Five pigs were inoculated intramuscularly (IM) with 1 mL of DMEM (Gibco, Waltham, MA, USA) containing 104 tissues culture infectious dosage (HAD50) of ASFV-DR21

Five pigs were inoculated intramuscularly (IM) with 1 mL of DMEM (Gibco, Waltham, MA, USA) containing 104 tissues culture infectious dosage (HAD50) of ASFV-DR21. isolated from samples collected in the DR (ASFV-DR21) was observed. Two groups of domestic pigs were inoculated either intramuscularly (IM) or oronasally (ON) with ASFV-DR21 (104 hemadsorbing dose-50% (HAD50)). A group of na?ve pigs (designated as the contact group) was co-housed with the ASFV-DR21 IM-inoculated animals to evaluate ASFV transmission and disease manifestation. Animals inoculated IM with ASFV-DR21 developed an acute disease leading to humane euthanasia at approximately day 7 post-inoculation (pi). Interestingly, animals inoculated via the ON route with ASFV-DR21 developed a heterogeneous pattern of disease kinetics. One animal developed an acute form of the disease and was euthanized on day 7 pi, another animal experienced a protracted presentation of the disease with euthanasia by day 16 pi, and the remaining two animals presented a milder form of the disease, surviving through the 28-day observational period. The contact animals also presented with a heterogenous presentation of the disease. Three of the animals presented protracted but severe forms of the disease being euthanized at days 14, 15 and 21 pi. The other two animals presented with a milder form of the disease, surviving the entire observational period. In general, virus titers in the blood of animals in all study groups closely followed the clinical presentation of the disease, both in length and extent. Importantly, all animals presenting with a prolonged form of the disease, as well as those surviving throughout the observational period, developed a strong ASFV-specific antibody response. These results suggest that ASFV-DR21, unless inoculated parenterally, produces a spectrum of clinical disease, with some animals experiencing an acute fatal form while others presented with a mild transient disease accompanied by the induction of a strong antibody response. At the time Rabbit Polyclonal to ACHE of publication, this is the first report characterizing the virulent phenotype of an ASFV field strain isolated from samples collected in the DR during the 2021 outbreak and provides information that may be used in developing epidemiological management measures to control ASF on the island of Hispaniola. gene (encoding for p72 protein) of ASFV as previously described [6]. Briefly, viral DNA was extracted using the MagMAX? Pathogen RNA/DNA kit (ThermoFisher Scientific, Waltham, MA, USA) on a KingFisher Flex automated extraction and purification system (ThermoFisher Methylene Blue Scientific, Waltham, MA, USA). Master mix was prepared using the TaqMan? Universal PCR Master Mix (Applied Biosystems, Waltham, MA, USA), and the following primers forward: 5-CTTCGGCGAGCGCTTTATCAC-3, reverse: 5-GGAAATTCATTCACCAAATCCTT-3 and probe: 5-FAM-CGATGCAAGCTTTAT-MGB NFQ-3. Assays were conducted on an Applied Biosystems 7500 Real-time PCR system. As performed, the sensitivity of detection is 1.28 DNA copies/reaction volume and 102.55 HAD50/mL [6]. 2.3. Animal Infections ASFV-DR21 virulence phenotype was assessed using 80C90 pound commercial Yorkshire crossbreed female swine [3]. Five pigs were inoculated intramuscularly (IM) with 1 mL of DMEM (Gibco, Waltham, MA, USA) containing 104 tissue culture infectious dose (HAD50) of ASFV-DR21. A second group of five pigs was oronasally (ON) inoculated by instilling in each animal 1 mL containing 104 HAD50 of ASFV-DR21 intranasally and another 1 mL of an equal concentration of virus via oral administration. A third group of five uninoculated pigs was co-housed with the IM-inoculated animals 24 h after inoculation to represent a co-housed viral transmission group. Animals from the IM-inoculated and uninoculated groups shared food and water in a single room with a floor surface of 110 square feet throughout the duration of the study. Clinical signs (anorexia, depression, fever, purple skin discoloration, staggering gait, diarrhea, and cough) and changes in body (rectal) temperature were recorded daily throughout the study period of 28 days. Clinical samples (blood, Methylene Blue serum, nasal swabs, and oral swabs) were collected on days 2, 4, 6, 8, 10, 12, 14, 19, 22, 25 and 28 post-inoculation for detecting the presence of the virus by Methylene Blue qPCR and virus titrations in swine macrophage cultures, as earlier described, and ASFV-specific antibodies by an enzyme-linked immunosorbent assay (ELISA) as described below. Animal infection studies were performed under biosafety level 3-agriculture conditions at the Plum Island Animal Disease Center (PIADC) animal facility following protocols approved by the PIADC Institutional Methylene Blue Animal Care and Use Committee (IACUC) of the US Departments of Agriculture and Homeland Security (protocol number 225.06-19-R, Methylene Blue approved 09-10-19). 2.4. Detection of Anti-ASFV Antibodies ASFV antibody detection was performed using an in-house ELISA as described previously [7]. Briefly, ELISA antigen was prepared from Vero cells infected with a Vero adapted strain of ASFV Georgia.