Characterization of the immune response of domestic fowl following immunization with proteins extracted from Dermanyssus gallinae

Dermanyssus gallinae is the most significant ectoparasite of European poultry egg laying production systems due to high costs of control and associated production losses as well as adverse effects on bird welfare. In this study, soluble proteins were extracted from unfed D. gallinae (DGE) using a urea-based detergent and ultra-filtration, passed through a 0.22 μm filter and blended aseptically with adjuvant. One group of laying hens was immunized with DGE and adjuvant (Montanide ISA 50 V) whilst another group (Control) received physiological saline and adjuvant. All birds were immunized on two occasions, 21 days apart. Antibody response to immunization was determined by ELISA and western blotting using immunoglobulins (Igs) extracted from egg yolk. DGE immunization of hens resulted in a significant (P < 0.05) IgY response compared to controls, although there was no significant difference in IgM response between treatments. A number of proteins were identified by western blotting using IgY antibodies from DGE immunized birds, most prominently at 40 and 230 kDa. Analysis of proteins from approximately corresponding bands on SDS-PAGE confirmed the identity of tropomyosin, whilst other proteins showed high sequence homology with myosin and actin from other arachnid and insect species. Immunization of hens with DGE resulted in a 50.6% increase in mite mortality (P < 0.001) 17 h after feeding when tested by an in vitro mite feeding model. Data in this study demonstrate that somatic antigens from D. gallinae can be used to stimulate a protective immune response in laying hens. Further work is needed to identify other proteins of interest that could confer higher protection against D. gallinae, as well as optimization of the vaccination and in vitro testing protocol.     

Searching for proteins of Mycobacterium avium subspecies paratuberculosis with diagnostic potential by comparative qualitative proteomic analysis of mycobacterial tuberculins

Accurate immunodiagnosis of bovine paratuberculosis is among others hampered by the lack of specific antigens. One of the most frequently used antigen preparations is purified protein derivative (PPD), also known as tuberculin. This crude extract has limitations when used in diagnostic assays due to the presence of cross-reactive antigens. The aim of the current study was to systematically analyze the qualitative protein composition of PPD of the major mycobacterial pathogens.One-dimensional gel electrophoresis followed by tandem mass spectrometry analysis of PPD from Mycobacterium avium subspecies paratuberculosis (MAP), Mycobacterium avium subspecies avium (MAA) and Mycobacterium bovis (MB) identified 156, 95 and 132 proteins, respectively. Comparative sequence analysis led to the selection of a MAP-specific protein (MAP1718c), and finally heterologous expression in Escherichia coli of this and other diagnostic candidate proteins (MAP3515c and MAP1138c (LprG)) enabled evaluation of their immunogenicity. Lymphocyte proliferation responses did not indicate substantial diagnostic potential of the antigens tested. In contrast serum antibody levels for MAP1138c in paratuberculosis infected cows (N = 20) were significantly higher (p < 0.01) than in control animals (N = 20), despite the conserved nature of this protein.In conclusion, this study showed that a combination of proteomics and genomics, starting from complex protein mixtures, present in tuberculins, can reveal novel proteins aiding the development of immunodiagnostics for mycobacterial diseases.     

西瓜、黄瓜、甜瓜等瓜类枯萎病研究进展

对瓜类枯萎病及其发病规律、抗病机理、分子标记在抗病育种中的应用和抗源新材料创造等方面的研究进行了综述和评价,介绍了国内外瓜类抗枯萎病育种取得的最新进展,并对瓜类抗枯萎病育种进行了讨论和展望。     

Effect of isoflavone genistein on activation and proliferation of mouse T cells in vitro

AIM: To study the effect of genistein on activation and proliferation of T cells, and explore the molecular mechanism of genistein. METHODS: Fluorescence conjugated monoclonal antibodies and flow cytometry were used to detect the express of CD69 and CD25 by activated T cells in vitro in response to Concanavalin (ConA )and Phorbol 12, 13-dibutyrate(PDB) or T cell proliferation stained by CFSE in response to PDB / Ionomycin or ConA. RESULTS: Genistein inhibited the expression of CD69 and CD25 in activated T cells in response to Con A in a concentration-dependent manner and in response to PDB in a high concentration. Genistein inhibited proliferation of T cells in both groups in a concentration-dependent manner. CONCLUSION: Genistein inhibited activation and proliferation of T cells in vitro in response to polyclonal stimulus, and it may hold potential as a new immunosuppressant.     

Diagnosis of brucellosis by serology

Serological diagnosis of brucellosis began more than 100 years ago with a simple agglutination test. It was realized that this type of test was susceptible to false positive reactions resulting from, for instance, exposure to cross reacting microorganisms. It was also realized that this test format was inexpensive, simple and could be rapid, although results were subjectively scored. Therefore, a number of modifications were developed along with other types of tests. This served two purposes: one was to establish a rapid screening test with high sensitivity and perhaps less specificity and a confirmatory test, usually more complicated but also more specific, to be used on sera that reacted positively in screening tests. This led to another problem: if a panel of tests were performed and they did not all agree, which interpretation was correct? This problem was further compounded by the extensive use of a vaccine which gave rise to an antibody response similar to that resulting from field infection. This led to the development of an assay that could distinguish vaccinal antibody, starting with precipitin tests. These tests did not perform well, giving rise to the development of primary binding assays. These assays, including the competitive enzyme immunoassay and the fluorescence polarization assay are at the apex of current development, providing high sensitivity and specificity as well as speed and mobility in the case of the fluorescence polarization assay.     

鸡白细胞表面抗原的分型

本实验选用京白鸡和选育中的红鸡作材料，采用同种免疫方法，以提纯的外周血白细胞作免疫原制备抗血清４０份，用微量淋巴细胞毒的试验方法作４０×４０的交叉棋盘反应，并用统计学的方法，借助电子计算机进行集群分析。结果从４０份抗血清中获得使用单价血清７种。这７种使用单价血清是相对单一的高效价狭抗体血清，可作为鉴别鸡白细胞抗原的分型试剂。本文对与这些使用单价血清相对应的抗原，根据有关鸡主要组织相容性抗原的国际统一命名法，作了暂时的命名。其标准化命名有待进一步研究确定。     

鸡毒支原体抗原在大肠杆菌中的表达

利用表达载体λgt11构建鸡毒支原体（MG）S6株基因文库。根据载体中lacZ基因插入失活的原理,重组子中大约69％为重组噬菌体。应用MG纯化抗体筛选文库,得到179个阳性克隆,不足重组噬菌体的1％。随机选择一个阳性克隆,收集其蛋白进行Western免疫印迹,检测到－38kD蛋白,表明在大肠杆菌中能全盛具有免疫学活性的MG蛋白,为MG的特异性血清学诊断提供良好工具。     

鸡毒支原体抗体识别抗原的基因克隆和序列分析

抗体筛选阳性克隆,用EcoRI酶切回收3．6kb外源片段,测序。序列用大肠杆菌密码进行读框分析,至少有6个开放放阅读框,而在MG可能分为3个开放阅读框,同源性分析,发现序列的某些碱基主要与肺炎支原体、生殖支原体、山羊支原体和莱氏无胆甾原体的某些地方同源性高、没有同其他细胞或病毒有同源性的地方,说明克隆的基因完全是一个新的基因片段。     

Comparison of three feline leukaemia virus (FeLV) point-of-care antigen test kits using blood and saliva

Feline leukaemia virus (FeLV) can be a challenging infection to diagnose due to a complex feline host-pathogen relationship and occasionally unreliable test results. This study compared the accuracy of three point-of-care (PoC) FeLV p27 antigen test kits commonly used in Australia and available commercially worldwide (SNAP FIV/FeLV Combo, Witness FeLV/FIV and Anigen Rapid FIV/FeLV), using detection of FeLV provirus by an in-house real-time polymerase chain reaction (qPCR) assay as the diagnostic gold standard. Blood (n = 563) and saliva (n = 419) specimens were collected from a population of cats determined to include 491 FeLV-uninfected and 72 FeLV-infected individuals (45 progressive infections [p27 and qPCR positive], 27 regressive infections [p27 negative, qPCR positive]). Sensitivity and specificity using whole blood was 63% and 94% for SNAP Combo, 57% and 98% for Witness, and 57% and 98% for Anigen Rapid, respectively. SNAP Combo had a significantly lower specificity using blood compared to the other two kits (P = 0.004 compared to Witness, P = 0.007 compared to Anigen Rapid). False-positive test results occurred with all three kits using blood, and although using any two kits in parallel increased specificity, no combination of kits completely eliminated the occurrence of false-positive results. We therefore recommend FeLV proviral PCR testing for any cat that tests positive with a PoC FeLV antigen kit, as well as for any cat that has been potentially exposed to FeLV but tests negative with a FeLV antigen kit, before final assignment of FeLV status can be made with confidence. For saliva testing, sensitivity and specificity was 54% and 100%, respectively, for all three test kits. The reduced sensitivity of saliva testing compared to blood testing, although not statistically significant, suggests saliva testing with the current generation of PoC FeLV antigen kits is unsuitable for screening large populations of cats, such as in shelters.     

A promising multiple-epitope recombinant vaccine against classical swine fever virus

Classical swine fever (CSF) is a highly contagious and often fatal disease of swine. It is caused by classical swine fever virus (CSFV), one of the members of the genus Pestivirus of the Flaviviridae family. The development of a safe and effective vaccine against the CSF is critical to pandemic control, this article shows a tandem-repeat multiple-epitope recombinant vaccine can protect pigs from CSFV challenge. That was composed as following: two copies each of glycoprotein E2 residues 693–707, 241–276 and 770–781, and two copies amino acid residues 1446–1460 of the non-structural protein NS2-3. In the challenge test, all of the swine vaccinated with Chinese vaccine strain (C-strain) were fully protected from a challenge with CSFV. However, after three successive vaccinations with the multiple-epitope recombinant vaccine, three out of five pigs were protected from challenge with CSFV (in terms of both clinical signs and viremia). These results demonstrate that multiple-epitope recombinant vaccine which carrying the major CSFV epitopes can induce a high level of epitope-specific antibodies and exhibit a protective capability that parallels induced by C-strain to a certain extent.