Interpreting Unexpected Feline Leukemia Virus Test Results
ACVIM 2008
John R. August, BVetMed, MS, MRCVS, DACVIM
College Station, TX, USA


The exclusion of feline leukemia virus (FeLV) infection is an important part of the comprehensive diagnostic examination of sick cats who have been at risk for infection. Similarly, many healthy cats are screened for FeLV as part of routine wellness examinations, irrespective of their risk of exposure.

In most circumstances, the test results make sense, and help to clarify the health status of the patient. From time to time, however, unexpected results are obtained, especially when screening tests are used. Often, these spurious results arise from technical errors. Less commonly, they may occur as the result of the complicated relationship between virus and the cat's immune system. Not all cats recover completely from infection or develop persistent viremia. Atypical infections may explain discordant test result patterns noted in some apparently healthy cats, or in patients with vague signs of illness.

In these unexpected situations, it is the responsibility of the attending veterinarian to clarify the unexpected results as accurately and as quickly as possible. Misinterpretation of test results, or delaying retesting unnecessarily, only leads to client distress and the provision of erroneous prognostic advice. The introduction of new diagnostic tests based on the detection of FeLV provirus will assist in the clarification of some unexpected or discordant test results.

Pathobiology of Feline Leukemia Virus Infection

FeLV is a single-stranded RNA virus with 3 subgroups. Only subgroup A is infectious and transmitted between cats; persistent infection predisposes cats to immunosuppressive, hematologic, and neoplastic diseases.1 The outcome of natural exposure to FeLV depends on the age of the cat, the dose of virus, the duration of primary viremia, and the quality of the cat's immune response. Possible outcomes of exposure include 1) self-limiting oropharyngeal infection; 2) transient viremia; 3) sequestered (atypical) infections; 4) latent infections; and 5) persistent viremia. The variable outcomes of exposure, the low prevalence of infection in the general population of feline patients, the differing biological bases for currently available diagnostic tests, and the opportunity for technical errors, all increase the likelihood for occasional test results that are spurious.

Stage 1 infection with FeLV is associated with viral replication in oropharyngeal tissue. The infection is terminated at this point in many adult immunocompetent cats, and goes no further. Because neither antigenemia nor viremia occur, tests to detect viral antigens in circulation are negative. Most of these cats become resistant to future challenge following this early effective immune response. Stage 2 infections, characterized by primary viremia, develop if an early effective virus neutralizing antibody response has not occurred. Small numbers of infected lymphocytes and monocytes are found in circulation, and antigenemia may be detected. Depression, fever, and lymphadenopathy may be noted in some cats during this phase of infection.1 Amplification of infection in systemic lymphoid tissues such as lymph nodes, spleen, thymus, and gastrointestinal tract characterizes Stage 3 of FeLV infection. Transient viremia usually lasts 3 to 6 weeks; bone marrow cells usually become infected after about 3 weeks. The more protracted the initial viremia, the more likely that latent infections will occur in bone-marrow cells of those cats capable of terminating the course of disease at this time, or that persistent viremia will occur. Stage 4 infections have become established in the bone marrow, and are characterized by the persistent infection of hematopoietic precursor cells. The release of infected neutrophils and platelets from the bone marrow characterizes Stage 5 of infection, with affected cats showing positive results on tests that detect cell-associated viremia. Finally, Stage 6 occurs when there is disseminated epithelial cell infection, including salivary and lacrimal glands, and urinary bladder, and the shedding of large amounts of infectious virus in secretions.2

Using this information, consider the following case studies. For each case, choose the most appropriate traditional diagnostic test, and the sequence of testing, in order to provide the highest quality information about the FeLV status of the patient. How may emerging FeLV tests be used to clarify these results?

Case Studies (All tests were run using whole blood taken from the medial saphenous vein)

Patient 1

A 3-year-old female spayed domestic shorthaired cat is presented for a yearly wellness examination. There are no other cats in the household. The cat spends 100 per cent of her time indoors, except for regular visits to the veterinarian. An ELISA test for FeLV is weakly positive (all previous yearly tests have been negative). What do you tell the owners? How would you clarify this test result to provide the owners with an accurate prognosis?

Patient 2

One of your clients has rescued a 12-week-old male kitten from an abandoned house where other feral cats are known to live. The kitten appears healthy, but an ELISA test for FeLV is strongly positive. What do you tell the owners? How would you clarify this test result to provide the owners with an accurate prognosis?

Patient 3

A 4-year-old neutered male domestic longhaired cat from a multiple-cat household is presented with a history of recurring low-grade fevers, accompanied by inappetence and hematopoietic dyscrasias. On physical examination, the mucous membranes appear pale. ELISA tests for FeLV and FIV are negative. To you, however, the cat still looks very suspicious for FeLV-associated disease. What do you tell the owners? How would you further evaluate this patient for retroviral disease?

Patient 4

A 6-year-old spayed female Siamese cat is presented for a semi-annual wellness examination. For the past 3 years, the cat has been persistently weakly positive on ELISA tests for FeLV, but persistently negative on IFA tests. The cat lives with two other adult cats, both of whom have been persistently ELISA-negative. None of the cats go outdoors. How do you explain these results to the owners?

Conventional Diagnostic Test Techniques for Feline Leukemia Virus

ELISA and Immunochromatographic (ICGA) Methods

These methods detect p27 antigen in blood or other fluids of the patient.3 The rate of production of p27 antigen is in excess of that needed for incorporation into complete FeLV virions, resulting in circulating p27 antigenemia detected by these tests.1 Compared to the immunofluorescent antibody (IFA) test, these methods are more likely to detect weak, early, or transient infections when viral antigens often are not present in circulating neutrophils and platelets. False positive results, which are often characterized as weak color reactions, can be reduced by using plasma or serum for testing, rather than whole blood. The incidence of false-positive test results is exacerbated when healthy cats, from a population in which the prevalence of disease is extremely low, are tested routinely.1 Healthy cats who test ELISA or ICGA positive (after false-positives have been excluded) and IFA negative on a single examination may be undergoing a transient regressive infection, the early stages of eventual persistent viremia (before the bone marrow has become infected), or may have an atypical sequestered infection. Differentiation of these groups may be accomplished by re-testing with ELISA or ICGA methods 12 weeks later; those cats who continue to test positive should undergo confirmatory testing at that time with an IFA test. New polymerase chain reaction tests to detect FeLV provirus in bone-marrow or circulating mononuclear cells may be useful in clarifying persistently discordant test results.4 Reverse discordancy, characterized by ELISA negative and IFA positive tests on blood samples, is considered to be the result of laboratory error.1 However, the author has observed a very small cohort of cats with hematopoetic dyscrasias with negative serum ELISA tests, but positive IFA tests on blood smears (usually buffy coat smears) and bone marrow. The biological basis of this test pattern is not clear; however, the infection in these cats may be highly cell-associated with little or no soluble antigenemia.

Immunofluorescent Antibody Test

The IFA test detects p27 antigen in infected neutrophils and platelets in blood or bone marrow smears. The test should not be used to screen cats for FeLV infection, because early and atypical infections will be missed. There is a strong correlation between IFA positive status and the ability to recover infectious virus from the patient's blood and saliva. Only a small percentage of cats with transient viremia will test IFA positive; positive results have a high correlation with persistent viremia.1 A subgroup of FeLV-infected cats with hematopoietic dyscrasias have negative ELISA/ICGA and IFA test results on blood, but positive IFA tests on bone-marrow smears, representing another atypical infection with FeLV in patients with incomplete immunity to the virus. Neither ELISA/ICGA or IFA tests detect latent infections, characterized by the complete absence of virus and p27 and by the presence of cells containing FeLV provirus in their genome. Table 1 summarizes the 6 stages of FeLV infection and the ability to detect p27 antigen in blood or secretions at each stage.

Table 1. Stages of FeLV infection and detection of p27 antigen.



ELISA test

IFA test


Stage 1





Stage 2

Primary viremia




Stage 3

Lymph tissue




Stage 4





Stage 5

Marrow viremia




Stage 6

Epithelial tissue




New Diagnostic Test Techniques for Feline Leukemia Virus

Recently, there has been renewed interest in the development of more sensitive techniques to identify cats with atypical infections and those with latent infections in which no virus antigen can be detected. Currently, PCR testing for FeLV provirus is considered to be the best method to exclude a diagnosis of FeLV infection in cats.4 Latent FeLV infections in the bone marrow may contribute to the pathogenesis of myelodysplastic diseases in some cats, and reactivation of latent infections in the mammary glands of breeding queens may perpetuate infections in catteries. Real-time PCR has identified proviral DNA in the blood of 9.3 per cent of cats with undetectable antigenemia that were submitted for testing, and in 12 of 14 cats with discordant results.4 The clinical and epidemiologic significance of provirus-positive and antigen-negative cats is not known.4 In another recent study, proviral DNA was detected by nested PCR in 25 per cent of blood samples submitted, in the absence of antigenemia.5

Because it can be difficult to obtain satisfactory blood samples from some fractious cats, and because previous ELISA tests for salivary FeLV p27 antigen are considered inaccurate, less invasive and more sensitive methods of detecting infection are being investigated. Recently, the use of PCR to detect FeLV RNA in saliva has been used to avoid some of these shortcomings.6 There was a high correlation between the detection of plasma p27 antigen by ELISA and the detection of salivary FeLV RNA in this cohort of cats. However, some cats in this study tested negative for p27 antigen and salivary FeLV RNA, yet tested positive for FeLV proviral DNA suggesting true latent infections.


1.  Hartmann K. In Greene CE (ed): Infectious Diseases of the Dog and Cat, ed 3, 2006, Elsevier, pp105-130.

2.  Lappin MR. In Rand J (ed): Problem-Based Feline Medicine, 2006, Elsevier, pp526-551.

3.  Barr MC. Semin Vet Med Surg (Small Anim) 1996;11(3)144.

4.  Pinches MDG, et al. J Fel Med Surg 2007;9:8.

5.  Arjona A, et al. J Fel Med Surg 2007;9:14.

6.  Gomes-Keller MA, et al. J Clin Microbiol 2006;44(3):916.

Speaker Information
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John August, BVetMed, MS, MRCVS, DACVIM
Texas A&M University
College Station, TX