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.     

Differentiation of feline immunodeficiency virus vaccination, infection, or vaccination and infection in cats

BACKGROUND: Serodiagnosis of feline immunodeficiency virus (FIV) is complicated by the use of a formalin-inactivated whole-virus FIV vaccine. Cats respond to immunization with antibodies indistinguishable from those produced during natural infection by currently available diagnostic tests, which are unable to distinguish cats that are vaccinated against FIV, infected with FIV, or both. HYPOTHESIS: An enzyme-linked immunosorbent assay (ELISA) detecting antibodies against formalin-treated FIV whole virus and untreated transmembrane peptide will distinguish uninfected from infected cats, regardless of vaccination status. ANIMALS: Blood samples were evaluated from uninfected unvaccinated cats (n = 73 samples), uninfected FIV-vaccinated cats (n = 89), and FIV-infected cats (n = 102, including 3 from cats that were also vaccinated). METHODS: The true status of each sample was determined by virus isolation. Plasma samples were tested for FIV antibodies by a commercial FIV diagnostic assay and an experimental discriminant ELISA. RESULTS: All samples from uninfected cats were correctly identified by the discriminant ELISA (specificity 100%). Of the samples collected from FIV-infected cats, 99 were correctly identified as FIV-infected (sensitivity 97.1%). CONCLUSIONS AND CLINICAL IMPORTANCE: With the exception of viral isolation, the discriminant ELISA is the most reliable assay for diagnosis of FIV. A practical strategy for the diagnosis of FIV infection would be to use existing commercial FIV antibody assays as screening tests. Negative results with commercial assays are highly reliable predictors for lack of infection. Positive results can be confirmed with the discriminant ELISA. If the discriminant ELISA is negative, the cat is probably vaccinated against FIV but not infected. Positive results are likely to represent infection.     

Efficacy and safety of a feline immunodeficiency virus vaccine

Fel-O-Vax FIV is an inactivated virus vaccine designed as an aid in the prevention of infection of cats, 8 weeks or older, by feline immunodeficiency virus (FIV). It contains two genetically distinct FIV strains. The efficacy of this vaccine was demonstrated in a vaccination-challenge study designed to meet various regulatory requirements for registering the vaccine. Eight-week-old kittens were vaccinated with an immunogenicity vaccine which contained minimal release levels of FIV antigens formulated with a proprietary adjuvant system. Twelve months later, all vaccinates and controls were challenged with a heterologous FIV strain. Following the vigorous challenge exposure, cats were monitored for FIV viremia. It was found that 16% of the vaccinated cats developed viremia while 90% of the controls became persistently infected with FIV, which demonstrated that the vaccine was efficacious and the protective immunity lasted for at least 12 months. The safety of the vaccine was demonstrated by a field safety trial in which only 22 mild reactions of short duration were observed following administering 2051 doses of two pre-licensing serials of Fel-O-Vax FIV to cats of various breeds, ages and vaccination histories. Thus, Fel-O-Vax FIV is safe and efficacious for the prevention of FIV infection in cats.     

Development of FIV-specific cytolytic T-lymphocyte responses in cats upon immunisation with FIV vaccines

Vaccine protection has been achieved in cats against experimental infection with feline immunodeficiency virus (FIV). Such protection has been attributed to FIV-specific humoral immunity, as well as cellular immunity of unknown mechanism(s). Since cell-mediated immunity plays a crucial role in the clearance of viral infections, this study evaluated the role of FIV-specific CTL in vaccine prophylaxis. Cats were immunised with inactivated FIV vaccines, reported to have >90% vaccine efficacy. Significant levels of specific CTL activity were detected following the third immunisation. CTL activity persisted for several months and could be enhanced through a booster immunisation. The levels of CTL activity were comparable to those induced by a recombinant canarypoxvirus based FIV vaccine. These results suggest a possible role for CTL-mediated immunity in vaccine protection against FIV infection in cats.     

Dual-subtype vaccine (Fel-O-Vax FIV) protects cats against contact challenge with heterologous subtype B FIV infected cats

Fel-O-Vax FIV is a dual-subtype vaccine consisting of inactivated whole viruses of subtype A (Petaluma strain) and subtype D (Shizuoka strain). The efficacy of this vaccine against heterologous subtype A strain challenge was demonstrated, but it is unclear whether the result reflects efficacy in the field. In this study, we evaluated the efficacy of this vaccine against contact challenge by exposing both vaccinated and unvaccinated control animals with cats infected with Aomori-2 strain belonging to subtype B, a subtype prevalent in many regions of the world. Nineteen specific-pathogen-free (SPF) cats were divided into a vaccinated group (six cats), an unvaccinated control group (eight cats), and a challenge group (five cats), and maintained in the same room. Cats were monitored for FIV proviral DNA by nested PCR and for FIV-specific antibody levels by ELISA. After 1 year of commingling, each cat in the vaccinated group was given a booster dose. In addition, the original challenge group was removed and replaced with another challenge group of SPF cats, which were inoculated with the Aomori-2 strain. FIV infection was confirmed in four of the eight animals in the unvaccinated control group by the 29th week in the second year of commingling. In contrast, all of the animals were negative in the vaccinated group. These findings confirmed the efficacy of this vaccine against heterologous stains classified as subtype B, and suggested that the vaccine exhibits broad efficacy against genetically diverse FIV.     

Effect of vaccination against feline immunodeficiency virus on results of serologic testing in cats

OBJECTIVE: To determine the effect of vaccination against FIV on results of serologic assays for FIV infection. DESIGN: Prospective clinical trial. ANIMALS: 26 specific-pathogen-free cats, 102 laboratory-reared cats (42 unvaccinated and uninfected, 41 vaccinated and uninfected, and 19 infected with FIV), and 22 client-owned cats infected with FIV. PROCEDURE: To determine the onset and duration of anti-FIV antibody production in cats following vaccination with a whole-virus vaccine, serum was obtained from the 26 specific-pathogen-free cats prior to vaccination and weekly for 10 weeks, then monthly for 52 weeks, after vaccination; serum was tested for anti-FIV antibodies with lateral flow and microwell plate ELISAs. To determine the diagnostic performance of serologic assays for FIV infection, plasma from uninfected, unvaccinated cats; uninfected, vaccinated cats; and FIV-infected cats was tested for FIV antibodies with the 2 ELISAs, a western blot assay, and an immunofluorescence antibody assay and for FIV antigen with an ELISA. RESULTS: Anti-FIV antibodies were detected in all 26 vaccinated cats 1 year after vaccination. Sensitivity of the antibody assays for FIV infection was high (98% to 100%). Specificity was high in unvaccinated cats (90% to 100%) but poor in vaccinated cats (0% to 54%). None of the vaccinated or infected cats had detectable FIV antigen in plasma. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that vaccination against FIV causes false-positive results for at least 1 year with currently available serologic assays for FIV infection. Negative FIV antibody assay results are highly reliable for detection of uninfected cats, but positive results should be interpreted with caution.     

Immunization-induced decrease of the CD4+:CD8+ ratio in cats experimentally infected with feline immunodeficiency virus.

In a previous experiment a group of 15 specified pathogen free (SPF) cats were experimentally infected with a Swiss isolate of feline immunodeficiency virus (FIV). A group of 15 SPF cats served as FIV negative controls. Nine cats of each group were vaccinated with a recombinant feline leukemia virus (FeLV) vaccine, six cats in each group with a placebo vaccine. All vaccinated cats developed high antibody titers to FeLV and were protected against subsequent FeLV challenge infection. In both control groups five of six cats became persistently infected with FeLV. Unexpectedly, the primary immune response to the vaccine antigen was significantly higher in the FIV positive group than in the FIV negative. The secondary response was stronger in the FIV negative cats. The goal of the present investigation was to further study the immune response in these 30 cats. They were immunized twice with the synthetic peptide L-tyrosine-L-glutamic acid-poly(DL-alanine)-poly(L-lysine) (TGAL) 21 days apart. Blood samples were collected on four occasions during the immunization process. They were tested for antibodies to TGAL, complete blood cell counts and CD4+, CD8+ and pan-T-lymphocyte counts. The following observations were made: (1) in contrast to the FeLV vaccine experiment, the primary immune response to TGAL was not significantly stronger in the FIV positive cats when tested by enzyme-linked immunosorbent assay (2). The absolute size of the CD4+ lymphocyte population was distinctly smaller in the FIV positive than in the FIV negative cats. The lowest CD4+ values were found in the dually FIV/FeLV infected cats. (3) A population of CD8+ lymphocytes was identified that was characterized by a distinctly weaker fluorescence. The size of this population increased in FIV positive and decreased in FIV negative cats during the TGAL immunization experiment. (4) The CD4+:CD8+ ratio increased in FIV negative cats during TGAL immunization from 1.9 to 2.3. In contrast, in FIV positive animals the CD4+:CD8+ ratio decreased significantly from 1.9 to 1.3 during the same period. From these and earlier data it was concluded that in short-term FIV infection the immune response to T-cell dependent antigens may be increased over that of the controls. Immune suppression develops gradually with duration of the infection. The significant drop of the CD4+:CD8+ ratio over a 5 week immunization period suggests that antigenic stimulation may accelerate the development of immune suppression in FIV positive cats. If this is a general feature, FIV infection may provide a particularly interesting model for studying the pathogenesis of AIDS.     

Vaccination of cats against feline immunodeficiency virus (FIV): a matter of challenge.

So far, most apparently successful immunodeficiency virus vaccines have only been tested against challenge with cell culture-adapted virus. However, even live priming of cats with feline herpesvirus (FHV) vectors expressing the FIV gag and env gene followed by inactivated booster vaccination with fixed FIX infected cell vaccine proved non-protective against infection with a primary FIV isolate. An effective FIV vaccine for field applications therefore is still not underway.     

A CELL CULTURE VACCINE AGAINST BOVINE EPHEMERAL FEVER

SUMMARY A vaccine was prepared from cell culture fluids harvested from the twelfth passage of the 919 strain of bovine ephemeral fever (BEF) virus in Vero cell cultures. Cattle were vaccinated subcutaneously with various combinations of strain 919 virus and adjuvants. Neutralising antibodies were assayed at various times after vaccination and some cattle were challenged by intravenous inoculation with the virulent 417WBC strain of BEF virus. Strain 919 virus of the third and twelfth passage levels in Vero cells produced neither fever, clinical illness nor detectable viraemia in 5 calves inoculated intravenously. Nor could viraemia be detected in 5 heifers receiving vaccine subcutaneously. When the vaccine was administered mixed with aluminium hydroxide adjuvant, the production of neutralising antibodies increased with an increase in the volume of vaccine from 2.5 ml to 10 ml and the response to 2 injections was significantly better than the response to a single injection. The neutralising antibody response was decreased when vaccine was diluted in phosphate buffered saline. The neutralising antibody response following 2 subcutaneous vaccinations with strain 919 virus mixed with aluminium hydroxide adjuvant was higher than that following intravenous inoculation with virulent virus. The vaccine-induced antibodies persisted for at least 12 months, and revaccination at this time led to an increase in the titre of neutralising antibody. Antibodies induced by a single subcutaneous administration of strain 919 virus mixed with Freund's complete adjuvant persisted for at least 40 weeks; those induced by vaccine containing Freund's incomplete adjuvant had virtually disappeared within 16 weeks. All these calves responded to vaccination with aluminium hydroxide-containing vaccine with increases in levels of neutralising antibodies. Of 26 vaccinated calves challenged with virulent BEF virus, 24 remained clinically normal. Two developed brief periods of pyrexia on the seventh day after challenge, but no other clinical signs. One of these calves had a viraemia that was demonstrated only by intravenous inoculation of a susceptible calf. The remaining calf had no detectable viraemia. All of 7 unvaccinated calves developed severe clinical BEF within 5 days of challenge. No disease attributable to the 919 virus occurred in 24 vaccinated pregnant heifers or their newborn calves.     

Transmission of feline leukaemia virus in the milk of a non-viraemic cat

The possibility of the transmission of feline leukaemia virus (FeLV) from latently infected cats was studied. Five female cats with latent infections were examined for evidence of transmission of the virus to their kittens. One of the cats infected members of four consecutive litters of kittens which subsequently became persistently viraemic and transmitted the virus to other susceptible kittens by contact. Shortly after birth its kittens were apparently FeLV-free since neither viral antigen nor infectious virus was detected in their blood and no virus was found in cell cultures made from aspirates of bone marrow. The kittens became viraemic from 45 days of age onwards at a time when their passively acquired colostral FeLV neutralising antibodies were no longer detectable. Transmission of the virus occurred via the milk since both FeLV antigen and infectious virus were found in milk samples taken six weeks after kittening and the virus was transmitted to a fostered kitten. Eleven weeks after the birth of the fourth litter the cat became viraemic. The intermittent presence of FeLV antigens detected by the Leukassay F test, but not infectious virus, in the plasma of this cat over the previous months and a low level of serum neutralising antibodies distinguished it from four other latently infected queens which did not transmit infection to their kittens. These factors may indicate a risk of milk transmission and reactivation of latent virus.     

Diagnosing feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV) infection: an update for clinicians

With the commercial release in Australia in 2004 of a vaccine against feline immunodeficiency virus (FIV; Fel‐O‐Vax FIV®), the landscape for FIV diagnostics shifted substantially. Point‐of‐care (PoC) antibody detection kits, which had been the mainstay for diagnosing FIV infection since the early 1990s, were no longer considered accurate to use in FIV‐vaccinated cats, because of the production of vaccine‐induced antibodies that were considered indistinguishable from those produced in natural FIV infections. Consequently, attention shifted to alternative diagnostic methods such as nucleic acid detection. However, over the past 5 years we have published a series of studies emphasising that FIV PoC test kits vary in their methodology, resulting in differing accuracy in FIV‐vaccinated cats. Importantly, we demonstrated that two commercially available FIV antibody test kits (Witness? and Anigen Rapid?) were able to accurately distinguish between FIV‐vaccinated and FIV‐infected cats, concluding that testing with either kit offers an alternative to PCR testing. This review summarises pertinent findings from our work published in a variety of peer‐reviewed research journals to inform veterinarians (particularly veterinarians in Australia, New Zealand and Japan, where the FIV vaccine is currently commercially available) about how the approach to the diagnosis of FIV infection has shifted. Included in this review is our work investigating the performance of three commercially available FIV PoC test kits in FIV‐vaccinated cats and our recommendations for the diagnosis of FIV infection; the effect of primary FIV vaccination (three FIV vaccines, 4 weeks apart) on PoC test kit performance; our recommendations regarding annual testing of FIV‐vaccinated cats to detect ‘vaccine breakthroughs’; and the potential off‐label use of saliva for the diagnosis of FIV infection using some FIV PoC test kits. We also investigated the accuracy of the same three brands of test kits for feline leukaemia virus (FeLV) diagnosis, using both blood and saliva as diagnostic specimens. Based on these results, we discuss our recommendations for confirmatory testing when veterinarians are presented with a positive FeLV PoC test kit result. Finally, we conclude with our results from the largest and most recent FIV and FeLV seroprevalence study conducted in Australia to date.     

New challenges for the diagnosis of feline immunodeficiency virus infection.

Vaccination of cats against feline immunodeficiency virus (FIV) with a whole-virus vaccine results in rapid and persistent production of antibodies that are indistinguishable from those used for diagnosis of FIV infection. There are no diagnostic tests available for veterinary practitioners at the present time to resolve the diagnostic dilemma posed by use of whole-virus vaccines for protection of cats against FIV. There is a great need for development of commercially available rapid diagnostic tests that conform to differentiation of infected from vaccinated animals standards.     

High prevalence of Borna disease virus in domestic cats with neurological disorders in Japan

A total of 15 (T-1–T-15) domestic cats with neurological disorders in Tokyo area were examined for association with Borna disease virus (BDV). None had detectable antibodies to feline immunodeficiency virus (FIV), feline leukemia virus, feline infectious peritonitis virus and Toxoplasma gondii, and only cat T-8 had detectable antibody to FIV. Serological and molecular epidemiological studies revealed a significantly high prevalence of BDV infection in these cats: antibodies against BDV p24 and/or p40 proteins in 10/15 (66.7%) and p24 and/or p40 RNA in peripheral blood mononuclear cells in 8/15 (53.3%). Further, in situ hybridization and immunohistochemistry analyses of the autopsied brain samples derived from one of the cats (T-15) revealed BDV RNA predominantly in neuronal cells in restricted regions, such as olfactory bulb and medulla of cerebrum. Thus, BDV is present in Japanese domestic cats with neurological disorders at a high prevalence.     

Vaccine efficacy of a cell lysate with recombinant baculovirus-expressed feline infectious peritonitis (FIP) virus nucleocapsid protein against progression of FIP

The Type II feline infectious peritonitis virus (FIPV) infection of feline macrophages is enhanced by a monoclonal antibody (MAb) to the S protein of FIPV. This antibody-dependent enhancement (ADE) activity increased with the MAb that showed a neutralizing activity with feline kidney cells, suggesting that there was a distinct correlation between ADE activity and the neutralizing activity. The close association between enhancing and neutralizing epitopes is an obstacle to developing a vaccine containing only neutralizing epitopes without enhancing epitopes. In this study, we immunized cats with cell lysate with recombinant baculovirus-expressed N protein of the Type I FIPV strain KU-2 with an adjuvant and investigated its preventive effect on the progression of FIP. Cats immunized with this vaccine produced antibodies against FIPV virion-derived N protein but did not produce virus-neutralizing antibodies. A delayed type hypersensitivity skin response to N protein was observed in these vaccinated cats, showing that cell mediated immunity against the FIPV antigen was induced. When these vaccinated cats were challenged with a high dose of heterologous FIPV, the survival rate was 75% (6/8), while the survival rate in the control group immunized with SF-9 cell-derived antigen was 12.5% (1/8). This study showed that immunization with the cell lysate with baculovirus-expressed N protein was effective in preventing the progression of FIP without inducing ADE of FIPV infection in cats.     

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.     

Immunologic responses in healthy random-source cats fed N,N-dimethylglycine-supplemented diets.

The immunomodulatory capacities of N,N-dimethylglycine (DMG) were examined in random-source cats. Blood mononuclear leukocytes of healthy adult cats that had negative results to tests for FeLV and feline immunodeficiency virus were exposed in vitro to various concentrations of DMG (10 to 1,000 micrograms/ml) and were evaluated for proliferative responses to T- or B-cell phytomitogens. Although increased, mean lymphocyte blastogenic responses to phytolectins in DMG-treated cultures did not differ significantly from responses of untreated cultures. For in vivo studies, cats were given a solution containing either 100 mg of DMG or a control solution without DMG orally at 8 AM and 6 PM for 40 consecutive days. On post-treatment day 24 and 25, mean blastogenic responses to phytolectins in DMG-treated and control cats inoculated 10 days earlier with an inactivated feline virus vaccine were similar. Cats given DMG and inoculated twice in a 3-week interval with a commercial vaccine containing inactivated feline herpesvirus-1 and feline calicivirus had significantly (P = 0.045) lower virus neutralizing serum antibody titers against feline herpesvirus-1, compared with titers of control cats, whereas feline calicivirus titers were similar in both groups. On day 25, mean serum interferon activity, induced after IV inoculation of Newcastle disease virus, was significantly (P = 0.021) lower in the DMG-treated cats. Results of this study of DMG in healthy cats failed to demonstrate enhancement of either specific or nonspecific immunity.     

Accuracy of polymerase chain reaction assays for diagnosis of feline immunodeficiency virus infection in cats

OBJECTIVE: To determine the sensitivity, specificity, and overall diagnostic accuracy of polymerase chain reaction (PCR) assays offered by commercial diagnostic laboratories for diagnosis of FIV infection in cats. DESIGN: Prospective clinical trial. ANIMALS: 124 cats. PROCEDURE: Blood was collected from cats that were neither infected with nor vaccinated against FIV, uninfected cats that were vaccinated with a licensed FIV vaccine, and cats experimentally and naturally infected with FIV representing subtypes A, B, and C. Coded blood samples were submitted to 3 laboratories in the United States and Canada offering PCR assays for diagnosis of FIV infection to veterinary practitioners. All laboratories tested fresh blood samples, and 1 laboratory also tested samples submitted as dried blood smears. The FIV infection status in all cats was confirmed by virus isolation. Sensitivity, specificity, and correct results were calculated for each PCR assay. RESULTS: Sensitivity ranged from 41% to 93%. Specificity ranged from 81% to 100% in unvaccinated cats and 44% to 95% in cats vaccinated against FIV. Correct results were obtained in 58% to 90% of 124 cats tested. All tests misidentified both uninfected and infected cats. False-positive results by all laboratories were higher in cats vaccinated against FIV than in unvaccinated cats, suggesting that vaccination interferes with the performance or interpretation of PCR assays used for diagnosis of FIV infection. CONCLUSIONS AND CLINICAL RELEVANCE: PCR assays used for diagnosis of FIV infection presently marketed to veterinary practitioners in North America vary significantly in diagnostic accuracy and did not resolve the diagnostic dilemma resulting from vaccination of cats against FIV.     

Detection of carriers of foot-and-mouth disease virus among vaccinated cattle

To investigate and optimise detection of carriers, we vaccinated 15 calves with an inactivated vaccine based on foot-and-mouth disease virus (FMDV) A Turkey strain and challenged them and two further non-vaccinated calves with the homologous virus four weeks later. To determine transmission to a sensitive animal, we put a sentinel calf among the infected cattle from 60 days post-infection until the end of the experiment at 609 days post-infection. Samples were tested for the presence of FMDV, viral genome, specific IgA antibodies, antibodies against FMDV non-structural (NS) proteins or neutralising antibodies. Virus and viral genome was intermittently isolated from probang samples and the number of isolations decreased over time. During the first 100 days significantly more samples were positive by RT-PCR than by virus isolation (VI), whereas, late after infection more samples were positive by virus isolation. All the inoculated cattle developed high titres of neutralising antibodies that remained high during the entire experiment. An IgA antibody response was intermittently detected in the oropharyngeal fluid of 14 of the 17 calves, while all of them developed detectable levels of antibodies to NS proteins of FMDV in serum, which declined slowly beyond 34 days post-infection. Nevertheless, at 609 days after inoculation, 10 cattle (60%) were still positive by NS ELISA. Of the 17 cattle in our experiment, 16 became carriers. Despite frequent reallocation between a different pair of infected cattle no transmission to the sentinel calf occurred. It remained negative in all assays during the entire experiment. The results of this experiment show that the NS ELISA is currently the most sensitive method to detect carriers in a vaccinated cattle population.     

Detection of transient and persistent feline leukaemia virus infections

A study was made of cats persistently or transiently viraemic with feline leukaemia virus (FeLV) following experimental oronasal infection. Cats of two ages were exposed to the virus. One group was infected when eight weeks old in the expectation that most of the cats would become persistently viraemic, and the second group when 16 weeks old, so that some would show signs of a transient infection and then recover. The periods following infection when virus was detectable in the blood and in the oropharynx were determined for each group. Three methods for detecting viraemia were compared: virus isolation, immunofluorescence on blood smears and an enzyme-linked immunosorbent assay (ELISA). There was good overall agreement among the three tests in detecting virus-positive cats. Virus was found sooner after infection by virus isolation than by the other methods, and virus appeared in the blood slightly sooner in cats which developed persistent viraemia than in transiently viraemic cats. Infectious FeLV was isolated from the oropharynx of all of the persistently viraemic cats, in most cases simultaneously with virus in the plasma. Virus was also isolated from the mouth of most transiently viraemic cats. Under field conditions such transient excretion of virus lasting only a few days would rarely be detected in a single sampling. This might explain how FeLV is maintained in free range urban cats in the absence of a large number of cats with persistent active FeLV infection. For routine diagnosis, immunofluorescence would appear to offer the best chance of differentiating transient and persistent infections by FeLV.