Testing of retail cheese, butter, ice cream and other dairy products for highly pathogenic avian influenza in the US

The recent outbreak of highly pathogenic avian influenza (HPAI) in dairy cows has created public health concerns about the potential of consumers being exposed to live virus from commercial dairy products. Previous studies support that pasteurization effectively inactivates avian influenza in milk and an earlier retail milk survey showed viral RNA, but no live virus could be detected in the dairy products tested. Because of the variety of products and processing methods in which milk is used, additional product testing was conducted to determine if HPAI viral RNA could be detected in retail dairy samples, and for positive quantitative real-time RT-PCR (qRT-PCR) samples to be tested for presence of live virus. Revised protocols were developed to extract RNA from solid dairy products including cheese and butter. The solid dairy product was mechanically liquified with garnet and zirconium beads in a bead beater diluted 1 to 4 with BHI media. This pre-processing step was suitable in allowing efficient RNA extraction with standard methods. Trial studies were conducted with different cheese types with spiked in avian influenza virus to show that inoculation of the liquified cheese into embryonating chicken eggs was not toxic to the embryos and allowed virus replication. A total of 167 retail dairy samples, including a variety of cheeses, butter, ice cream, and fluid milk were collected as part of nationwide survey. A total of 17.4% (29/167) of the samples had detectable viral RNA by qRT-PCR targeting the matrix gene, but all samples were negative for live virus after testing with embryonating egg inoculation. The viral RNA was also evaluated by sequencing part of the hemagglutinin gene using a revised protocol optimized to deal with the fragmented viral RNA. The sequence analysis showed all viral RNA positive samples were highly similar to previously reported HPAI dairy cow isolates. Using the revised protocols, it was determined that HPAI viral RNA could be detected in a variety of dairy products, but existing pasteurizations methods effectively inactivate virus assuring consumer safety.