Selecting the right test is important for obtaining accurate results when attempting to diagnose influenza or other diseases in pigs. Choosing the right sample and test depends on the timing of sample collection relative to when the pigs were infected. Since we don’t know precisely when individual pigs become infected, we have to rely on clinical signs to provide some clues to guide our approach.
A generic timeline for the clinical course of a respiratory disease is shown in Figure 1. The timing of this clinical course varies for different swine diseases. For example, the starting and ending of the clinical signs can occur at quite different times depending on the disease. Understanding the specific dynamics of the clinical course for a particular disease is an important first step in developing a diagnostic plan.
There are several terms that we use to describe the different phases of the clinical infection. The term "antigen" refers to the disease agent (e.g. bacteria or virus) that causes the disease. In the case of swine influenza, the antigen is the influenza virus.
Immediately after infection, the antigen cannot be detected because it hasn’t multiplied in the host yet. This phase before the antigen can be detected is called the "latent" period. In the case of influenza virus in pigs, the latent period can range from a few hours to a few days on an individual pig basis.
This phase can also be called the "prepatent" period, but that term is usually reserved for the same phase in parasite infestations. The latent period should not be confused with another term – “latency” – that applies to a unique dormant period after the patent period for herpes viruses when the virus is still present, no longer active, but still capable of becoming active again (called recrudescence) under certain circumstances.
The patent period is the phase when the antigen is present and can be detected. This is significant because detecting and, in particular, culturing virus is sometimes important. The patent period is over once the virus has been cleared and can no longer be detected. In the case of influenza virus, this is a relatively short period, lasting only three or four days on an individual pig basis.
"Antibodies" are produced by the body in response to immune stimulation by antigens. We can detect antibodies in serum samples by taking blood samples from exposed pigs. It takes a while for the body to develop antibodies to detectable levels. When antibodies have developed to detectable levels, we say the animal has seroconverted. In the case of influenza, seroconversion takes place at a minimum of 9-14 days after infection.
Antibodies can be present from maternal colostrum or arise from exposure to natural infection or vaccines. Knowing the age of the pig and vaccine and clinical histories are important for being able to interpret serology test results.
There is also a lag period following infection prior to the onset of clinical signs. This phase before clinical signs develop is called the "incubation" period. Clinical signs can be highly variable depending on the strain of virus, immune status of the pig and presence of other disease agents in the pigs. The incubation period isn’t always the same as the latent period. However, for most diseases, including influenza virus, there is a high correlation between the phase when fever is present and the patent period, the time when virus can be detected. If you’re trying to find flu virus, look for pigs with a fever and you will increase your odds considerably. Other clinical signs such as cough can linger for an extended period, especially if there are other respiratory disease agents present in the herd.
The timing and severity of when lesions occur can also be variable, depending on the same factors listed for clinical signs. The gross lung lesions can look quite similar to lesions caused by other respiratory diseases, so the initial appearance of the lung is not a very specific diagnostic tool. The microscopic lesions observed in lung sections with gross lesions that were fixed in formalin are more specific for influenza virus.
Influenza Virus Diagnostic Approach
The diagnostic approach for swine influenza depends on what you are trying to accomplish and when pigs were exposed and infected. If it has been a few weeks since the infection was believed to have occurred, serology is really the only tool available because the virus would have been cleared and the lesions resolved.
If there is active clinical disease currently in the herd, there are more options, again depending on the objective. If the objective is to detect the virus, using polymerase chain reaction (PCR) tests on oral fluid, nasal swab or lung samples from sick pigs will usually be the best course. We don’t absolutely need live virus to characterize the virus. Subtype testing and some virus sequencing can be done on the product produced in a positive PCR test reaction. Sequencing the virus is more likely to be successful on virus that has been isolated.
If there is a need for virus isolation (such as for use in a vaccine or for virus sequencing), however, lung or nasal swab samples are better because the rate of successful virus isolation from oral fluid samples is relatively low at this time (Table 1). Efforts are underway to improve the performance of influenza virus isolation on oral fluid samples, so this may change in the near future.
In a study reported this summer by Romagosa and others on pigs infected with influenza experimentally, “the probability of detecting influenza virus in oral fluids was 99% when within pen prevalence was higher than 18% and decreased to 69% when prevalence was 9%. Results indicated that pen-based collection of oral fluids is a sensitive method to detect influenza even when within pen prevalence is low and when pigs have been vaccinated, and highlight the potential use of oral fluids for influenza surveillance.”
Oral fluids are an excellent screening tool, but virus isolation and more sophisticated characterization are best done on samples collected as part of a thorough postmortem examination from euthanized pigs that were exhibiting clinical signs of respiratory disease or part of the susceptible population.
Reference: Romagosa A, Gramer M, Joo HS, Torremorell M. Sensitivity of oral fluids for detecting influenza A virus in populations of vaccinated and non-vaccinated pigs. Influenza Other Respi Viruses. 2011 Jul 21. doi: 10.1111/j.1750-2659.2011.00276.x.
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Jerry Torrison, DVM; Marie Gramer, DVM
University of Minnesota Veterinary Diagnostic Laboratory