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.     

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.     

Shedding of feline leukemia virus RNA in saliva is a consistent feature in viremic cats

The purpose of this investigation was to characterize the shedding pattern of feline leukemia virus (FeLV) RNA in saliva, and to correlate it with the proviral load in whole blood, viral load in plasma, levels of p27 in saliva and plasma, the isolation of infectious FeLV from saliva, and the titers of FeLV-specific antibodies of the IgG and IgA isotypes. We evaluated 24 experimentally FeLV-infected cats for these parameters using real-time RT-PCR and PCR, cell culture assay and sandwich ELISA. We observed that shedding of viral RNA in saliva was a consistent feature in viremic cats. Latently FeLV-infected cats, displaying a very low proviral load, did not shed infectious virus in saliva, but occasionally shed viral RNA. Consequently, salivary shedding of FeLV RNA may not necessarily indicate a transmission potential for susceptible cats. This study also confirmed previous results from our laboratory, showing that a negative result for p27 in plasma, or for viral RNA in plasma or saliva does not exclude FeLV infection, considering that blood cells from those cats contained provirus. We also showed that FeLV RNA and DNA were stable for more than 64 days in saliva samples stored at room temperature. We conclude that the detection of FeLV RNA in saliva may be a useful indicator of viremia, and that the detection of salivary viral RNA by RT-PCR could become a reliable tool for the diagnosis of FeLV infection, which is facilitated by the low invasive method of collection of the samples.     

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.     

Effects of treatment with cobra venom factor on experimentally induced feline leukemia

Five- to six-month-old specific-pathogen-free cats were exposed to cobra venom factor (CVF) alone (4 cats), Rickard feline leukemia virus (FeLV; 9 cats), or CVF and FeLV (6 cats). Host-virus relationships were evaluated by monitoring the development of viremia, production of antibody against feline oncornavirus-associated cell membrane antigen, and amount of circulating immune complexes (CIC). Exposure to CVF induced complement depletion, which lasted 8 to 15 days. However, complement depletion did not promote the development of persistent viremia nor alter the production of antibody to feline oncornavirus-associated cell membrane antigen or CIC. Results indicated that the complement system did not protect cats during their initial exposure to FeLV and that an intact complement system was not necessary for the development of antibody against feline oncornavirus-associated antigen or for the formation of CIC.     

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.     

Effects of feline leukemia virus infection on neutrophil chemotaxis in vitro

A procedure for measuring in vitro feline neutrophil chemotaxis was developed, using a modified Boyden chamber apparatus and 3-microns-pore polycarbonate filters. A pooled feline serum sample was used as the chemoattractant. Chemotaxis was evaluated in 5 groups of cats: group 1-specific-pathogen-free cats that had not been exposed to feline leukemia virus (FeLV); group 2-previremic, FeLV-infected, specific-pathogen-free cats; group 3-FeLV-viremic, subclinically affected cats; group 4-FeLV-viremic, clinically affected cats; and group 5-sick cats that were not infected with FeLV. Neutrophils from the viremic, clinically affected cats had significantly lower (P less than 0.025) chemotactic responses than did those from subclinically affected, viremic cats. Conversely, neutrophils from cats that were ill due to causes other than FeLV had the highest mean chemotactic values. Among the viremic, subclinically affected cats, a linear relationship was found between age and chemotaxis, indicating that impairment of neutrophil function may be greater in younger viremic cats. However, FeLV-infected cats can not be identified on the basis of neutrophil chemotaxis.     

Evaluation of efficacy and safety of an inactivated virus vaccine against feline leukemia virus infection.

An inactivated virus vaccine was developed for prevention of FeLV infection in domestic cats. When given in 2 doses, 3 weeks apart, to cats that were greater than or equal to 9 weeks old at the time of first vaccination, the vaccine prevented persistent viremia from developing in 132 of 144 (92%) vaccinates after oronasal challenge exposure with virulent FeLV. In contrast, persistent viremia developed after oronasal challenge exposure with FeLV in 39 of 45 (87%) age-matched nonvaccinated control cats. Transient viremia, indicated by early detection of p27 by ELISA in serum of cats protected from persistent viremia at 12 weeks after challenge exposure, was found in 10 of 132 (8%) vaccinates. Cats that were aviremic 12 to 16 weeks after challenge exposure were examined for reactivation of latent FeLV infection; 4 weekly doses of methylprednisolone were administered, followed by in vitro culture of bone marrow cells. Latent infection was readily reactivated in 6 of 8 (75%) nonvaccinated control cats that had been transiently viremic after challenge exposure. However, latent infection was reactivated in only 5 of 48 (10%) protected vaccinates, and in none of 38 vaccinates in which transient viremia had not been detected. In a safety field trial, only 34 mild reactions of short duration were observed after administration of 2,379 doses of vaccine to cats of various ages, breeds, and vaccination history, for a 1.43% reaction rate. Results indicate that the aforementioned inactivated virus vaccine is safe and efficacious for the prevention of infection with FeLV.     

Study of feline leukemia virus immunity.

Fifteen specific-pathogen-free cats were experimentally infected with FeLV; 8 cats recovered after transient or nondetectable viremia, and 7 cats became persistently viremic. Four additional cats served as noninfected controls. Antibodies to whole FeLV (ELISA and immunoblot [western] analysis), antibodies to fixed FeLV-infected cells, and virus-neutralizing antibodies were monitored for as long as 3 years after infection. As a group, cats that recovered after acute infection developed higher titer of these various antibodies than did cats that became persistently viremic. However, specific combination or titer of antibodies was not always found in recovered cats or in persistently viremic cats. Six cats that had recovered from acute FeLV infection nearly 3 years earlier were reinfected with the same virus. Three of the cats appeared to be resistant to reinfection, 2 cats became transiently viremic, and 1 cat became persistently viremic. Slight and transient anamnestic ELISA-detectable antibody response to whole virus was seen after reinfection; immunofluorescence- and western blot-detectable responses were not greatly enhanced. Five FeLV-recovered cats were monitored for 2 years; FeLV infection spontaneously recurred in 1 cat.     

Modifications of the immunofluorescence assay for feline leukemia virus group-specific antigens.

Observations and minor modifications are presented concerning the immunofluorescence assay for feline leukemia virus (FeLV) group-specific antigens (GSA) in blood cells of cats. Data are given regarding absorption of goat FeLV GSA antiserum in vivo in cats, absorption of the antiserum in vitro with feline blood cells, and the comparative efficacy of various chemical fixatives in preservation of FeLV GSA for immunofluorescent staining. The best results were obtained with in vitro absorption of antiserum and methanol fixation of FeLV GSA in blood smears.     

Protection against feline leukemia virus infection by use of an inactivated virus vaccine.

The protective immunity induced by 3 experimental FeLV vaccines were evaluated: Prototype inactivated FeLV vaccine developed from a molecularly cloned FeLV isolate (FeLV-FAIDS-61E-A); a mixture of immunodominant synthetic peptides corresponding to regions of the FeLV-Gardner-Arnstein-B (FeLV-GA-B) envelope proteins; and an adjuvant-disrupted but non-activated virus prepared from a non-cloned FeLV field isolate comprised of subgroup A and B viruses (FeLV-05821-AB). Included as controls were parallel groups of cats inoculated with adjuvants alone or with an established commercial FeLV vaccine. After each inoculation and after virulent virus challenge exposure, sera from all cats were assayed for ELISA-reactive antibody against purified FeLV, FeLV neutralizing (VN) antibody, and FeLV antigenemia/viremia--viral p27 antigen in serum and within circulating leukocytes. Immunity was challenged by oral/nasal exposure of vaccinated and control cats with FeLV-FAIDS-61E-A or FeLV-05821-AB, an infective, noncloned, tissue-origin, FeLV field isolate containing subgroup-A and -B viruses. Vaccine-induced immunity was assessed by comparing the postchallenge-exposure incidence of persistent viremia and the pre- and postchallenge exposure titers of VN and ELISA antibody in cats of the control and vaccine groups. The percentage of cats, that resisted development of persistent viremia after FeLV challenge exposure and the preventable fraction (PF) for the vaccine groups (which adjusts for the severity of the challenge and the degree of innate resistance in the controls) were as follows: adjuvant controls, 26%; FeLV-FAIDS-61E-A inactivated virus vaccine, 95% (PF = 93.2%); FeLV-GA-B peptide vaccine, 5% (-28.4%); FeLV-05821-AB noninactivated vaccine, 67% (55.4%); and commercial FeLV vaccine, 35% (12.2%). The prechallenge exposure mean VN antibody titer for each group was: less than 1:8 in the adjuvant controls; 1:43 in the FeLV-FAIDS-61E-A-vaccinated cats; less than 1:8 in the peptide-vaccinated cats; 1:38 in the noninactivated virus-vaccinated cats group; and 1:12 in the cats vaccinated with the commercial vaccine. Thus, induction of VN antibody in the vaccinated cats, although modest, appeared to be correlated with induction of protective immunity as defined by resistance to FeLV challenge exposure. Results of these studies indicate that inoculation of cats with an experimental inactivated virus vaccine prepared from a molecularly cloned FeLV isolate was most effective in stimulating protective immunity against heterologous and homologous FeLV challenge exposure.     

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.     

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.     

Real-time PCR investigation of feline leukemia virus proviral and viral RNA loads in leukocyte subsets

Cats exposed to feline leukemia virus (FeLV), a naturally occurring gammaretrovirus develop either progressive or regressive infection. Recent studies using analyses with enhanced sensitivity have correlated loads throughout FeLV with the clinical outcome, though remarkably, during the acute phase of infection, proviral and viral RNA burdens in the peripheral blood do not differ between groups. We hypothesized that viral loads in specific leukocyte subsets influence the infection outcome. Using a method established to determine the proviral and cell-associated viral RNA loads in specific leukocyte subsets, we evaluated viral loads in eleven FeLV-exposed specific pathogen-free (SPF) cats 2.5 years post-infection. Six cats had undergone regressive infection whereas five were persistently viremic. Aviremic cats had lower total proviral blood loads than the persistently infected cats and FeLV proviral DNA was shown to be integrated into genomic DNA in four out of four animals. Lymphocytes were predominantly infected vs. moncytes and granulocytes in aviremic cats. In contrast, persistently viremic cats were provirus-positive in all leukocyte subsets. The acute phase kinetics of FeLV infection were analyzed in two additional cats; an early lymphoreticular phase with productive infection in lymphocytes in both cats and in monocytes in one cat was followed by infection of the granulocytes; both cats became persistently infected. These results indicate that FeLV persistent viremia is associated with secondary viremia of bone marrow origin, whereas regressive cats only sustain a non-productive infection in low numbers of lymphocytes.     

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.     

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.     

Protection against feline leukemia virus challenge for at least 2 years after vaccination with an inactivated feline leukemia virus vaccine

Twelve cats were vaccinated at 8 and 11 weeks of age with a commercially available inactivated FeLV vaccine (Nobivac FeLV, Intervet/Schering-Plough Animal Health). Eleven cats served as age-matched, placebo-vaccinated controls. All cats were kept in isolation for 2 years after vaccination and were then challenged with virulent FeLV to evaluate vaccine efficacy and duration of immunity. Cats were monitored for 12 weeks after challenge for development of persistent viremia using a commercial FeLV p27 ELISA. Persistent viremia developed in all 11 (100%) of the control cats, whereas 10 of 12 (83%) vaccinated cats were fully protected from persistent viremia following challenge. The results demonstrate that the vaccine used in this study protects cats from persistent FeLV viremia for at least 2 years after vaccination.     

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.     

Fecal shedding of infectious feline leukemia virus and its nucleic acids: a transmission potential

Although it is assumed that fecal shedding of feline leukemia virus (FeLV) constitutes a transmission potential, no study has been performed showing that feces of infected cats can be a source of infection. In this study, we investigated fecal viral shedding of FeLV and its role in viral pathogenesis with the goal to improve infection control. FeLV RNA and DNA levels were determined in rectal swabs of experimentally infected cats by real-time PCR, and the results were correlated with proviral and viral loads in whole blood and plasma, respectively, and plasma p27 levels. All antigenemic cats shed FeLV RNA and DNA in feces. To determine whether the viral RNA detected was infectious, virus isolation from feces was also performed. Infectious virus was isolated from feces of antigenemic cats, and these results perfectly correlated with the isolation of virus from plasma. Na?ve cats exposed to these feces seroconverted, showing that infection through feces took place, but remained negative for the presence of FeLV provirus and p27 in blood, an outcome so far not described. Some of the organs collected after euthanasia were provirus positive at low copy numbers. From these results it is concluded that fecal shedding of FeLV plays a role in transmission, but it is probably of secondary importance in viral pathogenesis. Nevertheless, sharing of litter pans by susceptible and viremic cats could increase the environmental infectious pressure and appropriate measures should be taken to avoid unnecessary viral exposure.     

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.