Characterization of culture supernatant proteins from Brucella abortus and its protection effects against murine brucellosis

In this study, we characterized the secreted proteins of Brucella abortus into the enriched media under the bacterial laboratory growth condition and investigated the pathogenic importance of culture supernatant (CS) proteins to B. abortus infection. CS proteins from stationary phase were concentrated and analyzed using 2D electrophoresis. In MALDI TOF/TOF analysis, more than 27 proteins including CuZn SOD, Dps, Tat, OMPs, Adh, LivF, Tuf, SucC, GroEL and DnaK were identified. Cytotoxic effects of CS proteins were found to increase in a dose-dependent manner in RAW 264.7 cells. Upon B. abortus challenge into phagocytes, however, CS proteins pre-treated cells exhibited lower bacterial uptake and intracellular replication compared to untreated cells. Immunization with CS proteins induced a strong humoral and cell mediated immune responses and exhibited significant higher degree of protection against virulence of B. abortus infection compared to mice immunized with Brucella broth protein (BBP). Taken together, these results indicate that B. abortus secreted a number of soluble immunogenic proteins under laboratory culture condition, which can promote antibody production resulted in enhancing host defense against to subsequently bacterial infection. Moreover, further analysis of CS proteins may help to understand the pathogenic mechanism of B. abortus infection and host–pathogen interaction.     

Effect of topical anesthesia on evaluation of corneal sensitivity and intraocular pressure in rats and dogs

Objective To determine the effect of 0.5% proparacaine in tonometry by evaluating corneal touch threshold (CTT) and intraocular pressure (IOP). Animal studied Nine rats (18 eyes, Sprague–Dawley) and 10 dogs (20 eyes, Beagle) Procedures The IOP and CTT were measured in each eye before and after topical anesthesia with 0.5% proparacaine. The IOP was evaluated using Tonopen for dogs and Tonolab for rats. The corneal sensitivity was evaluated by CTT through a Cochet–Bonnet aesthesiometer. Results The mean IOP was not significantly changed in rats or dogs before and after topical anesthesia. However, after application of proparacaine, CTT was significantly increased in both animal groups compared with that before application of proparacaine. Conclusion From this study, topical anesthesia was found to significantly lower the corneal sensitivity but have little effect on IOP measurements. In ophthalmologic examination, topical anesthesia can be used to reduce corneal sensation without an effect on IOP.     

对饲养毛皮动物饲料的卫生检验与监督

当前饲养的毛皮动物种类较多 ,食性较杂。水貂、狐、貉、獾、鼬、水獭等以畜禽副产品、鱼类、肉类为主要饲料。麝鼠、海狸鼠、毛丝鼠、獭兔等以植物性饲料为主。毛皮动物的阿留申病、结核病、巴氏杆菌病、旋毛虫病等都是由肉类饲料传染的。因此 ,饲养毛皮动物的饲料 ,必须认真搞好检验与监督 ,防止各种疫病的发生。下面 ,就毛皮动物常用的动物、植物性饲料 ,如何搞好卫生检验与监督分述如下 :１畜禽副产品的检验１．１猪副产品。猪的传染病种类较多 ,是水貂阿留申病毒的携带者。用生的猪副产品饲喂动物 ,易引起阿留申氏病的地方性流行。因此…     

Dog erythrocyte antigens 1.1, 1.2, 3, 4, 7, and Dal blood typing and cross‐matching by gel column technique

Background: Testing for canine blood types other than dog erythrocyte antigen 1.1 (DEA 1.1) is controversial and complicated by reagent availability and methodology. Objectives: The objectives of this study were to use available gel column technology to develop an extended blood‐typing method using polyclonal reagents for DEA 1.1, 1.2, 3, 4, 7, and Dal and to assess the use of gel columns for cross‐matching. Methods: Dogs (43–75) were typed for DEA 1.1, 1.2, 3, 4, 7, and Dal. Methods included tube agglutination (Tube) using polyclonal reagents, a commercially available DEA 1.1 gel column test kit (Standard‐Gel) using monoclonal reagent, and multiple gel columns (Extended‐Gel) using polyclonal reagents. Blood from 10 recipient and 15 donor dogs was typed as described above and cross‐matched using the gel column technique. Results: Of 43 dogs typed for DEA 1.1, 23, 25, and 20 dogs were positive using Standard‐Gel, Extended‐Gel, and Tube, respectively. Typing for DEA 1.2 was not achievable with Extended‐Gel. For 75 dogs typed for DEA 3, 4, and 7, concordance of Extended‐Gel with Tube was 94.7%, 100%, and 84%, respectively. Dal, determined only by Extended‐Gel, was positive for all dogs. Post‐transfusion major cross‐matches were incompatible in 10 of 14 pairings, but none were associated with demonstrable blood type incompatibilities. Conclusions: Gel column methodology can be adapted for use with polyclonal reagents for detecting DEA 1.1, 3, 4, 7, and Dal. Agglutination reactions are similar between Extended‐Gel and Tube, but are more easily interpreted with Extended‐Gel. When using gel columns for cross‐matching, incompatible blood cross‐matches can be detected following sensitization by transfusion, although in this study incompatibilities associated with any tested DEA or Dal antigens were not found.     

Protective potential of an attenuated Pasteurella multocida,which expresses only the N-terminal truncated fragment of P. multocida toxin

Pasteurella multocida serogroup D causes progressive atrophic rhinitis in pigs and produces a potent, intracellular, mitogenic toxin known as P. multocida toxin (PMT), which is encoded by the toxA gene. Highly toxic to cells, PMT is a poor antigen and becomes more immunogenic after its native structure has been destroyed. Previously, we found that the N-terminal fragment of PMT (N-PMT) can induce a strong immune response that is protective against wild-type challenge. Here, an attenuated P. multocida mutant expressing only N-PMT was developed and its protective effect was evaluated. The mutant provides protective immune responses against bacterial and toxin challenges, and so is a good live vaccine candidate.     

A dominant antigenic epitope on SARS-CoV spike protein identified by an avian single-chain variable fragment (scFv)-expressing phage

Severe acute respiratory syndrome (SARS) is a newly emergent human disease, which requires rapid diagnosis and effective therapy. Among antibody sources, immunoglobulin Y (IgY) is the major antibody found in chicken eggs and can be used as an alternative to mammalian antibodies normally used in research and immunotherapy. In this study, phage-expressing chicken monoclonal scFv antibody was chosen and characterized with phage display antibody technology. Truncated fragments of SARS-CoV spike protein were cloned in pET-21 vector and expressed in BL-21 Escherichia coli (E. coli) cells. After purification, the purity of these recombinant spike proteins was examined on SDS-PAGE and their identity verified with Western blot analysis using anti-his antibodies and sera from convalescent stage SARS-CoV-infected patients. Using these bacteria-derived proteins to immunize chickens, it was found that polyclonal IgY antibodies in the egg yolk and sera were highly reactive to the immunogens, as shown by Western blot and immunocytochemical staining analysis. A phage displaying scFv library was also established from spleen B cells of immunized chicken with 5 x 10(7) clones. After four panning cycles, the eluted phage titer showed a 10-fold increase. In sequence analysis with chicken germline gene, five phage clones reacted, with large dissimilarities of between 31 and 62%, in the complementarity-determining regions, one dominant phage 4S1 had strong binding to fragment Se-e, located between amino acid residues 456-650 of the spike protein and this particular phage had significantly strong binding to SARS-CoV-infected Vero E6 cells. Based on the results, we conclude that generating specific scFv-expressing phage binders with the phage display system can be successfully achieved and that this knowledge can be applied in clinical or academic research.