The influence of temperature on the Russian isolate of African swine fever virus in pork products and feed with extrapolation to natural conditions

It is known that one of the ways to spread African swine fever (ASF) virus is alimentary way (e.g., through feeding animals with contaminated feed and slaughterhouse waste products not subjected to thermal treatment, not passed veterinary and sanitary examination, and derived from epidemiologically disadvantaged regions). A variety of preserved food, such as corned beef, canned meat, bacon, etc., is made of pork products and by-products. Persistence of the virus in these products is not fully understood. For the first time we carried out studies on infectious activity of ASF virus isolates circulating in the Russian Federation, in contaminated pig products, feed, as well as in samples intended for veterinary-sanitary supervision. For this purpose, the 3-4 month-old piglets of 20-30 kg body weight were intramuscularly challenged with virus-containing blood. For inoculation we used ASFV isolate Volgograd-Kalach 2012 with the initial infectious titer of 7.5 lg HAU50/cm3 obtained from the National Collection of the SSINRRIVV&M of RAAS. Three days after infection the animals were slaughtered, samples of internal organs and tissues were taken for virus isolation, and the pork was used for making corned beef, canned meat and salted lard. Blood, containing ASF virus, was also used for contamination of a compound feed and drinking water. To study effects of storage temperature on the virus infectious activity and genome detection, the samples were divided into aliquots and stored at 20-25 °С (room temperature), 4-6 °С (a common refrigerator); -16…-20 °С (freezer). The temperatures selected for the storage of samples conform to the natural weather conditions at various seasons (20-25 °С in the summer, -16…-20 °С in winter); 4-6 °С is generally maintained in storerooms or stern room in the winter months. Samples from the aliquots were taken with a 5 day intervals to determine the ASF virus titer and its genome fragments. Virus isolation was carried out in a primary culture of swine bone marrow cells for 5 days in two consecutive passages. Hemadsorption test (HAD) and real-time PCR were conducted for ASFV titer estimation and genome detection. As a result, the ASF virus was found in corned beef and lard stored at 20-25 °С for 16 days. At storage temperature of 4-6 °С the ASF virus was revealed in corned beef during the observation period (60 days). In frozen samples the ASFV infectious activity and DNA fragments were found throughout the entire study period (60 days). Thus, it is found that in food products and by-products from pigs slaughtered soon after ASF infecting the ASFV infectivity persists for a long time depending on the storage conditions and can pose a serious danger as a pathogen transmission factor. The data also allow to predict the temperature conditions conducive to maintaining circulation of the virus in АSF outbreaks.