Circulating markers of endothelial activation in canine parvoviral enteritis

Materials and methods

Animals

This 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 collection

A 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 evaluation

Thawed 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.