Using indirect ELISA to assess different antigens for the serodiagnosis of Fasciola gigantica infection in cattle, sheep and donkeys

An indirect enzyme-linked immunosorbent assay (iELISA) was evaluated for its diagnostic capability in detecting antibodies against Fasciola gigantica infection in cattle, sheep and donkeys sera using crude worm, excretory–secretory and glutathione S-transferase antigens prepared from adult liver fluke. Presence of F. gigantica worms at post-mortem examination of cattle, sheep and donkey’s livers was taken as a gold standard for the evaluation of the assay. The diagnostic sensitivity, specificity and accuracy percentages of iELISA were determined for each antigen. Excretory–secretory antigen gave the best results for the serodiagnosis of F. gigantica infection in cattle, sheep and donkeys using iELISA with diagnostic sensitivity percentages of 93.3%, 94.9% and 93.3%, respectively, while the specificity percentages were 96.7%, 97.2% and 96.3%, respectively, whereas the accuracy percentages were 95%, 96% and 95.7%, respectively. The diagnostic sensitivity percentages of iELISA using crude worm antigen were 96.7%, 100% and 93.3%, respectively, while the specificity percentages were 80%, 83.3% and 85.2%, respectively, whereas the accuracy percentages were 88.3%, 86.7% and 87%, respectively. The diagnostic sensitivity percentages of iELISA using glutathione S-transferase antigen were 66.7%, 71.8% and 60%, respectively, while the specificity percentages were 70%, 77.8% and 77.8%, respectively, whereas the accuracy percentages were 68.3%, 74.7% and 73.9%, respectively. Conclusively, excretory–secretory antigen dependent iELISA can be used as a reliable serodiagnostic test for F. gigantica infection in cattle, sheep and donkeys.     

The influence of age and Rhodococcus equi infection on CD1 expression by equine antigen presenting cells

There is a distinct age-associated susceptibility of horses to Rhodococcus equi infection. Initial infection is thought to occur in the neonatal and perinatal period, and only foals less than 6 months of age are typically affected. R. equi is closely related and structurally similar to Mycobacterium tuberculosis, and causes similar pathologic lesions. Protective immune responses to M. tuberculosis involve classical major histocompatibility complex (MHC)-restricted T cells that recognize peptide antigen, as well as MHC-independent T cells that recognize mycobacterial lipid antigen presented by CD1 molecules. Given the structural similarity between these two pathogens and our previous observations regarding R. equi-specific, MHC-unrestricted cytotoxic T lymphocytes (CTL), we developed 3 related hypotheses: (1) CD1 molecules are expressed on equine antigen presenting cells (APC), (2) CD1 expression on APC is less in foals compared to adults and (3) infection with live virulent R. equi induces up-regulation of CD1 on both adult and perinatal APC. CD1 expression was examined by flow cytometric analysis using a panel of monoclonal CD1 antibodies with different species and isoform specificities.

Results

Three CD1 antibodies specific for CD1b showed consistent cross reactivity with both foal and adult monocyte-derived macrophages (MDM). CD1b and MHC class II expression were significantly higher on adult MDM compared with foals. R. equi infected MDM showed significantly lower expression of CD1b, suggesting that infection with this bacterium induces down-regulation of CD1b on the cell surface. Histograms from dual antibody staining of peripheral blood mononuclear cells also revealed that 45–71% of the monocyte population stained positive for CD1b, and that the majority of these also co-expressed MHC II molecules, indicating that they were APC. The anti-CD1 antibodies showed no binding or minimal binding to bronchoalveolar lavage (BAL)-derived macrophages.

Conclusion

The CD1b isoform is evolutionarily conserved, and is present on equine MDM, as well as on circulating blood monocytes. The unique susceptibility of foals to R. equi infection may be due in part to lower expression of CD1 and MHC class II, as observed in this study. The data also suggests that infection with R. equi induces down-regulation of CD1b on equine MDM. This may represent a novel mechanism by R. equi to avoid detection and killing of infected cells by the immune system, similar to that observed when human APC are infected with M. tuberculosis.     

Immunohistochemical detection and localization of new type gosling viral enteritis virus in paraformaldehyde-fixed paraffin-embedded tissue

To determine the distribution and localization of new type gosling viral enteritis virus (NGVEV) in paraformaldehyde-fixed paraffin-embedded tissues of experimentally infected goslings, for the first time, an immunohistochemical (IHC) staining method was reported. Anti-NGVEV polyclonal serum was obtained from the rabbits immunized with purified NGVEV antigen, which was extracted by caprylic-ammonium sulphate method and purified through High-Q columns anion exchange chromatography. Three-day-old NGVEV-free goslings were orally inoculated with NGVEV-CN strain suspension as infection group and phosphate buffered saline solution (PBS) as control group, respectively. The tissues were collected at sequential time points between 0.5 and 720 h post inoculation (PI), and prepared for IHC staining and ultra-structural observation. The positive immunoreactivity could be readily detected in the lymphoid and gastrointestinal organs of infected goslings as early as 48 h PI, in the liver, kidney, pancreas and myocardium from 72 h, and in the cerebrum and cerebellum from 96 h, while it was hardly detected in the respiratory organs at any time. The positive staining reaction could be detected in NGVEV-infected goslings until 600 h PI, and no positive staining cell could be observed in the controls. The highest levels of viral antigen were found in the bursa of Fabricius (BF), thymus, proventriculus, gizzard and intestine tract, moreover, the liver, kidney, spleen, myocardium and pancreas were intensively and widely stained. The target cells had a ubiquitous distribution, especially included the epithelial cells, endothelial cells, superficial and crypt mucosal cells, glandular cells, fibrocytes, macrophages and lymphocytes, which served as the principal sites for antigen localization. The ultra-structural observation by transmission electron microscope (TEM) further indicated that NGVEV particles could be widely detected in the lymphoid and digestive organs of infected goslings from 72 h PI onwards. This work may be useful not only for offering a possibility of routine diagnosis of NGVE, but also for better understanding of the pathogenesis of the disease.     

东方田鼠血清筛选噬菌体展示抗原对小鼠日本血吸虫病免疫效果观察

本研究利用前期研究中东方田鼠血清筛选日本血吸虫成虫噬茵体展示cDNA文库获得的10个阳性克隆中的8个噬茵体克隆单独或联合免疫BALB/c小鼠,观察对日本血吸虫病的免疫预防效果,以筛选其中具有发展成疫苗的候选抗原编码基因.研究发现与空白对照组相比,展示血吸虫锌指蛋白的1号噬茵体克隆免疫组在两次实验中分别获得了32.10%和31.25%的减虫率、61.14%和47.31%的肝脏减卵率,虫体合抱率分别由92.59%、57.39%下降到69.09%、41.03%;其它噬茵体克隆单独免疫组或联合免疫组在第一次实验中均获得了8.02%～32.72%的减虫率和40.19%～69.53%的减卵率,但在第二次实验中未能得到验证.研究结果提示,日本血吸虫环状锌指蛋白(1号克隆)编码基因在疫苗方面具有重要研究价值.     

鸡大肠杆菌改良微量凝集试验抗原的制备及试验条件优化

本研究对微量凝集试验进行了改良,改进了抗原的制备方法,优选了检测鸡血清中大肠杆菌抗体的微量凝集试验的条件,经方阵试验,抗原最适浓度为70亿/ml,反应最适温度为37℃,最适反应时间为4～5h.结果表明改良后的微量凝集试验具有良好的敏感性和特异性,阳性结果和凝集图案清晰,易于观察.本试验具有敏感、特异、快速、简便、定性又定量的特点,便于推广应用.     

鸡新城疫病毒抗原超滤技术研究

应用分子筛过滤技术超滤浓缩鸡新城疫病毒,并就ＮＤＶ抗原量对鸡免疫应答的和了动态研究,试验结果显示,将ＮＤＶ对外开放尿囊毒浓缩４倍体积,ＨＡ效价提高了２个滴度,病毒回收率达１００％;病毒活性亦未发生改变。免疫１１天后,ＮＤ浓缩苗和ＮＤ－ＩＢＤ二联苗组鸡的ＮＤ－ＨＩ抗体水平远远高于免疫对照组,差异极显著。结果表明,所建超滤技术适用于ＮＤＶ怕的浓缩应用;适量增另ＮＤＶ怕量能够加快鸡的体液免疫应答,增强鸡     

鸡大肝和大脾病抗原在免疫器官的定位和分布

用免疫金－银法（ＩＧＳＳ）和免疫胶体细胞化学方法,对人工感染的大肝和大脾（ＢＬＳ）鸡鸡免疫器官作ＢＬＳ抗原（ＢＬＳＡｇ）定位,分布及其动态研究。结果感染后第１,７,１９周,在脾脏,盲肠扁桃体,胸腺,法氏囊,红骨髓和哈德尔氏腺组织中均检查到ＢＬＳＡｇ,６主要分布在淋巴细胞,网状细胞,网状上皮细胞,巨噬细胞,单核细胞、浆细胞的胞浆、内质网,线粒体和一些空泡内。ＢＬＳＡｇ阳性淋巴细胞早期主要分布在胸腺和     

鸡大肠杆菌(O50)菌毛抗原气雾免疫和菌毛油乳苗皮下免疫效果比较研究

比较了鸡大肠杆菌（Ｏ５０）菌毛抗原液气雾免疫与菌毛油乳苗皮下免疫的效果。油乳剂疫苗皮下免疫与菌毛液气雾免疫攻毒后的发病率分别是１３．３３％和２０％,病变差异不显著（Ｐ〉０．０５）,而阳性对照组攻麦发病率为８０％,病变差异显著。结果,鸡大肠杆菌菌毛液气雾免疫与菌毛油乳剂疫苗皮下注射免疫效果接近。     

丙肝病毒融合抗原基因导入烟草叶绿体及转化株同质化的研究

植物生物反应器的研究已成为当前基因工程领域的一个新生长点。 本实验用PCR方法, 从丙型肝炎病毒cDNA文库中克隆了非结构NS3区基因片段及核心抗原C区基因片段, 并在两段基因间加入连接肽SPGS的密码子序列, 构建成融合抗原基因NS3-C。 将该融合基因转入烟草叶绿体转化及表达载体中, 通过基因枪转化法得到转基因植株。 对N     

马立克氏病肿瘤细胞表面抗原的提取与鉴定

用MDVGA株感染1日龄SPF雏鸡,饲养至发病后获得MD肿瘤细胞,采用木瓜蛋白酶消化法从MD肿瘤细胞上提取马立克氏病肿瘤相关的表面抗原,经间接荧光抗体染色和间接ELISA鉴定,证明获得的粗提物中含有马立克氏病肿瘤相关的表面抗原,为进行马立克氏病肿瘤相关的表面抗原的进一步研究奠定了基础。