RESUMO
Osteoporosis is a multifactorial disease of high prevalence and has great impact on quality of life, because the effects on bone structure increase the risk of fractures, what may be very debilitating. Based on the observation that patients with depression have lower bone mineral density than healthy individuals, many studies have indicated that stress could be an aggravating factor for bone loss. This study evaluates the effect of a protocol of chronic mild stress (CMS) on parameters of bone assessment in male and female rats. Five 5-monh-old rats of each sex underwent a schedule of stressor application for 28 days. Stressors included cold, heat, restraint, cage tilt, isolation, overnight illumination, and water and food deprivation. Five rats of each sex were kept under minimum intervention as control group. The animals were weighed at beginning and end of the period, and after euthanasia had their bones harvested. Femur, tibia and lumbar vertebrae were analyzed by bone densitometry. Biomechanical tests were performed in femoral head and diaphysis. Trabecular bone volume was obtained from histomorphometric analysis of femoral head and vertebral body, as well as of femoral midshaft cross-sectional measures. Not all parameters analyzed showed effect of CMS. However, tibial and L4 vertebral bone mineral density and cross-sectional cortical/medullar ratio of femoral shaft were lower in female rats submitted to the CMS protocol. Among male rats, the differences were significant for femoral trabecular bone volume and maximum load obtained by biomechanical test. Thus, it could be confirmed that CMS can affect the balance of bone homeostasis in rats, what may contribute to the establishment of osteopenia or osteoporosis.(AU)
A osteoporose é uma doença multifatorial, de alta prevalência e que tem um grande impacto na qualidade de vida, principalmente porque os efeitos sobre a estrutura do osso aumentam o risco de fraturas, que podem ser muito debilitantes. Com base na observação de que pacientes com depressão têm menor densidade mineral óssea que indivíduos saudáveisââ, muitos estudos têm indicado que o estresse pode ser um fator agravante para a perda óssea. Este estudo avalia o efeito de um protocolo de estresse moderado crônico (EMC) em parâmetros de avaliação óssea em ratos machos e fêmeas. Cinco animais de cada sexo, com cinco meses de idade, foram submetidos a um cronograma de aplicação de estressores durante 28 dias. Os estressores incluídos foram: frio, calor, contenção, inclinação da gaiola, isolamento, iluminação durante a noite e privação de água e ração. Cinco animais de cada sexo foram mantidos com um mínimo de intervenção como grupo controle. Os animais foram pesador no início e no final do período, e após eutanásia tiveram seus ossos coletados. Fêmur, tíbia e vértebra lombar foram analisados por densitometria óssea. Testes biomecânicos foram realizados na cabeça e na diáfise do fêmur. Volume trabecular ósseo foi obtido a partir de análise histomorfométricas da cabeça do fêmur e do corpo vertebral, bem como medidas da seção transversal diáfise femoral. Nem todos os parâmetros avaliados sofreram efeito do protocolo de EMC. No entanto, a densidade mineral óssea da tíbia e da vértebra L4 e a razão osso cortical/medula da seção transversal da diáfise femoral foram menores nas fêmeas submetidas ao protocolo. Entre os ratos machos, as diferenças foram significativas no volume trabecular ósseo da cabeça femoral e na carga máxima obtida no teste biomecânico. Assim, confirma-se que o protocolo de EMC pode afetar o equilíbrio da homeostase óssea em ratos, o que pode contribuir para o estabelecimento de osteopenia ou osteoporose.(AU)
Assuntos
Animais , Ratos , Densidade Óssea , Osteoporose/veterinária , Padrões de Referência , Estresse Psicológico/complicações , Estresse Psicológico/fisiopatologia , Densitometria/veterinária , Modelos AnimaisRESUMO
Ten male, 12-month-old Jersey with intact spleens, serologically and parasitologically free from Babesia were housed individually in an arthropod-free isolation system from birth and throughout entire experiment. The animals were randomly divided into two groups. Five animals (group A) were intravenously inoculated with 6.6 X10(7) red blood cells parasitized with pathogenic sample of Babesia bovis (passage 7 BboUFV-1), for the subsequent "ex vivo" determination of the expression of adhesion molecules. Five non-inoculated animals (group B) were used as the negative control. The expression of the adhesion molecules ICAM-1, VCAM, PECAM-1 E-selectin and thrombospondin (TSP) was measured in bovine umbilical vein endothelial cells (BUVECs). The endothelial cells stimulated with a pool of plasma from animals infected with the BboUFV-1 7th passage sample had a much more intense immunostaining of ICAM-1, VCAM, PECAM-1 E-selectin and TSP, compared to the cells which did not received the stimulus. The results suggest that proinflammatory cytokines released in the acute phase of babesiosis may be involved in the expression of adhesion molecules thereby implicating them in the pathophysiology of babesiosis caused by B. bovis.
Dez bezerros machos, da raça Jersey, com 1 ano de idade com baços "in situ", sorológica e parasitologicamente livres de Babesia, foram mantidos em baias individuais no isolamento a prova de artrópodes do Depto de Veterinária desde o nascimento e ao longo de toda a experimentação. Os animais foram divididos aleatoriamente em dois grupos. Cinco animais (grupo A) foram inoculados por via intravenosa com 6,6 x10(7) hemácias parasitados com amostra patogênica de Babesia bovis (BboUFV - 1 7ª passagem) , para a determinação subseqüente "ex vivo" da expressão de moléculas de adesão . Cinco animais não inoculados (Grupo B ) foram utilizados como controlo negativo . A expressão de moléculas de adesão ICAM - 1, VCAM , PECAM - 1, E - selectina e trombospondina ( TSP ) foi medida em células endoteliais da veia umbilical de bovinos (BUVECs). As células endoteliais estimuladas com um pool de plasma proveniente de animais infectados com BboUFV - 1 7ª passagem tinham uma imunocoloração muito mais intensa de ICAM - 1 , VCAM , PECAM - 1 de E - selectina e de TSP , em comparação com as células que não receberam o estímulo . Os resultados sugerem que as citocinas pró-inflamatórias liberados na fase aguda da babesiose pode estar envolvida na expressão de moléculas de adesão , implicando , assim, elas na fisiopatologia da babesiose causada por B. bovis.
Assuntos
Animais , Bovinos , Babesia bovis/isolamento & purificação , Bovinos/imunologia , Bovinos/parasitologia , Molécula 1 de Adesão Intercelular/isolamento & purificação , Molécula 1 de Adesão de Célula Vascular/isolamento & purificação , Cordão UmbilicalRESUMO
A neosporose é reconhecida como uma das maiores causas de aborto e perdas neonatais em bovinos de leite e corte em todo o mundo. Nos últimos anos esta doença tem atraído o interesse de pesquisadores com foco na epidemiologia e métodos eficazes de diagnóstico desta doença. No presente estudo objetivou-se desenvolver e padronizar um teste Dot-ELISA para o diagnóstico sorológico de Neospora caninum com um peptídeo recombinate como antígeno, visando o desenvolvimento de um kit para diagnóstico a campo. O peptídeo recombinante (rNcGRA1) foi desenhado com base na metodologia de genética reversa de epítopos antigênicos originados de uma proteína de grânulos densos de N. caninum, e sintetizado pela GenScript (USA). Produzido mediante o processo fermentativo em leveduras Pichia pastoris KM71. Para a padronização do Dot-ELISA, membranas de nitrocelulose de 0.22µm foram sensibilizadas com 1µL do antígeno e posteriormente os soros foram diluídos em solução de lavagem e incubados durante 1 hora. A revelação foi feita mediante a adição de Proteína G marcada com peroxidase por 30 minutos, seguido da solução reveladora a base de 3,3-Diaminobenzidine (DAB). Logo após a padronização foram testados 44 soros bovinos diagnosticados por imunofluorescência indireta (RIFI), obtendo-se uma concordância nos resultados do teste de 95,5% e uma sensibilidade e especificidade de 100% e 92% respectivamente. Quanto ao Kit para diagnóstico a campo na Plataforma Tecnológica RapidFlow-Through Miriad®, o peptídeo rNcGRA1 apresentou marcações visíveis ao reagir com os soros positivos, e não apresentou marcações usando os soros negativos. Este estudo é o primeiro a utilizar peptídeos recombinantes e mostrar-se eficiente para o diagnóstico sorológico de bovinos naturalmente infetados por N. caninum...
Neosporosis is recognized as a major cause of abortion and neonatal loss in cattle worldwide, both for dairy cattle and beef cattle. In recent years this disease has attracted the interest of researchers and studying the epidemiology and effective methods of diagnosis of this disease. The present study aimed to develop and standardize on a Dot-ELISA for the serological diagnosis of Neospora caninum by using recombinant peptide as antigen for the development of a diagnostic kit used on the field. The recombinant antigen (rNcGRA1) was designed based on the method of reverse genetics derived antigenic epitopes of dense granules protein of N. caninum and synthesized by GenScript (USA). It was produced by the fermentation in yeasts Pichiapastoris KM71. The serological technique was used for the Dot-ELISA detection of IgG specific for N. caninum in which 0.22μm nitrocellulose membranes were sensitized with 1μL of antigen and subsequently the plasmas were diluted in a washing solution and incubated for 1 hour. The results will revealed by the addition of Protein G labeled with peroxidasse for 30 minutes, followed by the developing solution based on 3,3-Diaminobenzidine (DAB). Soon after standardization tested 44 bovine plasmas were diagnosed by indirect immunofluorescence assay (IFA), agreeing with the results on a 95.5% and a sensitivity and specificity of 100% and 92% respectively. In regard to the diagnostic kit for the Technology Platform Rapid Flow-Through Miriad®, the peptide presented rNcGRA1 visible markings to react with positive plasma, and showed no markings using the negative plasma. This study is the first to use recombinant peptides and prove to be efficient for the serological diagnosis of cattle naturally infected...
Assuntos
Animais , Bovinos , Bovinos/parasitologia , Ensaio de Imunoadsorção Enzimática/veterinária , Neospora/isolamento & purificação , Peptídeos/isolamento & purificação , Doenças Parasitárias em Animais/diagnóstico , Testes Imunológicos/veterináriaRESUMO
A dependência exclusiva de compostos químicos para o controle de Rhipicephalus (Boophilus) microplus tornou-se uma das maiores preocupações científicas e econômicas dos últimos anos, e como consequência, estão sendo realizadas pesquisas para o desenvolvimento de vacinas. O objetivo deste trabalho foi avaliar a resposta de linfonodos de bovinos imunizados a campo com o peptídeo rSBm7462 anti R. (B.) microplus. Foram utilizados 14 bovinos mestiços (Bos taurus x Bos indicus), com idades entre 4-10 meses, mantidos em duas propriedades rurais do norte do estado de Minas Gerais. Os animais receberam três imunizações do peptídeo rSBm7462, aplicados por via subcutânea, com intervalo de 30 dias. Após 15 dias de cada imunização, os linfonodos pré-escapulares foram coletados e fixados por 18 horas em formol. Posteriormente, foram incluídos em Paraplast e as amostras foram coradas pela técnica hematoxilina-eosina (HE) para a observação de eventos celulares. Para a identificação do antígeno nos linfonodos dos animais imunizados, foi realizada a técnica de imuno-histoquímica (IHQ) com o método peroxidase-anti-peroxidase (PAP). A resposta de linfonodos dos bovinos inoculados foi avaliada pelas análises de formação de centros germinais (CG), hiperplasia de cordões medulares (CM) e a presença do antígeno rSBm7462 em células PAP+, demonstrando que o peptídeo recombinante rSBm7462 induz uma resposta imune adaptativa T-dependente, caracterizada nos tecidos linfóides secundários pela formação de estruturas que conferem afinidade e memória imunológica.
Exclusive chemicals dependence for the control of Rhipicephalus (Boophilus) microplus has become one of the largest scientific and economical concerns in recent years, and as a result, research to vaccine development are being undertaken. The objective of this study was evaluating the lymph nodes response of cattle immunized at field with the rSBm7462 anti-R. (B.) microplus peptide. Fourteen crossbred cattle (Bos taurus x Bos indicus), aged 4-10 months, were used. The animals were maintained on two farms in the north of Minas Gerais state and received three immunizations with the peptide rSBm7462 applied subcutaneously at 30-day intervals. Pre-scapular lymph nodes were collected surgically 15 days after each immunization and fixed in formalin for 18 hours, then, they were embedded in Paraplast subsequently and the samples were stained with Hematoxylin-Eosin (HE) technique for cellular events observation. On the other hand, in order to antigens identifying in immunized animals lymph nodes, the immunohistochemistry (IHC) with peroxidase-anti peroxidase (PAP) method was performed. Lymph node response of cattle inoculated was evaluated by analysis of germinal centers (GC) formation, medullary cords hyperplasia (MC) and antigen rSBm7462 presence in PAP+ cells. This study shows that the recombinant peptide rSBm7462 induces a T-dependent adaptive immune response characterized on secondary lymphoid tissues by structure formation for affinity and immunological memory.
Assuntos
Animais , Bovinos , Bovinos/parasitologia , Linfonodos/fisiopatologia , Rhipicephalus , Vacinas Combinadas/administração & dosagem , Doenças Parasitárias/prevenção & controle , Vacinas de Subunidades AntigênicasRESUMO
The mucosal immunization consists on antigen administration in these surfaces it is a not invasive method, inductor of local and systemic immune response. This work evaluated the immune response of the synthetic vaccine anti-tick Rhipicephalus (Boophilus) microplus SBm7462®, via oral and nasal routes. Were used 60 BALB/c mice, divided in three groups of 20 animals each (I: oral immunization; II: nasal immunization; III: animals not immunized). Nine and 15 days after the first and the second immunization was collected, separately for each group, T cells from the immunized animals, cultivating them per 10 days. After the incubation, were determined the percentage of cellular viability and the specific nature of this T cells, which had held as memory T cell. The averages of T cell SBm7462-reagents had been submitted to the analysis of variance and comparison for Tukey test, 5% of probability. Group II presented higher cellular proliferation "in vitro". For ELISA test, were positive only two animals in group I and eight in group II. The oral and nasal routs alternatively viable for immunization with the synthetic peptide SBm7462®, however require greater number of doses to induce responses with high levels of immunoglobulins.
Assuntos
Imunização/métodos , Rhipicephalus/imunologia , Infestações por Carrapato/prevenção & controle , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologia , Vacinas/administração & dosagem , Animais , Imunidade Celular , Camundongos , Camundongos Endogâmicos BALB C , Mucosa Bucal , Mucosa Nasal , Vacinas de DNARESUMO
The vaccination represents optimum method evaluated with effective cost to prevent economic losses and to increase the duration and quality of life of the production animals. . Diverse vaccines are produced from the intestinal protein Bm86 of the Rhipicephalus. (B.) microplus. The knowledge of the conservation of the gene bm86 is very important to evaluate the vaccine efficiency and the possibility of reaction crossed between different species of ticks. Samples of R. (B.) microplus come from different localities had been sequenced. The analyses of multiple alignments of the sequences had been made through the BioEdit program 7.0.5.3 version and the verification of polymorphism for visual inspection. In this work the alignment of all was become fulfilled sequences using itself BLAST in the search for similarity. Similarity was observed enters the sequenced fragments of R. (B.) microplus with the sequence of the protein Rs86 de Rhipicephalus sanguineus and with protein HA98 of the tick Hyalomma anatolicum anatolicum. The results give molecular support to synthetic the vaccine use based in the gene bm86 (SBm7462®) to be used in different species of ticks.
Assuntos
Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Carrapatos/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Vacinas/imunologia , Animais , Sequência de Bases , Rhipicephalus sanguineus/imunologia , Vacinas/genética , Vacinas de DNARESUMO
Rhiphicephalus (Boophilus) microplus is one of the most important arthropods in veterinary medicine due economic losses and health problems caused in cattle production. The vaccination represents optimum method evaluated with effective cost to prevent economic losses and to increase the duration and quality of life of the production animals. A synthetic peptide, SBm 7462, derived from Bm86, has been shown great results in control of ticks. The construction and synthesis of one nucleotide sequence based on this peptide might be useful for design a DNA vaccine that has many advances than peptide vaccine. A gene, called seq1, was constructed with a three repetition of nucleotide sequence of SBm 7462. It was cloned into a pCIneo vector expression in mammals and injected in BALB/c mouse. When mice were inoculated with the expression cassette they did not response in ELISA. They elevated antibody titles only when vaccinated with the synthetic peptide SBm7462®. And, the best titles of immunoglobulins were seen when the SBm7462® was administered subcutaneously.
Assuntos
Genes Sintéticos , Rhipicephalus , Infestações por Carrapato/prevenção & controle , Vacinas de DNA/síntese química , Vacinas de Subunidades Antigênicas/genética , Animais , CamundongosRESUMO
Thirty Boophilus microplus strains from various geographic regions of Brazil, Argentina, Uruguay, Venezuela and Colombia were analyzed for the bm86 and bm95 gene. A fragment of cDNA of 794 base pairs of the parasite larvae, included between nucleotides 278-1071s, was amplified and cloned on the pGEM-T vector. Two random clones were sequenced for each population and the nucleotides 278-1071 and predicted amino acid sequences compared with the bm86 and bm95 genes. Variations from 1.76 to 3.65% were detected in the nucleotides sequence when compared with the homologous sequence of the bm86 gene and a 3.4-6.08% in the homologous amino acid sequence of the Bm86 protein. When the sequences obtained were compared with the bm95 gene, variations from 0.50 to 3.15% were detected. Variations from 1.14 to 4.56% were detected for the Bm95 protein homologous sequences in the deduced amino acid sequence. Only five of the 30 strains analyzed presented two different types of alleles expressed and the two alleles of the Alegre population and allele 1 of the Betim population were the most divergent of all those analyzed.
Assuntos
Ixodidae/genética , Glicoproteínas de Membrana/genética , Polimorfismo Genético , Proteínas Recombinantes/genética , Vacinas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases/genética , Bovinos/parasitologia , Clonagem Molecular , Ixodidae/classificação , Glicoproteínas de Membrana/química , Dados de Sequência Molecular , Filogenia , Proteínas Recombinantes/química , Alinhamento de Sequência , América do Sul , Vacinas/químicaRESUMO
The present study, describes the antigenic characterization of a Brazilian isolate of Anaplasma marginale with appendage (tail). A panel of monoclonal antibodies (McAbs) was produced and tested by the indirect fluorescent antibody test (IFAT), ELISA and Western blotting, and used to characterize two isolates of A. marginale (one with appendage and another without appendage). Among the clones produced, eight recognized antigenic proteins, with molecular weights varying from 18.4 to 66kDa. In Western blotting, the McAb reacted against a 45kDa antigen, which was shown, by the IFAT, to be located in the tail. Immunocytochemistry confirmed the tail specificity of the monoclonal reacting against the 45kDa antigen. The panel of McAb produced has a potential use in discriminating morphologically distinct A. marginale isolates. The present study, demonstrates the occurrence of antigenic diversity among Brazilian isolates of A. marginale.
