Circulating markers of endothelial activation in canine parvoviral enteritis

Materials and methodsAnimalsThis was a prospective observational clinical study in which dogs with CPV enteritis were compared with healthy control dogs at presentation. The CPV group were client owned dogs, between 6 weeks and 12 months of age, with clinical signs consistent with natural CPV infection including lethargy, anorexia, vomiting, diarrhoea and dehydration. Based on clinical suspicion, dogs were tested for CPV using the faecal CPV ELISA test (IDEXX SNAP Parvo Test, Netherlands) and confirmed with faecal electron microscopy. Dogs were excluded if they received treatment for CPV enteritis within the preceding 7 days. The healthy control group consisted of dogs that presented for routine procedures (vaccinations and sterilisations) and were aged matched to the CPV group. Control dogs were excluded if there was any history of illness for the preceding 14 days or if CPV was identified on faecal electron microscopy. For both groups, dogs had to weigh more than 3 kg. Dogs receiving medication known to influence inflammation or with co-morbidities identifiable on clinical examination or blood smear evaluation, with the exception of gastro-intestinal helminths, were excluded. The study was approved by Faculty and animal ethics committee of the University of Pretoria (REC089-18 and v090-18)Data and sample collectionA history, clinical examination, peripheral blood smear and faecal flotation was performed at presentation. A faecal sample was collected from all dogs and a CPV ELISA snap test was performed on the CPV group. Faecal samples from both groups were stored at 2 - 8⁰C and electron microscopy was performed within 72 hours of collection to confirm/exclude infection with CPV. Blood was collected atraumatically via jugular venepuncture, using the Vacutainer blood collection system, into EDTA and serum vacutainer tubes (Beckton Dickinson Vacutainer Systems, UK). A complete blood count (CBC) (Advia 2120i, Siemens, Germany) and central blood smear were performed within 30 minutes of collection. The EDTA and serum samples were then centrifuged. When possible, serum samples were refrigerated at 2-8⁰C and used to measure CRP (canine specific immunoturbidimetric CRP method, Cobas Integra 400 plus analyser) within 24 hours of collection. When analysis within 24 hours was not possible, the serum and EDTA plasma were stored at -80⁰C. Batch measurement of CRP was then performed at the end of the study period. Freezing and storage of serum do not significantly influence CRP concentrations (Aziz et al., 2003, Hillstrom et al., 2014). Endothelial adhesion molecule evaluationThawed EDTA plasma was used to measure ICAM-1, VCAM-1 and HMGB-1 using canine-specific ELISA sandwich enzyme immunoassays (USCN Life Science, Wuhan, China) (Kules et al., 2017, Baric Rafaj et al., 2013). Studies have reported no effect of freezing and thawing of samples when running these assays (Wang et al., 2015, Kavsak et al., 2008). The ELISA analyses included plate preparation and assay procedures performed according to the manufacturer’s recommended protocol. The colour change of the enzyme-substrate reaction was measured spectrophotometrically at a wavelength of 450 nm (Thermo Scientific Multiskan™ FC Microplate Photometer, ThermoFischer Scientific). The concentrations of the ICAM-1, VCAM-1 and HMGB-1 were determined by comparing the optical density of the samples to the standard curves. The detection ranges for the ICAM-1, VCAM-1 and HMGB-1 assays were 1.56-100 ng/mL, 3.12-200 ng/mL and 6.25-400 pg/mL, respectively. The concentrations read from the standard curve were multiplied by the dilution factor for VCAM-1 and HMGB-1. The intra-assay coefficient of variance was less than 10% and the inter-assay coefficient of variance less than 12% for all three immunoassays.

材料与方法
### 实验动物
本研究为前瞻性观察性临床研究，旨在对比就诊时确诊犬细小病毒性肠炎的患犬与健康对照犬。CPV组受试犬均为客户饲养的家犬，年龄介于6周至12月龄，临床表现符合自然感染犬细小病毒（CPV）的特征，包括嗜睡、厌食、呕吐、腹泻及脱水。基于临床疑似诊断，所有CPV组犬均接受粪便CPV酶联免疫吸附试验（ELISA）检测（采用荷兰IDEXX SNAP细小病毒检测试剂盒），并经粪便电子显微镜检查确认感染。若受试犬在入组前7天内接受过CPV肠炎治疗，则予以排除。

健康对照组犬均因常规诊疗（疫苗接种与绝育手术）就诊，且年龄与CPV组匹配。若对照犬在入组前14天内有患病史，或粪便电子显微镜检查检出CPV，则予以排除。两组受试犬体重均需超过3kg。此外，排除接受已知可影响炎症反应的药物治疗，或经临床检查及血涂片评估发现合并症（胃肠道蠕虫感染除外）的受试犬。本研究经比勒陀利亚大学医学院及动物伦理委员会批准，批准号为REC089-18与v090-18。

### 数据与样本采集
受试犬就诊时均完成病史采集、临床检查、外周血涂片检测及粪便浮集检查。采集所有受试犬的粪便样本，CPV组犬同时完成CPV ELISA快速检测。两组粪便样本均置于2~8℃环境保存，并于采集后72小时内完成电子显微镜检查，以确认或排除CPV感染。

采用真空采血管系统（英国贝克顿-迪金森公司Vacutainer采血系统）经颈静脉无创采血，分别收集乙二胺四乙酸（EDTA）抗凝血管与血清采血管内的血液样本。于采血后30分钟内完成全血细胞计数（CBC）检测（采用德国西门子Advia 2120i全自动血液分析仪）及中心血涂片制作与镜检。随后对EDTA抗凝血与血清样本进行离心处理。

若实验条件允许，血清样本置于2~8℃冷藏，并于采集后24小时内采用犬特异性免疫比浊法检测C反应蛋白（CRP）（采用罗氏Cobas Integra 400 Plus全自动生化分析仪）。若无法在24小时内完成检测，则将血清与EDTA血浆置于-80℃冻存。本研究终末期统一批量检测CRP浓度，已有研究证实冻存与冷藏不会显著影响CRP浓度（Aziz等，2003；Hillstrom等，2014）。

### 内皮黏附分子检测
采用解冻后的EDTA抗凝血血浆，通过犬特异性ELISA夹心酶免疫分析法检测细胞间黏附分子-1（ICAM-1）、血管细胞黏附分子-1（VCAM-1）及高迁移率族蛋白B1（HMGB-1），试剂盒购自武汉优宁维生命科学有限公司（USCN Life Science, Wuhan, China）（Kules等，2017；Baric Rafaj等，2013）。已有研究证实，该类检测样本的冻融操作不会对检测结果产生显著影响（Wang等，2015；Kavsak等，2008）。

ELISA检测严格按照试剂盒生产商推荐的操作流程完成板条制备与检测程序。采用Thermo Scientific Multiskan™ FC微孔板分光光度计（赛默飞世尔科技）在450nm波长下检测酶底物反应的颜色变化程度。通过将样本的光密度值与标准曲线对比，计算得到ICAM-1、VCAM-1及HMGB-1的浓度。三种检测的量程范围分别为：ICAM-1：1.56~100 ng/mL，VCAM-1：3.12~200 ng/mL，HMGB-1：6.25~400 pg/mL。将标准曲线读取的浓度值乘以VCAM-1与HMGB-1的稀释倍数即可得到最终浓度。三种免疫检测的批内变异系数均小于10%，批间变异系数均小于12%。