Diagnosing Different Courses of FeLV Infection

Although prevalence and importance of FeLV as a pathogen in cats have decreased—primarily because of effective testing, eradication programs, and routine use of FeLV vaccines, particularly in high-risk cats^2,5-8—recent studies have suggested a stagnation in this decrease in prevalence in several countries.^9-11 In a recent study in 30 European countries, FeLV prevalence was still ≈2% in all cats visiting veterinary clinics¹²; therefore, the importance of FeLV and its prevention should not be neglected.¹¹

FeLV is transmitted via close contact among cats, particularly in cats that live together or fight, and is commonly transmitted from infected queens to their kittens.^10,13 Viremic cats (ie, cats that are progressively infected or in the early phase of regressive infection; see FeLV Infection Courses) shed the virus mainly in saliva, but the virus can also be found in nasal secretions, milk, urine, and feces.^14-19 FeLV susceptibility is age-dependent; older cats are more resistant and rarely develop progressive FeLV infection, which is the most severe course.^20-22

After exposure, FeLV is found in local lymphoid tissues.^23,24 FeLV subsequently spreads via monocytes and lymphocytes (first viremia) into the periphery. During the first viremia, the virus can infect the bone marrow.^23-25 Following bone marrow infection, a second viremia can occur, with FeLV-containing neutrophils and platelets appearing in the blood.^25,26 Within 1 week of FeLV exposure, plasma viral RNA is usually detectable by reverse transcriptase PCR (RT-PCR), followed by proviral DNA (ie, a DNA copy of viral RNA integrated into the cat’s genome) detectable by PCR (without reverse transcription being necessary) a few days to weeks later, and finally by free (soluble) FeLV p27 antigen (virus core protein), which is detectable by ELISA (or other immunochromatographic or rapid immunomigration assays), usually after 3 to 6 weeks.^18,25,27-31

Ideally, FeLV status should be determined for every cat because infection can impact health status and requires long-term management.³² FeLV testing includes evaluation of different viral and immunologic parameters.⁶ FeLV diagnosis can be challenging because of variable infection dynamics secondary to interplay between host immune and viral factors. For example, the virus can be reactivated in a cat that has regressive FeLV infection with subsequent viremia^6,33-36; conversely, a cat that has persistent viremia can clear the virus from the blood years later.

FeLV courses of infection (ie, progressive, regressive, abortive, focal; Table) have been characterized in experimental infections, but natural FeLV infection cannot always be clearly stratified into one course.^33,34,37 FeLV clinical course is determined by virus and host immune interactions, particularly in the early phase of infection (generally the first 12 weeks).³⁸ Although the course of infection is typically determined in the early phase, lifelong host immune system and virus interactions can affect and change the course of infection later.^35,36,39,40 In FeLV-infected cats, the equilibrium between host and virus can be altered by several factors (eg, immunosuppression, coinfection, environmental changes) that can influence disease outcome and prognosis.^12,19,41,42

Approximately one-third of cats that live in multicat environments with FeLV shedding cats (high infectious pressure) develop persistent viremia and become progressively infected.⁴¹ Progressive infection is characterized by insufficient FeLV-specific immunity.^43,44 FeLV is not contained during early infection, and extensive viral replication occurs in the lymphoid tissues, bone marrow, and mucosal and glandular epithelial tissues.^23-25 Mucosal and glandular infection is associated with excretion of infectious virus mainly in saliva.^13-15 Progressively infected cats have shorter survival times and commonly succumb to FeLV-associated diseases.^{6,10,34,39,45,46}

Approximately one-third of cats that live in multicat environments with FeLV shedding cats develop regressive infection.⁴¹ Although these cats never have (or will eventually clear) viremia, FeLV provirus is integrated into the cat’s genome, resulting in lifelong infection (ie, FeLV provirus carrier state).^39,47 FeLV proviral DNA can be detected in the blood by PCR.^28,33 No antigen or culturable virus is present in the blood and the virus is not shed in saliva after these cats have undergone the initial infection phase and their immune system has suppressed the virus^26,28,33,48; therefore, these cats are not infectious to other cats except via blood transfusion or if reactivation occurs.^49,50

Regressive infection is characterized by an effective immune response and high antibody concentrations, and viral replication is contained prior to or at the time of bone marrow infection.^26,28,33 Although FeLV is integrated in the cat’s genome, viral shedding does not occur after viral replication is suppressed by the immune system.^26,28,33,48 Regressive infection can be distinguished from progressive infection by FeLV proviral DNA load and viral load in the blood, both of which decrease after an initial peak.^26,28,31 FeLV replication in cats with regressive infection can be reactivated and viremia can reoccur, particularly during immunosuppression, at which point cats become antigen-positive, shed virus, and can develop FeLV-associated diseases.^39,40,51 The risk for reactivation of viremia decreases with time^28,51-55; however, integrated provirus maintains its replication capacity, and reactivation is possible years (possibly lifelong) after initial exposure to FeLV.^39,40 In some cats, regressive infection can cause clinical problems (eg, lymphoma, bone marrow suppression).^56,57 In cats with regressive infection, vaccination is ineffectual because these cats have already developed a strong anti-FeLV immune response and reactivation is not prevented by vaccination.^{23,34,29,33,58}

Focal infection (ie, atypical infection) is considered very rare and occurs in cats that have FeLV infection restricted to certain tissue (eg, spleen, lymph nodes, small intestine, mammary  glands).^{6,17,41,55,59} These cats frequently have discordant and varying FeLV test results.^60,61 They do not shed the virus in saliva but can still transmit infection under certain circumstances; for example, a queen with focal FeLV infection of the mammary glands can transmit FeLV to her kittens via milk.¹⁷

Approximately one-third of cats that live in multicat environments with FeLV shedding cats develop abortive infection characterized by low-grade infection and immunity.^19,33,34,62 In these cats, direct virus detection methods produce negative results, and the only sign of FeLV exposure is the presence of FeLV-specific antibodies.⁶² Abortive infection is characterized by a strong immune response to the virus.³⁴ Cats test negative for culturable virus, antigen, viral RNA, and proviral DNA but have FeLV-specific antibodies.⁶² Cats with abortive infections do not shed infectious virus and do not develop clinical signs.^19,33,34,62

Diagnosing FeLV can be difficult because of the different courses of infection. In addition, interaction between the virus and immune system can change over time depending on various factors (eg, age, immune function, infectious pressure, pathogenicity of the virus strain, genetic variability of the virus over time).^41,42 The European Advisory Board of Cat Diseases (ABCD) has created a diagnostic algorithm (the ABCD FeLV diagnostic tree; Figure) outlining the diagnostic steps for FeLV.

Although FeLV infection tests can detect presence of the virus (eg, FeLV antigen, FeLV DNA) or antibodies, they cannot be used to diagnose lymphoma or leukemia (ie, forms of leukosis) and should not be called leukosis tests. These tests cannot determine whether a cat has FeLV-associated disease because clinical signs in FeLV-infected cats can be secondary or completely unrelated to FeLV.³⁵

Most available tests detect the virus directly, with the exception of recently introduced antibody tests (ie, indirect detection method).⁶³ Direct FeLV infection tests are not influenced by maternal antibodies, so kittens (including neonates) can be tested at any age. FeLV vaccinations do not cause a positive result in direct FeLV tests.^29,34,64 Tests can vary in diagnostic value, particularly point-of-care tests (POCTs) performed in-house, and results should be confirmed by other methods or by repeating the POCT, ideally from a different brand (see Figure and When to Immediately Repeat an FeLV Point-of-Care Test).^6,65-71

ELISAs and other immunochromatographic or rapid immunomigration assays that detect free (soluble) FeLV p27 antigen in blood are available as POCTs or laboratory tests (usually plate ELISAs).^65-72 POCTs generally have a good overall performance with only slightly varying diagnostic sensitivities and specificities. Laboratory tests that detect FeLV p27 antigen have similar sensitivities and specificities as compared with POCTs, but some also quantify the antigen load.^65,70 POCTs based on ELISA should be performed with serum or plasma, not whole blood. POCTs and laboratory tests for detection of free FeLV p27 antigen in blood should not be used with saliva because false-negative results are possible.^73-75 Antigenemia is present if test results are positive; antigenemia is generally a measure for viremia and, if persistent, is diagnostic for progressive infection. False-positive results have become more common because of decreased FeLV prevalence in many countries. Negative results are reliable because of low FeLV prevalence in most populations.^12,70,76 In the early phase of infection (within the first 3 weeks), antigen tests are commonly not yet positive.³⁰

Immunofluorescence assays (IFAs) detect intracellular p27 antigen on blood smears (in neutrophils and platelets) and provide positive results later (typically, ≈3 weeks later) than tests for free p27 antigen because intracellular FeLV p27 antigen can only be detected by IFAs in infected neutrophils and platelets after bone marrow becomes infected.^76-78 IFAs are therefore not recommended as screening tests because cats in the first weeks of viremia already shed FeLV. False-negative IFA results can occur, mainly in cats with neutropenia and thrombocytopenia. False-positive results can occur as a result of nonspecific staining, smears of inappropriate thickness, high background fluorescence, or interference when using anticoagulated blood.^79,80 IFAs require special processing, fluorescence microscopy, and highly experienced staff; thus, only results from experienced reference laboratories should be interpreted.⁷⁷

Virus isolation detects replicating virus in blood and requires culture of virus in feline cell lines.⁸¹ This is a sensitive test that can be used to detect FeLV infection during primary viremia; therefore, results can be positive early postinfection, even before tests for free p27 antigen. However, virus isolation is not practical for routine diagnosis because it is difficult and time-consuming to perform and requires special facilities; thus, it is not recommended as a screening test but can be used for confirmation of positive FeLV p27 antigen test results.

PCR detects proviral DNA (FeLV provirus) in blood that are viral nucleic acid sequences integrated in the cellular genome of cats.^28,31,82,83 Diagnostic values can vary because PCR methods are not standardized; only laboratories with adequate quality control should be used. PCR is generally a sensitive method because it amplifies FeLV sequences and can detect small amounts of DNA; it is also highly specific, which can lead to false-negative results, when minor variations in the viral genome prevent binding of the primers. Primers should therefore target highly conserved regions of the FeLV genome. PCR can also be performed on bone marrow or tissue instead of blood^{25,28,31,39,67,82} and can help resolve cases with discordant p27 antigen test results. It is the recommended confirmatory test for positive p27 antigen test results and the test of choice to detect regressive infection (positive PCR in combination with negative p27 antigen test).^28,29

RT-PCR detects viral RNA in blood and saliva. Viral RNA can be detected during viral replication; therefore, RT-PCR detecting viral RNA does not provide the same information as PCR detecting FeLV provirus (DNA).^18,29 RT-PCR is highly specific and sensitive but has the same methodologic advantages and disadvantages as PCR^14,15,43 and, therefore, should only be performed in specialized laboratories. RT-PCR performed on blood or saliva has different clinical significance. Positive RT-PCR in saliva indicates FeLV shedding, whereas strong positive RT-PCR in blood indicates viremia and progressive (or early regressive) infection, although low positive RT-PCR in blood also can occur in regressively infected cats and then serve as an indicator of future reactivation.³⁴ When performed on blood, RT-PCR is helpful in detecting FeLV infection in the early phase because it provides positive results earlier than do tests for free p27 antigen.^14,15,18,29 When performed on saliva, RT-PCR is a reliable indicator of antigenemia.¹⁵ The presence of FeLV-shedding cats living in a multicat environment can be ruled out when testing saliva (swabs), for which saliva samples from up to 10 cats can be pooled in the laboratory.¹⁴

The presence of FeLV antibodies in serum indicates previous exposure to the virus (or certain FeLV vaccines). FeLV antibody tests are positive in cats with regressive or abortive infection. These are the only tests that can identify abortive infection.^62,63 Determination of antibodies can also be used to quantify the immune response in cats with FeLV infection.^28,34,50,63 Antibody tests are not currently routinely used and are only performed in specialized laboratories, but they could be of future importance. A new POCT that detects antibodies against p15E antigen (ie, envelope transmembrane protein) has recently been commercialized in Europe; however, its diagnostic value has yet to be evaluated.

FeLV is an important infection still affecting many cats worldwide. Courses of infections differ among individual cats and can vary over time. The complex pathogenesis, variety in outcome, and availability of different tests make FeLV infection complicated and a challenge for clinicians.