FeLV and FIV: testing... diagnosing... preventing (Proceedings)

Simply stated, the goal of vaccination is to sensitize or prime the immune system such that it can generate a population of unique cells capable of mounting an effective immune response subsequent to infection by a pathogenic organism. But this is a FAR MORE COMPLEX story. In just the past 5 years, remarkable advances have been made in our knowledge of how the immune system actually achieves the ability to "learn" and "remember" key features of an infectious organism and then act to either minimize the consequences of infection ("non-sterile immunity") or, in some cases, eliminate the organism altogether and prevent infection ("sterile immunity")...it's more, much more, than just antibody!

Table 1: FeLV Testing in the Clinical Setting

Feline retrovirus infections, FeLV and FIV, perhaps illustrate this fact best. Clearly among the most complex infections affecting the cat, a retroviral infection demands an immune response that is robust and sustained if the infected cat is to survive long-term. Both innate immune responses (neutrophils, macrophages, and antigen presenting cells [APCs]) as well as adaptive immunity (B-lymphocytes and T-lymphocytes) are critical. Susceptibility to FeLV infection is greatest during the first 6 months of a cat's life. Natural resistance of adult cats to FeLV infection is well documented. In the immunologically naïve kitten, exposure to FeLV is likely to result in life-long infection. At issue, then, is how long will the cat live? Susceptibility to FIV infection, on the other hand, does not change with age. Risk among kittens is similar to that in adults.

Vaccination Against Feline Leukemia Virus Infection

Today, it does appear that the prevalence of FeLV within the domestic cat population has declined over the last decade. Two factors are most likely to have played a major role in this decrease: vaccination and in-hospital testing for FeLV antigen in sick cats and cats presented for routine vaccination. However, results of a recent survey of cats in the US suggest that the prevalence of FeLV is still as high as 2.3% of all cats (feral and non-feral). Clearly the need for routine testing and vaccination of susceptible cats still exists.

Of the 4 FeLV vaccines available today, 2 are vaccines are killed, whole-virus, vaccines (Schering-Plough and Fort Dodge), 1 is a subunit vaccine (Pfizer), and, most recently licensed is the recombinant FeLV vaccine administered transdermally (Merial). We have recently completed a study at NCSU in which ALL 4 (monovalent) leukemia vaccines were tested in cats. At 21 days post vaccination, NONE of the vaccines caused false + test results for p27 antigen in cats tested by the ELISA method

All FeLV vaccines, however, are NOT the same. The killed and subunit vaccines contain adjuvantand require a 1.0 ml dose administered parenterally. The immunity conferred by these products is associated with antibody production only. The recombinant FeLV vaccine is a non-adjuvanted, canarypox vectored vaccine. The total 0.25 ml dose is administered by the transdermal (or, needle-free) route. The vaccine induces both humoral immunity and cell-mediated immunity.

The recombinant FeLV vaccine immunizes by its ability to deliver 2 genes that express 2 important immunogenic proteins: p27 (gag) and gp70 (env). The canarypox virus is widely recognized as an ideal "vector-virus" for cats, dogs, and humans since it does not recognize mammalian tissues and therefore does not multiply (replicate) in the vaccinated animal (or person). Because the virus does not replicate, there is no risk of the canarypox virus being shed from vaccinated cats.

While the concept of transdermal vaccination is new to veterinary medicine, it is far more significant than simply offering veterinarians an alternative method of administering vaccine to cats. The transdermal route addresses one of the pre-eminent vaccination strategies in medicine today: to maximize vaccine safety and efficacy by exploiting our knowledge of how immunity is induced and how an effective immune response is maintained. By administering vaccine into the skin, rather than through the skin, antigen processing and lymphocytes activation by dendritic (Langerhans) cells, the antigen-processing cells abundant in the skin, results in an immune response is potent and sustained.

Vaccination of kittens should consist of 2 vaccine doses administered at a 2 to 3 week interval beginning as early as 8 or 9 weeks of age. A 2-dose regimen is required at the time of administration of the first vaccine, regardless of the cat's age. Cats presented for the second dose of vaccine at more than 4 weeks following the first dose should be given a third dose 2 to 3 weeks later. Administration of an annual booster is recommended. Regardless of the vaccine used during the initial 2-dose regimen, it is not necessary to use vaccine from the same manufacturer when administering subsequent annual boosters.

Vaccination Against Feline Immunodeficiency Virus (FIV)

In July 2002, the first licensed vaccine (Fort Dodge) against feline immunodeficiency virus (FIV) was introduced in the United States. Although few articles have addressed FIV in the clinical literature over the past few years, most clinicians would agree that the consequences of FIV infection in the individual cat are significant and justify the need for an FIV vaccine. Characterized by a long latent period, infected cats gradually experience deterioration of immune function associated with declining numbers of T helper lymphocytes (CD4+). [REF: Levy, 2000] The consequences are manifest as a wide spectrum of vague clinical features, none of which are diagnostically distinctive.

The principle serological test for FIV infection used throughout the world is the determination of FIV antibody in serum. The enzyme-linked immunsorbent assay (ELISA) and immunoblot (Western Blot) methods used to detect FIV antibody have become the mainstay for diagnosing infected cats and conducting surveys among populations of cats at risk for infection. Epidemiological studies using these tests have provided good evidence for horizontal transmission of FIV among cats and have identified adult male cats living outdoors as those at greatest risk of infection. Since the virus can be recovered from the saliva of infected cats, bite wounds sustained during fighting are believed to be a principle means of virus transmission. On the other hand, casual contact among infected and non-infected cats is an unlikely means of transmission. Although it appears possible that FIV can be sexually transmitted, as the virus has been recovered from the semen of infected cats, this mode of transmission appears to be uncommon in nature. Likewise, transmission from infected queen to fetus (vertical transmission) is possible, but rare. On the other hand, it is more likely that infected queens will transfer FIV antibody, not virus, via colostrum to nursing kittens. Since maternal FIV antibody may persist in kittens for several months, it is customary to disregard a "positive" FIV antibody test result in healthy kittens under 6 months of age.

The introduction and use of the killed FIV vaccine substantially changed the approach clinicians use to assess potentially infected cats. Of particular importance is the fact vaccination is known to be associated with development of FIV antibody that interferes with all FIV tests on the market today. In addition, it has recently been demonstrated that a vaccinated, seropositive queen will pass antibody to kittens (presumably through clostrum). FIV testing of kittens that nursed from FIV seropositive cats will cause a false positive test result. Until an alternative, reliable, and accessible laboratory test for FIV infection is made available, or an alternative (recombinant) vaccine is introduced, veterinarians have lost the ability to distinguish between a vaccinated cat and an infected cat.

The Vaccine

The current FIV vaccine is a killed, whole virus vaccine containing two virus subtypes, or clades, of FIV called clade A (Petaluma strain) and clade D (Shizuoka strain). The manufacturer recommends a vaccination schedule that entails administration of 3 doses initially, 2-3 weeks apart, followed by annual revaccination. Each 1 ml dose is administered subcutaneously to cats 8 weeks of age or older. The vaccine is adjuvanted.

Vaccine Efficacy and Safety

Based on experimental studies submitted to the USDA, the FIV vaccine has a good efficacy and safety profile. Efficacy (challenge) studies showed that 4 of 24 (16%) vaccinated cats were infected following challenge while 17 of 19 (90%) unvaccinated (control) cats were infected following challenge. This represents an 82% preventable fraction. In clinical safety studies, over 2000 doses were administered to 689 cats (299 were less than 12 weeks of age). Reactions such as pain at the injection site, lethargy, and fever, were reported in 1.1%. There were no fatalities reported.

...but what about the test?

FIV antibody testing has become the hallmark of serologic tests used to identify cats infected with FIV. In fact, FIV testing has become so widely used in practice that an advisory panel organized by the American Association of Feline Practitioners and Academy of Feline Medicine recently published revised guidelines on feline retrovirus testing and implications for managing cats determined to be positive for FIV antibody. [REF: Guidelines]. In summary, the advisory panel recommended that all cats be tested for both FeLV and FIV infection. With respect to FIV antibody testing, emphasis is placed on the importance of testing all sick cats regardless of negative results of previous FIV tests.

Regarding test selection, the advisory panel has published that ELISA and other immunochromatographic tests available in clinical practice, are the preferred screening tests to be used in the initial assessment of any sick cat. It is further recommended that all positive screening test results be confirmed by the Western blot test.

Here's the problem...all cats vaccinated with the killed FIV vaccine are expected to develop FIV antibodies following administration of the first dose. Antibodies are known to persist for at least 1 year. Vaccine-induced antibodies interfere with all antibody tests commercially available in the US and Europe:

In addition, kittens of vaccinated queens are likely to have a positive test result due to passively acquired vaccine-induced antibody. Negative test results for antibody may still be interpreted as negative for exposure and infection.

With the introduction of the FIV vaccine, and loss of the ability to identify FIV-infected cats in clinical practice, a substantial effort is underway to identify an alternative diagnostic test that is reasonably priced and accurate.

Alternative Testing

Virus isolation (VI) has been suggested as possible means of distinguishing vaccinated cats from infected cats. However, virus stability during transport, availability, and cost are such significant limiting factors that

VI is not a reasonable consideration for veterinarians in clinical practice. Isolating FIV from infected cats is well suited to experimental laboratories where the sample collection and virus isolation methods can be highly controlled.

On the other hand, polymerase chain reaction (PCR)-based tests for identification of RNA and proviral DNA have received considerable attention, subsequent to the release of the killed FIV vaccine, as "the" alternative test for detecting infected cats...whether or not they've received prior vaccination. While it is possible to identify FIV, in both vaccinated and unvaccinated cats, using PCR technology, the ability to provide widespread diagnostic services to practitioners through commercial laboratories has not yet been accomplished. In the long run, this may prove to be quite problematic given the nature of PCR technology.

PCR technology must not be viewed as simply another "new and improved" means of detecting FIV antibody. In fact, it doesn't detect antibody at all...but that's just the beginning. The "family" of feline immunodeficiency viruses, is varied and their expression, once they've infected a cat, is quite complex. That, combined with the inherent sensitivities of PCR-based test methods make turning a PCR test into the replacement test for FIV antibody, and doing so at the levels of reliability and consistency we have enjoyed, a major technological challenge.

Although most clinicians will not be especially interested in all of the technical and methodological issues pertaining to PCR testing, it is important to understand that PCR, discovered just within the last 20 years, is an exceptionally accurate method for rapidly manufacturing unlimited copies of DNA. In effect, PCR has made it possible to identify unique sequences of DNA even when the sample size is miniscule. Obviously, such technology would be of considerable value in diagnosing infection, particularly viral infections, where the virus quantity can be quite small and the genetic features of the virus quite distinct. Recently, the ability of a novel quantitative polymerase chain reaction (qPCR) method to detect proviral DNA in FIV-infected cats was described in 1999. While using the Taqman® PCR to detect FIV provirus is a significant fact, incorporating the technology into routine use in the clinical setting will take time.

Even if PCR testing for FIV does become commercially available in the near future, the clinician must understand and appreciate the fact that the incredible sensitivity of PCR method has direct and important implications on test results. For example, in-hospital PCR testing for FIV, or anything else, is simply not feasible today. An outside laboratory must analyze all specimens. In addition to contending with the risk of sample transport and contamination (with extraneous nucleic acid), it will become critical (at least it should be) for all laboratories offering PCR testing for FIV to use standardized, validated reagents and testing protocols. The process of validating test methods will require documenting the accuracy of PCR against various FIV field strains (variants of virus subtypes) seen in the United States. At this writing, the means of standardizing PCR tests for veterinary medicine simply doesn't exist. Then, with all that said, it's a matter of price.

FeLV and FIV: Testing vs. Vaccination

Clearly, one of the most significant technologic advances introduced into clinical practice within the last 10 years has been the ENZYME-LINKED IMMUNOSORBENT ASSAY, or ELISA. The ability to perform in-hospital, same-day screening for feline leukemia virus (FeLV), feline immunodeficiency virus (FIV), and canine parvovirus (CPV) are among the most important value-added services offered by the companion animal practitioner today.

The feline retroviral infections, FeLV and FIV, continue to represent a major threat to the health of the domestic cat population. Extensive surveys have consistently demonstrated that among sick cats presented to veterinarians, from 11% to 16% are FeLV-positive while from 6% to 9% are FIV-positive. Retrovirus infection is best prevented by eliminating opportunities for exposure of susceptible cats and kittens to infected cats. However, in the clinical setting, it is unreasonable to assume that recommendations for avoiding contact with FeLV/FIV–infected cats constitutes a practical approach to prevention. Fundamental to the strategic management of feline retrovirus infection is the opportunity to vaccinate at-risk cats against FeLV. However, routine FeLV vaccination of cats presented to the practice only represents one component of a prevention strategy. Since the available FeLV vaccines are unlikely to confer complete protection in all vaccinated cats and that there is currently no FIV vaccine available suggests that the risk of retrovirus infection among susceptible cats is still significant.

In accordance with current guidelines, it is recommended that the FeLV and FIV status of all cats seen by the practice be established. However, the commitment to perform routine screening of cats for retroviral infection raises important issues pertaining to interpretation of test results and follow-up actions needed to further manage those households with confirmed FeLV and/or FIV positive cats. Current testing recommendations outlined by the AAFP/AFM have recently been published and are ALTERNATIVES: A Veterinary Clinical Update. (A Supplement to Compendium on Continuing Education, published by Veterinary Learning Systems, Volume 19; Nbr. 12).

The current ELISA technology used for in-hospital retrovirus testing in cats is remarkably sensitive and accurate assuming proper technique and specimen handling are used. These same tests are recommended for routine screening of both healthy and sick cats presented to the practice.

Fundamental to the proper use of ELISA-based testing for the diagnosis of FeLV and FIV infected cats is an understanding that FeLV tests are designed to detect the p27 antigen (core antigen) while FIV tests detect the presence of antibody. In clinical practice, these facts have important implications. For example, the FeLV test can detect FeLV virus in the blood (serum or plasma) of young kittens; a positive test is consistent with infection. It is important to note that FeLV tests designed to detect the presence of virus in tears and/or saliva are also ELISA-based tests. However...these tests are significantly less sensitive/specific (ie, large numbers of false-positive and false-negative results) than tests utilizing blood, serum, or plasma. Saliva and tear tests should not be used for routine screening of individual cats. It should also be noted that neither maternal antibody nor recent FeLV vaccination interferes with the ELISA-based FeLV test. The reader is referred to Figure 1 for guidelines on interpreting the various test results in cats at various stages in the course of FeLV infection.

ELISA-based FIV tests are not reliable in kittens less than 6 months of age. Since antibody response to FIV infection requires weeks or months to become detectable, a negative test result could occur in an exposed, infected kitten that has not seroconverted. On the other hand, uninfected kittens from FIV infected queens may test positive as a result of having acquired maternal FIV antibody; detectable levels of maternal FIV antibody can persist for as long as 6 months.

Among healthy cats with a positive ELISA test for either FeLV or FIV, follow-up testing is recommended. The clinician should repeat the ELISA test in 1 to 3 months. As noted in Figure 1, waiting 1-3 months is justified in healthy cats with a positive test considering the possibility that the infection is early (transient) and a protective neutralizing antibody may still develop. Alternatively, the indirect fluorescent antibody (IFA) could be submitted in an attempt to confirm infection. As many as 80% of FeLV-positive cats will die within 3 years from illness associated with the infection.

The ELISA-based FIV antibody test is not a confirmatory test. Confirmation of infection is predicated on a positive Western blot assay. In contrast to FeLV, the FIV-infected cat may live for months or years with its infection; early detection and treatment of associated illness will enhance longevity and quality of life.

Cats infected with either FeLV or FIV are considered to be shedding virus. As such, strong precautions are recommended to prevent unnecessary exposure to healthy, susceptible cats.

Suggested Reading

1. Levy JK: CVT Update: feline immunodeficiency virus In, JD Bonagura (ed): Current Veterinary Therapy XIII. WB Saunders, Philadephia. pp. 284-288, 2000.

2001 Report of the American Association of Feline Practitioners and Academy of Feline Medicine Advisory Panel on Feline Retrovirus Testing and Management. [available from: AAFP/AFM, 530 Chruch Street, Suite 700, Nahsville, TN 37219].

3. Leutenegger CM, Klein D, Hofmann-Lehmann R, et al: Rapid feline immunodeficiency virus provirus quantitiation by polymerase chain reaction using the TaqMan® florigenic real-time detection system. Journal of Virological Methods. 78:105-116, 1999.

4. Zenger E: FIP, FeLV, and FIV: making a diagnosis. Feline Pract. 28:16-18, 2000.

5. Hartmann K: Feline immunodeficiency virus infection-an overview. Vet J. 155:123-157, 1998.

6. Hartmann K. Feline leukemia infection. In CE Greene (ed). Infectious Diseases of the Dog and Cat. Saunders-Elsevier, St Louis. pp. 105-131, 2006.