Entwicklung und Prüfung von Verfahren zum Nachweis des Virus der Bovinen Virusdiarrhoe in getrockneten Ohrgewebeproben mittels Antigen-ELISA und real time RT-PCR

摘要

Early detection and elimination of cattle persistently infected (PI) with bovine viral diarrhoea virus (BVDV) is a key element for eradication programs. Testing dried skin biopsies derived from ear tagging might be useful for detection of BVDV in newborn calves. The aims of this study were the development of methods for antigen solubilization and RNA preparation, the investigation of the stability of these viral components and the comparison of the analytical sensitivity of different tests. Commercial antigen capture ELISAs for NS3, Erns and mixed antigens, a blocking ELISA for BVDV antibodies and two real time RT-PCR assays for 5’UTR were used as BVDV specific tests.Ear biopsies were collected from nine BVDV antibody free PI animals (infected with BVDV-I CR4043) after slaughter and from twelve PI calves (fetal infection with BVDV-I PT810) after euthanasia at the age of 5-13 days in the time of colostral antibodies. For solubilization of the BVDV antigens the detergents dodecyl sulfate, sodium deoxycholate and EMPIGEN were inappropriate. Out of the suitable detergents (CHAPS, Triton X100, Nonidet P-40, Tween 20, n-Octyl-ß-D-glucopyranoside and Digitonin) Triton X100 in a concentration of 1% was chosen for antigen solubilization. Best RNA yield was obtained using a mixer mill and a guanidine thiocyanate containing buffer, followed by RNA isolation with Qiagen RNeasy® kits (Fibrous Tissue Mini Kit and Lipid Tissue Mini Kit). RNA isolation kits provided by Roche were less efficient.NS3-ELISAs were of low analytical sensitivity for ear biopsies, maternal antibodies led to negative results. In addition NS3 epitopes are very heat sensitive. In contrast Erns-ELISAs showed high analytical sensitivity. Samples of PI animals without maternal antibodies gave mean titers above 30. Maternal antibodies had limited effects. In samples of nine PI animals with colostral antibodies the lowest titer was 13. Relevant temperatures by sample drying and storage led to minor titer reductions. The real time RT-PCR resulted in a sensitivity more than 104 fold over the detection limit, even in calves in the time of neutralizing antibodies. Bacterial or fecal contamination before sample drying had no relevant influence on the stability of Erns and 5’UTR RNA.Erns-ELISAs and real time RT-PCR seem to be suitable for detecting BVDV in dried ear notch samples of PI animals. Before appliance in BVDV eradication programs, the diagnostic sensitivity and specificity of tests using ear biopsies have to be evaluated by studies in the field with an adequate number of samples and an appropriate monitoring.