirmar soroconversão. Para análise da infecção adquirida, machos de C. callosus seguiram o mesmo protocolo de infecção e tratamento das fêmeas grávidas, mas por tempos diferentes. Os cérebros maternos e de machos adultos, bem como os olhos dos fetos foram dissecados e embebidos em parafina para análise por imunohistoquímica usando anticorpo policlonal anti-T. gondii. Um dos olhos de fetos sacrificados no 19º dia foi utilizado para a detecção de DNA do parasita pela técnica de PCR em tempo real. A quantidade de parasitas estava significantemente reduzida em tecido cerebral das fêmeas tratadas com azitromicina quando comparados a SPAf ou com veículo. Nas regiões oculares fetais, não foram detectados parasitas nos animais tratados com azitromicina. Não houve diferença na infecção ocular fetal entre os animais tratados com a SPAf ou PBS. Na infecção adquirida, a azitromicina reduziu a quantidade de parasitas em vacúolos parasitóforos e estruturas como cistos. Nosso trabalho demonstrou a eficácia da azitromicina no combate à infecção ocular congênita, na redução da carga parasitária nos cérebros maternos e de machos adultos imunocompetentes mostrando ser uma possível alternativa no tratamento da toxoplasmose

Eficácia do tratamento da Azitromicina na transmissão congênita de Toxoplasma gondii em Calomys callosus

Trabalho de Conclusão de Curso (Graduação)Toxoplasma gondii é um protozoário parasita intracelular obrigatório capaz de infectar uma grande variedade de hospedeiros. Quando transmitidos durante a gestação, as conseqüências para o feto são geralmente graves, especialmente no sistema nervoso central, onde ocorre inflamação e necrose, deixando lesões irreversíveis. A droga mais utilizada no tratamento da toxoplasmose durante a gestação é a espiramicina, que tem diversos efeitos colaterais. O propósito deste estudo foi testar se o macrolideo azitromicina era capaz de reduzir a transmissão plancentária de T gondii em modelo experimental no roedor Calomys callosus. Fêmeas de C. callosus foram inoculadas por via oral com 20 cistos da cepa ME-49 de T gondii no dia da fertilização, determinado pela presença da rolha vaginal. Fetos e placentas foram coletados do 15° ao 20° dia de gestação, sendo que cérebro e olhos foram fixados em formalina e embebidos em parafina para análise imunohistoquímica usando anticorpo monoclonal contra TgSAG1. O cérebro e o figado, assim como a placenta, foram macerados e inoculados intraperitonealmente em camundongos Swiss para análise sorológica. No grupo controle, Toxoplasma foi detectado em todos os cortes de cérebro e em cortes oculares dos dias gestacionais 17 e 19, enquanto que no grupo tratado com azitromicina, só houve detecção de taquizoitas em cortes de cérebro do dia gestacional 16, sendo todos os cortes oculares negativos para a presença do parasita. No modelo estudado, o tratamento de fêmeas grávidas com azitromicina diminuiu a transmissão congênita de T. gondii

Seroprevalence and risk factors for Toxoplasma gondii infection in pigs in southern Piauí

This study is aimed to assess the prevalence and risk factors associated with T. gondii infection in pigs. We evaluated 143 pigs, in 10 randomly-chosen farms located in Southern Piauí. The pig's blood serum was analyzed through ELISA in detection of anti-T. gondii antibodies. A seroprevalence of 25.5% was observed in the pigs that reacted against T. gondii antigens. The data from the records demonstrated an association with some factors such as: age, diet, type of management, breed and presence of cats in the farms with a prevalence of T. gondii. With the exception of sex, all others features represent risk factors for T. gondii infection. Furthermore, our data contributed to the understanding of the T. gondii seroprevalence in pig farms located in Southern Piauí

Lysophosphatidylcholine Triggers TLR2- and TLR4- Mediated Signaling Pathways but Counteracts LPSInduced NO Synthesis in Peritoneal Macrophages by Inhibiting NF-κB Translocation and MAPK/ERK Phosphorylation

Made available in DSpace on 2015-09-28T13:02:39Z (GMT). No. of bitstreams: 2 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) igor_almeida_etal_IOC_2013.pdf: 680569 bytes, checksum: 68dcc939113bc5eb10c5deee819a7ff8 (MD5) Previous issue date: 2013Universidade Federal do Rio de Janeiro. Centro de Ciências da Saúde. Instituto de Bioquímica Médica. Programa de Biologia Molecular e Biotecnologia. Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular- INCT-EM. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Centro de Ciências da Saúde. Instituto de Bioquímica Médica. Programa de Biologia Molecular e Biotecnologia. Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular- INCT-EM. Rio de Janeiro, RJ, Brasil.University of Texas at El Paso. Department of Biological Sciences. The Border Biomedical Research Center. El Paso, Texas, USA.University of Texas at El Paso. Department of Biological Sciences. The Border Biomedical Research Center. El Paso, Texas, USA / Universidade de São Paulo. Faculdade de Medicina de Ribeirão Preto. Departamento de Biologia Celular e Molecular Patogênicos. Ribeirão Preto, SP, Brasil.University of Texas at El Paso. Department of Biological Sciences. The Border Biomedical Research Center. El Paso, Texas, USA.University of Texas at El Paso. Department of Biological Sciences. The Border Biomedical Research Center. El Paso, Texas, USA / Universidade de São Paulo. Faculdade de Medicina de Ribeirão Preto. Departamento de Biologia Celular e Molecular Patogênicos. Ribeirão Preto, SP, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunofarmacologia. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Centro de Ciências da Saúde. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Parasitologia Molecular. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Centro de Ciências da Saúde. Instituto de Bioquímica Médica. Programa de Biologia Molecular e Biotecnologia. Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular- INCT-EM. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunofarmacologia. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Centro de Ciências da Saúde. Instituto de Bioquímica Médica. Programa de Biologia Molecular e Biotecnologia. Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular- INCT-EM. Rio de Janeiro, RJ, Brasil.Background: Lysophosphatidylcholine (LPC) is the main phospholipid component of oxidized low-density lipoprotein (oxLDL) and is usually noted as a marker of several human diseases, such as atherosclerosis, cancer and diabetes. Some studies suggest that oxLDL modulates Toll-like receptor (TLR) signaling. However, effector molecules that are present in oxLDL particles and can trigger TLR signaling are not yet clear. LPC was previously described as an attenuator of sepsis and as an immune suppressor. In the present study, we have evaluated the role of LPC as a dual modulator of the TLR-mediated signaling pathway. Methodology/Principal Findings: HEK 293A cells were transfected with TLR expression constructs and stimulated with LPC molecules with different fatty acid chain lengths and saturation levels. All LPC molecules activated both TLR4 and TLR2-1 signaling, as evaluated by NF-қB activation and IL-8 production. These data were confirmed by Western blot analysis of NF-қB translocation in isolated nuclei of peritoneal murine macrophages. However, LPC counteracted the TLR4 signaling induced by LPS. In this case, NF-қB translocation, nitric oxide (NO) synthesis and the expression of inducible nitric oxide synthase (iNOS) were blocked. Moreover, LPC activated the MAP Kinases p38 and JNK, but not ERK, in murine macrophages. Interestingly, LPC blocked LPS-induced ERK activation in peritoneal macrophages but not in TLR-transfected cells. Conclusions/Significance: The above results indicate that LPC is a dual-activity ligand molecule. It is able to trigger a classical proinflammatory phenotype by activating TLR4- and TLR2-1-mediated signaling. However, in the presence of classical TLR ligands, LPC counteracts some of the TLR-mediated intracellular responses, ultimately inducing an anti-inflammatory phenotype; LPC may thus play a role in the regulation of cell immune responses and disease progression

Specific cell-mediated immune responses elicited by immunization with microneme proteins.

<p>Spleen cells were harvested from immunized (indicated as TgMICs) and control mice (PBS) on day 15 post the last antigen injection and cultured in the presence of medium only, STAg (10 μg/ml), or Concanavalin A (2 μg/ml) for 72 h. (A) Proliferation of spleen cells was measured by [3H]-thymidine incorporation assay. Each bar represents the average of four mice per group and is representative of three independent experiments. Statistical significance is denoted as *p < 0.05 compared to the control group. (B–D) Cytokine concentration was measured by ELISA in the supernatant of spleen cell cultures. Panels show the IL-12 (B), IFNγ(C), and IL-10 (D) concentrations. Each bar represents the mean ± SD of triplicate samples and the results are representative of three independent experiments. Statistical significance is denoted as *p < 0.05 compared to PBS-inoculated mice; # p < 0.05 compared to non-stimulated cells of the same group.</p

Number of brain cysts and survival of mice immunized with microneme proteins and infected with <i>T</i>. <i>gondii</i>.

<p>Mice immunized on days 0, 15, and 30 with the indicated preparations of TgMICs or with the vehicle (PBS) were challenged after one month (day 60) with <i>T</i>. <i>gondii</i> infection, provoked by gavage with cysts of the ME49 strain. (A) Cyst numbers were counted from whole brain homogenates of mice, harvested one month after challenge with 40 cysts. The results are expressed as means ± SD for each group. Significance is denoted as *p < 0.05, compared to the PBS group. (B) Survival curves of mice that were challenged with 80 cysts of the ME49 strain and observed daily for mortality. Data are representative of six experiments with similar results.</p