Course of feline leukemia virus infection and its detection by enzyme-linked immunosorbent assay and monoclonal antibodies

Monoclonal antibodies specific for 3 distinct epitopes of the species-specific determinants of feline leukemia virus (FeLV) p27 were used in an enzyme-linked immunosorbent assay (ELISA) for measurement of serum p27 in cats infected with FeLV. Group-specific antigen (GSA) of FeLV in peripheral blood leukocytes was also determined by an immunofluorescence assay. Antibodies to FeLV and the feline oncornavirus-associated cell membrane antigen (FOCMA) were also measured. Thirty-six cats were surveyed and assigned to 4 categories. Five developed persistent viremia (category 1), characterized by continuous expression of p27, GSA, and low antibody titers to FeLV and FOCMA. Eleven cats with transient viremia (category 2) and 13 cats that were never detectably viremic (category 3), as judged by absence of GSA and p27, developed increased antibody titers to FeLV and FOCMA. Seven cats were never viremic, as judged by the GSA in the peripheral blood leukocytes, but still had detectable serum p27 (category 4). Most category 4 cats developed high antibody titers against FOCMA and/or FeLV. Of 307 field cats examined, 7% of the healthy cats and 10% of the sick cats could be assigned to category 4. However, this difference was not significant (P greater than or equal to 0.05). Of 26 cats with neoplasms 2 (1 of 12 with lymphosarcoma) could be classified as category 4. Because virus could be isolated from 2 category 4 cats, they were considered immune carriers.     

Association of feline leukemia virus with lymphosarcoma and other disorders in the cat.

Two hundred fifty Boston cats with disorders such as lymphosarcoma, myeloproliferative disease, anemia, glomerulonephritis, pregnancy abnormalities, feline infectious peritonitis, toxoplasmosis, and various bacterial infections were examined for feline leukemia virus (FeLV) by immunofluorescence. Antibody titers against feline oncornavirus-associated cell membrane antigen (FOCMA) were tested in 133 of these cats. The tests for FeLV and FOCMA antibody were also conducted among healthy cats not known to have been exposed to FeLV, as well as among healthy cats from households where FeLV was known to be present. Most of the cats with lymphosarcoma and the other aforementioned disorders were infected with FeLV and low FOCMA antibody titers. Healthy cats known to have been exposed to FeLV were often viremic, but those that remained healthy were able to develop high FOCMA antibody titers. Healthy cats without known prior exposure to FeLV were unlikely to be viremic but often had detectable FOCMA antibody titers, indicating that some exposure occurs under natural conditions in the Boston area. The association of FeLV with infections other than lymphosarcoma was assumed to be caused by the immunosuppresive effect of FeLV, thus allowing development of disease.     

Safety and efficacy studies of live- and killed-feline leukemia virus vaccines.

The safety and the efficacy of several feline leukemia virus (FeLV) vaccines for 16-week-old kittens were determined. Vaccines were derived from an FL74 lymphoblastoid cell line that has been in continuous tissue culture passage for about 4 years. The vaccines were made from living virus, formaldehyde-inactivated whole FL74 cells, and formaldehyde-inactivated whole virus. The efficacy of each produced vaccine was determined by challenge exposure of vaccinated cats with virulent FeLV. The two formaldehyde-inactivated vaccines were found to be safe for use in kittens. Neither vaccine produce a significant feline oncornavirus-associated cell membrane antigen or virus-neutralizing antibody response, nor did they prevent infection with virulent FeLV. The inactivated whole-virus vaccine, however, did substantially decrease the proportion of kittens infected with virulent FeLV that became persistently viremic. In contrast, the whole FL74 cell vaccine did not reduce the number of infected kittens that became persistently viremic. The live-virus vaccine was found to be both safe and efficacious. About a half of the kittens vaccinated with live virus had transient bone marrow infection that lasted from 2 to 4 weeks. Viral antigen was not detected in peripheral blood, and infective virus was not shed in saliva, urine, or feces during the period that the vaccinal virus could be recovered from the bone marrow. In addition, there was no horizontal spread of vaccinal virus from vaccinated to non-vaccinated cagemates. Within several weeks, vaccinated kittens demonstrated no clinical or hematologic abnormalities and had high serum levels of feline oncornavirus-associated cell membrane antigen and virus-neutralizing antibody. Kittens vaccinated with living FeLV were resistant to infection with virulent virus.     

Exposure of cats to low doses of FeLV: seroconversion as the sole parameter of infection

In felids, feline leukemia virus (FeLV) infection results in a variety of outcomes that range from abortive (virus readily eliminated and never detectable) to progressive infection (persistent viremia and viral shedding). Recently, a novel outcome was postulated for low FeLV infectious doses. Naïve cats exposed to faeces of persistently infected cats seroconverted, indicating infection, but remained negative for provirus and p27 antigen in blood. FeLV provirus was found in some tissues but not in the bone marrow, infection of which is usually considered a necessary stage for disease progression. To investigate the impact of low FeLV doses on young cats and to test the hypothesis that low dose exposure may lead to an unknown pathogenesis of infection without involvement of the bone marrow, 21 cats were infected oronasally with variable viral doses. Blood p27, proviral and viral loads were followed until week 20 post-infection. Tissue proviral loads were determined as well. The immune response was monitored by measuring FeLV whole virus and p45 antibodies; and feline oncornavirus-associated cell membrane antigen (FOCMA) assay. One cat showed regressive infection (transient antigenemia, persistent provirus-positivity, and seroconversion) with provirus only found in some organs at sacrifice. In 7 of the 20 remaining cats FOCMA assay positivity was the only sign of infection, while all other tests were negative. Overall, the results show that FeLV low dose exposure can result in seroconversion during a presumed abortive infection. Therefore, commonly used detection methods do not detect all FeLV-infected animals, possibly leading to an underestimation of the prevalence of infection.     

Prevalence of disease in nonviremic cats previously exposed to feline leukemia virus

Feline leukemia virus status and antibody titer to feline oncornavirus-associated cell membrane antigen (FOCMA) were determined on plasma from 183 outpatient cats and 61 cats from 2 closed, FeLV-positive, multiple-cat households. Cats with FOCMA antibody titer had a significantly (P less than 0.02) higher prevalence of history of disease than did cats without FOCMA antibody. Diseases included upper respiratory tract infections, abscesses, ear infections, lower urinary tract infections, gastrointestinal disease, pneumonia, uterine infection, lymphadenopathy, fever of unknown origin, and bacterial infections. The FOCMA antibody titer was determined by use of an indirect fluorescent antibody test; titer greater than or equal to 1:16 was considered to be positive results. Lower mean FOCMA antibody titer was observed in young cats with history of disease (P less than 0.05) than in young cats without history of disease or in older cats with or without history of disease. Prevalence of FOCMA antibody titer was identical (38%) in young and adult cats, indicating cats likely were exposed to FeLV as kittens because a higher prevalence of FOCMA antibody titer in older cats would otherwise be expected.     

La leucose feline: Developpements recents sur l''etiologie et l''immunoprevention de la maladie

Feline leukemia is a useful model for malignant hematopoïetic tumor studies. It is caused by a type C, RNA virus, the Feline Leukemia virus (FeLV), transmitted horizontally, and widespread in the cat population.The presence of DNA sequences and virus specific RNA expression in cell cultures of SPF cats and cat embryos, indicates a vertical transmission may occur.These FeLV-related sequences in virus negative lymphosarcoma, almost from older cats, indicate that in certain FeLV related diseases the viral replication may not occur. An endogenous ecotropic feline virus may also explain this finding. The absence of FeLV gene expression in some lymphomatous cats—many older—suggest that, in these cats, spontaneous lymphoma may not be caused by FeLV.The widespread occurrence of feline xenotropic endogenous virus RD-114 gene, in feline lymphoma, suggest that expression of certain functions of this virus may be involved etiologically in the development of lymphoid tumors in the cat.Nevertheless, immunisation against FeLV would provide a good prevention against the main part of the feline lymphosarcomas and other FeLV-related diseases. Inactivated FeLV does not provide a good immunisation in young cats. By contrast a good protection against tumoral development is obtained by vaccination using the Feline oncogenic virus cell membrane antigen (FOCMA).     

Markers of feline leukaemia virus infection or exposure in cats from a region of low seroprevalence

Molecular techniques have demonstrated that cats may harbour feline leukaemia virus (FeLV) provirus in the absence of antigenaemia. Using quantitative real-time polymerase chain reaction (qPCR), p27 enzyme-linked immunosorbent assay (ELISA), anti-feline oncornavirus-associated cell-membrane-antigen (FOCMA) antibody testing and virus isolation (VI) we investigated three groups of cats. Among cats with cytopenias or lymphoma, 2/75 were transiently positive for provirus and anti-FOCMA antibodies were the only evidence of exposure in another. In 169 young, healthy cats, all tests were negative. In contrast, 3/4 cats from a closed household where FeLV was confirmed by isolation, had evidence of infection. Our results support a role for factors other than FeLV in the pathogenesis of cytopenias and lymphoma. There was no evidence of exposure in young cats. In regions of low prevalence, where the positive predictive value of antigen testing is low, qPCR may assist with diagnosis.     

Vaccination of cats experimentally infected with feline immunodeficiency virus, using a recombinant feline leukemia virus vaccine.

A group of 15 cats experimentally infected with a Swiss isolate of feline immunodeficiency virus (FIV) and a group of 15 FIV-negative control cats were inoculated with an FeLV vaccine containing recombinant FeLV-envelope. High ELISA antibody titer developed after vaccination in FIV-positive and FIV-negative cats. Vaccinated and nonvaccinated controls were later challenge exposed by intraperitoneal administration of virulent FeLV subtype A (Glasgow). Although 12 of 12 nonvaccinated controls became infected with FeLV (10 persistently, 2 transiently), only 1 of 18 vaccinated (9 FIV positive, 9 FIV negative) cats had persistent and 2 of 18 had transient viremia. From these data and other observations, 2 conclusions were drawn: In the early phase of FIV infection, the immune system is not depressed appreciably, and therefore, cats may be successfully immunized; a recombinant FeLV vaccine was efficacious in protecting cats against intraperitoneal challenge exposure with FeLV.     

Immunohistochemical identification of B and T lymphocytes in formalin-fixed, paraffin-embedded feline lymphosarcomas: relation to feline leukemia virus status, tumor site, and patient age.

The lymphocyte phenotype of 70 formalin-fixed, paraffin-embedded feline lymphosarcomas (LSAs) was determined immunohistochemically using a T cell polyclonal antibody, and a B cell monoclonal antibody. Forty-seven of 70 (67%) tumors were T cell, 19/70 (27%) were B cell, and 4/70 (6%) did not stain with either marker. Thirty-eight of 70 (54%) tumors were positive for feline leukemia virus (FeLV) antigen by immunohistochemistry (IHC), and 52/70 (74%) tumors were positive for FeLV DNA using the polymerase chain reaction (PCR). B cell tumors were as frequently FeLV-positive as T cell tumors using either IHC or PCR. Intestinal tumors were more likely to be B cell than T. The incidence of B and T cell tumors was not different among young (< or = 3 y), middle-aged (> 3 y to < or = 8 y), and old (> 8 y) cats. Both B and T cell tumors from old cats were FeLV-positive more often by PCR than by IHC. Feline leukemia virus DNA but not antigen, was detected in B cell tumors and intestinal tumors from cats > 8 y as often as it was detected in B cell tumors and intestinal tumors from cats < or = 8 y. Previously, most B cell and intestinal tumors from old cats were considered to be negative for FeLV. Here, the results suggest involvement of latent or replication-defective forms of the virus in such tumors from old cats. This study supports a role for FeLV in feline B cell as well as T cell tumorigenesis.     

Use of a subunit feline leukemia virus vaccine in exotic cats

Three adult bengal tigers, 2 immature white tigers, and 3 adult servals were vaccinated IM with three 1-ml doses of a subunit FeLV vaccine with dosage interval guidelines of the manufacturer. All cats had increased antibody titers to FeLV gp 70 capsular antigen and feline oncornavirus cell membrane-associated antigen during the vaccination trial. Three weeks after the third vaccination, 7 of the 8 cats had gp70 antibody titers greater than 0.2 (optical density), and all 8 cats had feline oncornavirus cell membrane-associated antigen antibody titers greater than 1:8.     

Immunogenicity and Efficacy of a Commercial Feline Leukemia Virus Vaccine

Twenty young adult specific pathogen-free cats were randomly divided into two groups of 10 animals each. One group was vaccinated with two doses of feline leukemia virus vaccine according to the manufacturer's recommendations. All 20 cats were challenge exposed oronasally (4 times over a 1-week period), beginning 3 weeks after immunization, with a virulent subgroup A strain of FeLV (CT600-FeLV). The severity of the FeLV infection was enhanced by treating the cats with methylprednisolone acetate at the time of the last FeLV exposure. Ten of 10nonvaccinated cats became persistently viremic compared with 0/10 of the vaccinates. ELISA antibodies to whole FeLV were present at high concentrations after immunization in all of the vaccinated cats, and there was no observable anamnestic antibody response after challenge exposure. ELISA antibodies to whole FeLV appeared at low concentrations in the serum of nonvaccinated cats after infection but disappeared as the viremia became permanently established. Virus neutralizing antibodies were detected in 3/10 vaccinates and 0/10 nonvaccinates immediately before FeLV challenge exposure, and in 8/10 vaccinates and 1/10 nonvaccinates 5 weeks later. Although vaccination did not consistently evoke virus neutralizing antibodies, it appeared to immunologically prime cats for a virus-neutralizing antibody response after infection. Active FeLV infection was detected in bone marrow cells taken 14 weeks after infection from 10/10 nonvaccinates and 0/10 vaccinates. Latent FeLV infection was not detected in bone marrow cells from any of the vaccinated cats 14 weeks after challenge exposure.     

Prevalence of feline immunodeficiency virus and other retroviral infections in sick cats in Italy.

Two hundred and seventy-seven sick pet cats living in Italy were tested for antibodies to feline immunodeficiency virus (FIV) and for feline leukemia virus (FeLV) antigen. Overall, 24% of the cats resulted positive for anti-FIV antibody and 18% for FeLV antigen. FIV was isolated from the peripheral mononuclear blood cells of ten out of 15 seropositive cats examined and from one out of eight saliva samples. No FIV isolations were obtained from six serum samples cultured. Feline syncytium forming virus (FeSFV) could be isolated from blood and/or saliva in ten out of 11 FIV seropositive cats examined, in six out of nine FeLV antigen positive cats, in two cats found positive for both infection markers, and in three out of 11 cats negative for both markers. Thus, the probability of isolating FeSFV was enhanced by infection with other exogenous retroviruses.     

Survey of the feline leukemia virus infection status of cats in Southern Germany

Most studies that investigate the prevalence of infections with feline leukemia virus (FeLV) are based on the detection of p27 antigen in blood, but they do not detect proviral DNA to identify the prevalence of regressive FeLV infections. The aim of the present study was to assess the prevalence and status of FeLV infection in cats in Southern Germany. P27 antigen enzyme-linked immunosorbent assay (ELISA), anti-p45 antibody ELISA, DNA polymerase chain reaction (PCR) of blood and RNA PCR of saliva were performed. Nine out of 495 cats were progressively (persistently) infected (1.8%) and six were regressively (latently) infected (1.2%). Cats with regressive infections are defined as cats that have been able to overcome antigenemia but provirus can be detected by PCR. Twenty-two unvaccinated cats likely had abortive infections (regressor cats), testing FeLV antigen- and provirus-negative but anti-p45 antibody-positive. Most of the FeLV-vaccinated cats did not have anti-FeLV antibodies. Both progressive, as well as regressive infections seem to be rare in Germany today.     

Feline immunodeficiency virus, feline leukaemia virus, Toxoplasma gondii, and intestinal parasitic infections in Taiwanese cats

A population consisting of 70 breeder cats, 43 clinical cases, and 16 feral cats was examined for the presence of Toxoplasma gondii, feline immunodeficiency virus (FIV), and feline leukaemia virus (FeLV). No oocysts of T. gondii were observed in 96 faecal samples; faecal samples were not available from the feral cats. Other intestinal parasites identified included Isospora felis (three cats), Isospora rivolta (five), Dipylidium canium (two), Toxocara cati (four), Toxascaris leonina (one), and Ancylostoma sp. (two). Using a kinetics-based enzyme-linked immunosorbent assay on 117 sera including all the feral cats, nine had antibody to T. gondii antigen, three for antigens to FIV, and seven to the p27 antigen of FeLV. Of the nine cats with antibody to T. gondii, only one was also infected with FIV.     

Influence of thymectomy on the susceptibility of cats to feline leukemia virus and lymphosarcoma.

Twelve cats were thymectomized at 5 weeks of age. Six of these cats were inoculated at 8 weeks of age and 6 at 4 months of age with the Rickard (R) strain of feline leukemia virus (FeLV), which produces a high incidence of thymic lymphosarcoma. Two groups of age-matched nonthymectomized cats were inoculated with the same FeLV-R stock. Thymectomy prior to FeLV infection had no influence on the induction of viremia or the incidence of lymphosarcoma. In the FeLV-inoculated nonthymectomized cats, lymphosarcoma developed in the thymus. In the thymectomized cats, lymphosarcoma developed in the intestine, mesenteric lymph nodes, and bone marrow, but the malignant lymphoblasts had surface markers characteristic of feline T lymphocytes. It was concluded that the presence of the thymus per se is not required for infection and oncogenesis by FeLV and that feline T lymphocytes may be transformed after peripheralization to other tissues.     

Antibody response of kittens after vaccination followed by exposure to feline leukemia virus-infected cats.

Protein (western) blot analysis and virus-neutralization assay were used to evaluate the antibody response of specific-pathogen-free kittens to FeLV vaccination and followed by natural exposure. Several kittens had barely detectable reactions to specific FeLV antigens prior to vaccination or exposure. Correlation was not found between protection against persistent viremia and antibody response after vaccination as measured by western blot analysis or virus neutralization assay. A statistically significant (P less than 0.01) difference in the antibody response against p27 antigen after natural exposure to FeLV was observed between persistently viremic kittens and transiently viremic or aviremic kittens. Measurable (P less than 0.05) virus neutralizing antibody titer after FeLV exposure was found only in a small number of kittens that were protected against persistent viremia. Lack of association between humoral response and vaccination-induced protection against persistent FeLV infection suggests an important role for cell-mediated immunity in such protection.     

Use of tears for diagnosis of feline leukemia virus infection

A comparison was made of the use of serum, tears, and saliva for the detection of feline leukemia virus (FeLV) infection in cats. Cotton swabs were used to collect saliva, and tear-test strips were used to collect tears. Specimens were analyzed by a commercially available ELISA. Using a 10- to 15-minute specimen incubation period, FeLV was detected in 70% of the saliva specimens and in 73% of the tear specimens from viremic (serum-positive) cats. Feline leukemia virus antigen was not detected in saliva and tear specimens from serum-negative cats. The sensitivity of the tear assay was improved by increasing the incubation time to 24 hours. Tear strips could be air-dried and stored at room temperature for up to 7 days without any appreciable loss of activity. Client-owned and experimentally infected laboratory cats were tested for FeLV, using air-dried tear-test strips and a 24-hour incubation period. Tears were positive (contained FeLV antigen) in 65 of 72 (90%) serum-positive cats and did not contain antigen in 46 of 46 (100%) serum-negative cats. Results of ELISA obtained from serum and tears also were compared with results obtained from indirect fluorescent antibody testing of blood smears. Results of indirect fluorescent antibody and ELISA compared favorably with each other and with the results of tear testing.     

Serological survey of Toxoplasma gondii, Dirofilaria immitis, Feline Immunodeficiency Virus (FIV) and Feline Leukemia Virus (FeLV) infections in pet cats in Bangkok and vicinities, Thailand

The seroprevalence of Toxoplasma gondii, Dirofilaria immitis (heartworm), feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) infections was examined using serum or plasma samples from 746 pet cats collected between May and July 2009 from clinics and hospitals located in and around Bangkok, Thailand. The samples were tested for heartworm, FIV, and FeLV using a commercial ELISA. Of the 746 samples, 4.6% (34/746) were positive for heartworm antigen, 24.5% (183/746) had circulating FeLV antigen, and 20.1% (150/746) had antibodies against FIV. In addition, the first 348 submitted samples were tested for T. gondii antibodies using a modified agglutination test (MAT, cut off 1:25); 10.1% (35/348) were seropositive. Of the 348 cats sampled for all four pathogens, 11, 10, and 1 were positive for T. gondii antibodies and FIV antibodies, FeLV antigen, or D. immitis antigen, respectively. Of the 35 T. gondii-seropositive cats, 42.9% (15/35) were co-infected with at least one of the other three pathogens. The presence of antibodies to FIV was significantly associated with both age and gender, while FeLV antigen presence was only associated with age. In the case of FIV, males were twice as likely to be infected as females, and cats over 10 years of age were 13.5 times more likely to be infected than cats less than 1 year of age. FeLV antigen was more common in younger cats, with cats over 10 years of age being 10 times less likely to be FeLV positive than cats under 1 year of age. This is the first survey for these four pathogens affecting feline health in Thailand.     

Selective impairment of humoral immunity in feline leukemia virus-induced immunodeficiency.

We used a panel of in vitro assays to investigate the nature of immune dysfunction in cats infected with FeLV-FAIDS, a naturally occurring, molecularly cloned feline leukemia virus (FeLV) isolate which induces a fatal immunodeficiency syndrome in infected cats. During the asymptomatic period preceding immunodeficiency disease, we were unable to detect any deficits in concanavalin A-induced blastogenesis, xenogeneic mixed-lymphocyte reaction assays, stimulation of lymphocytes by soluble protein antigen, and cytotoxic T lymphocyte assays. However, during this period humoral immune responses in the FeLV-FAIDS-infected cats were dramatically impaired. As early as 9 weeks after virus inoculation, the ability to mount either an IgM or IgG response to soluble protein antigens was lost. Neither B cell function, as assessed by lipopolysaccharide-induced blastogenesis or circulating B cell numbers, as assessed by immunofluorescence, differed between infected and control cats. These results suggest that FeLV-FAIDS infection may impair a subpopulation of T helper cells, that provides help for the production of antibody. Consistent with earlier observations of cats naturally infected with FeLV, our results indicate that early impairment of humoral immunity is an important component of the immunodeficiency syndrome induced by FeLV in cats.     

Long-term impact on a closed household of pet cats of natural infection with feline coronavirus, feline leukaemia virus and feline immunodeficiency virus

A closed household of 26 cats in which feline coronavirus (FCoV), feline leukaemia virus (FeLV) and feline immunodeficiency virus (FIV) were endemic was observed for 10 years. Each cat was seropositive for FCoV on at least one occasion and the infection was maintained by reinfection. After 10 years, three of six surviving cats were still seropositive. Only one cat, which was also infected with FIV, developed feline infectious peritonitis (FIP). Rising anti-FCoV antibody titres did not indicate that the cat would develop FIP. The FeLV infection was self-limiting because all seven of the initially viraemic cats died within five years and the remainder were immune. However, FeLV had the greatest impact on mortality. Nine cats were initially FIV-positive and six more cats became infected during the course of the study, without evidence of having been bitten. The FIV infection did not adversely affect the cats' life expectancy.