Processing and analysis of samples

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3.5 Processing and analysis of samples

Details of the methods for handling, processing and examining biological samples have been described. 4 A brief summary is provided here. Representative samples were taken from each fixed tissue and processed routinely for embedding in paraffin wax. Histological sections were cut at 5 μm, stained with hematoxylin and eosin, and examined for pathological changes using light microscopy. Samples in VTM were processed using conventional methods for viral RNA extraction using commercial nucleic acid extraction kits. Quantitative polymerase chain reaction qPCR was used to detect nucleic acids from organisms commonly associated with BRD. The qPCR was performed using a commercial kit QuantiTect virus +ROX vial kit, Qiagen Inc., Valencia, CA according to th e manufacturer’s instructions. Primer and probe sequences were sourced from available sequences for each of the pathogens of interest: BCoV, BoHV-1, BPIV-3, BRSV, BVDV, Mannheimia haemolytica, Pasteurella multocida, Mycoplasma bovis and Histophilus somni. To reduce the number of tests, required reactions were multiplexed as follows: BCoV and BPIV-3, BRSV and BVDV, M. haemolytica and P. multocida, H. somni and M. bovis. Bovine herpesvirus 1 was run as a single assay. Each run contained duplicate samples of a positive control either a virus isolate or a clinical extract that had previously been characterized, a negative control an extract of cell culture – grade fetal bovine serum, and a blank PCR-grade water. Runs were only considered valid if the positive control was amplified at the expected threshold cycle, and the negative and blank controls showed no amplification. Positive samples were those with a characteristic sigmoidal curve similar to the positive control, crossing the threshold before 40 cycles. Negative samples were those with no characteristic sigmoidal curve. Samples crossing the threshold after 40 cycles were regarded as suspicious for containing the nucleic acid of interest and were retested before classifying as negative or positive.

3.6 Determining the cause of death