Diagnostic assays or tests are tools used by producers and veterinarians to assess the disease and immune status of pigs.
Depending on the specific diagnostic assay, laboratory tests detect antibodies or proteins that are produced in large quantities by the pig in an immune response to an infection or the presence of an infectious agent.
Immunity is essentially the pig's resistance to infection. Diagnostic assays do not measure immunity. Rather, they look for evidence of disease exposure in response to vaccination, antibodies or infectious agents.
In terms of immunity, the underlying assumption is that prior infection or vaccination has stimulated an immune response that will provide some level of protection from disease symptoms in the future.
For these reasons, diagnostic tests are direct measures of exposure or vaccination, and indirect measures of immunity.
Population Measures of Immunity
Population testing provides a direct measure of the disease exposure pattern (or vaccination coverage) in a herd and an indirect measure of the herd's level of immunity. Population sampling over time provides a picture of the dynamics of infection in a herd and the basis for making sound management decisions regarding prevention and control of disease.
Sampling the same pigs over time, or taking samples from pigs of different ages within a production system, can provide useful information on the dynamic interaction between infection and immunity within the herd.
Understanding the rate at which a disease changes in a herd can help strategically plan the timing of therapeutic interventions, such as vaccination or medication.
For example, repeated samples can be used to determine when the levels of maternal antibodies in young pigs have declined to the point that they will no longer interfere with the pigs' immune response to vaccination.
Tests Not Perfect
While the use of diagnostic assays is helpful in understanding and identifying the disease exposure or vaccination success in a herd, the interpretation of test results can sometimes be perplexing.
The major source of confusion in test interpretation is the false belief that a diagnostic test is perfect. In fact, every test has some inherent level of error.
This just means that, like every other tool in your toolbox, you need to be aware of the limitations and appropriate applications of diagnostic tests. Even if the level of test error is low, it will inevitably occur, and some day an animal you expect to be negative will test positive, or vice versa. Understanding how the tests work can eliminate some of this confusion.
Understanding the Tests
Diagnostic assays either measure the immune response induced by infection or detect the pathogen itself. Most common diagnostic assays are based on detection of antibodies specific to the pathogen. Typically, serum is used as the sample, although thoracic fluid and colostrum are also used.
Antibody detection assays have distinct advantages in detection of a pathogen. The sample (usually blood) is easy to collect, handle, and store; antibodies are abundant in blood and can usually be detected for a long time after infection. With most diseases, antibodies are detectable long after the pathogen is not.
The pathogen may be detected by isolation (growing it in the laboratory), staining and visualization by microscopy. Recently, highly sensitive polymerase chain reaction- (PCR) based assays have become available. The assays differ from earlier tests in that they detect the genetic material of the pathogen, are generally able to detect low numbers of the pathogen, and are not limited by the requirement to grow the agent.
Regardless of the approach — antibody detection or pathogen detection — it is important to know the limitations of each assay and care must be taken to not over-interpret results.
Antibody Detection (Serology)
Serological assays are the most common diagnostic tool used in swine population medicine. When a pig is infected with a pathogen, the immune system responds to the invasion by activating specific immune cells and producing antibodies. Serology is based on antibodies detected in the bloodstream.
Many different antibodies are produced during an infection. Some are highly effective against the infectious organism; others are not. Therefore, the presence of antibodies indicates exposure to an organism, not necessarily protection against disease.
At best, diagnostic assays provide indirect evidence of prior exposure to an organism. Time between exposure to a pathogen and detectable levels of antibodies depends on the pathogen, the assay, and the ability of the immune system to respond.
For example, an organism like Mycoplasmal pneumonia, which is in the airway of the pig and not in close contact with the immune system, may take weeks to months to produce a measurable antibody response.
In contrast, swine influenza virus can induce the production of antibodies as quickly as five days after exposure.
Antibodies induced by vaccination are usually measurable within 10-14 days.
Single serum samples are a poor measure of herd health status. Sequential sampling, including samples collected several weeks apart, provides information on time of exposure to a pathogen and the level of disease within a herd. With a few exceptions, serological assays can't differentiate vaccination from infection.
An example of “differentiable” vaccine is the pseudorabies (PRV) vaccines developed in the 1980s and 1990s. The fact that antibodies against these vaccines could be differentiated from infection with wild-type virus greatly facilitated PRV eradication.
Assays Detect Pathogens
Antibodies often linger after the disease is gone. Antibody detection isn't solid proof that the pathogen producing the antibodies is linked to the latest disease problem.
In contrast, detection of a pathogen along with clinical disease is strong proof of its involvement.
Several different approaches for pathogen detection are available:
Fluorescent antibody and immunohistochemistry (IHC) combine immunology and pathology to detect pathogenic microoganisms in tissues. Most disease changes in tissues aren't unique to specific infectious agents.
However, pathologists can combine microscopic examination with the use of antibodies to confirm the presence of specific pathogens. In these procedures, the antibody is attached to the infectious agent, then the antibody is detected by reacting it with a fluorescent dye or other color-coded tag.
These assays are extremely useful because they are able to confirm the association of the pathogen within the diseased tissues — strong evidence that the pathogen caused the disease.
Polymerase chain reaction (PCR) has revolutionized our ability to directly detect pathogens. PCR assays detect the genetic material (DNA or RNA) of a pathogen. Unlike isolation/identification tests, PCR can detect an organism whether it is infectious or dead. By itself, PCR is merely a procedure to replicate nucleic acid. The entire process of extracting the genetic material, replicating the nucleic acid, and detecting the PCR product makes it a diagnostic assay.
PCR-based assays have been rapidly adapted to the detection of animal health pathogens. This is an area of very active research and refinement.
Currently, PCR can be used as a qualitative (Yes/No) assay for the presence of an organism, or as a quantitative assay for measuring the number of organisms present. Two of the more common assay formats are “nested” PCR and real-time PCR.
Discuss strengths and weakness of PCR assays with your veterinarian or laboratory diagnostician.
As with all tests, PCR-based assays aren't perfect. Both false negative and false positive results occur. In addition, unlike serology, PCR-based assays are highly “sample-dependent.” Different samples from the same animal can give very different results.
For example, a serum sample from a porcine reproductive and respiratory syndrome (PRRS) virus-infected boar may be PCR negative, while a semen sample from the same animal is positive. This occurs because the infectious agent targeted by a PCR-based assay isn't uniformly distributed throughout the body. For that reason, understanding the biology of the infectious agent is extremely important.
Restriction fragment length polymorphism (RFLP) is an assay sometimes used to compare the genetic makeup of a pathogen. Its most common use in swine health has been to compare PRRS viruses in epidemiological (presence of disease in a population) studies. The assay is based on the use of several enzymes that cut the viral genome at very specific genetic sequences.
In PRRS virus, the pattern of fragments gives a three-numbered result, 2-5-2, for example. Interpretation of RFLP results assumes that viruses with similar fragment patterns are genetically similar. However, no association has been shown between specific PRRS virus RFLP patterns and the severity of clinical disease. To a large degree, the use of RFLP is being replaced by genetic sequencing.
Genetic sequencing produces the genetic code of a pathogen for a specific segment of its genetic material. At present, sequencing is used primarily with viruses and is available through most diagnostic laboratories. When used with computer “free-ware” on the Internet, the genetic makeup of different virus isolates can be compared and/or genetic changes in isolates from a herd can be tracked over time.
But, sequencing has limitations, too. For most viruses, the exact genetic sequence responsible for virulence is unknown. Also, only a small portion of the virus' genetic material is sequenced and potentially important differences are not identified.
Sequencing can be helpful in tracking viral changes on an individual farm over time. But until the clinical significance of the genetic code is better understood, interpretation of sequences and application sequence results to intervention strategies will be problematic.
Five Steps to Monitoring Herd Immunity
Designing a plan to monitor herd immunity has never been easy. But it has become more complex as herds get larger and more highly stratified.
Designing a monitoring plan involves working through specific steps:
- Have a specific purpose for monitoring
The purpose may be to test for porcine reproductive and respiratory syndrome- (PRRS) induced disease by monitoring sow herd immunity, or to determine when to vaccinate young pigs by monitoring interfering maternal antibody levels. Whatever the purpose, you should be able to state it clearly, in a single, short sentence.
- Tailor the sampling plan to your purpose
In general, this involves determining how many animals will be tested, how frequently samples will be collected, and which test (s) will be run. The issues of sample number and sampling frequency are complex. A discussion of these issues in the 2003 PRRS Compendium (to be released by the National Pork Board in June 2003) may be helpful.
- Be prepared to interpret the results
Understand how the test works and what it measures. Some degree of variation in test results is expected, regardless of the laboratory. Establish a relationship with laboratory personnel and use their insights.
- Be prepared to respond to the results
Because bad things can happen to good people, you should have an action plan in place before you submit the samples.
- Put a pencil to it
Can the expense of monitoring be justified? Specifically, are the losses that will be avoided greater than the expense of monitoring? If the answer is “no”, then re-think your approach.