| In-house serologic test kits have continued to improve in reliability, ease of use, and types available. They include tests for infectious disease antigens and antibodies, hormones, and immunoglobulin levels. Many of these tests are ELISA that may be microwell or membrane-based, but other types including immunomigration and agglutination tests are also available. Sample requirements may be plasma, serum, whole blood, feces, or saliva, depending on the test format. |
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Feline Leukemia Virus (FeLV): |
| Serologic testing for FeLV is important in order to identify infected cats and prevent transmission of the virus (see
Feline Leukemia Virus and Related Diseases: Introduction). Testing is also recommended prior to vaccination against FeLV because vaccination of infected cats will not limit development or transmission of disease. Most currently available in-house diagnostic kits are designed to detect soluble FeLV-specific gag protein p27, which is produced in large amounts during viremia. The time between infection and the presence of detectable antigen varies, but is likely to be within 28 days.
Vaccination against FeLV will not yield a positive test result when antigen tests are used, nor will the presence of maternally-derived antibodies in kittens. Testing serum or plasma is considered more reliable than testing whole blood, saliva, or tears. Most test kits have positive and negative controls incorporated into the kit, so technical problems with running the test may be detected. There is a relatively high rate of false positive results with these tests of soluble
antigen, especially in a population of cats with a low incidence of disease. A true positive result may reflect either transient or persistent viremia, so clinical decisions should not be based on a single test result. Confirmation of positive results, especially in asymptomatic cats, should be pursued by testing for cell-associated antigen, eg, with an immunofluorescent antibody assay. |
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Feline Immunodeficiency Virus (FIV): |
| Because the concentration of FIV antigen in the blood of infected cats is often very low, in-house diagnostic tests are designed to detect anti-FIV antibodies rather than antigen. Test kits yield a positive or negative result for antibody rather than a quantitative result. Antibodies usually develop within 60 days of infection, but this time period is quite variable and a few infected cats fail to develop detectable antibody levels. Once present, antibodies appear to be present
for life, except for those transiently detectable in kittens that have maternally-derived antibodies. A kitten <6 mo of age that tests positive should be retested after 6 mo of age for this reason. If the test is still positive, the kitten is most likely infected. |
| With ELISA tests, the incidence of false positives is relatively high. Positive results, especially in asymptomatic cats, should be confirmed by another test such as Western blot. Currently, antibodies produced in response to vaccination with FIV vaccine cannot be reliably distinguished from those produced by natural infection, so results must be interpreted with knowledge of a cat’s vaccination history. In addition, kittens born to vaccinated queens are antibody-positive for a
variable length of time. PCR testing may be able to distinguish naturally-infected cats from vaccinated cats, but this method is unlikely to become practical for in-clinic laboratories. |
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Canine Parvovirus (CPV): |
| Both ELISA and immunomigration test kits are available for detection of CPV antigen in feces. These tests are fairly specific for the virus, but sensitivity is somewhat lower for several reasons. Fecal shedding only occurs for ~7-10 days, beginning on day 3-5 following exposure, so virus is not always detectable in dogs with clinical signs. Blood in the stool as well as the formation of antigen-antibody complexes may be associated with false negative results. |
| Because of interest in measuring serum antibodies to CPV to evaluate the need for revaccination and the presence of possibly interfering maternal antibodies, in-house tests for assay of anti-CPV antibody have become available. These tests are not intended to diagnose parvovirus infection and do not distinguish between natural exposure and vaccine-induced antibodies. Currently available test kits are ELISA for detection of IgG antibody in serum or plasma. They are
semiquantitative and use color changes in positive control wells compared to color changes in samples to determine relative antibody levels. The level of anti-CPV antibody that is protective against infection is not known, but results are interpreted based on protective titers measured by other methods. |
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Canine Distemper Virus: |
| In-house test kits for determination of antibodies to canine distemper virus are available in combination with CPV antibody testing. As with the CPV kits, these tests are semiquantitative ELISA for anti-canine distemper IgG in serum or plasma. They may be used to evaluate the need for revaccination and to determine the level of maternal antibody present in puppies. |
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|
Borrelia
burgdorferi
: |
| Lyme disease (
Borreliosis: Introduction) is caused by the tickborne spirochete
B burgdorferi
. In general, diagnostic tests for this infection have several potential problems, including relatively high levels of false positive results that may be difficult to distinguish from subclinical infections, persistence of antibodies following apparent resolution of clinical disease, interpretation of antibody titers in vaccinated dogs, and the fact that detectable antibody may not be present until 4-6 wk after exposure. There are several in-clinic ELISA test kits for
detection of anti-
B
burgdorferi
antibodies in serum or plasma. One of these tests uses a synthetic peptide derived from a
B
burgdorferi
-specific protein that is important in inducing a humoral immune response by 4-5 wk following infection in dogs. In addition to reducing the level of cross-reaction with other infectious agents because of its specificity, this test does not give a positive result to vaccine antigens and thus appears to distinguish vaccine-induced antibodies from those produced in response to natural infections. Current in-house tests are not quantitative; they give only a positive or
negative result and are not suitable for monitoring response to treatment. |
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|
Ehrlichia
canis
: |
|
Canine ehrlichiosis (
Ehrlichiosis and Related Infections) is a tickborne disease caused by a rickettsial organism. It can be associated with thrombocytopenia, anemia, and neutropenia as well as other nonspecific clinical signs. While identification of
Ehrlichia
morulae in leukocytes can be diagnostic for this infection, serologic assay for detection of antibodies is more common and has better sensitivity. In-house tests for ehrlichiosis are either qualitative (giving only a positive or negative result) or semiquantitative; those currently available are ELISA to be performed on serum, plasma, or whole blood. Newer tests that use recombinant analogs of major outer membrane proteins of
E
canis
are more reliable than those using whole
E
canis
proteins as the antigen. Because anti-
E
canis
antibodies may be very long-lived and may be present in subclinical infections, detection of these antibodies does not distinguish between exposure and
Ehrlichia
-induced illness and cannot reliably indicate success of response to therapy. |
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Brucella canis: |
| Infection with
B
canis
may be subclinical, or it may cause a variety of clinical signs including abortion, infertility, and diskospondylitis (see
Brucellosis in Dogs: Introduction). Infection most often occurs during mating; thus, testing of breeding animals is important in disease prevention. An in-house test for anti-
B
canis
antibody is a rapid slide agglutination test that includes a 2-mercaptoethanol (2-ME) step to reduce false-positive reactions by eliminating nonspecific IgM reactions. This assay detects serum antibody to surface antigens of the bacteria. If the first step (with no 2-ME) shows agglutination, the second step (with 2-ME) is performed to rule out a nonspecific reaction. Although this test is very convenient as a rapid screening test, some newer ELISA techniques performed
in reference laboratories have higher specificity and sensitivity, especially in the early stages of infection. |
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Heartworm Antigen—Dogs: |
| Serologic testing for heartworm antigen in dogs is more sensitive than screening for microfilaremia and, in addition, can detect occult infections. Heartworm antigen is first detectable at ~5 mo postinfection and will usually precede microfilaremia by a few weeks. There are several different antigen test formats available including ELISA with microwells, membrane ELISA, agglutination, and lateral flow membrane immunomigration. Serum or plasma may be used in all kits, and some
may be run using whole blood. Some test kits can be stored at room temperature, while others must be stored refrigerated and brought to room temperature before use. Batch testing and single sample testing are both available. All of the test kits have very high specificity. False-positive results are most often due to technical problems such as inadequate washing or failure to read the results at the optimal time. The manufacturer’s instructions should be closely followed with any
of the test formats. |
| Sensitivity varies from one test to another and is affected by worm load, worm gender (a female antigen is detected, thus male-only infections will not be detected), and maturity of female worms. None of these tests has 100% sensitivity. When unexpected false results are obtained on an antigen test, additional testing, using a different format, is recommended. |
| Following adulticide treatment, antigenemia should become undetectable within 4-5 mo. It can take more than a month for some adult heartworms to die, however, so a positive antigen test at 4-5 mo post-treatment does not necessarily indicate treatment failure and the test should be repeated 2-3 mo later. |
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Heartworm Antibody and Antigen—Cats: |
| Heartworm disease in cats is substantially different from the disease in dogs, and recommendations for serologic testing are consequently different. Cats tend to have a much lower worm burden—often only 1 or 2 worms. Cats also have single-sex infections more frequently than do dogs. Circulating microfilaremia is rare in cats, and microfilariae have a shorter life span. These differences are often attributed to a more effective feline immune response to heartworm
infection. Heartworms can, however, cause serious disease in cats. |
| Heartworm antigen tests are less sensitive for detection of infection in cats than they are in dogs. Antibody testing may be preferred because antibodies are produced to both male and female worms, and they are produced as early as 2 mo after infection, which is much earlier than antigen may be detected. The presence of antibody does not confirm feline heartworm disease, because transient exposure to larvae will stimulate production of antibody. Many early infections are
cleared spontaneously, and adult heartworms never develop. A combination of testing for heartworm antigen and anti-heartworm antibody is warranted in cats with clinical signs of heartworm disease when results of a single test are not conclusive. |
| Test kits for detection of anti-heartworm antibody in cats are marketed in several different formats. These can be run using either plasma or serum and are qualitative rather than quantitative. There are currently test kits specific for heartworm antigenemia in cats as well as those that can be used to test either canine or feline samples. |
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Canine Pregnancy Diagnosis—Relaxin: |
| The only known pregnancy-specific hormone in the bitch is relaxin, which is produced by the placenta when a fertilized egg is implanted. Relaxin is first detectable in the plasma around day 22-25 following fertilization. It is not present in pseudopregnant or nonpregnant bitches. Relaxin levels peak by about day 40-50 of gestation and drop at parturition, but they may remain detectable for up to 50 days during lactation. In-clinic test kits to measure relaxin include microwell
ELISA and immunomigration qualitative tests requiring serum, plasma, or whole blood. The assays appear to have very good specificity; that is, a detectable level of relaxin is not found in nonpregnant bitches. False negatives have been reported in some bitches carrying very small litters or in those with 1 or more nonviable puppies. |
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Canine Ovulation and Whelping Timing—Luteinizing Hormone: |
| In the bitch, ser- um luteinizing hormone (LH) is normally present in very low levels except for a dramatic rise just prior to ovulation. Ovulation occurs 2 days after this LH surge, and the LH level returns to baseline within 24-40 hr of peaking. Serum progesterone levels begin to rise at the time of the LH surge. A bitch will be fertile between days 4-7 following the LH surge with the most fertile period on days 5 and 6. In addition, the LH surge determines the gestation
period, with parturition occurring between day 64 and 66 following the surge. The LH surge may occur anywhere from 3 days before to 5 days after the onset of estrus behavior, so it cannot be reliably predicted by behavior. |
| Ovulation timing by measurement of LH requires daily testing, which usually begins when >50% of vaginal epithelial cells are cornified, based on vaginal cytology. There is occasionally a false LH peak that is not followed by ovulation, but it will also not be followed by an increase in progesterone. For that reason, assay of both LH and progesterone are recommended for most accurate ovulation detection. An elevated LH concentration that is not followed by increased
progesterone levels is considered a proestrus fluctuation and testing for ovulation should continue. |
| In-clinic kits to measure LH concentrations in serum are essentially qualitative, with a serum level of <1 ng/mL being read as negative and a positive test indicating LH concentration of >1 ng/mL. Current test kits have a short shelf life. |
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|
Canine Ovulation Timing—Progesterone: |
| Serum progesterone begins to increase following the LH surge with a concentration of 2.0-3.9 ng/mL on the day before ovulation and 4.0-10.0 ng/mL on the day of ovulation. Progesterone continues to increase and stays elevated throughout pregnancy or diestrus. As the rise in progesterone is more constant, compared to LH, daily testing is not necessary. It is recommended that testing begin in late proestrus and continue every 2-3 days until a high range is reached, indicating ovulation. |
| The test kits for in-clinic progesterone testing are semiquantitative ELISA with levels of <1 ng/mL, <2 ng/mL, or <4 ng/mL designated as pre-ovulatory, depending on the kit. Some kits are designed to give 2 additional progesterone ranges (intermediate and high), while others will indicate only pre-ovulatory and “ovulatory day or later” levels. In comparison with other test methods, the in-house test kits are less accurate, particularly in the range of roughly 1.5-10
ng/mL, which is the range of interest for earliest detection of ovulation. Accuracy is greater at higher progesterone levels. As mentioned above, measurement of both LH and progesterone is recommended for most accurate breeding management. |
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Thyroxine: |
| Assay of total serum thyroxine (T4) may be used as a screening test for canine hypothyroidism or as a diagnostic test for feline hyperthyroidism. (See also
the thyroid gland,
The Thyroid Gland : Introduction.) In addition, T4 concentrations are measured when monitoring therapy for hypo- or hyperthyroidism. An in-house ELISA test kit, which uses an additional instrument to read results, provides semiquantitative information about T4 concentration in canine and feline sera. The assay is run at 1 of 2 “dynamic ranges” depending on the predicted range of results. Although
there are currently few published studies comparing this assay with other test methods for T4, one comparison of this kit with a radioimmunoassay reported a fairly low correlation in comparing results using the 2 methods. |
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Foal Immunoglobulin (IgG): |
| Measurement of foal serum IgG concentration within the first 24 hr after birth can be useful in preventing disease related to failure of passive transfer of IgG from mare to foal in colostrum. Use of foal-side testing procedures can facilitate prompt diagnosis and treatment. A foal serum IgG level of >800 mg/dL is generally considered optimal, with <200 mg/dL indicating failure of passive transfer. Concentrations of 200-800 mg/dL are considered evidence of partial transfer. |
| Although radial immunodiffusion (RID) is considered the most accurate test for IgG concentration, it takes much longer (5-24 hr) than many of the other test methods and thus is not as useful as an indicator of the need for therapeutic intervention. More rapid screening tests include the zinc sulfate turbidity test, glutaraldehyde clot test, and ELISA test kits. The zinc sulfate test estimates IgG in serum based on its precipitation when added to a zinc-containing solution.
Turbidity generally becomes visible when there are 400-500 mg/dL IgG. This test takes ~1hr and may be performed with zinc sulfate solutions made in the clinic, or reagents for the test may be purchased as a test kit. The glutaraldehyde clot test requires the addition of 1 volume of serum to 10 volumes of 10% glutaraldehyde and examination of the tubes at timed intervals up to 60 min. The presence of IgG in the serum causes formation of a solid clot in the tube. Clot formation in
<10 min generally correlates to an IgG concentration of >800 mg/dL, while a positive reaction within 60 min is interpreted as >400 mg/dL of IgG. Both of these test methods use serum, rather than plasma, so time for the blood to clot and separation of serum must be added to the time needed to perform the test. ELISA test kits for in-house or foal-side testing can use either serum or whole blood as the sample and take ~10 min. They are semiquantitative with color changes
corresponding to IgG concentrations of <400 mg/dL, 400-800 mg/dL, or >800 mg/dL. Results, especially at high and low IgG levels, have been shown to correlate well with RID results. |
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|
Calf Immunoglobulin: |
| Measurement of IgG concentration in neonatal calf serum is important for the same reasons described for foals. IgG levels are, however, different from those in foals, with >1,000 mg/dL considered evidence of adequate passive transfer and <1,000 mg/dL indicating failure of passive transfer. As with foals, RID is the gold standard for accurate measurement of serum IgG, but the length of time required to complete this assay makes it less useful than other methods. Other
methods that have been used include zinc sulfate and sodium sulfite turbidity tests, glutaraldehyde coagulation test, measurement of total serum solids by refractometry, and a lateral flowthrough ELISA test kit. |
| The sodium sulfite and zinc sulfate turbidity tests are both based on the precipitation of high molecular weight proteins in these solutions. Serum is used as the sample and tests are read after 15-30 min of incubation. Results of these tests vary in sensitivity and specificity depending on the endpoint chosen, and there are some technical difficulties with reagents that can decrease test performance. The glutaraldehyde clot test is performed as in foals, with no clot formation
at 60 min indicating failure of passive transfer. |
| Measurement of serum total protein by refractometer is a fairly reliable indicator of adequate passive transfer in healthy, well-hydrated calves. A level of 5.2 g/dL is roughly equivalent to an IgG concentration of 1,000 mg/dL. |
| A commercially-available ELISA test kit uses serum as the sample and takes ~20 min. The assay is qualitative in that it indicates an IgG concentration of >1,000 mg/dL or <1,000 mg/dL. Sensitivity and specificity are reasonably good and this test is less influenced by factors such as calf dehydration or reagent instability than some other methods. Manufacturer recommendations for storing and using the kits must be followed. |
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