Evaluation of the effects of long-term storage of bovine ear notch samples on the ability of two diagnostic assays to identify calves persistently infected with bovine viral diarrhoea virus

Abstract

Research aimed at optimizing diagnostic laboratory procedures is central to the development of effective bovine viral diarrhoea virus (BVDV) control programmes. BVDV is a single-stranded RNA virus that crosses the placenta to infect foetuses resulting in reproductive losses due to foetal death or persistently infected (PI) calves that usually die early in life. Persistently infected animals are widely accepted to be the primary reservoir of BVDV and the largest source of BVDV infection. Persistently infected animals that survive calfhood are at risk of developing mucosal disease in later life which is a severe and usually fatal condition. In addition, persistently infected calves that become replacement heifers in the herd may experience significant morphological changes that occur in the ovaries which can result in impaired reproductive performance. This poses important challenges to overall animal/herd health and causes losses to the cattle industry. Long-term storage of bovine ear notch samples from calves persistently infected (PI) with BVDV may affect the ability of diagnostic assays to efficiently detect the virus. This study assessed the effects of 1) long-term storage of formalin-fixed samples at room temperature to detect BVD viral antigen with the aid of immunohistochemistry (IHC), 2a) long-term storage of fresh ear notch samples kept at -20°C, and 2b) long term storage of phosphate-buffered saline (PBS) ear notch supernatant kept at -20°C on the ability of an antigen-capture ELISA (AC-ELISA) to detect viral antigen. Previous studies have verified 100% sensitivity for both AC-ELISA on ear notch supernatant and immunohistochemical testing of ear notches to detect BVDV provided that samples are properly collected and stored. In this study, ear notch samples from seven animals were subjected to prompt formalin fixation and fresh samples to prolonged storage at -20°C. Frozen ear notches and ear notch supernatant yielded positive results on AC-ELISA for the duration of the study, i.e. 6 months, and OD values remained significantly within range. There was no significant difference between storing fresh ear notch samples and PBS ear-notch supernatant at -20°C. However, positive IHC staining on formalin-fixed ear notches started to fade away between day 17 and day 29 when stored at room temperature. We conclude that fresh ear notches could safely be stored at -20°C for a period of 6 months for detecting BVD viral antigen at a later stage. Copyright