How to detect pathogens in domestic animals quickly and accurately

Infectious diseases are not just a significant public health hazard for humans, they also pose a serious threat to the animals we raise. Bovine and porcine respiratory and enteric diseases in particular have a profound negative impact on animal health and are of great economic importance in commercial livestock production. However, diagnosis can be slow or nonexistent, and overuse of antibiotics leads to additional challenges such as resistance that can then spread to other species.

With a goal of improving animal health through pathogen control, Nicole Bakkegård Goecke, PhD, now a postdoc in veterinary clinical microbiology in the Department of Veterinary and Animal Sciences at the University of Copenhagen, focused her graduate work on the development of a faster and less expensive way to detect and diagnose disease that could also be implemented as a monitoring system aimed at disease prevention.

As a PhD student back in 2015 at the Danish National Veterinary Institute, Technical University of Denmark, which had a large diagnostic unit focused on identifying the cause of diseases in domestic animals and surveillance of the different variants of avian and swine flu, Goecke recognized an opportunity to improve the current sample analysis process, replacing a conventional low-throughput real-time PCR platform with a system that could handle a much higher capacity at a much lower cost.

The expense of the conventional system bled from the institute to the farmers. A high cost per sample due to the use of a larger volume of expensive reagents led many farmers to minimize their sample submissions, which in turn led to fewer animals getting diagnosed and a higher rate of uncontrolled disease spread.

This lack of pathogen identification resulted in the use of less specific treatments, the overuse of antibiotics and suboptimal vaccination programs since veterinarians did not have the right basis for which to correctly treat and target a specific virus or bacteria.

To address these challenges, Goecke developed a new approach to sample analysis and monitoring based on a high-throughput real-time PCR system used for detection and typing of pathogens. The purpose of the new platform would be to reduce the cost of analysis per sample, improving the likelihood of increased submissions and furthering the lab’s ability to test more samples and identify a wider variety of pathogens at once.

She chose to work with the microfluidics-based Biomark™ HD platform, with the ability to use nanoliter volumes of samples and reagents and run up to 9,216 individual reactions simultaneously. The chip-based format also allows for multiple configurations, such as 48 samples with 48 assays, 96 samples with 96 assays, 192 samples with 24 assays or 24 samples with 192 assays.

“A big benefit of this system is that you can easily add and remove assays since each one is run independently. You just have to ensure the assays can be run under the same PCR conditions and then can quickly move forward,” Goecke explains.

Once developed, the team at the Danish National Veterinary Institute applied this new system to the monitoring of Danish pig and cow health to gauge its performance and useability in the field.

High-throughput disease detection and monitoring

Goecke developed two high-throughput real-time PCR assays, one to detect all relevant pathogens causing respiratory and intestinal diseases in pigs and the other covering the same in calves. The capability to include so many viruses and bacteria in each assay reduced the risk of overlooking pathogens that could then spread among the herds.

The porcine-focused system was applied to 10 Danish pig production units over three months and three different age groups. The pigs were also tested monthly and monitored for clinical symptoms to determine if pathogen monitoring at the herd level could be a supportive tool for veterinarians to create a more objective basis for intervention. Results from the study showed a significant correlation between clinical symptoms and associated detected infection.

Based on this study, a new health monitoring system was created, called SOS (Swine Objective Surveillance), for systematic sampling and implementation of common guidelines for maintaining herd health. The system has been successfully running since February 2020, providing an overview of pathogens circulating in different age groups and serving as an efficient health check that keeps farmers one step ahead and enables the correct use of antibiotics and vaccines.

The bovine-focused system, developed in connection with the robust calves project, was used to analyze thousands of samples collected from 100 Danish herds, with results correlated to clinical symptoms. The breadth of the system enabled the team to better understand pathogens that are most prevalent in a given herd or region, and also allowed a broader analysis of emerging pathogens and where they might have originated from. For example, influenza D virus is an emerging virus in Denmark, having been detected for the first time in 2011 in the US. Implementation of the monitoring system uncovered new circulation of influenza D virus in certain herds, enabling the team to alert veterinary professionals and farmers of its presence and what to watch for in order to help prevent and identify outbreaks.

The projects also educated the teams on managing vaccination. When a handful of calves tested positive for a certain virus [bovine respiratory syncytial virus (BRSV)] but presented no symptoms, it was determined that the samples tested showed high similarity to the vaccine strain used in recent vaccination of the herd. This finding is important because vaccinated calves can excrete the virus for up to 12 days after vaccination, something to watch for when vaccinating with live viruses.

Creating a positive impact

This new capability to closely monitor multiple pathogens generates information on their distribution in a healthy herd and in an outbreak situation to create benchmarks and develop more specific treatments. These studies utilizing Goecke’s high-throughput system for pathogen detection demonstrate the possibilities for rapid and thorough analysis of bovine and porcine samples with the flexibility to use a variety of sample types. “The system enables testing of multiple samples for the presence of different pathogens in the same analysis and with reduced costs and turnover time,” Goecke says. “This not only enables continuous monitoring of pathogens and the dynamics of infections in herd animals, but also provides caretakers with an objective knowledge on the distribution of pathogens in the herd.”