Caracterização da resistência para acaricidas no carrapato Boophilus microplus*

Boophilus microplus  é um ectoparasita hematófago de bovinos, presente em áreas tropicais e subtropicais no mundo. É causador de enormes perdas econômicas pela espoliação que causa ao hospedeiro, além de ser transmissor de Anaplasma sp. e Babesia spp. O principal método de controle utilizado atualmente é o controle químico, que, embora eficiente, é caro, causa danos ao meio ambiente e deixa resíduos na carne. Além disso, é crescente o número de relatos que apontam um aumento das populações resistentes de carrapatos a diversos princípios químicos presentes nos acaricidas. As bases moleculares da resistência em Boophilus microplus ainda não são claramente conhecidas, mas muitos estudos indicam alguns dos mecanismos. Diferentes grupos de enzimas presentes em diferentes rotas metabólicas compõem os mecanismos de resistência e detoxificação celular, além de uma série de mutações que podem aumentar a tolerância a um determinado composto químico. Neste momento, outros tipos de controle, como o biológico e o imunológico estão sendo trabalhados para evitar a resistência nas populações de carrapato. No entanto, o controle biológico ainda não é viável para grandes rebanhos e a proteção induzida por vacinas não é suficiente para permitir o controle do B. microplus. O estudo dos mecanismos de resistência no carrapato pode ser de fundamental importância para seu controle em rebanhos

Porcine circovirus type 3: immunohistochemical detection in lesions of naturally affected piglets

This study aimed to evaluate the relationship between porcine circovirus type 3 (PCV3) viral load and histopathological findings in perinatal piglet tissues and to develop an immunohistochemical method for detecting the virus in lesions. The quantitative polymerase chain reaction (qPCR) cycle threshold (Ct) when amplifying PCV3 DNA and the area of perivascular inflammatory infiltrates in different organs [central nervous system (CNS), lung, heart, liver, spleen, and lymph nodes] were compared. To develop an immunohistochemistry technique, rabbit sera were produced against PCV3-capsid protein peptides selected using bioinformatic analyses. The assay was initially implemented using a tissue sample previously tested using qPCR and in situ hybridization to optimize the procedure and reagent dilutions. To evaluate immunohistochemistry performance, tissue samples from another 17 cases were analyzed using standardized parameters. The most common microscopic lesion was multisystemic periarteritis, with associated vasculitis, as the mesenteric vascular plexus is one of the most affected organs. Other tissues, such as the heart, lung, CNS, and skeletal muscle, were also affected. Comparison of the Ct values for different tissues showed no significant difference, except in lymphoid organs (spleen and lymph nodes), which had significantly higher viral loads than the CNS tissues. There was no correlation between Ct values and perivascular inflammatory infiltrates. PCV3 immunohistochemistry revealed granular immunolabeling, mainly in the cytoplasm of cells in the vascular mesenteric plexus, heart, lung, kidney, and spleen

Endocrinologia e controle da vitelogênese em carrapatos

Background:  :  :  : Ticks are distributed worldwide, with impacts on human and animal health. The cattle tick Rhipicephalus (Boophilus) microplus is the main parasite that affects livestock in tropical and subtropical regions of the world, causing large economical losses. Tick control methods are based on the application of chemical acaricides, which has resulted in selection of resistant ticks and a potential risk of environmental pollution and food contamination. Vaccines have showed to be a feasible tick control method that offers a cost-effective, environmental friendly alternative to chemical control. However, more than ten years after the commercialization of the first vaccine against ticks, the identification of tick-protective antigens remains a limiting step in the development of an efficient formulation that would avoid the use of chemical acaricides. So, the study of parasite biology and understanding physiological mechanisms could be a good strategy to find new targets for an efficient vaccine. Review: It was reviewed the main insights about the reproductive process in ticks, emphasizing the hormonal control of vitellogenesis and enzymes involved in vitellin processing during embryogenesis. The processes of vitellogenesis and embryogenesis have been studied in various organisms, particularly in cockroaches, flies and ticks. Although the roles of 20-hydroxyecdysone (20E) and juvenile hormone have been well characterized for vitellogenesis in insects, we know much less about the hormonal control of vitellogenesis in ticks. Initially, it was hypothesized that juvenile hormone was involved in tick vitellogenin-synthesis. However, more critical studies uncovered no evidence for the occurrence of juvenile hormone or juvenile hormone-like molecules in several tick species. Current research shows that in ticks, it appears that ecdysteroids, and not juvenile hormone, regulate the expression of the vitellogenin gene and the synthesis and release of vitellogenin protein into the hemolymph. In general, the carbohydrate, lipid and amino acid composition of tick vitellogenin is similar to that of insect vitellogenin. Once in the hemolymph, oocytes uptake vitellogenin through receptor-mediated  endocytosys. However, there are different strategies to control vitellogenin synthesis and uptake by ovary in ixodide ticks. In the oocytes, vitellogenin is partially processed in the endosomal compartment and then stored as vitellin, the main reserve of protein for embryo development, in specialized organelles, the yolk granules. Embryo development depends on the availability of yolk material stored into oocytes. So, the characterization of molecules involved in vitellogenesis and embryo development contribute to a better understanding of the tick parasite physiology. During embryogesesis, acidic enzymes are responsible for the availability of this material and embryo nutrition. The Vitellin-Degrading Cysteine Endopeptidase (VTDCE), Boophilus Yolk Pro-Cathepsin (BYC) and Tick Heme Binding Aspartic Proteinase (THAP) are enzymes involved in vitellin hydrolysis in R. microplus eggs. These enzymes are produced by gut and fat body and transported through the hemolymph to be internalized into the oocytes and then play their role in tick embryo nutrition. As VTDCE, BYC and THAP are involved in an important physiological process, their potential as targets in an anti-tick vaccine is an attractive research topic. With this objective, various enzymes have been tested in native or recombinant forms as candidate immunogens to a multiantigenic anti-tick vaccine. Conclusion: Significant advancements have been made in recent years on understanding the tick reproductive process, and some molecules that can be possible targets for development of new tick control strategies have been characterized

Perspectivas para o controle do carrapato bovino

O carrapato Boophilus microplus é um ectoparasita de bovinos, presente em áreas tropicais e subtropicais na América, África, Ásia e Austrália. O método atual para o controle do carrapato bovino Boophilus microplus é o uso de acaricidas. Porém, este método é caro devido aos custos das drogas e aos da mão de obra exigida para a sua aplicação. Também, existem relatos de carrapatos resistentes a diversos acaricidas. Além disso, os resíduos químicos na alimentação e a poluição ambiental são uma grande preocupação a ser considerada. Para superar estes problemas, métodos alternativos não-químicos incluem o uso de animais geneticamente resistentes, o desenvolvimento de vacinas, o gerenciamento de pastagem com alternância de espécies e o controle biológico. Neste momento, a proteção induzida por vacinas não é suficiente para permitir o controle do B. microplus. Entretanto, os resultados sugerem ser possível o uso combinado destes métodos para o controle do carrapato a fim de reduzir o uso de acaricidas químicos

Evaluation of ELISA procedures to detect von Willebrand Factor with monoclonal antibodies

The von Willebrand Factor is a multimeric glycoprotein responsible for the promotion of platelet adhesion and aggregation at sites of vascular injury, and for FVIII stabilization. Abnormalities on this protein are responsible for diverse types of von Willebrand Disease. In the present study, monoclonal antibodies against human von Willebrand Factor were developed as a means to improve von Willebrand Disease research and diagnosis. Monoclonal antibodies were tested for their ability to bind to purified and plasmatic von Willebrand Factor. Monoclonal antibodies vW22 and vW23 were found to bind only purified von Willebrand Factor, and monoclonal antibodies vW18 and vW21 were found to bind purified and plasmatic von Willebrand Factor. Antibodies vW18 and vW21 were used to perform a sandwich-enzyme-linked immunosorbent assay to detect and quantify von Willebrand Factor concentration in plasma samples from 143 coagulopathy patients and 12 healthy blood donors. The assay showed high performance, with strong correlation and agreement in results, when compared to electroimmunoassay (Rs = 0.843 and K = 0.691 with p<0.001) and a commercial ELISA (Rs = 0.930 and K = 0.819 with p<0.001). S-ELISA proved to be a useful tool in vWF quantification tests in Brazilian specialized laboratories as an alternative to imported tests.(Avaliação de procedimentos ELISA para detectar o Fator von Willebrand com anticorpos monoclonais). O Fator von Willebrand é uma glicoproteína multimérica responsável pela promoção da adesão e agregação de plaquetas nos locais de lesão vascular e pela estabilização do FVIII. Anormalidades na função ou estrutura desta proteína são responsáveis por diversos tipos de doença de von Willebrand. Para auxiliar na pesquisa e diagnóstico da doença de von Willebrand, este estudo desenvolveu anticorpos monoclonais contra fator von Willebrand humano. Os anticorpos monoclonais foram testados quanto à sua capacidade de se ligar ao fator von Willebrand purificado e plasmático. Os anticorpos monoclonais vW22 e vW23 ligaram-se somente ao fator von Willebrand purificado, e anticorpos monoclonais vW18 e vW21 ligaram-se tanto ao fator von Willebrand purificado como ao plasmático. Anticorpos vW18 e vW21 foram utilizados no desenvolvimento de um ELISA sandwich para detectar e quantificar a concentração de fator von Willebrand no plasma em uma amostra de 143 pacientes com coagulopatias e 12 doadores de sangue saudáveis. O teste mostrou alto desempenho, com forte correlação e concordância quando comparado com uma imunoeletrofose (Rs = 0,843 e K = 0,691 p <0,001) e um ELISA comercial (Rs = 0,930 e K = 0,819 p <0,001). O ELISA-S desenvolvido no presente trabalho mostrou um bom desempenho para ser usado como teste de quantificação vWF em laboratórios especializados no Brasil como alternativa para testes importados

Proteomic Analysis of Cattle Tick Rhipicephalus (Boophilus) microplus Saliva: A Comparison between Partially and Fully Engorged Females

The cattle tick Rhipicephalus (Boophilus) microplus is one of the most harmful parasites affecting bovines. Similarly to other hematophagous ectoparasites, R. microplus saliva contains a collection of bioactive compounds that inhibit host defenses against tick feeding activity. Thus, the study of tick salivary components offers opportunities for the development of immunological based tick control methods and medicinal applications. So far, only a few proteins have been identified in cattle tick saliva. The aim of this work was to identify proteins present in R. microplus female tick saliva at different feeding stages. Proteomic analysis of R. microplus saliva allowed identifying peptides corresponding to 187 and 68 tick and bovine proteins, respectively. Our data confirm that (i) R. microplus saliva is complex, and (ii) that there are remarkable differences in saliva composition between partially engorged and fully engorged female ticks. R. microplus saliva is rich mainly in (i) hemelipoproteins and other transporter proteins, (ii) secreted cross-tick species conserved proteins, (iii) lipocalins, (iv) peptidase inhibitors, (v) antimicrobial peptides, (vii) glycine-rich proteins, (viii) housekeeping proteins and (ix) host proteins. This investigation represents the first proteomic study about R. microplus saliva, and reports the most comprehensive Ixodidae tick saliva proteome published to date. Our results improve the understanding of tick salivary modulators of host defense to tick feeding, and provide novel information on the tick-host relationship

Germ band retraction as a landmark in glucose metabolism during Aedes aegypti embryogenesis

<p>Abstract</p> <p>Background</p> <p>The mosquito <it>A. aegypti </it>is vector of dengue and other viruses. New methods of vector control are needed and can be achieved by a better understanding of the life cycle of this insect. Embryogenesis is a part of <it>A. aegypty </it>life cycle that is poorly understood. In insects in general and in mosquitoes in particular energetic metabolism is well studied during oogenesis, when the oocyte exhibits fast growth, accumulating carbohydrates, lipids and proteins that will meet the regulatory and metabolic needs of the developing embryo. On the other hand, events related with energetic metabolism during <it>A. aegypti </it>embryogenesis are unknown.</p> <p>Results</p> <p>Glucose metabolism was investigated throughout <it>Aedes aegypti </it>(Diptera) embryonic development. Both cellular blastoderm formation (CBf, 5 h after egg laying - HAE) and germ band retraction (GBr, 24 HAE) may be considered landmarks regarding glucose 6-phosphate (G6P) destination. We observed high levels of glucose 6-phosphate dehydrogenase (G6PDH) activity at the very beginning of embryogenesis, which nevertheless decreased up to 5 HAE. This activity is correlated with the need for nucleotide precursors generated by the pentose phosphate pathway (PPP), of which G6PDH is the key enzyme. We suggest the synchronism of egg metabolism with carbohydrate distribution based on the decreasing levels of phosphoenolpyruvate carboxykinase (PEPCK) activity and on the elevation observed in protein content up to 24 HAE. Concomitantly, increasing levels of hexokinase (HK) and pyruvate kinase (PK) activity were observed, and PEPCK reached a peak around 48 HAE. Glycogen synthase kinase (GSK3) activity was also monitored and shown to be inversely correlated with glycogen distribution during embryogenesis.</p> <p>Conclusions</p> <p>The results herein support the hypothesis that glucose metabolic fate changes according to developmental embryonic stages. Germ band retraction is a moment that was characterized as a landmark in glucose metabolism during <it>Aedes aegypti </it>embryogenesis. Furthermore, the results also suggest a role for GSK3 in glycogen balance/distribution during morphological modifications.</p

Caracterização da resistência para acaricidas no carrapato Boophilus microplus*

Boophilus microplus  é um ectoparasita hematófago de bovinos, presente em áreas tropicais e subtropicais no mundo. É causador de enormes perdas econômicas pela espoliação que causa ao hospedeiro, além de ser transmissor de Anaplasma sp. e Babesia spp. O principal método de controle utilizado atualmente é o controle químico, que, embora eficiente, é caro, causa danos ao meio ambiente e deixa resíduos na carne. Além disso, é crescente o número de relatos que apontam um aumento das populações resistentes de carrapatos a diversos princípios químicos presentes nos acaricidas. As bases moleculares da resistência em Boophilus microplus ainda não são claramente conhecidas, mas muitos estudos indicam alguns dos mecanismos. Diferentes grupos de enzimas presentes em diferentes rotas metabólicas compõem os mecanismos de resistência e detoxificação celular, além de uma série de mutações que podem aumentar a tolerância a um determinado composto químico. Neste momento, outros tipos de controle, como o biológico e o imunológico estão sendo trabalhados para evitar a resistência nas populações de carrapato. No entanto, o controle biológico ainda não é viável para grandes rebanhos e a proteção induzida por vacinas não é suficiente para permitir o controle do B. microplus. O estudo dos mecanismos de resistência no carrapato pode ser de fundamental importância para seu controle em rebanhos

Ecology and Genetic Identification of Freshwater Turtles in Pakistan

Background: The turtle population plays an important role in sustaining the water ecosystem by minimizing pollution from water. The identification and molecular investigation of freshwater fauna is essential for conservation of the species that are near to extinction. The quality of water, type of flora, fauna, and environmental condition are the major factors that directly affect the distribution of freshwater turtles. Studies on the species diversity and habitat of freshwater turtle have not been focused previously in the region. The present study was the first conducted to estimate the habitat and genetic diversity of freshwater turtles using 12S rRNA (ribosomal RNA) gene in Pakistan.Materials, Methods &amp; Results: A total of 26 samples were collected from various localities using hand net, cast net, gills net, steel hooks, thick chemical wire, using chicken intestine and small fishes. The collected turtle specimens were morpho-taxonomically categorized into two genera, Lissemys punctata andersoni (n=13, 50%) and Nilssonia gangetica (n=13, 50%). The collected species showed an aggressive and active behavior in captivity during summer. Genomic DNA was extracted from collected specimens and used in PCR reaction by using specific primers for the amplification of short fragments of 12S rRNA gene. Analysis of generated sequences confirmed the existence of L. p. andersoni in the region. The generated sequences of L. p. andersoni correspond to Clad A and showed a close resemblance among different species of the genus Lissemys.Discussion: This study is the first investigation about the habitat and of the endemic turtle species L. p. andersoni and N. gangetica in Pakistan. The genetic identification followed by phylogenetic analysis based on 12S rRNA partial genes revealed a closest similarity with the sequences generated for the same species from the neighboring countries. This study provided information to conduct further molecular studies that are essential to provide significant genetic data about turtle species.Keywords: turtle, ecology, diversity, phylogeny, Pakistan