Revolutionising African Swine Fever Surveillance with Environmental DNA

This work presented in this study resonates with my science journey. Having worked on African Swine Fever (ASF) during my first postdoctoral experience in Tanzania, I witnessed firsthand the devastating impact this disease has on pig farming. The search for solutions was urgent, and at the time, we were exploring genomic approaches to tackle the ASV outbreaks.

This article highlights a promising new method—using environmental DNA—for ASF surveillance. The work was conducted in Italy-where ASF outbreaks have been reported. ASF outbreaks in northern Italy, in January 2022, led to the culling of nearly 120,000 pigs to contain the disease, threatening the nation’s €20 billion pork industry, including prized prosciutto production.

African Swine Fever is a highly contagious viral disease that has devastated wild and domestic pig populations across Eurasia since 2007. It poses a significant threat to agriculture and wildlife ecosystems, especially through its association with wild boars, which play a key role in maintaining and spreading the virus. Controlling ASF is challenging, making rapid and efficient detection methods essential for effective management and containment of outbreaks.

The Urgency of Addressing African Swine Fever

ASF’s ongoing spread highlights the need for new surveillance methods. Traditional techniques involve directly sampling animals, which is invasive, time-consuming, costly, and risky because it requires close contact with potentially infected wildlife. In areas with many wild boars, monitoring becomes even more difficult. Therefore, non-invasive, cost-effective, and reliable surveillance tools are urgently needed to track the virus. Environmental DNA (eDNA) is a groundbreaking tool for monitoring. It consists of genetic material collected from environmental samples like water, soil, or air—without needing to capture or see the organisms themselves. This technique shows great promise for detecting diseases in various ecosystems. By analysing eDNA, researchers can identify specific species and their pathogens, making it invaluable for disease surveillance.

Research Objectives and Questions

The main goal of this study was to develop and validate an eDNA sampling method suitable for muddy water and soil environments to detect ASF virus (ASFV) and wild boar DNA. The researchers aimed to answer:

1. Can eDNA effectively detect ASFV and wild boar DNA in natural, muddy environments?
2. What are the best conditions and methods to maximise eDNA recovery from challenging samples like muddy water and soil?
3. How reliable and consistent is eDNA compared to traditional methods for early ASFV detection?

Methodology: From Field to Laboratory

The research took place in La Mandria Regional Park near Turin, Italy, spanning about 2,700 hectares. This park is home to various hoofed animals, including wild boars, red deer, roe deer, and fallow deer, with a high density of wild boars (about 15 per square kilometre). Importantly, the park is free of ASF and has no pathogen management restrictions, making it ideal for testing the method.

Four mudholes in the park were randomly selected and monitored with camera traps to confirm wild boar use. On sampling day, seven litres of muddy water were collected from each mudhole using a pump. To prevent contamination between samples, the tubes were cleaned with a 20% bleach solution between collections. Additionally, small soil samples (5 millilitres) were collected from each site, and a special buffer (Buffer AVL™) was added to deactivate any potential ASFV while preserving the DNA.

Laboratory Procedures and Sample Preparation

In the lab, researchers created a synthetic piece of ASFV DNA based on known sequences. They prepared four different dilutions of this synthetic DNA, each with varying amounts, and added them to separate water and soil samples. After a 12-hour incubation at room temperature, the water samples were filtered using fine filters (0.1 μm). Buffer AVL™ was added to help recover the DNA. The soil samples were shaken and centrifuged to separate sediments, and then the DNA was purified using a special kit (DNeasy PowerSoil Pro Kit).

qPCR Assays for ASFV and Wild Boar Detection

To detect ASFV and wild boar DNA, the researchers used quantitative Polymerase Chain Reaction (qPCR), a technique that amplifies DNA to detectable levels. For ASFV, they used iTaq Universal SYBR Green Supermix with specific primers (short DNA sequences that initiate amplification). For wild boar DNA, they used TaqMan™ Universal PCR Master Mix with appropriate primers. Each test was conducted three times to ensure accuracy, and a sample was considered positive if at least two out of three tests exceeded the limit of quantification (the smallest amount that can be reliably measured).

Key Findings: eDNA Proves Its Worth

The study showed promising results, demonstrating that eDNA can effectively detect ASFV and wild boar DNA in challenging environments.

• ASFV Detection: All water and soil samples spiked with synthetic ASFV DNA tested positive. Soil samples gave more consistent results than water samples, possibly because DNA is better preserved in soil.
• Wild Boar DNA Presence: Wild boar DNA was found in almost all water and soil samples, except for one soil sample that didn’t meet the required detection limit in two out of three tests. This suggests that eDNA is effective at detecting wild boars even without recent direct sightings.
• DNA Preservation: Soil samples not only preserved ASFV DNA better but also had higher concentrations of wild boar DNA, indicating that soil might be a more reliable medium for long-term eDNA monitoring.

Broader Impact: Beyond ASF

This research has implications beyond African Swine Fever. Using eDNA techniques could help monitor many wildlife diseases and support biodiversity conservation. To make the most of eDNA in managing wildlife diseases, future studies should:

1. Field Validation: Test the eDNA methods in various real-world settings to assess their robustness and adaptability.
2. Improved Molecular Techniques: Develop advanced tests that can differentiate between DNA from wild and domestic pigs for more precise monitoring.
3. Integration with Other Systems: Combine eDNA data with traditional monitoring methods and technologies to create comprehensive disease surveillance networks.

Integrating eDNA into disease surveillance is a major step forward in managing wildlife health. As ASF continues to challenge regions across Eurasia and beyond, innovative methods like eDNA sampling offer the tools needed to monitor and combat the disease effectively. Ongoing research in this field will not only help control ASF but also lay the groundwork for managing other wildlife diseases, ensuring the preservation of animal populations and agricultural stability.