RESUMO
beta-N-Acetylhexosaminidase (HEX, E.C. 3.2.1.52) from larvae of the ixodid tick Boophilus microplus was purified to capillary zone electrophoresis homogeneity, and characterized. Enzyme purification was carried out by sequential liquid chromatography on Sephadex G-200, p-aminobenzyl-N-acetyl-beta-D-thioglucosamine affinity, and Mono-Q FPLC columns. Purification was about 1600-fold, with a yield of 10%, as determined with p-nitrophenyl-N-acetylglucosaminide as substrate. The enzyme presented optimum pH 4.7, and optimum temperature 65 degrees C. The molecular weight of non-denatured enzyme was estimated as 127,000 by gel filtration chromatography, and 60,000 in SDS-PAGE. The tick hexosaminidase presented glycosyl residues, as evidenced by binding to Concanavalin-A. Among several p-nitrophenyl glycosides tested as substrate, HEX was active only on p-nitrophenyl-N-acetylglucosaminide and p-nitrophenyl-N-acetylgalactosaminide. The purified enzyme presented immunogenicity in rabbit, and the correspondent antibodies inhibited about 90% of its original, in vitro activity.
Assuntos
Carrapatos/enzimologia , beta-N-Acetil-Hexosaminidases/química , Animais , Anticorpos/farmacologia , Bovinos , Cromatografia , Eletroforese Capilar , Estabilidade Enzimática , Glicosídeos/metabolismo , Cinética , Larva/enzimologia , Metais/farmacologia , Especificidade por Substrato , Carrapatos/embriologia , Carrapatos/parasitologia , beta-N-Acetil-Hexosaminidases/antagonistas & inibidores , beta-N-Acetil-Hexosaminidases/isolamento & purificaçãoRESUMO
A polyclonal antibody (anti-HEX) was developed against a soluble N-acetylhexosaminidase (HEX) isolated from larval extracts of Boophilus microplus. Purified hexosaminidase was strongly inhibited by the IgG fraction of this antibody. The antibody inhibited the hexosaminidase activity of other sources, such as haemolymph and larval membranes. The antibody reacted with different antigens in the tick haemolymph, but did not recognize any antigen in saliva, as seen by immunoblot analysis. The anti-HEX was inoculated into fully engorged B. microplus females, resulting in a decrease in oviposition of approximately 26%, relative to the effect of pre-immune IgG. These data show the potential of the use of this tick enzyme as an antigen in vaccine development.
Assuntos
Anticorpos/imunologia , Doenças dos Bovinos/prevenção & controle , Infestações por Carrapato/veterinária , Carrapatos/fisiologia , beta-N-Acetil-Hexosaminidases/imunologia , Animais , Western Blotting/veterinária , Bovinos , Cromatografia em Agarose/veterinária , Cromatografia em Gel/veterinária , Compostos Cromogênicos/química , Eletroforese em Gel de Poliacrilamida/veterinária , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Hemolinfa/enzimologia , Imunoglobulina G/imunologia , Oviposição/imunologia , Coelhos , Infestações por Carrapato/prevenção & controle , Carrapatos/imunologia , beta-N-Acetil-Hexosaminidases/análiseRESUMO
An aspartic proteinase precursor, herein named BYC (Boophilus Yolk pro-Cathepsin) was isolated from eggs of the hard tick, Boophilus microplus. As judged by electrophoresis on sodium dodecyl sulfate polyacrylamide slab gel (SDS-PAGE), purified BYC presented 2 bands of 54 and 49 kDa, bearing the same NH2-terminal amino acid sequence. By Western blot analysis, BYC was also found in the haemolymph, indicating an extraovarian site of synthesis. Several organs were incubated in culture medium with [35S]methionine, and only the gut and fat body showed synthesis of BYC polypeptides. Protein sequencing of both the NH2-terminal and an internal sequence obtained after cyanogen bromide (CNBr) cleavage of BYC revealed homology with several aspartic proteinase precursors. Incubation at pH 3.5 resulted in autoproteolysis of BYC, which produced the mature form of the enzyme, that displayed pepstatin-sensitive hydrolytic activity against haemoglobin. Western blot analysis using anti-BYC monoclonal antibodies showed proteolytic processing of BYC during embryogenesis and suggested activation of the enzyme during development. A role of BYC in degradation of vitellin, the major yolk protein of tick eggs, is discussed.
Assuntos
Ácido Aspártico Endopeptidases/isolamento & purificação , Precursores Enzimáticos/isolamento & purificação , Carrapatos/enzimologia , Tecido Adiposo/enzimologia , Sequência de Aminoácidos , Animais , Ácido Aspártico Endopeptidases/química , Western Blotting , Cromatografia DEAE-Celulose , Ovos , Eletroforese em Gel de Poliacrilamida , Precursores Enzimáticos/química , Feminino , Hemoglobinas/metabolismo , Hemolinfa/enzimologia , Intestinos/enzimologia , Túbulos de Malpighi/enzimologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Coelhos , Carrapatos/crescimento & desenvolvimentoRESUMO
The capacity of the Boophilus Yolk pro-Cathepsin (BYC) to induce a protective immune response in cattle against Boophilus microplus infestation was tested by vaccination experiments and by inoculation of monoclonal antibody (MAb) against BYC into fully engorged tick females. In immunization experiments the measurement of various biological parameters demonstrated a partial protection against B. microplus. A continuous decrease in the levels of specific antibodies was observed over 11 months when six bovines were maintained in field conditions. The inoculation of the MAb into tick females produced a dose-dependent decrease in oviposition and survival of the ectoparasite compared to the control.
Assuntos
Ácido Aspártico Endopeptidases/imunologia , Precursores Enzimáticos/imunologia , Carrapatos/imunologia , Animais , Anticorpos Monoclonais , Western Blotting/veterinária , Bovinos , Eletroforese em Gel de Poliacrilamida/veterinária , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Imunização Passiva/veterinária , Insetos Vetores , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Four monoclonal antibodies (mAbs) against extracts of embryo and gut tissue obtained from fully engorged Boophilus microplus were produced. The mAb BrBml reacted with different instars and tissues, the BrBm2 recognized only antigens present in gut extract and the mAbs BrBm3 and BrBm4 recognized vitellin. The effect of inoculation of these mAbs into fully engorged Boophilus microplus females was also evaluated. The mAbs BrBm1 and BrBm2 caused a decrease in oviposition of approximately 50% and 70%, respectively, and the mAbs BrBm3 and BrBm4 did not affect reproductive efficiency. This assay may be useful as a low-cost test to provide preliminary information on the possible effects of anti-tick antibodies in damaging ticks before attempting cattle vaccination experiments.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Controle de Ácaros e Carrapatos/métodos , Carrapatos/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/farmacologia , Antígenos/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Camundongos , Camundongos Endogâmicos BALB C , Reprodução/efeitos dos fármacos , Carrapatos/efeitos dos fármacosRESUMO
The aim of the present work was to quantify the passage of bovine immunoglobulins into the hemolymph of the tick Boophilus microplus during the feeding process and to determine their antibody activity. The knowledge is of paramount importance when vector control or blocking of disease transmission is attempted by vaccination of cattle. Approximately 2% of bovine immunoglobulin present in the serum as determined by competitive ELISA was demonstrated in hemolymph of B. microplus and antibody activity against an antigen of B. microplus in the hemolymph of ticks fed on bovine immunized with the antigen purified from tick eggs was detected by Western blot assay. The antibody reactivity detected against the B. microplus antigen showed that functional antibodies are present in the hemolymph of fully engorged ticks for at least 48 h after completing the parasitic life cycle.
Assuntos
Anticorpos/sangue , Doenças dos Bovinos , Hemolinfa/imunologia , Imunoglobulinas/sangue , Infestações por Carrapato/veterinária , Carrapatos/imunologia , Vacinação , Animais , Bovinos , Ensaio de Imunoadsorção Enzimática , Infestações por Carrapato/imunologia , Infestações por Carrapato/prevenção & controleRESUMO
The major components of protein extracts from the cattle tick Boophilus microplus eggs and larvae of various ages were characterized by molecular sieving chromatography, ion exchange chromatography and SDS-PAGE. The fractions analysed showed a changing chromatographic pattern development. A serum raised against the components of a fraction showing characteristics of vitellin strongly reacted in Western blots with the major peptides of extracts from eggs, larvae, gut and ovary. Comparison of patterns obtained by electrophoresis in non-denaturing PAGE, stained with Coomassie blue or with benzidine/hydrogen peroxide, revealed that the major proteins of these extracts are haemoproteins, possibly in different aggregation states or heterogeneous in composition.
Assuntos
Proteínas do Ovo/metabolismo , Carrapatos/metabolismo , Animais , Bovinos , Cromatografia por Troca Iônica , Sistema Digestório , Feminino , Larva , Óvulo/metabolismo , Peptídeos/metabolismo , Carrapatos/crescimento & desenvolvimentoRESUMO
One therapeutic and one persistent efficacy study were conducted in Brazil to evaluate doramectin at a dose rate of 200 micrograms kg-1 against induced infestations of the single host tick, Boophilus microplus. Doramectin was highly effective in eliminating established tick populations from cattle and also in preventing infestation by the parasite. In the therapeutic study, 12 calves were infested three times a week along the dorsal line with 2500 recently hatched larvae, for a total of 11 times before treatment. Animals were allocated to two groups on the basis of uniformity of established engorged tick burdens. Six calves were treated with doramectin and six received saline solution. From Day -3 to Day 21 post-treatment, individual collections of detached engorged female ticks were made from each calf. In the persistent efficacy study, 12 calves were allocated to two groups of six animals. Six calves were treated with doramectin and six received saline solution. From Day 1 to Day 17 post-treatment, each animal was infested three times a week along the dorsal line with 2500 recently hatched Boophilus microplus larvae, for a total of nine times. From Day 18 to Day 42 post-treatment, daily collections of detached engorged female ticks were made from individual animals. In the therapeutic study, efficacy (reduction of collected engorged female ticks) progressed from 51% at 24 h post-treatment (p.t.) to at least 99% at 4 days p.t., and reached 100% at 8 days p.t. With the exception of one tick that did not lay eggs, recovered from one animal at 11 days p.t., no more ticks were recovered from doramectin-treated calves for the duration of the experiment. For the first 6 days after treatment, only a few detached engorged ticks were collected from treated animals, and their oviposition and hatchability declined rapidly. In the persistent efficacy study, doramectin treatment was highly efficacious in preventing the establishment of Boophilus microplus populations for 20 days after the first ticks completed their cycle in the non-treated group. The oviposition and hatchability of the few ticks that completed their life cycle in the doramectin group were severely reduced.
Assuntos
Doenças dos Bovinos/tratamento farmacológico , Inseticidas/uso terapêutico , Ivermectina/análogos & derivados , Infestações por Carrapato/veterinária , Animais , Bovinos , Avaliação de Medicamentos , Feminino , Injeções Subcutâneas/veterinária , Inseticidas/administração & dosagem , Ivermectina/administração & dosagem , Ivermectina/uso terapêutico , Masculino , Distribuição Aleatória , Infestações por Carrapato/tratamento farmacológico , Carrapatos , Fatores de TempoRESUMO
Two kinds of small extrachromosomal nucleic acid elements were found in the bovine babesias, Babesia bovis and B. bigemina. One element with an apparent size of 5.5 kilobase pairs (kbp) is a double stranded RNA related to virus like particles. Another molecule is a double stranded DNA with a molecular size of about 6.2 kbp. Southern blot comparison of restriction DNA fragments of the latter molecule, which is present in both B. bovis and B. bigemina is described.
Assuntos
Babesia/genética , DNA/genética , Herança Extracromossômica , RNA de Cadeia Dupla/genética , Animais , Babesia bovis/genética , DNA de Protozoário/genética , Vírus de RNA , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
Babesia bovis is an intraerythrocytic protozoan that causes bovine babesiosis. Agarose gel electrophoresis of nucleic acids extracted from two isolates of B. bovis reveals, besides bulk DNA, an ethidium bromide-stainable band at about 5.5 kb. Further characterization of the latter with DNase I, RNase and mung bean nuclease suggested it to be a double-stranded RNA. Sonicated parasites were fractionated in a CsCl buoyant density gradient. A sample containing the 5.5-kb RNA was analysed under an electron microscope and a virus-like particle was observed.
Assuntos
Babesia/microbiologia , RNA Viral/análise , Animais , Babesia/genética , Babesiose/genética , Bovinos , Centrifugação com Gradiente de Concentração , Desoxirribonuclease I , RNA de Cadeia Dupla/análise , Ribonuclease PancreáticoRESUMO
A entrada de animais infestados por carrapatos nas regiöes livres, nos meses de veräo, provoca surtos de tristeza parasitária nos animais nativos, que normalmente se encontram sem proteçäo para esta enfermidade. Nos veröes de 1987 e 1988, ocorreram vários focos isolados de carrapatos em Santa Vitória do Palmar, regiäo indene do extremo sul do Brasil. O presente trabalho teve como objetivo colaborar no esclarecimento do assunto, chegando os autores à conclusäo de que o carrapato ali existente é o Boophilus microplus, naturalmente livre de Babesia spp
Assuntos
Animais , Babesiose , Infestações por Carrapato , Carrapatos , BovinosRESUMO
Se presentan 2 pacientes con varices colonicas. Se hace una breve revision de la literatura, la cual incluye un total de otros 33 casos. La mayoria de los pacientes tienen hipertension portal. El motivo de consulta mas frecuente es el sangramiento rectal. La endoscopia es el metodo mas util para el diagnostico. El tratamiento que mejores resultados parece dar en los casos de hemorragia masiva es la anatomosis porto-cava