Testing kittens for FeLV and FIV (Proceedings)

Feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) are retroviruses that represent two of the most important infectious diseases of cats worldwide. The American Association of Feline Practitioners recommends the retrovirus status of all cats should be known and has published guidelines for retrovirus testing and management (Levy, Richards et al. 2001).

Diagnosis of FIV

The most common method of diagnosis of FIV infection is screening for FIV antibodies using an enzyme-linked immunosorbent assay (ELISA). Patient-side kits are commonly used in veterinary clinics and in shelters. Screening for viral antigen is not possible since the amount of circulating virus is low after the acute stage of infection. FIV produces a persistent, life-long infection so that the detection of antibodies is judged sufficient for diagnosis. Most cats will produce antibodies to FIV within 60 days of exposure, but the length of time to seroconversion can be much longer in some cats.

False-positive results can occur with ELISA testing, so it is recommended that the Western blot be used as a confirmatory test, especially in cats considered at low risk of infection. A recent study showed that the sensitivity and specificity for FIV was very high in unvaccinated cats using a commercially available ELISA test kit (SNAP® FIV/FeLV Combo, IDEXX Laboratories, Westbrook, Maine) (Levy, Crawford et al. 2004). A small number of cats will fail to produce detectable levels of antibodies after infection and these cats will have false negative results with both ELISA and Western blot testing.

Positive FIV antibody tests in kittens under 6 months of age must be interpreted carefully. Kittens born to infected queens may acquire FIV antibodies in colostrum(MacDonald, Levy et al. 2004). Since it is uncommon for kittens to acquire infection from the queen, most kittens that test positive are not truly infected and will test negative when re-evaluated at 6 months of age or older. Kittens that test positive for FIV antibody when over 6 months of age are considered to be infected. A negative FIV antibody test is generally reliable at any age, especially in a low-risk patient.

Due to potential test interference, it is tempting to delay testing kittens for FIV until over 6 months of age. However, most kittens test negative and can be reliably considered clear of infection. While FIV infection of kittens is uncommon, it has been known to occur. Infected kittens could be a source of infection for other cats if they are not identified and isolated. Compliance of both owners and veterinarians with retroviral testing recommendations is low, so that delaying testing of newly acquired kittens would potentially result in many cats that never undergo FIV testing at all (Goldkamp, Levy et al. 2008).

The release of the first vaccine against FIV (Fel-O-Vax FIV®, Fort Dodge Animal Health) has complicated the ability of veterinary practitioners to diagnose FIV infections. Vaccinated cats produce antibodies that cannot be distinguished from antibodies due to natural infection using currently available tests (Levy, Crawford et al. 2004). Antibodies due to vaccination persist for more than one year, and are also acquired by kittens nursing on vaccinated queens (MacDonald, Levy et al. 2004). Maternally derived vaccine-associated antibodies may persist in kittens for longer than 8 weeks, giving another reason to interpret positive FIV antibody tests in young kittens with caution and to recommend re-testing. Cats at risk of FIV infection, such as outdoor cats, should be tested periodically throughout their lives if they are not FIV vaccinated. It is also recommended that cats should be tested before vaccination against FIV.

In some patients, it may be difficult to determine if a positive FIV antibody test means the cat is truly infected with FIV, is vaccinated against FIV but not infected, or is vaccinated against FIV and also infected. Polymerase chain reaction (PCR) has been promoted by some commercial laboratories as a method to determine a cat's true status. In order to be useful to practitioners, PCR tests should be able to detect a wide array of genetically divergent FIV strains and laboratories must maintain stringent quality control. Published research indicates that PCR tests offered by commercial laboratories may be unreliable, with misidentification of both FIV-infected and uninfected cats (Bienzle, Reggeti et al. 2004; Crawford, Slater et al. 2005).

Recently, a discriminant ELISA detecting antibodies against formalin-treated FIV whole virus and untreated transmembrane peptide was shown to have high sensitivity (97.1%) and specificity (100%) for distinguishing uninfected from infected cats, regardless of vaccination status (Levy, Crawford et al. 2008). Should a discriminant ELISA become commercially available, a new testing strategy could be devised where existing commercial FIV antibody tests would be used as screening tests and positive results would be confirmed with the discriminant ELISA. If the discriminant ELISA is negative, the cat is probably vaccinated against FIV but not infected. Positive results with the discriminant ELISA are likely to represent infection.

Diagnosis of FeLV

Diagnosis of FeLV relies on detection of the core antigen p27 in peripheral blood. ELISA test kits detect soluble circulating antigen and are recommended for routine in-clinic use. They may be used with whole blood, serum or plasma, although the test kit should be checked for the manufacturer's recommendations on sample type. Tests performed on tears and saliva are less reliable and are not recommended. ELISA tests can detect infection early, during primary viremia. Most cats will test positive on ELISA within 1 month of exposure, although detection of antigenemia may take much longer in some cats. Immunofluorescent antibody (IFA) tests on smears from blood or bone marrow detect p27 antigen within infected neutrophils and platelets and are recommended as confirmatory tests. IFA tests do not detect infection until secondary viremia is established due to infection of bone marrow (6 to 8 weeks after initial infection).

Ideas on possible outcomes of infection with FeLV are currently undergoing re-evaluation. In the past, it was believed that about 1/3 of cats became persistently viremic and about 2/3 would clear infection. New research using PCR technologies suggests that most cats remain infected for life following exposure to FeLV. However, they may revert to a non-viremic state that is termed regressive infection. In regressive infections, there is no antigen present in the blood and virus cannot be cultured from blood. But FeLV proviral DNA can be detected in blood using PCR (Pepin, Tandon et al. 2007). The significance of PCR-positive but antigen-negative regressive infections is not yet clear. These cats are unlikely to shed infectious virus in saliva, but may transmit proviral DNA via blood transfusion if used as a blood donor. Prior to the advent of PCR technology, the term "latency" was used for antigen-negative cats where virus could not be cultured from blood, but could be cultured from bone marrow or other tissues. It now appears that "latency" is a phase through which cats pass during regressive infection.

Kittens can be tested at any age, as passively acquired maternal antibody does not interfere with testing for viral antigen. Newborn kittens infected via FeLV-positive queens may not test positive for weeks to months after birth. While it may be tempting to test only a queen and not her kittens in an attempt to conserve resources, it is inappropriate to test one cat as a representative for others. If a queen or any one of her litter of kittens tests FeLV-positive, all should be considered potentially infected and isolated, with follow up testing to resolve status. Susceptibility to FeLV infection is age-related, with the highest infection rates in very young kittens. Shelters sometimes test pooled blood samples from litters of kittens in order to save money, but this practice should be discouraged as the reliability of this method is unknown.

Kittens or cats that test negative but have a known or suspected exposure to FeLV should be retested no earlier than 1 month after exposure to rule out false negative test results obtained during incubation of the virus. Periodic testing of cats at ongoing risk of FeLV infection is justified and is not generally compromised by vaccination. However, blood collected immediately following vaccination may contain detectable FeLV antigens from the vaccine, so samples should be collected prior to FeLV vaccination (Levy J, unpublished data). It is not known how long this test interference persists.

Since the consequences of a positive test are significant and false positive test results can occur, confirmatory testing with IFA is recommended, especially in low-risk patients (Hartmann, Werner et al. 2000). Some cats may be only transiently viremic and may revert to ELISA-negative status (regressive infection). However, a positive IFA test at any time on blood or bone marrow generally indicates a cat is persistently infected.

Discordant test results occur when results of ELISA and IFA testing do not agree and may make it difficult to determine the true FeLV status of a cat. Most typically, this is an ELISA-positive and IFA-negative cat. Discordant results may be due to the stage of infection, the variability of host responses, or technical problems with testing. The status of the cat with discordant results may eventually become clear by repeating both tests in 60 days and yearly thereafter until the test results agree. Unfortunately, a significant number of these patients have persistently discordant test results and the cat's true status may not be known. Cats with discordant test results are best considered as potential sources of infection for other cats until their status is clarified.

PCR is offered by a number of commercial laboratories for the diagnosis of FeLV. PCR detects viral nucleic acid sequences (RNA or DNA) and can be performed on blood, bone marrow and tissues. PCR tests for FeLV are usually positive within one week of FeLV exposure. When performed by a well-equipped and well-trained laboratory, PCR can be the most sensitive test methodology for FeLV and could help resolve cases with discordant test results and detect regressive infections. Independent evaluation of commercial PCR testing for FeLV (or FIV) is not routinely performed nor are labs required to be licensed or regulated, so that veterinarians may not be able to ascertain the diagnostic efficacy of a test offered by a particular laboratory.

Recently, a real-time quantitative PCR assay was used to screen 597 Swiss cats for FeLV. Surprisingly, 10% of cats negative for FeLV p27 antigen by ELISA were positive by PCR (Hofmann-Lehmann, Huder et al. 2001). However, the proviral loads of these cats were 300-fold lower than for ELISA-positive cats. One possible explanation for the PCR-positive, ELISA-negative cat is that such cats are truly infected with FeLV but were able to overcome antigenemia (regressive infection). It is also possible that these cats were in the early stages of FeLV-infection and had not yet become ELISA-positive. Research such as this suggests it may be very difficult to determine the true status of cats that appear to be transiently infected and recovered.

References

Bienzle, D., F. Reggeti, et al. (2004). "The variability of serological and molecular diagnosis of feline immunodeficiency virus infection." Can Vet J 45(9): 753-7.

Crawford, P. C., M. R. Slater, et al. (2005). "Accuracy of polymerase chain reaction assays for diagnosis of feline immunodeficiency virus infection in cats." J Am Vet Med Assoc 226(9): 1503-7.

Goldkamp, C. E., J. K. Levy, et al. (2008). "Seroprevalences of feline leukemia virus and feline immunodeficiency virus in cats with abscesses or bite wounds and rate of veterinarian compliance with current guidelines for retrovirus testing." J Am Vet Med Assoc 232(8): 1152-8.

Hartmann, K., R.-M. Werner, et al. (2000). "Comparison of different practice tests for rapid diagnosis of feline immunodeficiency and feline leukemia virus infection (ESVIM abstract)." J Vet Intern Med 14(2): 232.

Hofmann-Lehmann, R., J. B. Huder, et al. (2001). "Feline leukaemia provirus load during the course of experimental infection and in naturally infected cats." J Gen Virol 82(Pt 7): 1589-96.

Levy, J., J. Richards, et al. (2001). "Feline retrovirus testing and management." Comp Contin Edu Pract Vet 23(7): 652-657.

Levy, J. K., P. C. Crawford, et al. (2008). "Differentiation of feline immunodeficiency virus vaccination, infection, or vaccination and infection in cats." J Vet Intern Med 22(2): 330-4.

Levy, J. K., P. C. Crawford, et al. (2004). "Effect of vaccination against feline immunodeficiency virus on results of serologic testing in cats." J Am Vet Med Assoc 225(10): 1558-61.

MacDonald, K., J. K. Levy, et al. (2004). "Effects of passive transfer of immunity on results of diagnostic tests for antibodies against feline immunodeficiency virus in kittens born to vaccinated queens." J Am Vet Med Assoc 225(10): 1554-7.

Pepin, A. C., R. Tandon, et al. (2007). "Cellular segregation of feline leukemia provirus and viral RNA in leukocyte subsets of long-term experimentally infected cats." Virus Res 127(1): 9-16.