Genetic diversity, changing clinical picture make IAV-S detection challenging
By Phillip Gauger, DVM, MS, PhD
Iowa State University
Influenza A virus in swine (IAV-S) remains among the top health challenges facing the US swine industry and, worse yet, it may be on the increase.1
Based on porcine respiratory samples submitted to the Iowa State University’s Veterinary Diagnostic Laboratory (VDL), the number of influenza-associated swine respiratory cases has been rising over the past 7 years (Figure 1).
IAV-S is currently the second-most common cause of porcine respiratory disease diagnosed as a single agent or co-infection at the VDL.2 This is based on detection of the virus and associated microscopic lung lesions (Figure 2). As pork producers are well aware, IAV-S is also an important component of porcine respiratory disease complex.
The three IAV-S subtypes endemic in swine around the world are H1N1, H1N2 and H3N2. Although influenza is a zoonotic disease that can be transmitted from swine to humans, let’s remember that human-origin influenza viruses can be transmitted to swine. The first H1N1 in US swine, isolated from pigs in 1930, originated from the 1918 human Spanish flu pandemic.3 H3N2 is a reassortant IAV-S that was initially transmitted from humans to swine in 1998.4
Since that time, human-influenza viruses have continued to influence the IAV-S strains circulating in swine, including the 2009 H1N1 pandemic in people that was subsequently transmitted to pigs.5,6
As additional IAVs have been transmitted from people to pigs, genetic reassortment and mutations have contributed to a remarkable increase in the number of genetically different strains circulating in swine.7
A few factors that make the situation more complicated:
- The virus is transmitted easily.
- Large numbers of pigs are moved long distances for feeding and processing, enabling transmission of endemic IAV-S strains to other groups of pigs at distant locations.
- There are a few pig farms in regions with low pig density that have been able to prevent influenza in their herds, but the infection is otherwise considered ubiquitous and can be detected in most US swine.
Clinical presentation can differ
Many of us in the pork industry remember the epidemic influenza infections that were common years ago. Pigs presented with a deep, paroxysmal or “barking” cough that quickly affected most pigs in the herd. There was concurrent respiratory distress, or “thumping,” due to the severity of the pneumonia. Morbidity was usually high, but death loss was low and pigs quickly recovered as immunity kicked in and the infection cleared.
Today, the typical clinical expression of influenza varies. Pigs may present with subtle, less-severe clinical signs characterized by mild coughing that may be influenced by the strain of IAV or the immunity of the affected population.
Any age of swine can become infected with IAV, including suckling piglets. IAV-S infection at a young age may present as a subtle but persistent, nagging cough in a low percentage of litters scattered throughout the farrowing room. Producers don’t typically associate this type of cough with influenza, and the disease may be overlooked or not included on their list of differentials. When the same group of piglets is weaned and moved to the nursery, the cough may become more prevalent as the virus is transmitted to susceptible piglets at the same time maternal antibody wanes.
In other cases, declining maternal antibody in the group can result in predictable influenza outbreaks that occur during the late-nursery or early-grower production phase. The clinical expression of influenza may be less dramatic at this age but can persist into the finisher phase, moving slowly throughout the group — unlike the rapid onset and clearance observed years ago. Despite the clinical presentation of IAV-S, production and economic losses are significant.
The high prevalence of IAV-S throughout the US and its ease of transmission make the infection difficult to prevent with biosecurity alone. In addition, the genetic diversity of IAV-S has sometimes made it difficult for commercial IAV-S vaccines to keep up with circulating strains of the virus and is why producers may use autogenous vaccines. More recently, however, the industry has access to commercial vaccines that contain four to five strains of IAV-S, which is considered an important development.8
Detecting active infection
There’s no question the presence of new or emerging strains and the variable clinical presentations of the disease make detection, diagnosis and monitoring of IAV-S more challenging.
Detecting active infection and diagnosing clinical disease associated with IAV-S can be difficult due to the relatively narrow time course of infection or due to the low prevalence of IAV-S that may be present in a population. In addition, choosing which pigs to sample and proper sample collection are incredibly important, which is why a veterinarian should be involved to ensure the most appropriate procedures are used.
Samples appropriate for detecting IAV-S in live pigs — antemortem samples — include individual nasal swabs or snout wipes.
I’m often asked what type of swabs to use for detecting a virus such as IAV. Diagnostic labs prefer a synthetic swab (polyester, rayon or dacron) with a plastic shaft. About 1 to 2 ml of a liquid transport media is necessary to prevent desiccation of the swab. Don’t use a gel-based transport media.
Snout wipes are less commonly used but when they are, 2-inch x 2-inch cotton gauze is wiped across the nose of individual pigs and placed into 5 mls of liquid transport media. Try not to cross-contaminate the wipes between pigs to maintain the integrity of the sample.
Detecting influenza in swine is most productive during the early course of infection, when pigs are shedding the most virus. Clinical disease and shedding start 24 hours after infection and last for only about 5 to 8 days. The transmission of IAV-S through a population of pigs occurs at a variable rate depending on the age of pigs, the IAV-S strain, prior immunity, the presence of co-infections and other environmental factors.
When collecting individual samples from a population of nursery or grow/finish pigs, it’s important to select animals that represent the clinical signs of the population. For instance, if respiratory disease is observed, collect samples from pigs that appear lethargic and coughing. Samples should be collected from at least 10 to 30 affected pigs per room or barn, depending on the expected prevalence.
However, selecting the appropriate pig for sampling is difficult when respiratory disease is subtle, as is typical of suckling pigs that have a mild, periodic but nagging cough in the farrowing house. In these cases, I’ve had success detecting influenza in nasal swabs from affected suckling pigs with a fever (104° F or 40° C) or by collecting samples from the entire affected litter rather than focusing only on individual coughing pigs, which occurs when there’s already been lung damage and virus shedding may have started to diminish.
The prevalence of IAV-S is low in suckling pigs — often 2% or less — so samples need to be collected from a large number of piglets.9 Nasal swabs should be collected from at least 60 pigs per farrowing room. Some diagnostic tests can be pooled in groups of five to reduce the testing cost.
Oral fluid sampling
Oral fluid samples are also appropriate for population monitoring of nursery, grower or finisher pigs, and it’s a noninvasive method, which is a plus. It’s generally easy to perform, although collecting oral fluids from pigs younger than 3 weeks of age (suckling pigs) may be difficult and require training to be successful.
A 5/8-inch cotton rope is hung in a pen at shoulder height of the pigs, which are allowed to chew on strands of the rope for 20 to 30 minutes. The end of the rope is placed in a plastic bag and squeezed to allow the fluids to collect into the bottom of the bag. The saliva is then poured into a conical tube that should be tightly secured to prevent leakage during transport to a lab. Samples must be handled with great care to prevent cross-contamination. They should be chilled as quickly as possible after collection and shipped with ice packs.
Oral fluids have the advantage of representing several pigs in a pen/population that chewed on strands of the cotton rope. This helps detect IAV-S that may be circulating unevenly in pigs within the population.
Oral fluids should be collected when clinical signs are present, using one rope per 150 animals, and the ropes should be evenly spaced apart throughout the barn.
Finally, if mortality occurs or pigs can be euthanized, postmortem samples can be collected. Fresh lung samples are submitted for virus detection but only from pigs that aren’t chronically affected with co-infections and preferably from pigs that haven’t been treated with antibiotics. Figure 3 describes the appropriate sampling types for detection of IAV-S.
| Nasal discharge
|1 – 5 Days
|6 – 8 Days
|10 – 14 Days
Figure 3. Sampling types for detection of IAV-S at different stages of the disease
Serology for the diagnosis of IAV-S is of questionable value due to the endemic status of IAV-S and the use of vaccines. For example, ELISA tests (enzyme-linked immunosorbent assays) will not differentiate between antibodies induced by natural infection, vaccines or maternal antibody.10 Therefore, ELISA tests should be used for specific diagnostic questions that are needed to confirm the presence of IAV-S antibody.
Another serological test is hemagglutination inhibition, also known as HI. It also detects antibodies to IAV-S that are good indicators of protection. Unfortunately, IAV-S diversity has made HI difficult to interpret because different strains of IAV-S may not cross-react in this type of test. Therefore, it’s important to test serum from swine using an IAV-S that is either circulating in the herd or used in the farm-specific vaccine, if appropriate. Field samples need to be tested against a panel of IAV-S strains representing different H1 and H3 viruses to improve test sensitivity.
To monitor IAV-S in a herd, polymerase chain reaction testing is performed with genetic sequencing on positive samples, which will not only reveal whether IAV-S is present but also pinpoint the circulating subtype and genetic cluster. It is important for veterinarians and producers to know which virus is circulating in their swine, which has increased the need for genetic sequencing.
Whatever tests a producer and veterinarian decide upon, it’s important to maintain records of the results for comparison with future tests. You’ll want to know if the circulating IAV-S virus has changed, which obviously will affect the vaccination plan.
Influenza has been recognized as a respiratory pathogen in swine for over 90 years. Although the virus continues to change and IAV-S management remains a huge challenge for the swine industry, diagnostic methods continue to improve.
In the future, new vaccine platforms are likely to be approved that may significantly affect our ability to control IAV in swine. In addition, veterinarians and swine producers will need to monitor the IAV-S circulating in their production systems to detect new or emerging strains of the virus so that vaccines can be updated when necessary. Participation in the USDA IAV-surveillance program is one method available for producers that has helped monitor the presence of IAV-S in US swine.
Editor’s note: The opinions and recommendations presented in this article are the author’s and are not necessarily shared by the editors of Pig Health Today or its sponsor.
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2. Personal communication between Dr. Gauger and the VDL.
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Posted on January 4, 2018