Bovine immunisation with a recombinant peptide derived from synthetic SBm7462® (Bm86 epitope construct) immunogen for Rhipicephalus microplus control.

RHIPICEPHALUS MICROPLUS: is the most important ectoparasite of livestock in tropical and subtropical areas around the world. Research focused on developing an efficient vaccine for cattle tick control is a high priority. The aim of this study was to evaluate the rSBm7462® peptide (Bm86-B and T cell epitopes) regarding its properties of immunogenicity, protective effect in cattle and efficacy against R. microplus. This peptide was produced by a fermentative process in the yeast culture system of Komagataella (Pichia) pastoris strain Km 71. The vaccination assay was conducted in a tick-free area using non-splenectomised Holstein Friesian calves, separated into immunisation and control groups. These animals individually received the recombinant peptide and the inoculum without peptide using saponin as an adjuvant at three time points. The calves were challenged 21 days after the last immunisation using 4500 larvae per animal. An indirect ELISA was used to identify the IgG kinetics of serum samples from the calves studied. The qPCR was performed to determine the cytokine gene expression from the total RNA of the cultured PBMCs. Histomorphometry of the germinal centres (GCs) was performed measuring slides with haematoxylin-eosin staining of surgically removed lymph nodes from immunised calves. The antibody response showed a significant induction of high-affinity IgGs in calves immunised with the recombinant peptide in comparison to the controls. The kinetics of antibodies in immunised calves showed a significant increase during the experiment. This increase in high-affinity IgGs correlated with a gradual increase of the GC diameter following each peptide vaccination. Cytokine expression profiles demonstrating an adaptive immune response in calves immunised with rSBm7462® confirmed the T-dependent response. Vaccine efficacy was calculated at 72.4 % following the analysis and fecundity of collected adult female ticks, compared between control and vaccinated groups. These findings demonstrate that this new recombinant peptide is an option for control of R. microplus infestations.

Intestinal changes and performance parameters in ticks feeding on calves immunized with subunits of immunogens against Rhipicephalus microplus.

We describe the intestinal changes and biological parameters of the tick species Rhipicephalus microplus exposed to the immune response of calves vaccinated with two subunits of immunogens. The first group of Bos taurus calves was immunized with a synthetic peptide (SBm7462), whereas the second group received an inoculum for synthetic control. The third group was immunized with a recombinant peptide (rSBm7462); an inoculum was injected into a fourth group of calves for recombinant control. Each formulation was administered to these calves during three times at intervals of 30 days. At 21 days after the last immunization, the calves were challenged using a total of 4500 larvae per animal. Indirect ELISA was realized to identify the kinetics of IgGs from samples of calves studied. Naturally detaching ticks were collected for analyses of biological performance and histological changes in the midgut. We dissected randomly detached ticks. The midgut of each of these ticks was removed and processed routinely for histology, stained with hematoxylin-eosin (H&E) and slow Giemsa. Slides were also subjected to immunohistochemistry. The antibody response showed significant induction of high-affinity IgGs in calves immunized with both peptides in comparison to calves of the control groups. Histological changes included damage of the intestinal epithelium in ticks fed on immunized hosts and intense immunostaining in midgut cells, using the serum of calves immunized with recombinant peptide. There were significant differences in all biological performing parameters of ticks detached from vaccinated calves in comparison with ticks of the control groups. We identified reductions of 87.7 and 93.5% in engorged ticks detached from calves immunized with a synthetic and recombinant peptides, respectively, a 28 and 8.60% lower egg mass in groups immunized with synthetic and recombinant peptides, respectively, and a 38.4% reduction of the value of nutrient index/tick in the group immunized with the recombinant peptide. Our findings show that the immune response induced by small peptides in cattle can modify the digestion and metabolism of ticks fed on vaccinated animals, resulting in changes in tick performance.

Effects of chronic mild stress on parameters of bone assessment in adult male and female rats / Efeito do estresse moderado crônico sobre parâmetros de avaliação óssea em ratos adultos machos e fêmeas

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 saudáveis​​, 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)

Babesia bovis: expression of adhesion molecules in bovine umbilical endothelial cells stimulated with plasma from infected cattle / Babesia bovis: expressão de moléculas de adesão em células endoteliais de cordão umbilical de bovinos estimuladas com plasma de bovinos infectados

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.

Desenvolvimento e padronização do Dot-ELISA usando peptídeos recombinantes para o diagnóstico sorológico de Neospora caninum / Development and standardization of Dot-ELISA using recombinant peptides for serological diagnosis of Neospora caninum

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...

Resposta de linfonodos em bovinos inoculados a campo com a vacina recombinante rSBm7462 anti Rhipicephalus (Boophilus) microplus) / Lymph nodes response of cattle inoculated at field with a recombinant vaccine rSBm7462 against Rhipicephalus (Boophilus) microplus

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.

[Molecular analysis of the antigenic sequence (SBm7462®) from Bm86 of the Rhipicephalus (Boophilus) microplus and similarity with Rhipicephalus sanguineus and Hyalomma anatolicum anatolicum]. / Análise molecular da sequência antigênica (SBm7462®) da BM86 doRhipicephalus (Boophilus) microplus E similaridade com Rhipicephalussanguineus E Hyalomma anatolicum anatolicum

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.

Polymorphism of the bm86 gene in South American strains of the cattle tick Boophilus microplus.

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.