African Swine Fever Virus Load in Hematophagous Dipterans Collected in Outbreaks from Romania: Risk Factors and Implications.

African swine fever (ASF) is a contagious viral disease of swine that causes significant economic damage. The summer peaks and river courses have triggered the hypothesis that vectors may be involved in the transmission of the virus. In temperate climates, insect numbers increase in the late summer. Low temperatures and frosts decrease the number of active insects. Their presence is strongly associated with the nearby wetlands or swamps around the farms. The aim of our study was to evaluate the risk factors associated with the presence of ASFV DNA in hematophagous dipterans and to analyze the relevance of Ct values obtained following RT-PCR analysis of the positive samples in ASF outbreaks in Romania, as an indication for the viral load. The current study included 99 pools of stable flies () and 296 pools of biting midges ( spp.), collected in June-September 2020, from 30 outbreaks of ASF in domestic swine from backyard farms (BF), type A farms (TAF), and commercial farms (CF). All extracted DNA was tested for the presence of the ASFV genome using a real-time PCR protocol. Ct values of 39.53 and below were considered as positive (min: 18.19; median: 31.41; max: 39.53). The blood meal source was identified in the hematophagous insects by using a PCR protocol targeting the mitochondrial gene cytochrome c oxidase subunit 1. Data were analyzed using R software v. 4.0.5. In total, 3,158 insects ( = 198 and  = 2960) were collected in 23 farms of the 30 outbreak farms. Ten species of biting midges were identified. The total number of insect pools showed significant differences according to the month of sampling, with a higher number of pools collected in August and September. Overall, 137 pools out of the 395 examined were positive for the presence of ASFV DNA. There was a higher viral DNA load in farms where pigs were present at the moment of sampling compared to farms where pigs were already culled, in compared to spp. and in CF and TAF compared to BF.