The main objective of this project was to evaluate the feasibility of utilizing swine oral fluids as a reliable aggregate sample type for early detection of African swine fever (ASF). At present, the recommended diagnostic samples for ASF diagnosis are individually collected whole blood, tonsils, spleen, hemorrhagic lymph nodes, etc. However, for the surveillance of North American swine herds for rapid detection of ASF such individual sampling methods are not feasible. Oral fluid is a promising alternative sample type that has been used effectively to detect and monitor the spread of common swine viral pathogens such as porcine reproductive and respiratory virus and swine influenza virus. Previously, it has been shown that African swine fever virus (ASFV) genomic material can be detected in swine oral fluids collected from small groups of pigs when multiple pigs in the pen are infected with ASFV. The objective of this project was to explore the potential of detecting ASFV genomic material in oral fluids collected from pens similar in size to commercial hog farms in North America when the disease prevalence is at its lowest.

The two specific objectives of the proposal were to:

1. Estimate the likelihood of detection of ASFV as a function of within pen prevalence
2. Compare the timeline of clinical disease progression via observation with the timeline of ASFV detection in oral fluids and individual samples

In order to achieve these objectives, we infected four groups of pigs (each 20-25 pigs)-two with the ASFV Georgia 2007/1 (group 1 & 2) strain and two (group 3 & 4) with the ASFV Malta’78 strain. In each experiment, one randomly selected pig was infected with the virus and released into the pen as the seeder pig. Aggregate oral fluid samples were collected daily, and individual oropharyngeal swabs and whole blood samples were collected every other day. In addition, clinical parameters such as rectal temperatures, appetite and behavior were observed and recorded.

In experiment #1, the seeder pig remained active and alert until 4 days post-infection (dpi), depressed on 5 dpi and found dead on 6 dpi. In experiment #2, the seeder pig was alert until 5 dpi and found dead on 7 dpi. In experiment #3 and #4, seeder pigs died on 6 and 10 dpi respectively. Whole blood samples are the gold standard for ASFV, and the seeder pig tested positive in all cases by 1-3 dpi. ASFV genome was detected in oral fluids collected from pens as early as 3-5 days post infection (dpi) in all experiments, when the ASF pen prevalence was is at the lowest (4% for ASFV Georgia 2007/1 and 5% for ASFV Malta’78). At this time, no evidence of ASFV infection in contact pigs were observed. It is important to note that oral fluid contains significantly less virus than whole blood. At times, the viral load in oral fluid approaches the assay’s limit of detection, raising concerns about the potential for false negatives under field conditions.

Using oral fluid, it was possible to detect the presence of ASFV in the pen at least 3 days prior to the death of seeder pigs. Upon the seeder pig’s death, there were at least 2 days before contact pigs started to show mild clinical signs. The disease slowly spread through the pig pens, and it took from 10-18 days before significant mortality, indicating that possible ASF infection was observed in the pens. On the farm, this means that at the time of detection, the infected pig may not be clinically sick or only mildly affected and could be easily missed by the farm staff.

These results show that oral fluids can serve as a very effective sample type that can be easily and passively collected in the commercial swine industry. We were able to detect the presence of ASF virus in a pen containing up to 25 pigs when only one of the pigs in the pen was infected. In an outbreak situation, oral fluids when used with other recommended sample types, could greatly strengthen early detection methods.

Key Findings:
• ASFV genome was detected in oral fluids as early as 3-5 days post introduction of an ASF infected pig into a pen containing 19-24 pigs.
• At the time of the first detection in oral fluid, only the seeder pig had developed mild fever, while the other contact pigs were clinically normal.
• Upon death of the seeder pig, it took at least 2 days for the contact pigs to develop fever and clinical signs.
• Whole blood samples remain the gold standard for ASFV detection, as virus can be detected 1-3 days earlier in blood than oral fluid and blood contains significantly more virus, ensuring detection by PCR.
• Oral fluid contains significantly less virus than whole blood. At times, the viral load in oral fluid approaches the assay’s limit of detection, raising concerns about the potential for false negatives under field conditions. Evaluation of oral fluid samples from an ASFV outbreak are critical to fully understand risk.
• Upon death of the seeder pig, it took at least 2 days for the contact pigs to develop fever and clinical signs.