By Geoff Geddes
Editor’s note: The following piece was written for Swine Innovation Porc. For more information, contact Leslie Walsh at lwalsh@swineinnovationporc.ca.
If we diagnosed African Swine Fever (ASF) like we do the common cold, you could just look for pigs that are sneezing or blowing their nose. Unfortunately, ASF is much harder to detect and far more lethal for the hog industry.
In light of that reality, the timing was perfect for a presentation on ASF diagnosis during the 2021 Banff Pork Seminar. The talk was one of six that comprised an ASF webinar presented by Swine Innovation Porc (SIP) called, “African Swine Fever: How is Canada Getting Prepared?”
Using their virtual platform, swine sector experts offered a glimpse of what the pork industry is doing to prevent and prepare for an outbreak of ASF in Canada. Moderated by Stewart Cressman, Chair, SIP, the session included information on ASF research priorities in Canada, risk mitigation, emergency depopulation preparedness, research activities in the U.S. and Canada’s efforts to face the threat of ASF.
“My objective is to provide a brief overview of our efforts to evaluate alternative sample types for ASF diagnosis,” said Aruna Ambagala, a research scientist with the Canadian Food Inspection Agency’s (CFIA) National Centre for Foreign Animal Disease (NCFAD) in Winnipeg. Ambagala is head of the Mammalian Disease Unit and the World Organisation for Animal Health (OIE) Reference Lab for Classical Swine Fever (CSF) at the NCFAD.
“Because of the non-specific clinical signs, laboratory diagnosis is essential for ASF in Canada and the U.S.,” said Ambagala. “We have state-of-the-art diagnostics, but we continue to work on improving them as we go.”
How ASF is detected in carcasses
In regard to ASF diagnosis, there are two approaches: detecting the agent, which is the virus, and looking at the antibodies to the virus.
“With the agent, we are examining the genomic material of ASF, which you can do using a real-time PCR screening test available at NCFAD and at labs that are part of the Canadian Animal Health Surveillance Network,” said Ambagala.
The Canadian Animal Health Surveillance Network (CAHSN) is a network of federal, provincial and university animal health laboratories across Canada. Used to detect ongoing outbreaks, this test spots the presence of ASF in an animal and takes two to four hours once the samples are received in the lab. The initial result will be confirmed within 24 hours by sequencing, followed by virus isolation and whole genome sequencing, all at NCFAD. Virus isolation can take up to 15 days to complete.
“The other method of ASF diagnosis is scrutinizing the antibodies for evidence of past exposure to the virus,” said Ambagala. “You can see the antibodies for months or years afterwards in the serum samples through an enzyme-linked immunosorbent assay test at NCFAD.”
The enzyme-linked immunosorbent assay (ELISA) is an immunological assay commonly used to measure antibodies, antigens, proteins and glycoproteins in biological samples. NCFAD can handle a large number of samples, and because the specificity of the test is not optimal, they must perform complementary tests to augment it. Samples currently accepted for these additional tests can be whole blood and serum samples.
“If a pig is dead, we request a full post-mortem and submit tissue samples such as tonsils, spleen, liver, kidney or lung,” said Ambagala. “The most important sample for detection of ASF and other viruses like CSF are the tonsils, and bone marrow is requested if the carcass is decomposed.”
Test limitations and work-arounds
Of course, there are limitations to these sampling efforts. Early detection of ASF is critical, and diagnosis based on clinical signs is difficult. This is especially true during initial stages of the infection, when symptoms may not be an obvious indicator of the virus.
“In light of the limitations, we need to maintain some sort of surveillance program. This can be problematic, as individual sampling is quite labour-intensive and costly when you need to handle individual animals or conduct a full post-mortem to collect samples,” said Ambagala.
To overcome the cost and time hurdles, researchers are exploring alternative sample types for two different scenarios: groups of live animals and herds that have a high mortality rate.
“For group samples, we are looking at oral fluids and processing fluids,” said Ambagala. “In the case of samples from carcasses, we need ones that can be collected quickly and safely from dead pigs without a full post-mortem.”
All samples must be validated and compared to the ‘gold standard’ sampling types that include whole blood, tonsils and spleen. To do so, scientists examine the viral load of the samples as determined by real-time PCR and virus isolation.
Liquid gold: oral fluids test best
What makes oral fluids attractive for sampling?
“Oral fluids are a mixture of saliva coming from major and minor salivary glands, so they are a very rich sample that we can test for both pathogens and antibodies,” said Ambagala. “These fluids have also been proven to detect Porcine Reproductive and Respiratory Syndrome [PRRS], Porcine Circovirus type 2 [PCV2] and Swine Influenza Virus [SIV]. Multiple research projects confirm that the ASF genome and antibodies can be detected in oral fluids upon infection.”
Additionally, oral fluids are an easy sample to collect. Pigs are naturally attracted to a hanging rope, and once they chew on it for 30 minutes, the sample can be taken. Of particular interest to both farmers and consumers, collection of oral fluids is non-invasive, humane and feasible to do on-farm.
Validation of oral fluids is necessary
Like other sample types, oral fluid testing must be validated to confirm its efficacy. NCFAD, in collaboration with the Plum Island Animal Disease Center of New York, is performing experimental inoculations to show that ASF can be detected in oral fluids early enough to be used for surveillance.
“In a preliminary experiment at NCFAD, we inoculated four pigs in a pen with ASF and found we could identify the oral fluid before the animals showed real clinical signs,” said Ambagala. “We then repeated that experiment on a larger scale with two tests at NCFAD and two at Plum Island. We used ASFV Georgia – the strain of the virus currently circulating in Europe and Asia – at NCFAD, and we used Malta/78 ASFV – a moderately virulent strain – at Plum Island.”
The researchers then introduced an infected pig into a larger pen of 24 pigs and collected daily samples from all animals, observing how the virus spread in the pen and how early they could detect it in oral fluids. The results confirmed the power and potential of oral fluids in relation to ASF.
“The specific purpose of the oral fluid would be for early detection of ASF in commercial herds and, if all goes well, could be considered for claiming disease freedom in compartments during an outbreak,” said Ambagala.
Going forward, the plan is to conduct field evaluations on oral fluids in Vietnam, and the researchers are currently running some additional evaluations in the lab.
“We are also looking in more detail at the diagnostic sensitivity, specificity and measurement of uncertainty of the oral fluid samples, and we are trying different instrumentation and technicians. Because these samples can be tricky to work with, we are planning to conduct a reproducibility experiment where we test the same oral fluid samples at two labs to ensure our detection is accurate,” said Ambagala.
At the same time, the researchers are collaborating with Iowa State University to improve the sensitivity of ASF detection in oral fluids. They will soon be starting another project with Kansas State University that evaluates commercial PCR kits for spotting ASF in those fluids.
Additional detection method options
As a second option for detecting ASF, researchers are examining processing fluid, which is the serosanquinous fluid collected at routine castration and tail docking between the ages of four and 14 days. Serosanguinous fluids are composed of clotted or diluted red blood cells mixed with serum.
“With processing fluids, we are targeting breeding herds and suckling pigs – two groups that are not well suited to oral fluid testing, because they won’t go after the rope that is used to collect oral fluids,” said Ambagala. “If we can target these groups with this sample type, we will expand our ability to screen our swine herds, doing so with much less strain on producer finances and human resources.”
A third alternative to routine post-mortem sample types is the use of superficial lymph nodes. This sample can be easily collected with little to no bleeding and minimal environmental contamination, takes just one or two minutes to collect and is readily accessible. Initial testing with lymph nodes shows great promise for detection of ASF, and scientists plan to expand this work.
As detection improves, so does industry resiliency
Detecting ASF is one piece of the puzzle that should help Canada put up a strong defense against the virus. Engaging stakeholders from across the value chain and continuing to share information is the industry’s best chance at successfully remaining ASF-free.
As Stewart Cressman observed following the presentation, this project “highlights the collaboration that exists on these files across international borders.”
ASF detection may never be as simple as diagnosing the common cold, but if it can be done cheaply, easily and in a humane manner, that is nothing to sneeze at.
