There is a wide variety of diagnostic tests available to practitioners for infectious and immune-mediated diseases.
There is a wide variety of diagnostic tests available to practitioners for infectious and immune-mediated diseases. Many of these tests detect antibodies, which are produced by the immune system in response to stimulation. However, misunderstandings and controversies sometimes arise when antibody tests are interpreted. Antigen tests would seem more straightforward but also may cause confusion. The following is a review of how the immune system reacts to potential pathogens along with information on the use (and misuse) of some of the diagnostic tests.
To protect against microbial invasion, animals have three basic defenses: physical barriers, innate immunity, and acquired immunity. Barriers include the skin, self-cleaning processes such as coughing and urination, and normal flora. Innate immunity is the second layer of defense, and is a quick response system for destroying invaders that are recognized as foreign. Inflammation, defensins, and lysozymes are features of innate immunity and are mediated by macrophages, dendritic cells, and neutrophils. Acquired (specific) immunity is the "smart" system that can recognize and remember invaders. It has a slower onset than the innate system but is effective for a longer period of time due to immunologic "memory". The acquired immune system can be divided into humoral (antibodies in body fluids that destroy pathogens) and cell-mediated (specialized immune cells that seek out and destroy other infected cells). Diagnostic immunology generally focuses on the antibody response to infection.
Lymphocytes originate from stem cells and are processed in primary lymphoid organs. Lymphocytes mature into cells with specific functions: B cells, T cells, and NK (natural killer) cells. B cells are found in the spleen, bone marrow, Peyer's patches (lymphoid organs in the wall of the small intestine), and mainly in lymph nodes. Very few B cells circulate in the bloodstream. Each B cell has a large number of identical receptors and can only bind and respond to a single antigen (Ag). When the receptors bind an antigen, the B cell produces antibodies (Ab) specifically to bind and help destroy the invader. Co-stimulation of B cells leads to maturation into plasma cells, which are simply B cells terminally differentiated into Ab "factories".
Antibodies are proteins secreted by the plasma cells. They are classified as immunoglobulins, and each plasma cell can manufacture and release up to 10,000 molecules per second. The lifespan of the cells varies from a few days to over a year, but even after an Ab-secreting plasma cell dies the antibodies persist until slowly catabolized. Long-lived plasma cells are a form of "memory" cells and are found in lymph nodes and the bone marrow. If the animal is exposed to the same Ag, a rapid secondary immune response occurs with a shorter lag period due to memory. This explains why antibodies can be detected many years after an infection or vaccination, and theoretically some memory cells persist for the life of the animal.
Antibodies are classified into 5 classes (isotypes): IgG, IgM, IgA, IgE, and IgD. IgG plays the major role and is found in the highest concentrations in blood. IgM has the second highest concentration and is secreted first (primary immune response) by plasma cells when Ag is recognized. IgA is secreted at epithelial surfaces and helps with local immunity in the respiratory, GI, urinary, and other body systems. IgE is primarily bound to mast cells and basophils and only circulates in very low concentrations. IgD has been identified in some but not all mammals. For diagnostic purposes, it is important to remember that IgM is secreted early in an immune response (days to weeks) and then wanes, while IgG is slower to be produced (usually several weeks) and persists much longer.
Laboratory assays can use specific Ab to detect an antigen, either associated with an infectious agent or an important molecule. Also, Ab can be detected to determine if an animal has been exposed to a certain organism. The measurement of Ag-Ab interactions for diagnostic purposes is called serology. Common tests include immunofluorescence (direct or indirect fluorescent antibody), enzyme-linked immunosorbent assay (ELISA), Western blotting, immunochromatography, precipitation, agglutination, and neutralization. With all tests, false negatives and false positives occur which can be reported as sensitivity and specificity. A test with high sensitivity has few false negatives, while a test with high specificity has few false positives. No test achieves 100% sensitivity and specificity. When using diagnostic tests on animals it is necessary to know the prevalence of the infection, which is used to calculate positive and negative predictive values. Prevalence data is largely unmeasured and unknown for diseases important to veterinarians due to the lack of a central reporting database.
An antibody titer is defined as the amount or concentration that gives a positive test result by using double dilutions. The higher the number, the more Ab is present which usually (but not always) correlates with infection and/or protection. One dilution higher or lower is not significant – for example, a titer reported as 1:400 means that the actual Ab level is between 1:200 and 1:800. As different laboratories use different assays, techniques, and reference ranges, it is impossible to directly compare results.
Antigens may also be detected with immunologic assays. Concentrations of antigen may be reported but more often "positive" and "negative" results are given. Nonserologic techniques, such as identification of organisms with cytology or culture, are often more diagnostic.
The following is not intended to be a comprehensive diagnostic guide, but instead as a description of important things to look for and how to avoid misinterpretation of results.
Neutralizing Ab is first detected 10-20 days post-infection. IgM increases first, followed by IgG. Therefore, an unvaccinated puppy with clinical signs suggestive of acute distemper will typically have a high IgM titer with a low or negative IgG titer. If the dog survives the disease, IgM levels decrease over 3 months while IgG increases and stays persistently high. Vaccination also stimulates Ab production, so a dog recently vaccinated for distemper may show a similar pattern. Fluorescent Ab testing may detect viral Ag in conjunctival swabs or blood smears, but only in the first 2-3 weeks of infection. If CSF is obtained, most but not all dogs will have a higher Ab titer in the CSF compared with serum. RT-PCR may be run on buffy coat cells, serum, blood, or CSF. Interpretation of a single high IgG titer is difficult – it can mean exposure to distemper, current or previous infection, or vaccination at any time in the past. A 4-fold or greater increase in antibody titers (IgM, IgG, or both) 2-3 weeks apart is supportive of exposure/infection but is also seen with recent vaccination.
Fecal Ag detection along with typical clinical signs and lab results (leukopenia) is the most common way to diagnose parvoviral enteritis. Within a few days of infection, dogs will shed viral Ag and ELISA tests are available in-clinic and at reference labs. Quantitation is not reported except for "weak" or "strong" positive based on color change. There is no correlation between the quantity of Ag and the severity or prognosis. False negatives occur if there is sufficient Ab production to bind Ag (since Ag/Ab complexes are not detected by test kits). False positives may be due to fecal shedding without true infection (exposure) or recent vaccination with a modified live virus. It would appear that vaccination leads to false positive (usually "weak" positive) results from 3-10 days later. Ab titers are not used for clinical diagnosis but may be measured (HI or VN assays) to indicate response to vaccine or exposure to the virus.
E. canis is difficult to identify and culture, so Ab testing is routinely performed to detect exposure. After a tick transmits the organisms to a dog, an Ab response occurs 7-28 days later, and if untreated Ab levels peak at around 80 days. IgG is typically measured with IFA testing. A negative titer does not rule out ehrlichiosis (in the early stages), and a positive titer does not diagnose the disease (as many dogs are exposed and maintain persistently high Ab levels). Recommendations in older literature suggested retesting Ab+ dogs from 6 weeks to 9 months after treatment to see if the organisms had been "cleared". More recent studies and clinical experience have demonstrated that positive Ab titers continue for many months and even years after exposure. There is apparently no clinical value in retesting Ab levels in dogs that have recovered from ehrlichiosis. An in-clinic ELISA test (SNAP 4Dx Test, IDEXX) is available for detection of Ab titers of 1:500 or greater, but if there are no clinical signs or laboratory abnormalities suggestive of ehrlichiosis, a positive test only means exposure (weeks, months, or years previously). Treatment is not indicated simply because a dog tests positive to antibodies. Western blotting may be used if ELISA Ab results are equivocal. PCR testing for the actual organism is done at some reference labs and should be more accurate than Ab tests. However, both false negative and false positive results are possible with PCR and the assay isn't standardized at all labs. Real-time PCR testing for E. canis is offered at some reference labs, often as part of a panel testing for other tick-borne organisms.
Previously known as E. equi, this organism is transmitted by Ixodes sp. ticks and is prevalent in areas that are endemic for Lyme disease. In some affected dogs, morulae can be found in neutrophils on blood smears. However, E. ewingii have similar morulae and cannot be differentiated. PCR testing is currently recommended for definitive diagnosis of the organism. Serology is available, and an in-clinic ELISA (SNAP 4Dx) detects antibodies qualitatively. Reference labs may use IFA or Western blot. Cross-reactivity occurs with A. platys, the organism that causes infectious cyclic thrombocytopenia in dogs. As with E. canis titers, a positive result only indicates previous exposure, not necessarily active or acute infection. A combination of clinical signs, laboratory findings (thrombocytopenia, lymphopenia, occasionally anemia) positive serology, and PCR testing is necessary to prove infection.
Rocky mountain spotted fever
As with E. canis, the rickettsial organism is not routinely identified or cultured. Diagnostic labs may offer IgM and IgG testing, and in an acute infection IgM levels are elevated. IgG levels rise more slowly, usually 2-3 weeks after infection. Paired titers that demonstrate a 4-fold or greater increase are considered diagnostic. After treatment or resolution of the infection, Ab titers decrease after 3-5 months. Real-time PCR tests are available at some reference labs.
In acute infections, spirochetes may be seen in urine samples (with darkfield microscopy) or cultured. However, there are false negatives and a delay in getting these results back. Ab testing by microscopic agglutination is the most common assay performed by reference labs. In the first 7-10 days after infection, Ab levels are zero or low, then increase over 2-4 weeks. Paired serum samples may be useful to detect a rise in Ab. A single high titer with compatible clinical signs and laboratory tests is usually diagnostic. However, previous vaccination leads to low-to-moderate increases in Ab titers. Each serovar (canicola, icterohaemorrhagiae, pomona, grippotyphosa, bratislava, autumnalis are among those offered by labs) has its own Ab titer. If one serovar titer is higher than the rest, it is considered the source of the infection. However, this has not been fully validated, and cross-reactions among serovars do occur. Vaccine-induced titers are usually in the range of 1:200 or less, while acute infections result in 1:800 or higher. Each lab has its own reference ranges that should be consulted. Titers are not useful for detecting resolution of infection, as they will stay elevated for months or years. PCR testing on whole blood and urine is offered at some labs, although samples should be collected before antibiotics are given (may cause false negative result on PCR or culture).
Infections caused by Borrelia burgdorferi are common in some areas of the U.S. and rare to nonexistent in others. As with other tick-borne diseases, identifying or culturing the organism is difficult and subject to false negatives. Ab testing is used in both humans and dogs to detect previous exposure. An in-clinic ELISA (SNAP 4Dx) is available as well as various tests at reference labs (IFA, Western blot, C6 quantitative Ab levels). A very important fact to remember is that none of these are tests for Lyme disease. Many dogs living in endemic areas are exposed to ticks that carry Borrelia yet they do not have any clinical signs or lab results that indicate infection. An accurate diagnosis of Lyme disease should take into account history, physical exam, lab testing, response to treatment, and ruling out other infections and diseases. C6 Ab levels are generally detected 3-5 weeks after exposure, and both experiments and clinical experience demonstrate that Ab titers persist for months or years. Recommendations to run a C6 quantitative Ab level 6 months after infection are based on limited experimental studies and have not been validated. As with ehrlichiosis, treatment is not indicated solely because of a positive Ab test. Concerns about Lyme nephropathy have led some practitioners to look for proteinuria or treat all Ab+ dogs, but there is no consensus. Vaccination for Lyme disease does not interfere with C6 Ab testing but will cause positive results with older ELISA and all IFA tests. PCR testing may be available but is not standardized. Borrelia organisms localize in skin and joint tissue, so whole blood is not an appropriate specimen to submit for culture or PCR. Synovial fluid from joint taps may increase the chances of detecting the organisms but false negatives are still possible.
The organism may be identified by cytology or histopathology of affected tissues. Serologic testing (agar gel immunodiffusion or RIA for antibodies) is not definitive but may support a diagnosis. Antigen testing is available (MiraVista Diagnostics) using urine or serum specimens (EDTA cannot be used). Cross-reactions can occur with histoplasmosis or other fungal infections.
As with blastomycosis, cytology is diagnostic. Serology (AGID, CF) is supportive but false positives and false negatives are common. Antigen testing (MiraVista Diagnostics) using urine, serum, or fluid from bronchoalveolar lavage is recommended but cross-reactions can occur.
Cytology of affected tissues may be performed. A latex agglutination test for capsular antigen is offered at some reference labs. Any positive titer is significant.
If affected tissues are accessible, cytology may demonstrate organisms. Antibodies can be detected by tube precipitin (primarily IgM) and complement fixation (IgG). Antigen testing (MiraVista Diagnostics) using urine, serum, or other sterile body fluid is recommended but false-positives and false-negatives can occur. The Coccidiomycosis Serology Laboratory at UC Davis provides diagnostic and consultation services.
Antinuclear antibody (ANA) testing was once recommended to help diagnose systemic lupus erythematosus. However, false positives and false negatives are common so it is rarely useful. Dogs with ehrlichiosis, bartonellosis, and leishmaniasis may test positive. Neoplastic and other immune-mediated diseases also are known to cause positive results.
Rheumatoid factors may be detected in cases of rheumatoid arthritis. False positives and negatives are possible. A combination of criteria is used in both humans and dogs for diagnosis instead of a single test.
Coomb's testing for immune-mediated hemolytic anemia is a direct antiglobulin test to detect autoagglutination. A positive result along with compatible clinical signs is usually diagnostic. Agglutination is seen with other diseases as well so it is not 100% sensitive or specific. Different methods are used by different laboratories and results cannot be directly compared. Recent evidence suggests that testing should be done with both monovalent and polyvalent antisera at 4°C and 37°C.