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
动物实验
本研究为一项前瞻性观察性临床研究，对比了临床表现为CPV肠炎的犬与健康的对照犬。CPV组为宠物主人的犬只，年龄介于6周至12个月之间，临床症状与自然CPV感染相符，包括倦怠、厌食、呕吐、腹泻及脱水。基于临床怀疑，犬只通过粪便CPV ELISA检测（IDEXX SNAP Parvo Test，荷兰）进行CPV检测，并通过粪便电子显微镜进行确诊。若犬只在过去7天内接受过CPV肠炎的治疗，则予以排除。健康对照组由接受常规程序（疫苗接种和绝育手术）的犬只组成，其年龄与CPV组相匹配。若对照犬在过去14天内有任何疾病史或粪便电子显微镜检测出CPV，则予以排除。两组犬只的体重均需超过3公斤。排除接受已知可影响炎症的药物治疗的犬只，以及临床检查或血液涂片评估中可识别的合并症犬只，除非为胃肠道寄生虫。本研究已获得比勒陀利亚大学学院及动物伦理委员会的批准（REC089-18和v090-18）。
数据与样本收集
在首次就诊时进行病史采集、临床检查、外周血液涂片和粪便浮选。从所有犬只采集粪便样本，并在CPV组进行CPV ELISA快检。两组粪便样本均储存于2-8℃条件下，并在采集后72小时内进行电子显微镜检查，以确认/排除CPV感染。通过颈静脉穿刺，使用Vacutainer血液采集系统，将血液采集至EDTA和血清真空管中（贝克顿·迪金森Vacutainer系统，英国）。在采集后30分钟内进行完整的血细胞计数（CBC）和中心血涂片。随后对EDTA和血清样本进行离心。尽可能将血清样本冷藏于2-8℃条件下，并在采集后24小时内使用Cobas Integra 400 plus分析仪（西门子，德国）测量CRP（犬特异性免疫浊度法CRP，Cobas Integra 400 plus分析仪）浓度。如24小时内无法进行分析，则将血清和EDTA血浆储存于-80℃条件下。研究期末进行CRP浓度的批次测量。血清的冷冻和储存对CRP浓度无显著影响（Aziz等，2003，Hillstrom等，2014）。
内皮粘附分子评估
解冻的EDTA血浆用于测量ICAM-1、VCAM-1和HMGB-1，采用犬特异性ELISA夹心酶联免疫测定（USCN Life Science，武汉，中国）（Kules等，2017，Baric Rafaj等，2013）。研究表明，在运行这些检测时，样品的冷冻和解冻无任何影响（Wang等，2015，Kavsak等，2008）。ELISA分析包括板制备和按制造商推荐方案进行的检测程序。在450 nm波长下通过分光光度法测量酶-底物反应的颜色变化（Thermo Scientific Multiskan™ FC微孔板分光光度计，ThermoFischer Scientific）。通过将样本的光密度与标准曲线进行比较，确定ICAM-1、VCAM-1和HMGB-1的浓度。ICAM-1、VCAM-1和HMGB-1检测的检测范围分别为1.56-100 ng/mL、3.12-200 ng/mL和6.25-400 pg/mL。从标准曲线读取的浓度乘以VCAM-1和HMGB-1的稀释因子。
所有三种免疫测定的内测变异系数均小于10%，而间测变异系数小于12%。