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.     

Humoral immune response to feline immunodeficiency virus in cats with experimentally induced and naturally acquired infections.

Sera from cats with naturally acquired and experimentally induced feline immunodeficiency virus (FIV) infections were tested by immunoblot analysis, radioimmunoprecipitation assay (RIPA), and a complex trapping/blocking ELISA. In sequentially obtained samples from experimentally inoculated cats, antibodies against the envelope protein gp120 and the core protein p15 were the first to appear, as indicated by results of RIPA, using lysates of FIV-infected lymphocytes. Antibodies could be detected as early as 2 weeks after infection, followed by a response against p24, p43, and p50. By immunoblot analysis, p24 and p15 were the first proteins detectable between postinoculation weeks 3 and 5; an anti-envelope response was never found by use of this assay, but was found by RIPA. Using the latter test, most sera of naturally infected cats were found to recognize the major core protein p24 in addition to 1 or more minor core proteins. All 40 sera tested precipitated the envelope protein; 3 reacted exclusively with it. A complex trapping/blocking ELISA was developed to quantitate the anti-p24 response. Sera from healthy FIV-infected cats were shown to have higher anti-p24 titer than did those from diseased cats.     

Incidence of feline immunodeficiency virus reactive antibodies in free-ranging lions of the Kruger National Park and the Etosha National Park in southern Africa detected by recombinant FIV p24 antigen.

Lion sera from the Kruger National Park (KNP) dating back to 1977 and from the Etosha National Park (ENP), obtained from 1989 to 1991, have been analysed by ELISA and Western blot analyses using a genetically engineered antigen representing the p24 structural protein of feline immunodeficiency virus (FIV). It was concluded that some 83% of 98 KNP lion sera reacted with the p24 antigen, while none of 28 ENP lion sera reacted. A few other KNP felids (cheetahs and genets) gave samples that did not react with the FIV p24 antigen. For the KNP lions, apart from a lower prevalence in cubs (50%), no particular trends were demonstrated in terms of age, sex, date or origins of the samples. In Western blot and radio-immunoprecipitation analyses the lion sera reacted with the engineered p24 antigen, as well as with the p15 and p24 gag proteins and the p50 gag precursor protein from FIV, indicating that the agent is probably a lentivirus related to FIV. The ELISA with the engineered p24 antigen required less serum and appears to be more sensitive at detecting FIV-reactive antibodies than assays with available commercial kits.     

Seroepidemiological and clinical survey of feline immunodeficiency virus infection in northern Italy

Four hundred and thirty-nine feline serum samples from cats with different living conditions in the north of Italy were tested for antibodies to feline immunodeficiency virus (FIV) and for antigen of Feline Leukemia Virus by enzyme-linked immunosorbent assay. A Western blot technique was also used on the positive sera in order to confirm the presence of specific antibodies to FIV. The Western blot enabled the detection of a false positive serum. The prevalence of FIV infection in this population was 12.5% and among the seropositive cats a greater proportion was male (74.5%) than female (25.5%). A correlation between the clinical status and the evolution of the pathology is described together with a score based on the severity of the stomatitis in infected cats. The Western blot patterns of positive samples were then compared with the stage of the pathology. Statistical analysis on the distribution of FIV in stray cats, cats with garden and courtyard access and strictly house-confined cats showed a highly significant risk of the infection in the first group.     

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.     

马传染性贫血病病毒核心蛋白基因的克隆和表达

根据美国马传染性贫血病病毒（EIAV）Wyoming株基因序列,设计扩增EIAVgag和p26基因的引物,用PCR法能忠实的分别扩增出全长gag和p26基因。经用杆状病毒表达系统对所获gag和p26基因进行克隆和重组,获得了高效稳定表达Gag和p26蛋白的重组杆状病毒（rBVs）。含有gag和p26基因的重组杆状病毒感染昆虫Sf21细胞,经无血清昆虫细胞培养基增殖和纯化,每升培养物能获得2mgCag或12mgp26蛋白。     

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.     

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.     

Enhancement after feline immunodeficiency virus vaccination.

Cats were vaccinated with one of the three preparations: purified feline immunodeficiency virus (FIV) incorporated into immune stimulating complexes (ISCOMs), recombinant FIV p24 ISCOMs, or a fixed, inactivated cell vaccine in quil A. Cats inoculated with the FIV ISCOMs or the recombinant p24 ISCOMs developed high titres of antibodies against the core protein p24 but had no detectable antibodies against the env protein gp120 or virus neutralising antibodies. In contrast, all of the cats inoculated with the fixed, inactivated cell vaccine developed anti-env antibodies and four of five had detectable levels of neutralising antibody. However, none of the vaccinated cats were protected from infection after intraperitoneal challenge with 20 infectious units of FIV. Indeed there appeared to be enhancement of infection after vaccination as the vaccinated cats become viraemic sooner than the unvaccinated controls, and 100% of the vaccinated cats became viraemic compared with 78% of the controls. The mechanism responsible for this enhancement remains unknown.     

重组马传染性贫血病病毒核心蛋白抗原在 A GID 和ELISA中应用的评价

用昆虫杆状病毒表达系统获得的马传染性贫血病病毒（ Ｅ Ｉ Ａ Ｖ） 核心蛋白（ Ｇａｇ） 和 Ｐ２６ 蛋白, 作为免疫琼脂双扩散（ Ａ Ｇ Ｉ Ｄ） 和酶联免疫吸附试验（ Ｅ Ｌ Ｉ Ｓ Ａ） 抗原。对７６ 份已知马传贫非特异性血清进行检查, 同时与市售 Ａ Ｇ Ｉ Ｄ 和 Ｅ Ｌ Ｉ Ｓ Ａ 试剂盒作比较。证明, 用表达蛋白作抗原的 Ａ Ｇ Ｉ Ｄ 和 Ｅ Ｌ Ｉ Ｓ Ａ 检测结果均为阴性反应, 而用市售 Ａ Ｇ Ｉ Ｄ 试剂盒检查有５４ 份马血清出现非特异性反应, 市售 Ｅ Ｌ Ｉ Ｓ Ａ 试剂盒检查也出现了非特异性反应, Ｏ Ｄ 值比表达抗原 Ｅ Ｌ Ｉ Ｓ Ａ 高３５ 倍。初步证明在 Ａ Ｇ Ｉ Ｄ 和 Ｅ Ｌ Ｉ Ｓ Ａ 法中, 表达抗原优于常规马传贫病毒抗原。     

Comparison of six in-house tests for the rapid diagnosis of feline immunodeficiency and feline leukaemia virus infections

Six rapid tests for the diagnosis of feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV) infections which have recently been introduced in Europe for use in small animal practice were compared. Eight hundred serum samples were tested and those 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. The specificity and sensitivity of each test and the quality of the results produced were compared.     

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.     

Prevalence of feline leukemia virus and antibodies to feline immunodeficiency virus in cats in Norway.

Serum samples from 224 Norwegian cats were analyzed for the presence of feline leukemia virus (FeLV) p27 common core antigen, and for antibodies to feline immunodeficiency virus (FIV). Ninety specimens originated from the serum bank at the central referral clinic at the Norwegian College of Veterinary Medicine, which had been collected during the years 1983-1989; 67 sera were submitted from veterinarian practitioners; while 67 sera originated from cats presented for euthanasia. The cats were classified into one "healthy" and one "sick" group. Only 2.2% of sick cats and 1.2% of healthy cats showed FeLV antigenemia, a finding which is lower than which has been reported from many other countries. The prevalence of FIV antibodies was 10.1% in sick cats and 5.9% in healthy cats. Antibodies to FIV was most prevalent in male cats (14.7%) than in female cats (2.1%), and more prevalent among domestic cats (12.0%) compared to pedigree cats (2.4%). Antibodies to FIV in the cats demonstrated increasing prevalence with increasing age. It may be concluded that FeLV causes minor problems in Norwegian cats, while FIV is present in a similar prevalence to what is reported from other countries.     

马传染性贫血病毒p26蛋白单克隆抗体的制备

为制备马传染性贫血病毒(EIAV)的单克隆抗体(MAb),本研究以原核表达并纯化的EIAV p26蛋白作为免疫原免疫8周龄的BALB/c小鼠,利用淋巴细胞杂交瘤技术,建立抗EIAV衣壳蛋白p26抗体的杂交瘤细胞.应用重组p26蛋白为抗原建立ELISA筛选方法,获得两株能够稳定分泌特异性MAb的杂交瘤细胞株,分别命名为E11和F8.经试验证明,这些抗p26MAb均能够与感染驴胎皮肤细胞的EIAV和经SDS-PAGE分离的EIAV总蛋白中的p26蛋白结合.这两株p26蛋白MAb的获得将为EIAV的检测及鉴别诊断奠定基础.     

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.     

Diagnosis of feline leukemia virus and feline immunodeficiency virus infections

Feline leukemia virus is an oncogenic retrovirus that can result in a wide variety of neoplastic and non-neoplastic diseases, including immunosuppression. Diagnosis of FeLV infection can be achieved by several methods, including virus isolation; IFA assay of a peripheral blood smear; and detection of a viral protein (called p27) by ELISA testing of whole blood, plasma, serum, saliva, or tears. Commercially available ELISA kits have revolutionized FeLV testing and have become very popular as "in-house" procedures. This article discusses the interpretation of ELISA results and compares them with IFA assay findings. Feline immunodeficiency virus is a lentivirus that causes immunosuppression, but not neoplasia, in cats. It originally was called feline T-lymphotropic lentivirus. Differentiating FIV infection from the immunosuppressive type of FeLV infection requires virus isolation or serology. The most rapid method for diagnosis of FIV infection is ELISA testing for antiviral antibody.     

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.     

Differential responses of Equus caballus and Equus asinus to infection with two pathogenic strains of equine infectious anemia virus

Most in vivo studies with equine infectious anemia virus (EIAV) have been performed in horses and ponies (Equus caballus) with little published information available detailing the clinical responses of donkeys (Equus asinus) to infection with this virus. Consequently, donkeys were inoculated with two strains of EIAV (EIAV(PV) and EIAV(WY)) which have been documented to produce disease in E. caballus. Four ponies, 561, 562, 564 and 567 and two donkeys, 3 and 5 were infected with EIAV(PV) and one horse (94-10) and one donkey (4) were infected with EIAV(WY). Although the horse and ponies all experienced clinical signs of disease, which in some cases were severe, the donkeys remained asymptomatic throughout a 365-day observation period, except for mild transient reductions in platelet counts. The results from serological assays, virus isolation from plasma and detection of plasma-associated viral RNA by RT-PCR, indicated that initial replication of EIAV(PV) and EIAV(WY) was lower in donkeys than in horses and ponies. This conclusion was confirmed using competitive RT-PCR, in which viral RNA levels in the plasma of EIAV(PV)-infected ponies was up to 100,000-fold higher than in infected donkeys during the first 20 days post-infection (dpi). Similar results were obtained in the EIAV(WY)-infected animals, in which viral RNA burdens in the donkey at 20 dpi were 1000-fold less than in the horse. However, infection of donkey and horse monocyte-derived macrophage cultures with EIAV(PV) demonstrated that these cells in vitro were equally susceptible to virus-induced cytopathic effects and yielded similar levels of progeny virus. This result suggests that factors other than host cell permissiveness mediate the clinical differences observed between horses and donkeys infected with EIAV(PV) or EIAV(WY).     

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.