N‐Terminal Pro‐C‐Natriuretic Peptide and Cytokine Kinetics in Dogs with Endotoxemia

Background

Serum N‐terminal pro‐C‐natriuretic peptide (NT‐proCNP) concentration at hospital admission has sufficient sensitivity and specificity to differentiate naturally occurring sepsis from nonseptic systemic inflammatory response syndrome (SIRS). However, little is known about serum NT‐proCNP concentrations in dogs during the course of sepsis.

Objective

To determine serum NT‐proCNP and cytokine kinetics in dogs with endotoxemia, a model of canine sepsis.

Samples

Eighty canine serum samples.

Methods

Eight healthy adult Beagles were randomized to receive Escherichia coli lipopolysaccharide (LPS, 5 μg/kg) or placebo (0.9% NaCl) as a single IV dose in a randomized crossover study. Serum collected at 0, 1, 2, 4, and 24 hours was stored at −80°C for batch analysis. Serum NT‐proCNP was measured by ELISA and 13 cytokines and chemokines by multiplex magnetic bead‐based assay.

Results

Serum NT‐proCNP concentrations did not differ significantly between LPS‐ and placebo‐treated dogs at any time. When comparing serum cytokine concentrations, LPS‐treated dogs had higher interleukin‐6 (IL‐6), IL‐10, TNF‐α and KC‐like at 1, 2, and 4 hours; higher CCL2 at 1, 2, 4, and 24 hours; and higher IL‐8 and CXCL10 at 4 hours compared to placebo‐treated dogs. There were no differences in serum GM‐CSF, IFN‐γ, IL‐2, IL‐7, IL‐15 or IL‐18 between LPS‐ and placebo‐treated dogs.

Conclusions and Clinical Importance

Serum NT‐proCNP concentration does not change significantly in response to LPS administration in healthy dogs. Certain serum cytokine and chemokine concentrations are significantly increased within 1–4 hours after LPS administration and warrant further investigation as tools for the detection and management of sepsis in dogs.     

猪圆环病毒2型ORF2基因截断表达及ELISA抗体检测方法的初步建立

为建立以重组PCV2Cap蛋白为包被抗原的间接ELISA检测方法,构建了猪圆环病毒2型（PCV2）ORF2基因原核表达质粒pET28a-ORF2。SDS-PAGE显示,在0.1mmol/L IPTG和37℃条件下诱导4h,重组Cap蛋白高效表达。Western blotting证实该蛋白能够被PCV2阳性血清特异性识别。以纯化的蛋白为抗原建立了检测PCV2抗体的间接ELISA方法。结果表明,抗原最适包被质量浓度为2mg/L;血清最佳稀释度为1∶100;酶标二抗最适浓度为1∶2 000,该方法的敏感性为86.96%,特异性为100%。用该方法对河南省152份猪血清样品进行检测,与间接免疫荧光（IFA）的符合率为85.53%（130/152）,与商品化的韩国金诺PCV2ELISA试剂盒的符合率为88.16%（134/152）。本试验成功建立了PCV2血清抗体间接ELISA方法,具有较高的敏感性和特异性,可用于大规模的血清学检测。     

某猪场猪瘟免疫程序的调整及免疫效果评价

为了更好地预防和控制猪瘟,找出适合四川某猪场的猪瘟免疫程序,笔者根据实际情况对免疫程序进行了调整。然后用ELISA和IHA法对免疫程序调整前后的不同日龄猪血清进行了抗体检测,结果显示:调整前用ELISA法检测到3日龄、25日龄、50日龄、90日龄和120日龄猪只的猪瘟抗体阳性率分别为80%、54.54%、77.78%、80%和95%,用IHA法测得的阳性率分别为100%、63.64%、77.78%、90%和95%;调整后用ELISA法测得的猪瘟抗体阳性率分别为100%、80%、77.27%、77.78%和95%,用正向IHA法测得的抗体阳性率分别为97.73%、94.44%、96.15%、100%和100%。调整前用ELISA和IHA测得的平均阳性率为77.46%、85.28%,调整后则变为86.01%、97.66%,虽然均符合农业部颁布的猪群猪瘟抗体阳性率应不低于70%的标准,但免疫程序调整后的抗体阳性率明显高于调整前,可见调整后的免疫程序更适合于该猪场。     

A型口蹄疫病毒VP1蛋白间接ELISA检测方法的建立

以原核表达经纯化复性制备的A型口蹄疫病毒(FMDV)VP1蛋白代替传统病毒抗原包被,建立快速、安全、有效的A型FMDV抗体ELISA检测方法。对前期制备的A型VP1蛋白进行Western-Blot检测,结果表明,VP1蛋白能够特异性识别A型FMDV阳性牛血清,可作为检测抗原建立检测A型FMDV抗体的方法。以最佳质量浓度1mg/L VP1蛋白为检测抗原,方阵滴定法确定最佳检测血清稀释度为1∶50[V(血清)∶V(封闭液)],最佳的酶标二抗稀释度为1∶2 000[V(酶标二抗)∶V(封闭液)],建立A型FMDV抗体ELISA检测方法。该方法的敏感性为94.32%,特异性为99.09%,批内与批间重复试验变异系数均小于8%。采用该方法检测201份临床血清,与液相阻断ELISA试剂盒的符合率达92.54%。研制的VP1-ELISA试剂盒特异、敏感、稳定、操作简便,可用来监控A型口蹄疫抗体水平。     

Serotonin stimulates ovarian maturation and spawning in the black tiger shrimp Penaeus monodon

Serotonin (5-hydroxytryptamine, 5HT) has been reported to induce ovarian maturation and spawning in the crayfish Procambarus clarkii and white Pacific shrimp Litopenaeus vannamei. The aim of this study was to explore the role of exogenous 5HT on the reproductive performance of the black tiger shrimp Penaeus monodon. 5HT solution was injected into domesticated P. monodon broodstock at 50 μg/g body weight and ovarian maturation and spawning were recorded. The presence of 5HT in the ovary and oviduct of P. monodon was also studied by immunohistochemistry and its levels in the ovary by enzyme link immunoabsorbance assay (ELISA). The 5HT-injected P. monodon developed ovarian maturation and spawning rate at the level comparable to that of unilateral eyestalk-ablated shrimp. Hatching rate and the amount of nauplii produced per spawner were also significantly higher in the 5HT-injected shrimp, compared to the eyestalk-ablated shrimp. 5HT-positive reactions were found in the follicular cells of pre-vitellogenic oocytes, in the cytoplasm of early vitellogenic oocytes and on the cell membrane and cytoplasm of late vitellogenic oocytes. 5HT in the ovary was present at 3.53 ± 0.26 ng/mg protein level in previtellogenic stage and increased to 17.03 ± 0.57 ng/mg protein level in the mature stage of the ovary. The results suggest a significant role of 5HT, possibly directly on the ovary and oviduct, on the reproductive function of female P. monodon.     

Development of an enzyme-linked immunosorbent assay (ELISA) for vitellogenin measurement in greenback flounder Rhombosolea tapirina

Vitellogenin (Vtg) was purified from the plasma of 17-estradiol (E₂)-injected male greenback flounder,Rhombosolea tapirina. The molecular weight of the native Vtg was estimated by gel filtration as 540 kD. SDS-PAGE and Western blotting analyses indicated that this protein consisted of three bands with molecular weights of 155, 104, 79 kD, respectively. A polyclonal antibody against the highest molecular weight band of putative Vtg was generated in sheep and an indirect antibody-capture competitive enzyme-linked immunosorbent assay (ELISA) was developed. The assay was validatedfor plasma Vtg measurement in greenback flounder. Serial dilutions of plasma from vitellogenic females parallelled the standard Vtg curve, whereas no cross-reaction was observed with the plasma of males in the ELISA. The Vtg ELISA was used to assess the induction of Vtg by E₂ in vivo in males. The induction of Vtg in greenback flounder showed a time- and dose-dependent response as in other species. In E₂-treated fish, detectable levels of Vtg were first found at 48 h, and reached a peak at 96 h post-injection. Plasma levels of Vtg increased as the E2 dose increased with a threshold of 0.1 mg kg^–1.     

Development of a whole-body cortisol extraction procedure for determination of stress in golden shiners, <Emphasis Type="Italic">Notemigonus crysoleucas</Emphasis>

Baitfish such as golden shiners are subjected to stress during harvesting, grading, and transport. Their small size makes it difficult to measure the stress response with the biological indicator cortisol using conventional assay methods for plasma. This paper examines the development and validation of methods for whole-body cortisol extraction from individual baitfish. Three types of extracts were tested: (1) an ethyl ether unaltered extract (UA); (2) an extract reconstituted in phosphate buffered saline (PBS); (3) an extract that had been increased in volume by the addition of food-grade vegetable oil (VO). These extracts were evaluated using validation tests with radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA). The UA extract produced inadequate volumes of extract for multiple assays and could not be used for the determination of cortisol in a single fish. The PBS reconstitution method failed the precision recovery of serial dilutions (62.3%), linearity (R ²: 0.7864), and parallelism validation tests. The VO volume-boosting method passed all validation tests [intra-assay coefficent of variation (%CV): 16.3 for ELISA and 5.9 for RIA; inter-assay %CV: 10.3; spiked recovery: 102.0%; dilution recovery: 93.0%; linearity R ²: 0.9435; log of serial dilutions was parallel] and provided enough extract for multiple assays from an individual baitfish. Based on these results, we conclude that the VO volume-boosting method presents a means for determining cortisol from individual baitfish using either RIA or ELISA assays.     

以偶联多肽为抗原建立检测O型猪口蹄疫 病毒抗体的ELISA方法

以偶联多肽为抗原建立检测O型猪口蹄疫病毒抗体的ELISA 方法。通过FMDV 多肽序列的筛选、化学 合成及反应原性鉴定,将多肽与非蛋白结构多聚体载体进行偶联。利用偶联多肽作为抗原,通过方阵滴定确定偶联 多肽、血清及HRP 的最佳工作浓度,确定方法的阴阳性临界值,然后进行特异性试验、敏感度测定及临床猪血清样 本的免疫效果评估等。结果表明,偶联多肽具有良好的反应原性,最佳包被浓度为1.0 滋g/mL,血清最佳稀释度为1: 32,HRP 最佳稀释度为1:8 000,方法特异性好,敏感度高,操作方便省时,能够对FMDV 的自然感染进行检测,也可 对FMDV 免疫后的猪血清抗体水平进行评估。     

重组水泡性口炎病毒基质蛋白制备及ELISA检测方法的建立

水泡性口炎由水泡性口炎病毒(VSV)引起。VSV基因组编码5个结构蛋白,其中基质蛋白(matrix protein,M)是其重要毒力因子,可阻遏宿主细胞mRNA由胞核向细胞质的转运,并抑制感染病毒的细胞产生I型干扰素IFNα/β,使病毒得以快速繁殖。本实验克隆了印第安那型VSV的M基因,在大肠杆菌表达系统中表达制备重组M蛋白,以M蛋白为抗原建立了检测特异M蛋白抗体的间接ELISA方法。采用建立的ELISA方法分析了VSV感染小鼠体内M蛋白抗体的变化规律。     

全氟辛烷磺酸酶联免疫检测方法的研究

以人工合成的全氟辛烷磺酸免疫原免疫新西兰大白兔,制备多克隆抗体,用辛酸-硫酸铵沉淀法纯化抗体,选择包被抗原浓度为0.1μg/mL,抗体稀释度为1∶8 000,建立酶联免疫(ELISA)检测方法。交叉反应试验结果表明,除全氟癸烷磺酸和全氟己烷磺酸的交叉反应率偏高(16.2%,14.22%)外,与其他全氟化合物交叉反应率低于10%,即该抗体有较好的特异性。PFOS检测限为10μg/L,该ELISA方法的最佳工作范围在10~1 000μg/L之间,在此范围内平均回收率为101.62%,平均变异系数为2.4%,表明该方法准确度和精密度高,便于在线检测,可应用于对水体中全氟辛烷磺酸残留的酶联免疫检测。