Susceptibility of phlebotomine sandflies (Diptera: Psychodidae) collected in the field, to alpha-cypermethrin in four municipalities endemic to leishmaniasis

Although the chemical control against leishmaniasis began in 1953 in Brazil, little information is available on how this strategy has affected populations of phlebotomine sandflies in the field. The objective of this study was to analyze the susceptibility profile of four populations of phlebotomine sandflies to the insecticide alpha-cypermethrin. Sandflies collected in field in four Brazilian municipalities were evaluated using CDC bottles in different concentrations of alpha-cypermethrin. A total of 1,186 phlebotomine sandflies were used in the bioassays. The LD50 ranged from 1.48 to 2.57 ug/mL in the field populations. For a dose of 5 ug/mL of alpha-cypermethrin, the LT50 and LT95 ranged from 17.9 to 27.5 minutes, and LT95 from 39.7 to 61.5 minutes, respectively. All the populations studied were highly susceptible to the insecticide alpha-cypermethrin. Routine studies are needed to detect changes in sandflies susceptibility to insecticides

Influência da expressão e educação físico motora no desenvolvimento da criança do 1º ciclo do ensino básico : contextos, perspetivas e participantes

A formação global da criança, na qual se integra a formação motora, deve ocupar um lugar de relevância na escola atual. Neste espaço, os alunos constroem o seu próprio conhecimento, e ao vivenciarem as aprendizagens de forma lúdica, potenciam o desenvolvimento de outras competências. O presente Relatório Final de Estágio encontra-se dividido em duas partes distintas: as reflexões críticas das práticas em contexto e o trabalho de investigação. Na primeira parte é apresentada uma apreciação crítica das práticas em contexto prático, nomeadamente na educação pré-escolar e do ensino do 1.º CEB. Na segunda parte damos a conhecer o trabalho de investigação, baseado num estudo de natureza quantitativa realizado no contexto de 1.º CEB de um Agrupamento de Escolas de Aveiro e de um Agrupamento de Escolas de Viseu que visa compreender a influência da Expressão e Educação Físico-Motora no desenvolvimento global da criança. Para o efeito, recorremos a autores de referência e à legislação em vigor, assim como, em termos empíricos, as respostas aos questionários dirigidos aos professoras do 1.º CEB. Os resultados encontrados apontam para o seguinte conjunto de principais conclusões: 1.º os professores atribuem muita importância às práticas motoras, constituindo-se como um fator fundamental para o desenvolvimento integral e harmonioso da criança; 2.º a Expressão e Educação Físico-Motora não têm sido uma realidade nas escolas do estudo, os professores apresentam como principais causas: a falta de condições de materiais, espaciais e de equipamentos como também a necessidade de formação, quer a nível da formação inicial quer a nível da formação contínua; 3.º os agrupamentos, as escolas e os professores devem estar implicados nos diferentes contextos nos quais circula a criança e que são promotores de formação e desenvolvimento, tanto ao nível dos quadros letivos e curriculares como também nos de enriquecimento curricular e nos projetos mais lúdicos e de recreação; 4.º a Expressão e Educação Físico-Motora deve assumir claramente o seu carácter formativo, onde a iniciativa e responsabilidade na organização deve ir ao encontro das aspirações das crianças e de toda a comunidade educativa.The overall formation of the child, in which the motor formation is integrated, must occupy a place of relevance in the present school. In this space, students construct their own knowledge, and when they experience learning in a playful way, they foster the development of other skills. This Final Internship Report is divided into two distinct parts: the critical reflections of the practices and the research work. The first part presents a critical appraisal of practices in a practical context, namely in pre-school education and 1st cycle education (1st CEB). In the second part it is presented the research work, based on a quantitative study carried out in the context of 1st CEB in two schools from Aveiro and Viseu Portuguese Grouping of Schools that aims to understand the influence of physical-motor expression in the overall development of the child. To this end, authors of reference and legislation in force, as well as, in empirical terms, the answers to the questionnaires addressed to the teachers of the 1st CEB were used as methodology to reach the desirable goal. The results lead to the following set of main conclusions: 1) teachers attach great importance to motor practices, constituting as a fundamental factor for the integral and harmonious development of the child; 2) physical-motor expression have not been a reality in the schools of the study, the main causes of teachers are: the lack of material, space and equipment conditions as well as the need for training, both in training Initial or continuing training; 3) groups, schools and teachers must be involved in the different contexts in which the child circulates and are promoters of training and development, both in terms of school and curricular frameworks as well as curriculum enrichment and more playful projects and recreation; 4) expression and physical-motor education must clearly assume its formative character, where the initiative and responsibility in the organization must meet the aspirations of the children and the entire educational community

A vaccine therapy for canine visceral leishmaniasis promoted significant improvement of clinical and immune status with reduction in parasite burden.

Herein, we evaluated the treatment strategy employing a therapeutic heterologous vaccine composed of antigens of Leishmania braziliensis associated with MPL adjuvant (LBMPL vaccine) for visceral leishmaniasis (VL) in symptomatic dogs naturally infected by Leishmania infantum. Sixteen dogs received immunotherapy with MPL adjuvant (n = 6) or with a vaccine composed of antigens of L. braziliensis associated with MPL (LBMPL vaccine therapy, n = 10). Dogs were submitted to an immunotherapeutic scheme consisting of 3 series composed of 10 subcutaneous doses with 10-day interval between each series. The animals were evaluated before (T0) and 90 days after treatment (T90) for their biochemical/hematological, immunological, clinical, and parasitological variables. Our major results showed that the vaccine therapy with LBMPL was able to restore and normalize main biochemical (urea, AST, ALP, and bilirubin) and hematological (erythrocytes, hemoglobin, hematocrit, and platelets) parameters. In addition, in an ex vivo analysis using flow cytometry, dogs treated with LBMPL vaccine showed increased CD3+ T lymphocytes and their subpopulations (TCD4+ and TCD8+), reduction of CD21+ B lymphocytes, increased NK cells (CD5?CD16+) and CD14+ monocytes. Under in vitro conditions, the animals developed a strong antigen- specific lymphoproliferation mainly by TCD4+ and TCD8+ cells; increasing in both TCD4+IFN-?+ and TCD8+IFN-?+ as well as reduction of TCD4+IL-4+ and TCD8+IL-4+ lymphocytes with an increased production of TNF-? and reduced levels of IL-10. Concerning the clinical signs of canine visceral leishmaniasis, the animals showed an important reduction in the number and intensity of the disease signs; increase body weight as well as reduction of splenomegaly. In addition, the LBMPL immunotherapy also promoted a reduction in parasite burden assessed by real-time PCR. In the bone marrow, we observed seven times less parasites in LBMPL animals compared with MPL group. The skin tissue showed a reduction in parasite burden in LBMPL dogs 127.5 times higher than MPL. As expected, with skin parasite reduction promoted by immunotherapy, we observed a blocking transmission to sand flies in LBMPL dogs with only three positive dogs after xenodiagnosis. The results obtained in this study highlighted the strong potential for the use of this heterologous vaccine therapy as an important strategy for VL treatment

A vaccine therapy for canine visceral leishmaniasis promoted significant improvement of clinical and immune status with reduction in parasite burden.

Herein, we evaluated the treatment strategy employing a therapeutic heterologous vaccine composed of antigens of Leishmania braziliensis associated with MPL adjuvant (LBMPL vaccine) for visceral leishmaniasis (VL) in symptomatic dogs naturally infected by Leishmania infantum. Sixteen dogs received immunotherapy with MPL adjuvant (n = 6) or with a vaccine composed of antigens of L. braziliensis associated with MPL (LBMPL vaccine therapy, n = 10). Dogs were submitted to an immunotherapeutic scheme consisting of 3 series composed of 10 subcutaneous doses with 10-day interval between each series. The animals were evaluated before (T0) and 90 days after treatment (T90) for their biochemical/hematological, immunological, clinical, and parasitological variables. Our major results showed that the vaccine therapy with LBMPL was able to restore and normalize main biochemical (urea, AST, ALP, and bilirubin) and hematological (erythrocytes, hemoglobin, hematocrit, and platelets) parameters. In addition, in an ex vivo analysis using flow cytometry, dogs treated with LBMPL vaccine showed increased CD3+ T lymphocytes and their subpopulations (TCD4+ and TCD8+), reduction of CD21+ B lymphocytes, increased NK cells (CD5?CD16+) and CD14+ monocytes. Under in vitro conditions, the animals developed a strong antigen- specific lymphoproliferation mainly by TCD4+ and TCD8+ cells; increasing in both TCD4+IFN-?+ and TCD8+IFN-?+ as well as reduction of TCD4+IL-4+ and TCD8+IL-4+ lymphocytes with an increased production of TNF-? and reduced levels of IL-10. Concerning the clinical signs of canine visceral leishmaniasis, the animals showed an important reduction in the number and intensity of the disease signs; increase body weight as well as reduction of splenomegaly. In addition, the LBMPL immunotherapy also promoted a reduction in parasite burden assessed by real-time PCR. In the bone marrow, we observed seven times less parasites in LBMPL animals compared with MPL group. The skin tissue showed a reduction in parasite burden in LBMPL dogs 127.5 times higher than MPL. As expected, with skin parasite reduction promoted by immunotherapy, we observed a blocking transmission to sand flies in LBMPL dogs with only three positive dogs after xenodiagnosis. The results obtained in this study highlighted the strong potential for the use of this heterologous vaccine therapy as an important strategy for VL treatment

Toll receptors type-2 and CR3 expression of canine monocytes and its correlation with immunohistochemistry and xenodiagnosis in visceral leishmaniasis.

The aim of the present study was to investigate TLR2 expression in peripheral blood monocytes from dogs naturally infected with Leishmania (Leishmania) infantum to determine whether it correlates with CD11b/CD18 (CR3) expression, and to evaluate the potential of dogs as sources of infection using phlebotomine xenodiagnosis. Forty eight dogs were serologically diagnosed with L. infantum infection by indirect immunofluorescence antibody test (IFAT) and enzyme linked immunosorbent assay (ELISA). Parasitological exams from bone-marrow aspirates were positive by PCR analysis. All dogs were clinical defined as symptomatic. Ear skin tissue samples were obtained for immunohistochemistry (IHQ) analysis. The potential of these dogs as a source of infection using phlebotomine xenodiagnosis (XENO) was evaluated. Flow cytometry was carried out on peripheral blood mononuclear cells using superficial receptors including CD14, CD11b, TLR2 and MHCII. IHQ ear skin tissue parasite load and XENO where done where we found a strict correlation (r = 0.5373). Dogs with higher expression of MFI of CD11b inside CD14 monocytes were represented by dogs without parasite ear tissue load that were unable to infect phlebotomines (IHQ⁻/XENO⁻). Dogs with lower expression of MFI of CD11b inside CD14 monocytes were represented by dogs with parasite ear tissue load and able to infect phlebotomines (IHQ⁺/XENO⁺) (p = 0,0032). Comparable results were obtained for MFI of MHCII (p = 0.0054). In addition, considering the population frequency of CD11b⁺TLR2⁺ and CD11b⁺MHCII⁺, higher values were obtained from dogs with IHQ⁻/XENO⁻ than dogs with IHQ⁺/XENO⁺ (p = 0.01; p = 0.0048, respectively). These data, together with the TLR2 and NO assays results (CD11b⁺TLR2⁺ and NO with higher values for dogs with IHQ⁻/XENO⁻ than dogs with IHQ⁺/XENO⁺, led to the conclusion that IHQ⁻/XENO⁻ dogs are more resistant or could modulate the cellular immune response essential for Leishmania tissue clearance

Antibodies from dogs with canine visceral leishmaniasis (CVL) recognise two proteins from the saliva of lutzomyia longipalpis.

The saliva of the sand fly Lutzomyia longipalpis, a major vector of Leishmania, exhibits pharmacological and immunomodulatory activities that may facilitate entry and establishment of parasites into the vertebrate host. Salivary gland components of the sand fly are, therefore, potential candidates in the development of a vaccine against human leishmaniasis. With the objective of identifying sand fly saliva proteins that could be used to immunise animals against canine visceral leishmaniasis, we have evaluated anti-saliva antibody reactivity using serum samples collected from dogs naturally infected with Leishmania chagasi. Two proteins with molecular weights of 28.6 and 47.3 kDa were recognised by dog antibodies in Western blot assays. Protein bands were excised from an SDS-PAGE gel and the sequences determined by mass spectrometry. The proteins were identified as LuLo-D7 and Lulo YELLOW, respectively. The significance of these findings in the context of the development of multicomponent vaccination experiments is discussed

Vibrational spectroscopic characterization of the phosphate mineral althausite Mg2(PO4)(OH,F,O) - Implications for the molecular structure

Natural single-crystal specimens of althausite from Brazil, with general formula Mg2(PO4)(OH,F,O) were investigated by Raman and infrared spectroscopy. The mineral occurs as a secondary product in granitic pegmatites. The Raman spectrum of althausite is characterized by bands at 1020, 1033 and 1044 cm-1, assigned to ν1 symmetric stretching modes of the HOPO33- and PO43- units. Raman bands at around 1067, 1083 and 1138 cm-1 are attributed to both the HOP and PO antisymmetric stretching vibrations. The set of Raman bands observed at 575, 589 and 606 cm-1 are assigned to the ν4 out of plane bending modes of the PO4 and H2PO4 units. Raman bands at 439, 461, 475 and 503 cm-1 are attributed to the ν2 PO4 and H2PO4 bending modes. Strong Raman bands observed at 312, 346 cm-1 with shoulder bands at 361, 381 and 398 cm-1 are assigned to MgO stretching vibrations. No bands which are attributable to water were found. Vibrational spectroscopy enables aspects of the molecular structure of althausite to be assessed

Flow cytometry analysis considering distinct phenotypical kinetics of monocytes (SSC^intermediateCD14^hight+) taking into account the non-infected dogs (NID), immunohistochemistry (IHQ) and xenodiagnosis (XENO) results.

<p>Panel A and B represent geometric mean fluorescent intensity (MFI) of CD11b and MHC II, inside previously select monocyte subpopulation, taking into consideration NID (n = 5) <i>versus</i> double positive XENO⁺/IHQ⁺ (n = 21) <i>versus</i> double negative XENO⁻/IHQ⁻ (n = 16) results. Panel C and D represent the percentage of fluorescent cells within the population CD11bFITC⁺.TLR2PE⁺ and CD11bFITC⁺.MHCIIPE⁺ considering NID (n = 5) <i>versus</i> double positive IHQ⁺/XENO⁺ (n = 21) <i>versus</i> double negative IHQ⁻/XENO⁻ (n = 16) results. MFI and Parent frequencies of constituent groups: CD11b - NID (mean =  24.016, 50^th percentile = 22.350), Xeno⁺/IHQ⁺ (mean = 10.83, 50^th percentile = 9.700), Xeno⁻/IHQ⁻ (mean = 19.23, 50^th percentile = 15.320); MHC class II - NID (mean = 14.32, 50^th percentile = 14.04), Xeno⁺/IHQ⁺ (mean = 30.35, 50^th percentile = 29.03), Xeno⁻/IHQ⁻ (mean = 61.64, 50^th percentile = 68.23); CD11b⁺.TLR2⁺ - NID (mean = 0.3276, 50^th percentile = 0.2500), Xeno⁺/IHQ⁺ (mean = 0.2687, 50^th percentile = 0.1410), Xeno⁻/IHQ⁻ (mean = 0.4806, 50^th percentile = 0.4620); CD11b⁺.MHCII⁺ - NID (mean = 0.4492, 50^th percentile = 0.4600), Xeno⁺/IHQ⁺ (mean = 0.3851, 50^th percentile = 0.4200), Xeno⁻/IHQ⁻ (mean = 0.5765, 50^th percentile = 0.5755). Significant differences, at a level of 5% of probability (p<0.05), between cohorts are identified by the lower case letter (a, b and c) through Kruskal-Wallis and One-way ANOVA</p

Quantitative study by Real Time PCR (RT-PCR) of the skin tissue parasite load.

<p>Dogs naturally infected with <i>Leishmania (L.) chagasi</i> of double positive XENO⁺/IHQ⁺ (n = 17) <i>versus</i> double negative XENO-/IHQ- (n = 16) groups were analyzed. The load was carried out using RT-PCR with primers specific for a simple-copy gene of DNA polymerase of <i>Leishmania chagasi.</i> Xeno⁺/IHQ⁺ (mean =  5.440.292,60), Xeno⁻/IHQ⁻ (mean =  1.495,217). Significant differences, at a level of 5% of probability, between cohorts were identified by the lower case letter (a) through Mann-Whitney Test.</p