Evaluation of a new in‐clinic test system to detect feline immunodeficiency virus and feline leukemia virus infection

Background: Many in‐house tests for the diagnosis of feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) infection are licensed for use in veterinary practice. A new test with unknown performance has recently appeared on the market. Objectives: The aims of this study were to define the efficacy of a new in‐clinic test system, the Anigen Rapid FIV Ab/FeLV Ag Test, and to compare it with the current leading in‐clinic test, the SNAP Kombi Plus FeLV Antigen/FIB Antibody Test. Methods: Three‐hundred serum samples from randomly selected healthy and diseased cats presented to the Clinic of Small Animal Medicine at Ludwig Maximilian University were tested using both the Anigen Rapid Test and the SNAP Kombi Plus Test. Diagnostic sensitivity, specificity, and positive and negative predictive values were calculated for both tests using Western blot as the gold standard for verification of FIV infection and PCR as the gold standard for FeLV infection. Results: The presence of antibodies against FIV was confirmed by Western blot in 9/300 samples (prevalence 3%). FeLV DNA was detected by PCR in 15/300 samples (prevalence 5%). For FIV infection the Anigen Rapid Test had a sensitivity of 88.9%, specificity of 99.7%, positive predictive value of 88.9%, and negative predictive value of 99.7%. For FeLV infection, the Anigen Rapid Test had a sensitivity of 40.0%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 96.9%. Diagnostic accuracy was similar to that of the SNAP Kombi Plus Test. Conclusion: The new Anigen Rapid FIV Ab/FeLV Ag Test performed very well and can be recommended for use in veterinary practice.     

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.     

Feline immunodeficiency virus status of Australian cats with lymphosarcoma

OBJECTIVE: To determine the FIV status of Australian cats with lymphosarcoma and relate this to patient characteristics, tumour characteristics (tissue involvement, histological grade and immunophenotype), haematological and serum biochemical values and FeLV status of affected cats. DESIGN: Prospective study of 101 client-owned cats with naturally-occurring lymphosarcoma. PROCEDURE: Western blot analysis, ELISA and immunochromatography were used to detect FIV antibodies in serum from cats with lymphosarcoma. RESULTS: On the basis of Western blot analysis (which was considered the most accurate method for determining FIV status), 50/101 (50%) of cats with naturally-occurring lymphosarcoma were positive for FIV antibodies. Of these 50 cats, 35 had tumours of B-cell phenotype, 13 had T-cell tumours and 2 had tumours classified as non-B/non-T. Tumours from eight of these FIV-positive cats contained FeLV gene sequences, including a 9-month-old cat with FeLV antigenaemia. Compared with FlV-negative cats with lymphosarcoma, FIV-positive cats were more likely to be domestic crossbreds (P = 0.004), male (P = 0.048) and have atypical (especially nasal) forms of lymphosarcoma (P = 0.09). Only 39 of 107 (36%) blood or sera tested using ELISA were positive for FIV antibodies (including 5 false-positives). CONCLUSIONS: The prevalence of FIV infection was considerably higher in our cohort of cats compared with series of lymphosarcoma cases from the Northern hemisphere. A positive FIV status was strongly associated with lymphosarcoma in Australian cats and it is possible that this infection may predispose to the development of lymphoid neoplasia. The presence of FIV infection would have been underestimated if commercial kits alone had been used for serology.     

Serologic prevalence of selected infectious diseases in cats with uveitis.

Serologic evidence of infection by Toxoplasma gondii, feline leukemia virus, feline coronaviruses, or feline immunodeficiency virus (FIV) is commonly found in cats with uveitis. Serum samples from 124 cats with uveitis were assayed by use of ELISA for the detection of T gondii-specific immunoglobulin M (IgM), IgG, and circulating antigens (Ag), as well as an ELISA for feline leukemia virus Ag, an ELISA for antibodies to FIV, and an indirect fluorescent antibody assay for antibodies to feline coronaviruses. Serologic evidence of infection by 1 or more of the infectious agents was detected in 83.1% of the samples. Serologic evidence of T gondii infection, defined as the detection of T gondii-specific IgM, IgG, or Ag in serum, was found in 74.2% of the samples. The seroprevalence of T gondii infection was significantly greater in cats with uveitis than in healthy cats from a similar geographic area. Serum samples from cats with serologic evidence of both T gondii and FIV infections were more likely to contain T gondii-specific IgM without IgG than samples from cats with serologic evidence of T gondii infection alone. Cats with serologic evidence of FIV and T gondii coinfection had a higher T gondii-specific IgM titer geometric mean and a lower T gondii-specific IgG titer geometric mean than did cats with serologic evidence of T gondii infection alone. Serologic evaluation for T gondii infection should include assays that detect IgM, IgG, and Ag, particularly in cats coinfected with FIV.     

Quality of different in-clinic test systems for feline immunodeficiency virus and feline leukaemia virus infection

Many new diagnostic in-house tests for identification of feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV) infection have been licensed for use in veterinary practice, and the question of the relative merits of these kits has prompted comparative studies. This study was designed to define the strengths and weaknesses of seven FIV and eight FeLV tests that are commercially available. In this study, 536 serum samples from randomly selected cats were tested. Those samples reacting FIV-positive in at least one of the tests were confirmed by Western blot, and those reacting FeLV-positive were confirmed by virus isolation. In addition, a random selection of samples testing negative in all test systems was re-tested by Western blot (100 samples) and by virus isolation (81 samples). Specificity, sensitivity, positive and negative predictive values of each test and the quality of the results were compared.     

Feline immunodeficiency virus diagnosis after vaccination

Prior to the widespread use of vaccination for the control of feline immunodeficiency virus (FIV) infection, diagnosis was made by the detection of antibodies against FIV. A number of commercial animal side tests perform quite well for this determination, with positive predictive values between 91 and 100% and negative predictive values between 96 and 100%. Furthermore, results of these tests could be confirmed by western blot analysis of FIV test-positive sera. Currently, a killed whole virus FIV vaccine has been made available to practitioners. Vaccinated cats seroconvert by ELISA and western blot, making presently available diagnostic tests, which rely on antibody detection, useless in cats after vaccination. The advisory panels of the American Association of Feline Practitioners and Academy of Feline Medicine both recommend testing for feline leukemia virus antigen and FIV antibody before vaccination.     

Detection of antibodies to Borna disease virus in Turkish cats by using recombinant p40.

Recombinant p40 produced by baculovirus was used in an ELISA to screen samples of serum taken from 80 cats in Istanbul. The sera were also analysed for feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV). Antibodies to Borna disease virus- (BDV) p40 were detected in 34 (42-5 per cent) of the 80 cats. Seventy-three per cent of the sera which were positive for FIV and 26 per cent of the sera which were negative for FIV had antibodies to BDV. There was no difference in the percentage of sera which were positive for BDV between the cats that were positive or negative for FeLV. Three of the cats had neurological disease and two of these had antibodies to BDV. Six sera with low, medium or high optical densities (ODS) by ELISA were analysed by Western blotting. Only the sera with medium and high ODS reacted specifically with p40 at a dilution of 1 in 1,000.     

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.     

Prevalences of feline leukaemia virus and feline immunodeficiency virus infections in cats in Sydney

Objective To determine prevalences of feline leukaemia virus (FeLV) and feline immunodeficiency virus (FIV) infections in ‘healthy’ cats that, through acute misadventure or other circumstance, were presented to veterinary practitioners. Prevalences of FeLV and FIV in this population were compared to those in a population of predominantly sick cats. Design and procedures Serum specimens were obtained over a 2-year period from 200 cats oldeer than 1 year of age presented to veterinary clinics for routine procedures, including cat fight injuries or abscesses, vehicular trauma, neutering, dental scaling, vaccination, grooming or boarding. An additional 894 sera were obtained over approximately the same period from specimens submitted by veterinarians to a private clinical pathology laboratory, mainly from sick cats suspected of having immune dysfunction, but including some sera from healthy cats being screened prior to FeLV vaccination. FIV antibody and FeLV antigen were detected in samples using commercial enzyme immunoassays. Results Amongst 200 ‘healthy’ cats, the prevalence of FeLV infection was 0 to 2%, and the prevalence of FIV was 6.5 to 7.5%, depending on the stringency of the criteria used to define positivity. FIV infection was significantly more prevalent in cats which resided in an inner city environment (P = 0.013). Of the 894 serum specimens submitted to the laboratory by practitioners, 11/761 (1.4%) were FeLV positive, while 148/711 (20.8%) were FIV positive. The prevalence of FIV was significantly higher in these predominantly ‘sick’ cats than in cats seen for routine veterinary procedures (P < 0.00001), while there was no difference in the prevalence of FeLV (P = 0.75) Conclusions The prevalence of FeLV and FIV in healthy cats may have been substantially overestimated in some previous Australian surveys. FeLV infection would appear to be a rare cause of disease in Australian cats. The higher prevalence of FIV positivity in sick as opposed to healthy cats infers that FIV infection contributes to the development of disease.     

Densitometric analysis of Western blot assays for feline immunodeficiency virus antibodies

Western blot (WB) strips for antibodies directed to feline immunodeficiency virus (FIV) were analysed using reflectance densitometry by a semiautomatic densitometer. This method was used to quantify the antibody responses to different FIV proteins in both vaccinated and naturally or experimentally-infected cats. In order to increase reproducibility, reagents and protocols were accurately standardised and internal controls were added. In a first format, an internal control band consisting of feline IgG was added to each blot to minimise the effect of band intensity variation. In a second format, antibody concentrations were calculated from the ratio of the densities produced by test sera and by positive and negative standard sera. The sera under scrutiny were also examined by standard enzyme-linked immunosorbent assay (ELISA) and the results obtained compared with those of the corresponding WB. A statistically significant positive correlation was found between the results obtained with the two methods, and this was especially evident when ELISA titres were compared to corrected WB values (P = 0.001). Densitometric analysis of WB assays allowed to quantify the antibodies against FIV proteins and might be useful to investigate possible humoral immune correlates of protection in FIV vaccination studies and antibody production in the early phase of infection. The quantitation of antibodies to Gag and Env FIV antigens might be used to obtain further informations on the course of FIV disease, as previously demonstrated in human immunodeficiency virus-1 (HIV-1) infections.     

Tumor necrosis factor alpha levels in cats experimentally infected with feline immunodeficiency virus: effects of immunization and feline leukemia virus infection.

Tumor necrosis factor alpha (TNF alpha) levels were determined by enzyme-linked immunosorbent assay (ELISA) and by cell culture bioassay in supernatants of lipopolysaccharide-stimulated feline monocyte cultures and in cat serum samples. There was a good correlation between the results obtained by the two methods. From the fact that TNF alpha was neutralized quantitatively by antibodies to human TNF alpha in feline monocyte supernatants and in feline sera, it was concluded that feline TNF alpha immunologically cross-reacts with human TNF alpha and that the human TNF alpha ELISA can be used to quantitate feline TNF alpha. During the first 6 months after experimental feline immunodeficiency virus (FIV) infection no differences in serum TNF alpha values were observed between infected and non-infected cats. TNF alpha levels increased significantly after primary vaccination with a feline leukemia virus (FeLV) vaccine in FIV infected cats over those in the non-infected controls. During secondary immune response TNF alpha levels rose transiently for a period of a few days in both the FIV positive and the FIV negative cats. After FeLV challenge, TNF alpha levels increased in all animals challenged with virulent FeLV for a period of 3 weeks. This period corresponded to the time necessary to develop persistent FeLV viremia in the control cats. It was concluded from these experiments that in the asymptomatic phase of FIV infection no increased levels of TNF alpha are present, similar to the situation in asymptomatic HIV infected humans. Activation of monocytes/macrophages in FIV infected cats by stimuli such as vaccination or FeLV challenge readily leads to increased levels of TNF alpha.     

Seroprevalence of feline immunodeficiency virus, feline leukaemia virus and Toxoplasma gondii in stray cat colonies in northern Italy and correlation with clinical and laboratory data

Stray cat colonies in urban and rural areas of Lombardy, northern Italy, were surveyed for seroprevalence of feline immunodeficiency virus (FIV) antibodies, feline leukaemia virus (FeLV) antigen and Toxoplasma gondii IgG. Of 316 cats tested, 6.6% were positive for FIV and 3.8% were positive for FeLV infection; 203 cats were tested for T gondii IgG antibodies and a prevalence of 30.5% was detected. Statistical analysis tested the influence of provenience, age, gender, health status and laboratory results on seroprevalence and found male gender and adult age were risk factors for FIV infection. FIV-infected cats were more likely to have a decreased red blood cell count than FIV seronegative cats. No predictors were significantly associated with FeLV and T gondii seropositivity. Colony cats in this study posed a limited risk for retrovirus infection to pet cats allowed outdoors, whereas toxoplasmosis exposure was comparable with the worldwide data.     

Retrospective serologic survey for the presence of feline immunodeficiency virus antibody: a comparison of ELISA and IFA techniques.

A total of 878 samples from the New York State Diagnostic Laboratory (NYSDL), dating from January 1984 to May 1987, were examined to detect antibodies to feline immunodeficiency virus (FIV). We used 2 screening methods; an indirect immunofluorescence assay (IFA) and an enzyme-linked immunosorbent assay (ELISA). Of these, 211 samples were from cats that tested negative for feline leukemia virus (FeLV) and exhibited disease signs consistent with immunodeficiency disease; 19 (9.0%) serum samples were determined to be positive. An additional 508 samples were from cats that tested FeLV-negative and were asymptomatic; 6 (1.2%) sera were determined to be positive. The final 159 samples were from FeLV-positive cats and included symptomatic and asymptomatic animals; this population of cats produced 6 (3.8%) positives. Additionally, 521 samples from the Cornell Feline Health Center (CFHC) serum bank, dating back to 1966, were tested to determine the earliest sample in which FIV antibodies could be detected. Five (2.7%) 1971 and 3 (3.3%) 1969 CFHC samples tested positive. The IFA for FIV antibody proved to be a sensitive (97.4%) and specific (100%) test. The ELISA also had high sensitivity (100%) and specificity (99.6%); however, the IFA proved to be more specific than the ELISA when assaying FeLV-positive cats.     

Prevalence of feline immunodeficiency virus infection in domesticated and feral cats in eastern Australia

Serum samples from 340 pet cats presented to three inner city clinics in Sydney Australia, 68 feral cats from two separate colonies in Sydney, and 329 cattery-confined pedigree and domestic cats in eastern Australia, were collected over a 2-year period and tested for antibodies directed against feline immunodeficiency virus (FIV) using immunomigration (Agen FIV Rapid Immunomigration test) and enzyme-linked immunosorbent assay methods (Snap Combo feline leukaemia virus antigen/FIV antibody test kit, IDEXX Laboratories). Western blot analysis was performed on samples in which there was discrepancy between the results. Information regarding breed, age, gender, housing arrangement and health status were recorded for all pet and cattery-confined cats, while the estimated age and current physical condition were recorded for feral cats. The FIV prevalence in the two feral cat populations was 21% and 25%. The majority of FIV-positive cats were male (60-80%). The FIV prevalence in cattery-confined cats was nil. The prevalence of FIV in the pet cat sample population was 8% (27/340) with almost equal prevalence in 'healthy' (13/170) and 'systemically unwell' (14/170) cats. The age of FIV-positive pet cats ranged from 3 to 19 years; all FIV-positive cats were domestic shorthairs with outside access. The median age of FIV-positive pet cats (11 years) was significantly greater than the median age of FIV-negative pet cats (7.5 years: P<0.05). The prevalence of FIV infection in male pet cats (21/172; 12%) was three times that in female pet cats (6/168; 4%; P<0.05). With over 80% of this pet cat population given outside access and continued FIV infection present in the feral population, this study highlights the need to develop rapid, accurate and cost-effective diagnostic methods that are not subject to false positives created by concurrent vaccination against FIV. This is especially important in re-homing stray cats within animal shelters and monitoring the efficacy of the new vaccine, which has not been challenged against Australian strains. The absence of FIV within cattery-confined cats highlights the value in routine screening and indoor lifestyles. This study provides cogent baseline FIV prevalences in three cat subpopulations which can be used for appraising potential disease associations with FIV in Australia.     

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.     

Serological survey of Toxoplasma gondii,feline immunodeficiency virus and feline leukaemia virus in urban stray cats in Belgium

Three hundred and forty-six serum samples taken between 1998 and 2000 from urban stray cats in the city of Ghent were tested for antibodies to Toxoplasma gondii and feline immunodeficiency virus (FIV), and antigens of feline leukemia virus (FeLV). Of these 346 samples, 243 (70.2 per cent) were seropositive for Tgondii. Thirty-nine cats (11.3 per cent) had antibodies against FIV and 13 (3.8 per cent) had circulating antigens of FeLV. Fewer of the female cats had FIV and heavier cats had a higher seroprevalence of FIV. Exact logistic regression showed that cats that were infected with FIV were more likely to be infected with T gondii (P = 0.04), and the cats with FIV had a higher titre of Tgondii antibodies than FIV-negative animals. However, FeLV was not associated with either T gondii or FIV.     

Epidemiologic observations on feline immunodeficiency virus and Toxoplasma gondii coinfection in cats in Baltimore, Md

Five hundred eighty-five serum samples obtained between 1980 and 1981 from a diverse population of cats were tested by use of an indirect immunoperoxidase assay for antibodies to feline immunodeficiency virus (FIV). Results of 14 of the samples were positive (prevalence, 2.4%). The FIV-positive cats were markedly older than the overall population and frequently were coinfected (57%) with Toxoplasma gondii. The Toxoplasma titers of the FIV-positive cats were significantly (P less than 0.03) higher than those of the FIV-negative cats. The FIV-positive cats were not coinfected with FeLV. Our findings suggested that FIV-associated immunosuppression may be a factor in active Toxoplasma infection in adult cats.     

Epizootiologic association between feline immunodeficiency virus infection and feline leukemia virus seropositivity

Five hundred twenty-one feline serum samples submitted to the Texas Veterinary Medical Diagnostic Laboratory between Nov 1, 1988, and Jan 31, 1989 were tested for antibody to feline immunodeficiency virus (FIV) by use of an ELISA. The prevalence of FIV infection in this population was 11.3% (95% confidence interval: 8.6 to 14.0%). Serologic test results for FeLV were available for 156 of the 521 cats. A significant (P = 0.008) association between FIV infection and FeLV seropositivity was observed; FeLV-positive cats were nearly 4 times more likely to be seropositive for FIV than were FeLV-negative cats. The association remained statistically significant (P = 0.021) after adjusting for age and gender, using multiple-logistic regression analysis.     

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.