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

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.