Development of a baculovirus expression system for soluble porcine tumor necrosis factor receptor type I and soluble porcine tumor necrosis factor receptor type I-IgG fusion protein

Tumor necrosis factor-alpha (TNF-alpha) is a key mediator of inflammatory responses and gram-negative bacterial sepsis, but the role that it plays during Salmonella enterica species bacterial infections in swine has not yet been elucidated. To facilitate studies on the role of TNF-alpha on the pathology associated with Salmonella infections in pigs, recombinant soluble porcine TNF receptor type I (rspTNF-RI) and soluble TNF receptor type I fused to the Fc region of porcine IgG1 (rspTNF-RI-IgG) were expressed in insect cells using a baculovirus expression system. The proteins were secreted into the cell culture media and purified by anti-soluble porcine TNF-RI antibody and protein G affinity chromatography, respectively. The yield of protein using this method was approximately 1.5mg rspTNF-RI and 4mg rspTNF-RI-IgG/L of cell culture medium. In in vitro assays, rspTNF-RI-IgG was approximately 10-fold (0.97 vs. 10.00pmol/ml) more effective than rspTNF-RI at completely inhibiting the cytotoxic activity of 500U of recombinant porcine TNF-alpha on 3 x 10(4) WEHI 164 murine fibrosarcoma, clone 13, cells. Compared to previously described methods, this method yields significantly more biologically active rspTNF-RI.     

Temporal Mycobacterium paratuberculosis infection in T-cell receptor (TCR)-alpha and TCR-delta-deficient mice

Mycobacterium paratuberculosis is the causative agent of paratuberculosis (Johne's disease), a chronic inflammation of the terminal portion of the ileum in ruminants. The predominance of cell-mediated immunity in early stages of the disease suggests that T lymphocytes are essential to protect the host from infection with M. paratuberculosis. In this study, we investigated the role of alphabeta and gammadelta T cells in resistance to M. paratuberculosis infection using a T-cell receptor (TCR) knockout mouse model. Weanling TCR-alpha-deficient, TCR-delta-deficient, and C57BL/6 control mice (5-6 weeks of age) were acclimated for 2 weeks and then inoculated intraperitoneally with 10(8)CFU/ml of M. paratuberculosis (either strain 19698 or strain Ben). Groups of mice within each treatment group were euthanized at 1, 3 and 6 months post-inoculation. Sections of spleen, liver, ileum and mesenteric lymph node were prepared for bacterial culture and histologic examination. At all time points of infection and regardless of bacterial strain, TCR-alpha-deficient mice had higher levels of M. paratuberculosis colonization in their tissues compared to TCR-delta-deficient mice or C57BL/6 control mice. Lesions were located predominately in the liver and the ileum, depending upon period of infection, and lesion scores were higher for TCR-alpha-deficient mice compared to the other treatment groups. These results suggest that alphabeta T cells play a major role in resistance to infection with M. paratuberculosis and that gammadelta T cells may play a lesser role and potentially confound protective immune responses.     

Expression and characterization of a recombinant soluble form of bovine tumor necrosis factor receptor type I

A recombinant soluble bovine tumor necrosis factor receptor type I (sboTNF-RI) was expressed in the methylotrophic yeast Pichia pastoris and evaluated for its ability to inhibit bovine tumor necrosis factor alpha (TNF-alpha) cytotoxicity. A cDNA encoding the extracellular domain of bovine TNF-RI was placed under the control of the powerful and tightly regulated alcohol oxidase1 (AOX1) gene promoter of the pPICZa A vector and the resulting construct integrated into the 5' region of the alcohol oxidase genes of GS115 and KM71 strains of Pichia. Soluble bovine TNF-RI was secreted into the medium following induction of the AOX1 gene promoter with methanol, and purified to greater than 95% purity by ion-exchange chromatography. In in vitro assays, the purified recombinant sboTNF-RI will block the cytolytic activity of bovine TNF-alpha on WEHI 164 cells clone 13 by 50% when used at a concentration of 170 microg/ml, and by nearly 90% when used at a concentration of 310 microg/ml. Results of this study suggest that recombinant sboTNF-RI may have therapeutic value as a TNF inhibitor in cattle with coliform mastitis.     

HspX is present within Mycobacterium paratuberculosis-infected macrophages and is recognized by sera from some infected cattle

A portion of the gene encoding HspX has been previously identified as a sequence specific to Mycobacterium avium subspecies paratuberculosis (hereafter referred to as M. paratuberculosis) based on DNA hybridization experiments. In this study, rabbit antisera were raised against a recombinant protein of HspX fused to the Escherichia coli maltose binding protein (MBP/HspX). Immunoblots of lysates of M. paratuberculosis-infected macrophages probed with the rabbit antisera showed that HspX was present within infected macrophages of bovine and murine origin. This observation was confirmed by immunofluorescence microscopy of infected macrophages. Lysates of E. coli expressing HspX without the MBP fusion partner were loaded onto preparative SDS-PAGE gels and used to determine whether infected cattle generated a humoral immune response to the antigen. Sera from four of 24 paratuberculous cows (17%) detected HspX. No reactivity was present in sera from control cows. While HspX may be immunogenic during infection in some cows, the protein is not secreted and it does not stimulate cell-mediated immunity. Collectively, these data give a preliminary characterization of the first described M. paratuberculosis protein identified within infected macrophages.     

美国、加拿大生物安全三级动物实验室考察报告

美国、加拿大对ABSL-3的设计均为三层。下方的尸体、污物处理层和上方的设备层及中间的动物实验室层。设计比较科学，人性化，其高度均达6m以上。配备有索道和电葫芦，以供动物移动使用；电动解剖台可按电钮升降调节解剖台的高度，动物尸体处理由Renderer cooker尸体处理器经高压、粉碎、干燥成肉陷状颗粒，粪便及污水收集在三个5000L容量的高压灭菌处理器中高压灭菌处理，设施内的灭菌采用环保的过氧化氢消毒设备。     

Evaluation of a commercial ELISA for diagnosis of paratuberculosis in cattle

OBJECTIVE: To evaluate sensitivity and specificity of a new ELISA for antibodies against Mycobacterium avium subsp paratuberculosis. DESIGN: Cross-sectional observational survey. SAMPLE POPULATION: Serum samples from 590 cattle that were infected with M avium subsp paratuberculosis and 723 cattle that were not infected. PROCEDURE: Serum samples were tested by use of an ELISA for antibodies against M avium subsp paratuberculosis. RESULTS: Sensitivity of the test varied from 15.4 to 88.1%, depending on the clinical stage and bacterial shedding status of the cattle. CONCLUSIONS AND CLINICAL RELEVANCE: Results obtained with use of the new ELISA agreed favorably with those of a previous ELISA. Practitioners must be aware of variability in the sensitivity of the test, which depends on the clinical and shedding status of the cattle, because this may affect interpretation of test results.     

Transcutaneous ultrasound over the right flank to diagnose mid- to late-pregnancy in the dairy cow

Objective   To investigate the practical application and image quality of transcutaneous ultrasonography across the right flank for the diagnosis of mid- to late-gestation in cattle.
Procedure   Dairy cows of known pregnancy status were used to establish criteria for the identification of mid- to late-stage pregnancy by transcutaneous ultrasonography. Factors involved in the practical application of transcutaneous ultrasound for routine use in pregnancy diagnosis in dairy cattle were also examined.
Results   Transcutaneous ultrasound, using a 3.5-MHz sector transducer applied over the caudodorsal right flank fold, allowed rapid and detailed visualisation of bovine pregnancy (i.e. fetus, fetal fluids, placentomes and/or fetal membranes) and could be used to establish criteria for the diagnosis of pregnancy. The commonly available cattle holding facilities of the herringbone dairy, rotary herringbone dairy, AI race or crush, and walk-through dairy all allow safe access to the right flank of a dairy or beef cow. However, transcutaneous scanning over the right flank is difficult in modern rotary turnstile dairies.
Conclusion   A 3.5-MHz sector transducer applied over the caudodorsal right flank fold allows detailed visualisation of the bovine fetus in most types of cattle-holding facilities. Further investigation of the sensitivity and specificity of this technique over the entire gestation period and its possible use in ageing of the bovine fetus is merited.