Talita Bandeira Roos

Effect of Bacillus cereus var. Toyoi and Saccharomyces boulardii on the immune response of sheep to vaccines

Abstract In this study we evaluated the effects of Bacillus cereus var. Toyoi and Saccharomyces boulardii on the immune response of lambs to Escherichia coli K88ab and Bovine Herpes Virus type 5 (BoHV-5) vaccines. Thirty, 3-month-old lambs were randomly grouped in three lots of 10 each and vaccinated at days 0 and 30 of the experiment. They grazed on the same pasture and were fed ad libitum twice a day with commercial sheep feed supplemented with either B. cereus var. Toyoi at a concentration of 1×106 viable spores gr−1, S. boulardii at a concentration of 1×106 CFU gr−1, or non-supplemented feed. Blood samples were collected at weekly intervals over eight weeks and antibody titres were analysed by ELISA. The mean seroconversions against E. coli and BoHV-5 of the fed probiotics groups were higher (p<0.001) than the controls. Both probiotics enhanced the humoral immune response of lambs to the vaccines.

Expression of Neospora caninum NcSRS2 surface protein in Pichia pastoris and its application for serodiagnosis of Neospora infection

Abstract Neospora caninum is considerd a major cause of abortion in cattle worldwide. The antigenic domain of NcSRS2 in N. caninum is an important surface antigen present in the membrane of this parasite. In the present study, the Pichia pastoris expression system proved to be a useful tool for the production of recombinant protein. The truncated NcSRS2 gene (by removal of the N-terminal hydrophobic sequence), was cloned in the vector pPICZalphaB, and integrated on the genome of the methylotrophic yeast P. pastoris. Subsequently, the NcSRS2 protein was expressed, purified, and characterized using naturally infected cattle sera and Mab 6xhistag. The recombinant protein NcSRS2 was present in the supernatant of the culture, where later it was concentrated and purified using ammonium sulfate (∼100 mg/ml). An indirect immunoenzymatic assay (ELISA) was performed using cattle sera from endemic N. caninum area.

Cloning and expression of a truncated form of envelope glycoprotein D of Bovine herpesvirus type 5 in methylotrophic yeast Pichia pastoris.

Meningoencephalitis caused by Bovine herpesvirus type 5 (BoHV-5) is responsible for heavy economic losses in the cattle industry. As in other Alphaherpesviruses, the envelope glycoprotein IV (gD), which mediates penetration into host cells, is one of the major candidate antigens for a recombinant vaccine, since it induces a strong and persistent immune response. The DNA coding for a truncated form of BoHV-5 gD (tgD) has been cloned into the Pichia pastoris expression vector pPICZalphaB to allow protein secretion into the medium. After induction with methanol, a approximately 55kDa protein was obtained. Enzyme deglycosylation with Endo H showed a smaller size band in SDS-PGAE, with approximately 50kDa, suggesting that tgD has N-linked oligosaccharides and that it is not hyperglycosylated. The approximately 55kDa protein was recognized by several polyclonal antibodies, including polyclonal antibody anti-tgD and polyclonal antibodies of different animal species immunized with BoHV-5 and BoHV-1. This is the first report of BoHV-5 gD expression in yeast. It was shown that the recombinant truncated form of BoHV-5 gD has antigenic and immunogenic properties similar to the native BoHV-5 gD. Expression of tgD as a secreted protein allows simple and inexpensive purification methods that can be used for further studies to evaluate its immunogenicity in cattle.

The immune modulation of Bacillus cereus var. Toyoi in mice immunized with experimental inactivated Bovine Herpesvirus Type 5 vaccine

In recent years, there been significant progress toward develop more efficient vaccines. Different compounds with adjuvant capacity have been tested; however, no compound has emerged that suitable for universal use. Several efforts have been made to produce effective vaccines against Bovine herpesvirus 5 (BoHV-5), an important cattle pathogen. In this study we examine the use the probiotic Bacillus cereus var. Toyoi as a potential adjuvant to improve BoHV-5 vaccine efficacy. We observed in the supplemented animals a systemic enhanced IgG antibody response toward Th1, and increased IFN-γ, IL-12 and IL-10 cytokines mRNA levels. These results suggest that this probiotic could provide a promising means of improving vaccine efficacy, particularly those vaccines that rely on a cell-mediated immune response.

Use of ELISA based on NcSRS2 of Neospora caninum expressed in Pichia pastoris for diagnosing neosporosis in sheep and dogs.

Neosporosis is a disease caused by the protozoon Neospora caninum that leads to significant economic losses in many countries. In the present study, we report on use of the recombinant protein NcSRS2 of N. caninum expressed in Pichia pastoris in an indirect immunoenzymatic assay (ELISA) for diagnosing neosporosis infection in sheep and dogs. We observed that the ELISA test yielded specificity of 94.5% and sensitivity of 100% for sheep and specificity of 93.3% and sensitivity of 100% for dogs. We observed that the sensitivity was higher than shown by the indirect fluorescent antibody test, and this was confirmed by means of Western blot. The results from this study suggest that the recombinant protein expressed in P. pastoris is a suitable antigen for use in immunodiagnosis to detect N. caninum in two important species exposed to this parasitosis.

Validating the Efficiency of a Simplex PCR and Quantitative SYBR Green qPCR for the IdentificationÃÂ of Salmonella spp. DNA

This study aimed to perform a comparative validation of the efficiency of quantitative SYBR Green qPCR and simplex PCR identification of Salmonella spp. DNA. For this, the samples of DNA from the Salmonella typhimurium were diluted up to 10-5 in duplicate. The results showed that the same primers were effective for both simplex PCR and qPCR. It was possible to detect the bovine species up to a dilution of 10-1 using simplex PCR. For all dilutions, it was possible to obtain qPCR amplification with a minimum Ct value of 15.13 for the 10-1 dilution for Salmonella spp. Next, the SYBR Green qPCR amplicons were separated using agarose gel electrophoresis for confirmation of the amplified fragment size. The superiority of qPCR over multiplex PCR was validated in terms of sensitivity, even with the use of SYBR Green dye, suggesting the possible use for quality control in foodstuffs.

Efeito do jejum e da administração de insulina sobre os parâmetros metabólicos de ovelhas em confinamento

Os parâmetros bioquimicos sanguineos variam amplamente, sendo os niveis de glicose, ureia e corpos cetonicos utilizados para o diagnostico de condicoes patologicas em ovinos, e o aumento do conhecimento das respostas metabolicas, importante para a otimizacao de programas de nutricao. O objetivo do presente estudo foi avaliar a resposta metabolica de ovelhas confinadas submetidas ao jejum, tratamento com insulina e ao jejum associado ao tratamento com insulina. Para a realizacao deste experimento foram utilizadas 8 ovelhas mantidas em confinamento. As ovelhas foram divididas em quatro grupos, sendo estes: grupo controle, que nao recebeu tratamento; grupo insulina, que recebeu a administracao de insulina durante os 3 dias do experimento; grupo jejum, que foi submetido a jejum por 3 dias e o grupo jejum e insulina, que foi submetido ao jejum e administracao de insulina. Foram realizadas coletas de sangue durante os 3 dias do experimento para analise dos niveis de glicose e ureia, e insulina no ultimo dia. No final do terceiro dia do experimento, tambem foi realizada analise dos niveis de proteina total, albumina, beta-hidroxibutirato, triacilglicerideos, colesterol, magnesio, fosforo e fator de crescimento semelhante a insulina tipo I. Nao foram observadas alteracoes clinicas durante o periodo experimental. Nao houve variacoes relevantes nos marcadores analisados entre os grupos, o que demonstra uma grande capacidade de adaptacao dos animais as variacoes nutricionais de curto prazo, as quais os animais foram submetidos neste experimento. Portanto, ovelhas confinadas adaptadas a um padrao alimentar, sao capazes de resistir a variacoes de curto periodo no padrao alimentar, nao apresentando mudancas consideraveis nos niveis dos marcadores metabolicos estudados.