Mechanisms of acaricide resistance in ticks

Background: In several countries, including Brazil, the livestock industry plays a key role in the country's economy. Brazil has the second largest bovine herd in the world and the biggest commercial herd. Ticks are an ongoing problem for both large operation cattle producers and small family farmers. Rhipicephalus microplus causes expressive losses in cattle breeding, since it occurs in important beef production zones like South America, Africa, and Oceania. Some of the negative consequences of tick infestation to cattle breeding are anemia, loss in milk and beef production, and transmission of Babesia bovis and B. bigemina. Significant losses are caused by the cattle tick (R. microplus) in several regions of the world, costing around US$ 3.3 billion per year to the Brazilian livestock industry alone. The tick control methods are mainly based on synthetic acaricides. However, the improvement of current tick control requires the identification of new molecular targets in tick physiology and development of molecule compounds to target important physiology pathways. The strategies proposed to address this issue are expand the knowledge about the molecules involved in the detoxification of chemicals to enhance the efficacy of the acaricides as well as to develop new compounds for chemical control. Review: Tick control is currently based on chemical acaricides; however, effective control and prevention of tick infestation remain distant goals. In recent decades, a progressive decrease in the efficiency of acaricides due to drug resistance has been observed. Acaricide resistance is an evolutionary adaptation, which implies the existence of behavioral and physiological mechanisms that allow the survival of resistant individuals. Four resistance mechanisms are described: behavioral resistance, reduced drug penetration, target site insensitivity and increased drug detoxification. Augmented drug detoxification may be due to increased activity of enzymes or transporters due to increased gene expression or mutations in some genes. Research focus on mechanisms of acaricide resistance in ticks characterized detoxification pathways based on (1) increased activity of enzymes (cytochrome p450, esterase and GST) which play a role in biochemically altering acaricides towards decreased toxicity and, (2) enhanced excretion of the modified less toxic compounds. To bypass the current problems, a better understanding of the biology, physiology, and molecular biology of the mechanisms of resistance to acaricides is fundamental to prolong their efficiency in controlling ticks. Moreover, identifying the genes and proteins associated with resistance can support in the development of more sensitive diagnostic methods to identify acaricide resistance, as well as improving control strategies. Discussion: In the last years, many researchers have been studying resistance mechanisms and important advances have been made which showed that, in several tick species, ABC transporters, esterases, P-450 cytochromes and glutathione-S-transferases participate in acaricide resistance. The characterization of the alterations in the targets in tick physiology and identification of new drugs with potential to tick control are crucial goals to increase tick control

Molecular characterization of Paramphistomum cervi in buffaloes

Background: Paramphistomiasis (Rumen fluke disease) in ruminants is a major health problem, characterized by coarse hair, weakness, loss of appetite, weight retardations, intestine ulcers, inter-mandibular inflammation, causing substantial economic losses, and high mortality. In tropical and subtropical regions, the disease was neglected but has recently emerged as an important cause of production losses. While documented reports on Paramphistomum cervi, Paramphistomum ichikawai and Paramphistomum are limited in Asian countries and paramphistomosis has been considered the major health and economic problem in several countries. The present study aimed to identify paramphistomoid flukes that infects buffaloes with the goal of characterization of prevalence in Pakistan and its comparison with neighbor countries. Materials, Methods & Results: In 2018, a total of 178 slaughtered buffaloes aged four to six years were examined. After an immediate postmortem examination of each buffalo, flukes were collected from their infected rumen and reticulum using sterilized forceps and placed in a saline solution. DNA was extracted from adult Paramphistome species using the standard phenol chloroform method and used for amplification of partial fragment of 18S rRNA sequences using specific pair of primer. After amplification and sequencing of 18S rRNA partial fragment, the generated sequences were assembled and trimmed to remove any primer contaminations. Twenty-three randomly selected and morphologically identified adult Paramphistomum were used in species-level identification using specific primers for partial fragment of 18S rRNA sequences. The cleaned sequences (810 bp) were used to identify similar sequences using BLAST on the NCBI website. The GenBank retrieved sequences and new Paramphistomum species isolated sequences were aligned using CLUSTAL in the BioEdit Sequence...

Control of redox homeostasis in tick blood feeding

Introduction: Ticks are hematophagous ectoparasites that cause significate economical losses. Acaricide application is themain method to tick control. However, it causes environmental contamination and selects resistant ticks. The immunological control represents a suitable method to replace or complement acaricide application. During its life cycle, female ticksingest large amounts of blood, which contains toxic components able to damage biomolecules. Understanding which molecular mechanisms and proteins are involved in avoid damages caused by blood intake in ticks and other hematophagousarthropods could help to found potential candidate antigens to compose an anti-tick vaccine.Review: Hemoglobin comprises almost 20% of mammalian blood proteins, its hydrolysis during tick digestion increasestotal free heme that can potentially generate reactive oxygen species (ROS), which easily oxidize lipids, proteins and DNA,modifying they structure and function. Lipids are more susceptible to high ROS levels. It can lead to membrane instabilityand cell death. Protein modifications caused by ROS can promote the protein loss of function and cell disturbance, however,it can also work as post-translational modifications, acting in cell signaling processes. DNA presents more efficient protective mechanisms against ROS, but damages can lead to double helix rupture. Oxidative stress is defined as a disturbance inthe balance between the production and elimination of ROS, in favor of ROS production, leading to a disruption in redoxhomeostasis and/or molecular damage. Despite the well-recognized heme oxidative power and its already demonstratedcytotoxicity, ticks are able to feed on blood, controlling the redox homeostasis without causing oxidative stress. This occurs because ticks developed physiological adaptations to transport, store, metabolize and secrete toxic components fromthe diet. The strategies, such as heme compartmentalization in specialized organelles...

Control of redox homeostasis in tick blood feeding

Introduction: Ticks are hematophagous ectoparasites that cause significate economical losses. Acaricide application is themain method to tick control. However, it causes environmental contamination and selects resistant ticks. The immunological control represents a suitable method to replace or complement acaricide application. During its life cycle, female ticksingest large amounts of blood, which contains toxic components able to damage biomolecules. Understanding which molecular mechanisms and proteins are involved in avoid damages caused by blood intake in ticks and other hematophagousarthropods could help to found potential candidate antigens to compose an anti-tick vaccine.Review: Hemoglobin comprises almost 20% of mammalian blood proteins, its hydrolysis during tick digestion increasestotal free heme that can potentially generate reactive oxygen species (ROS), which easily oxidize lipids, proteins and DNA,modifying they structure and function. Lipids are more susceptible to high ROS levels. It can lead to membrane instabilityand cell death. Protein modifications caused by ROS can promote the protein loss of function and cell disturbance, however,it can also work as post-translational modifications, acting in cell signaling processes. DNA presents more efficient protective mechanisms against ROS, but damages can lead to double helix rupture. Oxidative stress is defined as a disturbance inthe balance between the production and elimination of ROS, in favor of ROS production, leading to a disruption in redoxhomeostasis and/or molecular damage. Despite the well-recognized heme oxidative power and its already demonstratedcytotoxicity, ticks are able to feed on blood, controlling the redox homeostasis without causing oxidative stress. This occurs because ticks developed physiological adaptations to transport, store, metabolize and secrete toxic components fromthe diet. The strategies, such as heme compartmentalization in specialized organelles...(AU)

The dynamics of energy metabolism in the tick embryo / A dinâmica do metabolismo energético em embriões de carrapato

The cattle tick Rhipicephalus (Boophilus) microplus is an ectoparasite capable of transmitting a large number of pathogens, causing considerable losses in the cattle industry, with substantial damage to livestock. Over the years, important stages of its life cycle, such as the embryo, have been largely ignored by researchers. Tick embryogenesis has been typically described as an energy-consuming process, sustaining cell proliferation, differentiation, and growth. During the embryonic stage of arthropods, there is mobilization of metabolites of maternal origin for the development of organs and tissues of the embryo. Glycogen resynthesis in late embryogenesis is considered as an effective indicator of embryonic integrity. In the cattle tick R.(B. (B.) microplus, glycogen resynthesis is sustained by protein degradation through the gluconeogenesis pathway at the end of the embryonic period. Despite recent advancements in research on tick energy metabolism at the molecular level, the dynamics of nutrient utilization during R. (B.) microplus embryogenesis is still poorly understood. The present review aims to describe the regulatory mechanisms of carbohydrate metabolism during maternal-zygotic transition and identify possible new targets for the development of novel drugs and other control measures against R. (B.) microplus infestations.(AU)

O carrapato bovino Rhipicephalus (B.) microplus é um ectoparasita capaz de transmitir diversos patógenos, sendo responsável por grandes perdas na pecuária pelos danos causados ao gado. Atualmente, muitos estudos têm negligenciado fases importantes do ciclo de vida deste parasita, como a fase embrionária. A embriogênese é classicamente descrita como um processo que demanda um consumo de energia, possibilitando a proliferação celular, diferenciação e crescimento. Além disso, em artrópodes, o estágio da embriogênese é caracterizado pela mobilização de metabolitos de origem materna para o desenvolvimento de novos tecidos e órgãos. A ressíntese de glicogênio no final da embriogênese tem sido descrita em diversas espécies de artrópodes, sendo considerada um indicador de integridade do embrião. No caso do R. (B.) microplus a ressíntese de glicogênio é sustentada pela degradação de proteínas durante a gliconeogênese, no terço final da embriogênese. Apesar dos recentes avanços, no estudo molecular e do metabolismo energético, os mecanismos envolvidos na dinâmica da utilização de diferentes substratos energéticos durante a embriogênese do carrapato R. (B.) microplus ainda é pouco entendido. Diante deste panorama, estudos que descrevam a regulação destes mecanismos e da associação do metabolismo de carboidratos com a transição materno zigótica, pode auxiliar na busca de novos alvos para o desenvolvimento de novos acaricidas e outras intervenções para o controle infestações de R. (B.) microplus.(AU)

Enfermedad de von Willebrand de tipo 1 en un perro de la raza de San Bernardo - presentación clínica y el perfil de la hemostasia / Type 1 von Willebrand disease in St. Bernard dog - clinical presentation and hemostasis profile / Doença de von Willebrand tipo 1 grave em cão da raça São Bernardo - apresentação clínica e perfil de hemostasia

La enfermedad de von Willebrand (EvW) es el resultado de una deficiencia cuantitativa y/o cualitativa del factor de von Willebrand (FvW). Esta clasificado como un defecto extrínseco de las plaquetas, donde el fallo de respuesta hemostática se atribuye a la deficiencia de un factor del plasma necesario para la función normal de las plaquetas. Este estudio tiene como objetivo describir sistemáticamente el diagnóstico de la EvW tipo 1, tomando como ejemplo el reporte de un caso de un perro que presenta intensa epistaxis bilateral tratado en el hospital veterinario.(AU)

The von Willebrand disease (vWD) results from a quantitative and / or qualitative deficiency of von Willebrand factor (vWF). It is classified as a platelet- extrinsic defect, wherein the hemostatic response failure is attributed to deficiency of a plasma factor required for normal platelet function. This study aims to describe systematically the diagnosis of vWD type 1 exemplified in a case report of a dog treated at a veterinary teaching hospital due to intense bilateral epistaxis.(AU)

A doença de von Willebrand (DvW) resulta de uma deficiência quantitativa e/ou qualitativa do fator de von Willebrand (FvW). É classificada como um defeito extrínseco à plaqueta, em que a falha da resposta hemostática é atribuída à deficiência de um fator plasmático necessário para a função plaquetária normal. Este estudo visa descrever de maneira sistemática o diagnóstico da doença de (DvW) tipo 1 exemplificada em um relato de caso de um cão atendido em hospital veterinário de ensino devido a uma intensa epistaxe bilateral.(AU)

Enfermedad de von Willebrand de tipo 1 en un perro de la raza de San Bernardo - presentación clínica y el perfil de la hemostasia / Type 1 von Willebrand disease in St. Bernard dog - clinical presentation and hemostasis profile / Doença de von Willebrand tipo 1 grave em cão da raça São Bernardo - apresentação clínica e perfil de hemostasia

La enfermedad de von Willebrand (EvW) es el resultado de una deficiencia cuantitativa y/o cualitativa del factor de von Willebrand (FvW). Esta clasificado como un defecto extrínseco de las plaquetas, donde el fallo de respuesta hemostática se atribuye a la deficiencia de un factor del plasma necesario para la función normal de las plaquetas. Este estudio tiene como objetivo describir sistemáticamente el diagnóstico de la EvW tipo 1, tomando como ejemplo el reporte de un caso de un perro que presenta intensa epistaxis bilateral tratado en el hospital veterinario.

The von Willebrand disease (vWD) results from a quantitative and / or qualitative deficiency of von Willebrand factor (vWF). It is classified as a platelet- extrinsic defect, wherein the hemostatic response failure is attributed to deficiency of a plasma factor required for normal platelet function. This study aims to describe systematically the diagnosis of vWD type 1 exemplified in a case report of a dog treated at a veterinary teaching hospital due to intense bilateral epistaxis.

A doença de von Willebrand (DvW) resulta de uma deficiência quantitativa e/ou qualitativa do fator de von Willebrand (FvW). É classificada como um defeito extrínseco à plaqueta, em que a falha da resposta hemostática é atribuída à deficiência de um fator plasmático necessário para a função plaquetária normal. Este estudo visa descrever de maneira sistemática o diagnóstico da doença de (DvW) tipo 1 exemplificada em um relato de caso de um cão atendido em hospital veterinário de ensino devido a uma intensa epistaxe bilateral.

A revision of two distinct species of Rhipicephalus: R. microplus and R. australis / Uma revisão sobre duas espécies distintas de Rhipicephalus: R. microplus e R. Australis

Rhipicephalus (Boophilus) species are monoxenous ticks with seasonal distribution in tropical and subtropical regions. For many years, Rhipicephalus microplus was considered as a single species; however, further analysis split these ticks into two distinct species. Because R. microplus and R. australis share similar attributes, it is hard to discriminate these two species and explain the changes in the classification of these parasites over the past decades. The reappearance of R. australis is an outcome of new research, which has afforded to better characterize these probably cryptic species. Evidence based on morphological features, the lack of conspecificity, microsatellite markers, mitochondrial 12S and 16S ribosomal DNA, and mitochondrial genome supports the re-classification of R. microplus as different species. Therefore, populations of R. microplus from Australia, Cambodia, Philippines, Indonesia, New Caledonia, Borneo, New Guinea, Tahiti and parts of Southeast Asia were recently reinstated as R. australis . Moreover, a better knowledge on the speciation between these two species could pave the way to important advances in tick control strategies.(AU)

As espécies pertencentes ao gênero Rhipicephalus (Boophilus) são carrapatos monoxenos de distribuição sazonal em regiões tropicais e subtropicais. Por muito anos, duas espécies de carrapatos foram consideradas como Rhipicephalus microplus. Contudo, estudos recentes reclassificaram esse carrapato em duas espécies: R. microplus e R. australis . Em razão de diversas semelhanças entre R. microplus e R. australis, distinguir essas duas espécies torna-se uma tarefa árdua, o que explica as mudanças de classificação dessas espécies nas últimas décadas. O reaparecimento da espécie R. australis surge com novas pesquisas, resultado de uma melhor caracterização dessas prováveis espécies crípticas. Evidências baseadas em análises das características morfológicas, na ausência de co-especificidade, em marcadores de microssatélites, no DNA ribossomal mitocondrial 12S e 16S, assim como no genoma mitocondrial, suportam a re-classificação de R. microplus como duas espécies distintas. Nesse sentido, populações de R. microplus da Austrália, Camboja, Nova Caledônia, Bornéo, Filipinas, Nova Guiné, Indonésia e Taiti foram recentemente renomeadas como R. australis . Além disso, um melhor entendimento sobre a especiação e localização dessas duas espécies pode trazer avanços importantes para melhorar as estratégias de controle desses carrapatos.(AU)

A PCR for differentiate between Anaplasma marginale and A. centrale

Background: Anaplasma marginale ssp. centrale (A. centrale) exhibits low pathogenicity and therefore is used as a live vaccine against bovine anaplasmosis in several countries. During production of the vaccine, accidental contamination with Anaplasma marginale (A. marginale) is a risk that can jeopardize the entire batch of vaccine. Due to limitation of microscopic examination to detect low levels of parasitaemia, the present study aims to standardize a polymerase chain reaction assay using primers for the msp4 gene of Anaplasma sp. for detecting and differentiating with greater sensitivity and specificity the species of A. centrale and A. marginale in blood samples from experimentally infected cattle.Materials, Methods & Results: The DNA extraction was performed from frozen blood. Erythrocytes infected with known A. centrale, A. marginale served as positive control and the erytrocytes infected with Babesia bovis and Babesia bigemina served as the negative control polymerase chain reaction. PCR was standardized from annealing temperature variations of the primers, magnesium chloride concentration, amounts concentration of primers and DNA concentration of rickettsiae. By PCR method, it was analyzed the DNA from blood samples of 13 cattle positive to A. marginale by microscopic examination from smear stained with Giemsa. The PCR assay was specific for A. centrale and A. marginale, presented 100% identity without presenting cross-reactivity with other bovine hemoparasites. The detection limits of the PCR were 0.25 pg and 0.125 pg of DNA for detection of A. centrale and A. marginale DNA respectively.Discussion: A. marginale is an obligate intracellular bacterium that infects bovine erythrocytes causing extravascular hemolysis and anemia being considered the main agent of bovine anaplasmosis.[...](AU)

A PCR for differentiate between Anaplasma marginale and A. centrale

Background: Anaplasma marginale ssp. centrale (A. centrale) exhibits low pathogenicity and therefore is used as a live vaccine against bovine anaplasmosis in several countries. During production of the vaccine, accidental contamination with Anaplasma marginale (A. marginale) is a risk that can jeopardize the entire batch of vaccine. Due to limitation of microscopic examination to detect low levels of parasitaemia, the present study aims to standardize a polymerase chain reaction assay using primers for the msp4 gene of Anaplasma sp. for detecting and differentiating with greater sensitivity and specificity the species of A. centrale and A. marginale in blood samples from experimentally infected cattle.Materials, Methods & Results: The DNA extraction was performed from frozen blood. Erythrocytes infected with known A. centrale, A. marginale served as positive control and the erytrocytes infected with Babesia bovis and Babesia bigemina served as the negative control polymerase chain reaction. PCR was standardized from annealing temperature variations of the primers, magnesium chloride concentration, amounts concentration of primers and DNA concentration of rickettsiae. By PCR method, it was analyzed the DNA from blood samples of 13 cattle positive to A. marginale by microscopic examination from smear stained with Giemsa. The PCR assay was specific for A. centrale and A. marginale, presented 100% identity without presenting cross-reactivity with other bovine hemoparasites. The detection limits of the PCR were 0.25 pg and 0.125 pg of DNA for detection of A. centrale and A. marginale DNA respectively.Discussion: A. marginale is an obligate intracellular bacterium that infects bovine erythrocytes causing extravascular hemolysis and anemia being considered the main agent of bovine anaplasmosis.[...]

Valores de Referência para tempos de coagulação em uma população de cães de Porto Alegre, RS, Brasil / Reference values for coagulation time of dogs in Porto Alegre, RS, Brazil

Background: Prothrombin time (PT) and activated partial thromboplastin time (PTT) are screening tests for coagulopathies in dogs. Both tests measure the clotting ability by the activation of different parts of the coagulation cascade. These tests vary widely in terms of reference parameters, mainly due the considerable diversity of reagents and analyzers available. In addition, there are many variations inherent to different populations, and little has been published about coagulation reference parameters for the local dog population. The main objective of the present study was to determine a clotting time reference range of a dog population in Porto Alegre, Rio Grande do Sul, Brazil.Materials, Methods & Results: Hemostatic reference range was determined from citrated plasma of 268 clinically heathy dogs of both genders. The animals did not present bleeding diathesis or thrombocytopenia history. All dogs were previously submitted to clinical examination (cardiopulmonary auscultation, abdominal palpation, and rectal temperature) and laboratory screening (complete blood count, creatinine, albumin, and alanine aminotransferase). PT and PTT of 71 and 258 samples were measured, respectively. Blood samples were collected into 2.7 mL 3.2 % sodium citrate tubes (9 parts blood : 1 part citrate) by vacuum. Blood samples were centrifuged; the plasma was harvested and stored at -30 °C upon analyses. All analyses were performed using the viscosity detection method in semi-automatic coagulometer according to manufacturers guidelines. The reference ranges were determined in accordance with the American Society for Veterinary Clinical Pathology. The PT and PTT reference ranges were between 6.06 to 9.32 s and between 15.25 to 24.57 s, respectively.Discussion: The increased activity of the extrinsic pathway clotting factor generates narrow values in the PT assay, in comparison to PTT results.[...](AU)

Valores de Referência para tempos de coagulação em uma população de cães de Porto Alegre, RS, Brasil / Reference values for coagulation time of dogs in Porto Alegre, RS, Brazil

Background: Prothrombin time (PT) and activated partial thromboplastin time (PTT) are screening tests for coagulopathies in dogs. Both tests measure the clotting ability by the activation of different parts of the coagulation cascade. These tests vary widely in terms of reference parameters, mainly due the considerable diversity of reagents and analyzers available. In addition, there are many variations inherent to different populations, and little has been published about coagulation reference parameters for the local dog population. The main objective of the present study was to determine a clotting time reference range of a dog population in Porto Alegre, Rio Grande do Sul, Brazil.Materials, Methods & Results: Hemostatic reference range was determined from citrated plasma of 268 clinically heathy dogs of both genders. The animals did not present bleeding diathesis or thrombocytopenia history. All dogs were previously submitted to clinical examination (cardiopulmonary auscultation, abdominal palpation, and rectal temperature) and laboratory screening (complete blood count, creatinine, albumin, and alanine aminotransferase). PT and PTT of 71 and 258 samples were measured, respectively. Blood samples were collected into 2.7 mL 3.2 % sodium citrate tubes (9 parts blood : 1 part citrate) by vacuum. Blood samples were centrifuged; the plasma was harvested and stored at -30 °C upon analyses. All analyses were performed using the viscosity detection method in semi-automatic coagulometer according to manufacturer’s guidelines. The reference ranges were determined in accordance with the American Society for Veterinary Clinical Pathology. The PT and PTT reference ranges were between 6.06 to 9.32 s and between 15.25 to 24.57 s, respectively.Discussion: The increased activity of the extrinsic pathway clotting factor generates narrow values in the PT assay, in comparison to PTT results.[...]

Characterization of Rhipicephalus microplus glycogen synthase kinase 3 Beta protein by inhibition assays

Background: Rhipicephalus microplus is a monogenetic, hematophagous ectoparasite that has a large economic impact due to associated losses in the cattle industry. Glycogen synthase kinase 3 is a highly conserved and ubiquitously expressed protein in several species. It has been identified as GSK-3 isoform in the cattle tick, and is involved in the modulation of glycogen synthase activity, as a regulator of glycogen synthesis with a role in energy metabolism of R. microplus. It is a fundamental kinase for embryo development, and is directly linked with R. microplus reproductive process. Thus, the aim of this study was to investigate the role of GSK-3 in R. microplus physiology by inhibiting its activity.Materials, Methods & Results: In vitro and in vivo assays were carried out to test the effects of immunological and chemical GSK-3 inhibition. Synthetic peptides were designed by an in silico analysis of the protein antigenic index. Rabbit antibodies were raised against the designed synthetic peptides based on R. microplus GSK-3 sequence (anti-SRm0218 and anti-SRm86100). To access if the inhibition of GSK-3 would result in a decrease in fertility and hatching, the purified IgG from rabbit sera was used to feed partially engorged tick females. Results show that the antibodies were not capable of affect egg production. The same antibodies were used in an in vitro...(AU)

Characterization of Rhipicephalus microplus glycogen synthase kinase 3 Beta protein by inhibition assays

Background: Rhipicephalus microplus is a monogenetic, hematophagous ectoparasite that has a large economic impact due to associated losses in the cattle industry. Glycogen synthase kinase 3 is a highly conserved and ubiquitously expressed protein in several species. It has been identified as GSK-3 isoform in the cattle tick, and is involved in the modulation of glycogen synthase activity, as a regulator of glycogen synthesis with a role in energy metabolism of R. microplus. It is a fundamental kinase for embryo development, and is directly linked with R. microplus reproductive process. Thus, the aim of this study was to investigate the role of GSK-3 in R. microplus physiology by inhibiting its activity.Materials, Methods & Results: In vitro and in vivo assays were carried out to test the effects of immunological and chemical GSK-3 inhibition. Synthetic peptides were designed by an in silico analysis of the protein antigenic index. Rabbit antibodies were raised against the designed synthetic peptides based on R. microplus GSK-3 sequence (anti-SRm0218 and anti-SRm86100). To access if the inhibition of GSK-3 would result in a decrease in fertility and hatching, the purified IgG from rabbit sera was used to feed partially engorged tick females. Results show that the antibodies were not capable of affect egg production. The same antibodies were used in an in vitro...

Metabolismo de biomoléculas na embriogênese do carrapato Rhipicephalus (Boophilus) microplus / Metabolism of biomolecules in the embryogenesis of the tick Rhipicephalus (Boophilus) microplus

Background: The hard tick Rhipicephalus (Boophilus) microplus is an hematophagous ectoparasite that causes important economic losses in the cattle raising. The control of the parasite is usually accomplished through the use of acaricides. Even though these chemicals substances present effectiveness, their continuous use can lead to the selection of resistant parasites. When not used properly acaricides can cause environmental damages in addition to risks for animal and human health. Because of these drawbacks there are efforts to the development of alternatives control methods. The immunological control is a promissing method due to target specifi city, which increases the environmental and animal safety. The development of such approach relies on the discovery and characterization of molecules involved in the metabolism of the parasite different life stages. The embryogenesis is the stage where the metabolites of maternal origin are mobilized through catabolic and anabolic pathways necessary for the development of the embryo. The study of the substrates and enzymes involved in metabolic pathways of the embryogenesis allows the directed search to potential antigens to the development of an anti-R. microplus vaccine. Review: The embryo, during its development, passes through the stages of a sincicial blastoderm, celular blastoderm and segmentation, and both morfological changes and energy sources mobilization are related. Lipids are the major energetic source to the cellular blastoderm formation. Up to cellularization stage and after embryo segmentation, sugars are the main metabolite required. It supports that tick embryogenesis occurs with two distinct phases regarding glucose utilization by the embryo. In the initial phase, until the formation of the celular blastoderm, there is the utilization of the maternal energetic source stored in oocytes, like glycogen. After segmentation, until larval hatching, the embryo performs gluconeogenesis through non-glucidic compounds to obtain the energy input required for its metabolism. Therewith occurs the activation of the glycolytic and gluconeogenic pathways to provide glucose to the developing embryo. Due to the high levels of glucose available, it is suggested that the insulin pathway is active and be conserved in R. microplus, as previously described for other organisms. Glycogen accumulation observed throughout tick embryogenesis could be a response to an endogenous insulin-like signal. During gluconeogenesi's amino acids stored in the storage proteins of the yolk granules are enzymatically mobilized to supply the metabolic energy required for the embryo development. In this context, three peptidases involved in the degradation of yolk proteins in eggs and one peptidase in larvae were characterized, and demonstrate the importance of the use of amino acids in this phase of the tick's life. Another metabolite involved in the development of the embryo is water, whose homeostasis must be mantained in the parasitic and environmental stages of the R. microplus's life. The function of water maintenance in eggs is particularly important for embryo development. Conclusion: The study of the energetic pathways important to the metabolism of R. microplus's embryo is a promissing way for development of control methods. In this context different enzymes involved in providing energetic substrates for the embryogenesis have been indentified, characterized and can be used as targets in the immunological control of the tick.Proteinases characterized from R. microplus eggs, BYC and VTDCE, were tested as antigens to immunize bovines and provided to be immunoprotective against the parasite. Other enzymes also showed a potential to be used as targets in an anti-R.microplus vaccine.

Peptídeos antimicrobianos em Rhipicephalus (Boophilus) microplus / Antimicrobial peptides in Rhipicephalus (Boophilus) microplus

Background: The arthropod Rhipicephalus (Boophilus) microplus is a hematophagous ectoparasite that transmits a wide number of microorganisms to their host such as bacteria Anaplasma marginale. Anaplasmosis is responsible for serious damages to livestock due to mortality caused in herds, decrease in milk production and weight gain and expenses with prevention and control. Is an enzootic disease in temperate, subtropical and tropical countries. In these regions, cattle contamination may occur biologically by ticks, mechanically by flies or iatrogenically. The immune system of invertebrates has multiple mechanisms, but it is simpler than the immune system of vertebrates, however the ticks have a wide variety of protection mechanisms, including production of antimicrobial peptides (AMPs) which act directly against invading pathogens. To this date, only a few AMPs have been described in R. microplus, and little is known about the activity of these AMPs against A. marginale. Review: The tick R. microplus has several mechanisms to protect itself against invading microorganisms. Besides a protective cuticle and epithelia lining which are part of the first line of defense against pathogens, there are intermediate compounds of melanization, coagulation, phagocytosis, encapsulation, nodule formation, reactive oxygen species, proteins such as cystatins and additionally a vast repertoire of antimicrobial peptides (AMPs). However, the Anaplasma sp. have developed evolutionary mechanisms to be able to adapt and survive in this arthropod which is the main biological vector this pathogen. The AMPs can be expressed constitutively by the immune system, induced by infection, or by the recognition of surface components of microorganisms such as lipopolysaccharides (LPS) and peptidoglycan (PNG). However, through evolutionary events, the Anaplasma marginale lost genes encoding these components characteristic of the cell wall of Gram-negative bacteria, and thus, is likely that the major surface proteins (MSPs) are involved in its strengthening as the resistance to AMPs. Although the mechanisms of action of AMPs have not been fully elucidated, models are proposed to demonstrate how the interactions between lipid bilayer and AMP happen. More than 1,000 AMPs have been described in several groups of eukaryotes. In particular, amphibian peptides account for 592 of total AMPs representing a rich source of these molecules. Additionally, another 166 AMPs were isolated from insects. However, in R. microplus, few studies have described the existence of AMPs. The known R. microplus antimicrobial peptides are defensin and ixodidin (both isolated from hemocytes), the microplusin (isolated from female hemolymph and eggs), VTDCE ( isolated gut and ovary), and other two peptides characterized as fragments of bovine hemoglobin, Hb 33-91 and Hb 98-114, (isolated from engorged female gut). Conclusion: Since the silencing of genes encoding AMPs expressed in R. microplus decreases the number of A. marginale, it is suggested that this bacteria could adapt to support the tick immune defense mechanisms generating a symbiotic relationship, a evidence that the expression of AMPs can be manipulated by the pathogen to assist in its multiplication by a mechanism not yet defined, thus the Anaplasma sp. and the tick vector can live together allowing the bacteria transmission by the host. More studies about antimicrobial peptides expressed in R. microplus against invading microorganisms are necessary in order to improve the comprehension of its immune system and its competence for bovine anaplasmosis as a vector.

Endocrinologia e controle da vitelogênese em carrapatos / Endocrinology and control of tick vitellogenesis

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.

Endocrinologia e controle da vitelogênese em carrapatos / Endocrinology and control of tick vitellogenesis

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.(AU)

Detecção do vírus da diarreia viral bovina em carrapatos Rhipicephalus (Boophilus) microplus alimentados em bovino persistentemente infectado / Detection of bovine viral diarrhea virus in Rhipicephalus (Boophilus) microplus ticks fed on persistently infected cattle

Bovine viral diarrhea virus (BVDV) is one of the main agents that cause economical losses in cattle worldwide. Congenitally infected calves that are born persistently infected (PI) to BVDV are the main sources of infection to susceptible cattle. Direct contact is the most important form of transmission, but indirect contact can also spread BVDV, not only inside herds, but also between them. Transmission of BVDV by haematophagous insects has been proven experimentally, but the role of ticks in the transmission of BVDV has never been investigated. Ticks can heavily infest cattle raised in tropical areas and Rhipicephalus (Boophilus) microplus is the most important among them. The present experiment was carried out to investigate the role of R. microplus ticks in the transmission of BVDV, experimentally infecting PI calf with ticks. Material, Methods and Results: Three calves were used in the experiment: one PI calf was identified from a natural.

Detecção do vírus da diarreia viral bovina em carrapatos Rhipicephalus (Boophilus) microplus alimentados em bovino persistentemente infectado / Detection of bovine viral diarrhea virus in Rhipicephalus (Boophilus) microplus ticks fed on persistently infected cattle

Bovine viral diarrhea virus (BVDV) is one of the main agents that cause economical losses in cattle worldwide. Congenitally infected calves that are born persistently infected (PI) to BVDV are the main sources of infection to susceptible cattle. Direct contact is the most important form of transmission, but indirect contact can also spread BVDV, not only inside herds, but also between them. Transmission of BVDV by haematophagous insects has been proven experimentally, but the role of ticks in the transmission of BVDV has never been investigated. Ticks can heavily infest cattle raised in tropical areas and Rhipicephalus (Boophilus) microplus is the most important among them. The present experiment was carried out to investigate the role of R. microplus ticks in the transmission of BVDV, experimentally infecting PI calf with ticks. Material, Methods and Results: Three calves were used in the experiment: one PI calf was identified from a natural.(AU)