No abstract

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.

No 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.

No abstract

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.

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 )10 6 viable spores gr (1 , S. boulardii at a concentration of 1 )10 6 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 B0.001) than the controls. Both probiotics enhanced the humoral immune response of lambs to the vaccines.

Rathke’s pouch tissue, from rat embryos of 11–15 days’ gestation, was microsurgically removed and transplanted into the hypothalamus of hypophysectomized adult females. The hosts were sacrificed 4 weeks later; brain and target organs were preserved for histological examination. Plasma samples were taken for the radioimmunoassay (RIA) of follicle stimulating hormone (FSH) and luteinizing hormone (LH). The implanted tissue invariably developed along certain lines. Undifferentiated primitive cells were found associated with nervous tissue, dense connective tissue, cartilage and glandular cells. In every age group, some implants became invasive, forming massive growths in the brain. These tumorous properties were principally associated (p < 0.05) with tissue from 12-day embryos. Pituitary primordia from all ages demonstrated the ability to develop into functional adenohypophyseal tissue. Target organ stimulation indicated a secretion of corti-cotropin, thyrotropin and somatotropin. FSH and/or LH were detected by RIA in the plasma of 61 % of the test animals. We suggest that this system offers unique opportunities for future investigations into the mechanisms that determine whether embryonic epithelial tissue will remain under normal growth control or will become tumorous or even cancerous.