TNF-TNFR1 Signaling Enhances the Protection Against Neospora caninum Infection.

Neospora caninum is a protozoan associated with abortions in ruminants and neuromuscular disease in dogs. Classically, the immune response against apicomplexan parasites is characterized by the production of proinflammatory cytokines, such as IL-12, IFN-γ and TNF. TNF is mainly produced during the acute phases of the infections and binds to TNF receptor 1 (CD120a, p55, TNFR1) activating a variety of cells, hence playing an important role in the induction of the inflammatory process against diverse pathogens. Thus, in this study, we aimed to evaluate the role of TNF in cellular and humoral immune responses during N. caninum infection. For this purpose, we used a mouse model of infection based on wildtype (WT) and genetically deficient C57BL/6 mice in TNFR1 (Tnfr1-/-). We observed that Tnfr1-/- mice presented higher mortality associated with inflammatory lesions and increased parasite burden in the brain after the infection with N. caninum tachyzoites. Moreover, Tnfr1-/- mice showed a reduction in nitric oxide (NO) levels in vivo. We also observed that Tnfr1-/- mice showed enhanced serum concentration of antigen-specific IgG2 subclass, while IgG1 production was significantly reduced compared to WT mice, suggesting that TNFR1 is required for regular IgG subclass production and antigen recognition. Based on our results, we conclude that the TNF-TNFR1 complex is crucial for mediating host resistance during the infection by N. caninum.

Formulation and Validation of Recombinant Antigens CFP10 and ESAT6 for Tuberculosis Diagnosis

Abstract The rapid and accurate diagnosis of tuberculosis (TB), especially considering limited resources, is still a challenge. Development of new methodologies and tests are needed to overcome several disadvantages of the available standard tests. We evaluated the diagnostic potential of two antigens specific for Mycobacterium tuberculosis, the CFP10 and ESAT6 recombinant proteins, and developed stable formulations thereof. Sensitivity and specificity of the delayed-type hypersensitivity (DTH) skin testing and the induction of gamma interferon production (IFN-γ) by lymphocytes, as a non-invasive test, were evaluated using the CFP10 and ESAT6 protein formulations. The recombinant proteins produced by our group presented a high DTH response and the ability to differentiate between tuberculosis infection, BCG vaccination, and the contact with non-tuberculous mycobacteria (NTM). The production of IFN-γ by stimulation with individual and combined proteins was detected in a panel of 40 individuals and showed a specificity of 100% and a sensitivity of 90% when the two proteins were used together. Lyophilized formulations were stable under all conditions, while soluble formulations were stable under freezing at -20 ºC and -80 ºC. The proposed formulations containing the ESAT6 and CFP10 recombinant antigens constitute satisfactory tools for TB testing, suitable to be developed and implemented in a large-scale trial.

Serological evidence of Toxoplasma gondii infection in Melanosuchus niger (Spix, 1825) and Caimam crocodilus (Linnaeus, 1758).

Toxoplasma gondii is a protozoan with worldwide prevalence, known to affect a large variety of warm-blooded hosts. However, its ability to induce long-lasting infections in cold-blooded animals remains unclear. The most likely source of infection is through consumption of meat containing tissue cysts or by ingestion of food or water contaminated with oocysts. The current global climate change trend and the progressive degradation of natural habitats are prone to alter the distribution of ectotherm populations over a short period of time, which may favor contact between these animals and the protozoan. In association, alligator meat is considered a delicacy in many regions and its consumption has been previously related to a diversity of foodborne diseases. In that sense, we proposed in this study to search for specific antibodies against T. gondii in serum samples of two common species of alligators from the Brazilian fauna (Melanosuchus niger and Caimam crocodilus). We obtained the serum samples from 84 alligators from the Araguaia region, which were tested by agglutination assays that do not require species-specific secondary antibodies (Modified Agglutination Test - MAT; Indirect Hemagglutination Assay - IHA). From the 84 samples tested, eight (9.5%) were positive by MAT. From those, seven (87.5% of MAT+, 8.3% of the total) were also positive by IHA, reassuring a probable exposure of these animals to the parasite. Direct parasite detection in muscle fragments of one serologically reactive alligator did not yield positive results. Our results provide serological evidence that Brazilian alligators may be exposed to T. gondii and further studies should be performed to elucidate whether alligators are natural hosts of this ubiquitous protozoan parasite.

In silico and in vitro Evaluation of Mimetic Peptides as Potential Antigen Candidates for Prophylaxis of Leishmaniosis.

Antigen formulation is the main feature for the success of leishmaniosis diagnosis and vaccination, since the disease is caused by different parasite species that display particularities which determine their pathogenicity and virulence. It is desirable that the antigens are recognized by different antibodies and are immunogenic for almost all Leishmania species. To overcome this problem, we selected six potentially immunogenic peptides derived from Leishmania histones and parasite membrane molecules obtained by phage display or spot synthesis and entrapped in liposome structures. We used these peptides to immunize New Zealand rabbits and determine the immunogenic capacity of the chimeric antigen. The peptides induced the production of antibodies as a humoral immune response against L. braziliensis or L. infantum. Next, to evaluate the innate response to induce cellular activation, macrophages from the peptide mix-immunized rabbits were infected in vitro with L. braziliensis or L. infantum. The peptide mix generated the IFN-γ, IL-12, IL-4 and TGF-ß that led to Th1 and Th2 cellular immune responses. Interestingly, this mix of peptides also induced high expression of iNOS. These results suggest that the mix of peptides derived from histone and parasites membrane molecules was able to mimic parasites proteins and induce cytokines important to CD4+ T cell Th1 and Th2 differentiation and effector molecule to control the parasite infection. Finally, this peptide induced an immune balance that is important to prevent immunopathological disorders, inflammatory reactions, and control the parasite infection.

Parasite detection in sand from bays on the north coast of São Paulo state, Brazil

Soil contamination by protozoan parasites and soil-transmitted helminths (STH) is common in beach sand due to a number of factors such as pets, pluvial water, garbage, etc. These pathogens may cause many diseases in humans and animals and become a public health problem. Thus, systematic evaluation and inspection are necessary to develop control strategies regarding public contamination. For this purpose, our aims were to evaluate the parasitic profile of sandy soils on an urban beach and an untouched beach and correlate this with environmental and seasonal characteristics in Ubatuba, Brazil, in two seasons (winter and summer). 132 soil samples were collected for parasite analysis utilizing Rugai's method and the sedimentation adapted method. Our results showed positivity in 62% of the samples for at least one parasite in the urban beach and no parasitic structures on the untouched beach. The positivity was higher in summer (85%) than in winter (51.7%). All samples were positive for both, helminths and protozoa. Seasonal influence was noted regarding the presence of STH while for protozoa this influence was not observed. The parasitic structures most found were larvae of hookworms (35%) and Toxocara eggs (31.7%). We also noted the presence of Strongyloides sp, Ascaris lumbricoides, coccidia, Dipylidium caninum, Entamoeba hystolitica/dispar, Endolimax nana, Entamoeba coli, Giardia sp, Toxascaris leonina, Trichuris sp and Dibothricephalus latus. There was a positive correlation between temperature and the presence of STH in the sand samples. Most of the collection sites on the urban beach presented dogs or canine traces and garbage in both seasons. There was an association between the presence of dogs or their traces and parasitic structures. In conclusion, seasonality, urbanization and the presence of pets on beaches can potentially favor environmental contamination by parasites increasing the risk of transmission of zoonotic and parasitic diseases.

Inducible Nitric Oxide Synthase is required for parasite restriction and inflammatory modulation during Neospora caninum infection.

Neospora caninum infection is an important cause of neuromuscular disease in dogs and abortion in cattle, leading to significant economic losses in beef and dairy industries. The protective immunity against apicomplexan parasites, specifically Toxoplasma gondii and N. caninum, is typically achieved by inducing an IL-12-driven Th1 immune response. IL-12 stimulates IFN-γ production, which activates Inducible Nitric Oxide Synthase (iNOS) and promotes consequent Nitric Oxide (NO) synthesis, classically described as one of the main effector mechanisms for parasite elimination. Here, we aimed to evaluate the role played by iNOS during N. caninum infection. Our results show that N. caninum infection in C57BL/6 wild type (WT) mice induce NO production in vivo and in vitro. In agreement, iNOS deficient mice, as well as WT mice treated with iNOS inhibitor aminoguanidine, succumbed during acute infection with a dose lethal to 50 % of the WT mice, and presented significant increase in parasite load when submitted to sub-lethal infection protocols. Interestingly, the lack of control of parasite proliferation observed in iNOS-/- mice was associated with notable CNS inflammation and increased production of the main systemic proinflammatory cytokines (IL-12, IFN-γ, IL-6, TNF and IL-17A). Taken together, our findings show that iNOS plays an important role in restricting N. caninum replication, while also modulates the inflammatory process induced by the infection.

Cyclooxygenase (COX)-2 Inhibitors Reduce Toxoplasma gondii Infection and Upregulate the Pro-inflammatory Immune Response in Calomys callosus Rodents and Human Monocyte Cell Line.

Toxoplasma gondii is able to infect a wide range of vertebrates, including humans. Studies show that cyclooxygenase-2 (COX-2) is a modulator of immune response in multiple types of infection, such as Trypanosoma cruzi. However, the role of COX-2 during T. gondii infection is still unclear. The aim of this study was to investigate the role of COX-2 during infection by moderately or highly virulent strains of T. gondii in Calomys callosus rodents and human THP-1 cells. C. callosus were infected with 50 cysts of T. gondii (ME49), treated with COX-2 inhibitors (meloxicam or celecoxib) and evaluated to check body weight and morbidity. After 40 days, brain and serum were collected for detection of T. gondii by real-time PCR and immunohistochemistry or cytokines by CBA. Furthermore, peritoneal macrophages or THP-1 cells, infected with RH strain or uninfected, were treated with meloxicam or celecoxib to evaluate the parasite proliferation by colorimetric assay and cytokine production by ELISA. Finally, in order to verify the role of prostaglandin E2 in COX-2 mechanism, THP-1 cells were infected, treated with meloxicam or celecoxib plus PGE2, and analyzed to parasite proliferation and cytokine production. The data showed that body weight and morbidity of the animals changed after infection by T. gondii, under both treatments. Immunohistochemistry and real-time PCR showed a reduction of T. gondii in brains of animals treated with both COX-2 inhibitors. Additionally, it was observed that both COX-2 inhibitors controlled the T. gondii proliferation in peritoneal macrophages and THP-1 cells, and the treatment with PGE2 restored the parasite growth in THP-1 cells blocked to COX-2. In the serum of Calomys, upregulation of pro-inflammatory cytokines was detected, while the supernatants of peritoneal macrophages and THP-1 cells demonstrated significant production of TNF and nitrite, or TNF, nitrite and MIF, respectively, under both COX-2 inhibitors. Finally, PGE2 treatment in THP-1 cells triggered downmodulation of pro-inflammatory mediators and upregulation of IL-8 and IL-10. Thus, COX-2 is an immune mediator involved in the susceptibility to T. gondii regardless of strain or cell types, since inhibition of this enzyme induced control of infection by upregulating important pro-inflammatory mediators against Toxoplasma.

Prevalence of intestinal parasites in child day care centers: epidemiological significance

Intestinal parasites are among the most commonly found pathogens in humans and are considered a public health problem. The frequency of intestinal parasites was evaluated in children and staff at two day care centers in the city of Ituiutaba, Brazil and health encouragement activities were developed. For parasite detection in stool samples the Ritchie's technique and the Hoffman-Pons-Janer, Baermann-Moraes, Willis and Ziehl-Neelsen methods were applied. Of 140 children, 88 (62.9%) were positive, where Giardia intestinalis cysts were present in 65 children (51.1%), Entamoeba coli in 22 (17.3%), Endolimax nana in 12 (9.4%), Entamoeba histolytica/E. dispar in 9 (7%), Hymenolepis nana eggs were found in 5 (4%), hookworms in 4 (3.1%), Ascaris lumbricoides and Enterobius vermicularis in 3 (2.3%) and Strongyloides stercoralis, Trichuris trichiura, Cryptosporidium spp. and Iodamoeba butschlii in one child (0.8%). Among the 41 employees examined, 10 (24.3%) were positive, E. coli cysts were found in 6 samples (35.2%) and E. nana in 5 (29.4%), followed by E. histolytica/E. dispar in 4 (23.6%) and G. intestinalis and E. vermicularis eggs in (5.9%). The data show the high frequency of intestinal parasites in fecal samples analyzed and demonstrate the importance of adopting preventive measures such as health and education involving the community

Dectin-1 Compromises Innate Responses and Host Resistance against Neospora caninum Infection.

Neospora caninum is an intracellular protozoan parasite that has drawn increasing interest due to its association with worldwide repetitive bovine abortions, which cause billionaire losses to the meat and dairy industries annually. Innate immunity plays an important role in infection control, and N. caninum activates the production of inflammatory mediators through toll-like receptors, NOD-like receptors, and mitogen-activated protein kinase signaling pathways. Advances in the knowledge of initial host-parasite interactions are desirable for the design of control measures against the infection, obliterating its pathogenesis. In that sense, we here aimed to describe the role of the innate C-type lectin receptor Dectin-1 during the infection by N. caninum. With that intent, we observed that the absence of Dectin-1, observed in genetically depleted (Dectin-1-/-) mice or competitively inhibited by an inert agonist [laminarin (LAM)], rescued 50% of the mice infected with lethal doses of N. caninum. Dectin-1-/- and LAM-treated mice also presented a reduction in the parasite load during acute and chronic phases, associated with decreased inflammatory scores in the central nervous system. Among all the cell phenotypes that migrated to the initial site of infection, dendritic cells and macrophages gained subpopulations with high Dectin-1 surface expression. The impairment of the receptor in these cells led to a decreased parasite burden, as well as augmented production of IL-12p40. We also found that Dectin-1+ cells produced less reactive oxygen species (ROS) at the initial site of the infection, while mice deficient in NADPH oxidase isoform 2 (NOX2-/-) were not able to control parasite replication and produce IL-12p40, even upon LAM treatment. Interestingly, the absence of functional Dectin-1 did not alter the susceptibility of mice against closely related Toxoplasma gondii. In conclusion, the gathered data suggest that Dectin-1 is involved in the parasite-induced downmodulation of ROS, and other key molecules triggered for the control of N. caninum infection and are a promising target for future development of protocols intended for intervention against neosporosis.

PREVALENCE OF CHAGAS DISEASE AMONG BLOOD DONOR CANDIDATES IN TRIANGULO MINEIRO, MINAS GERAIS STATE, BRAZIL / Prevalência de casos de doença de Chagas em candidatos a doação de sangue na região do Pontal do Triângulo Mineiro, Brasil

Despite public health campaigns and epidemiological surveillance activities, Chagas disease remains a major health problem in Latin America. According to data from the World Health Organization, there are approximately 7-8 million people infected with Trypanosoma cruzi worldwide, a large percentage of which in Latin America. This study aims to examine the serological profile of blood donors in blood banks of Hemominas hematology center, in the town of Ituiutaba, Minas Gerais State, Brazil. The study sample consisted of 53,941 blood donors, which were grouped according to gender and age. Sample collections were performed from January 1991 to December 2011, and 277 donors (0.5%) were considered serologically ineligible due to Chagas disease. Analysis of data showed no significant difference between genders. As for age, the highest proportion of ineligible donors was from 40 to 49 years (30%), and there was a positive correlation between increasing age and the percentage of patients seropositive for Chagas disease. Therefore, adopting strategies that allow the safe identification of donors with positive serology for Chagas disease is essential to reduce or eliminate indeterminate serological results.

A doença de Chagas, apesar das campanhas de saúde pública e das ações de vigilância epidemiológica, ainda constitui-se um sério problema de saúde na América Latina. De acordo com dados divulgados pela Organização Mundial de Saúde, existem cerca de 7 a 8 milhões de pessoas infectadas com Trypanosoma cruzi em todo o mundo, principalmente na América Latina. Este estudo tem por objetivo analisar o perfil sorológico de doadores de sangue dos bancos de sangue do Hemominas de Ituiutaba, Minas Gerais. Os doadores também foram separados de acordo com o sexo e a idade. A amostra do estudo foi composta por 53.941 doadores de sangue durante o período de janeiro de 2001 a dezembro de 2011. Duzentos e setenta e sete doações (0,5%) foram considerados sorologicamente inaptas para a doença de Chagas. Quanto à idade, a maior proporção de doadores impróprios foi de 40 a 49 anos (30%). Os dados não revelaram diferença significativa entre os sexos (p < 0,05). Houve correlação positiva entre o aumento da idade e o percentual de pacientes soropositivos para doença de Chagas. É imprescindível a adoção de estratégias que permitam a identificação segura de um doador com sorologia positiva para doença de Chagas, tentando assim minimizar ou eliminar resultados sorológicos indeterminados.

PREVALENCE OF CHAGAS DISEASE AMONG BLOOD DONOR CANDIDATES IN TRIANGULO MINEIRO, MINAS GERAIS STATE, BRAZIL.

Despite public health campaigns and epidemiological surveillance activities, Chagas disease remains a major health problem in Latin America. According to data from the World Health Organization, there are approximately 7-8 million people infected with Trypanosoma cruzi worldwide, a large percentage of which in Latin America. This study aims to examine the serological profile of blood donors in blood banks of Hemominas hematology center, in the town of Ituiutaba, Minas Gerais State, Brazil. The study sample consisted of 53,941 blood donors, which were grouped according to gender and age. Sample collections were performed from January 1991 to December 2011, and 277 donors (0.5%) were considered serologically ineligible due to Chagas disease. Analysis of data showed no significant difference between genders. As for age, the highest proportion of ineligible donors was from 40 to 49 years (30%), and there was a positive correlation between increasing age and the percentage of patients seropositive for Chagas disease. Therefore, adopting strategies that allow the safe identification of donors with positive serology for Chagas disease is essential to reduce or eliminate indeterminate serological results.