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A field study undertaken in Australia compared the antibody responses induced in client-owned cats that had been vaccinated using two inactivated whole feline leukaemia virus (FeLV) vaccines, the monovalent vaccine Fel-O-Vax® Lv-K and the polyvalent vaccine Fel-O-Vax® 5. Serum samples from 428 FeLV-uninfected cats (118 FeLV-vaccinated and 310 FeLV-unvaccinated) were tested for anti-FeLV neutralising antibodies (NAb) using a live virus neutralisation assay to identify 378 FeLV-unexposed (NAb-negative) and 50 FeLV-exposed (NAb-positive; abortive infections) cats, following by anti-surface unit (SU) FeLV-A and FeLV-B antibody ELISA testing. An additional 42 FeLV-infected cats (28 presumptively regressively infected, 14 presumptively progressively infected) were also tested for anti-SU antibodies. NAb-positive cats displayed significantly higher anti-SU antibody ELISA responses compared to NAb-negative cats (p < 0.001). FeLV-unexposed cats (NAb-negative) that had been vaccinated less than 18 months after a previous FeLV vaccination using the monovalent vaccine (Fel-O-Vax® Lv-K) displayed higher anti-SU antibody ELISA responses than a comparable group vaccinated with the polyvalent vaccine (Fel-O-Vax® 5) (p < 0.001 for both anti-FeLV-A and FeLV-B SU antibody responses). This difference in anti-SU antibody responses between cats vaccinated with the monovalent or polyvalent vaccine, however, was not observed in cats that had been naturally exposed to FeLV (NAb-positive) (p = 0.33). It was postulated that vaccination with Fel-O-Vax® 5 primed the humoral response prior to FeLV exposure, such that antibody production increased when the animal was challenged, while vaccination with Fel-O-Vax® Lv-K induced an immediate preparatory antibody response that did not quantitatively increase after FeLV exposure. These results raise questions about the comparable vaccine efficacy of the different FeLV vaccine formulations and correlates of protection.

Feline leukemia virus (FeLV) is a retrovirus that globally affects both domestic and wild cats, leading to the development of leukemia, lymphoma, and immunosuppression. However, it is important to note that the daily antigen test may yield false negative results. In this study, we successfully developed the first colorimetric loop-mediated isothermal amplification (LAMP) associated with neutral red (NR-LAMP) for the detection of FeLV. The NR-LAMP assay exhibited high sensitivity and efficiency compared to the routine polymerase chain reaction (PCR) reference method. To ensure specificity, a novel LAMP primer set was custom-designed based on the gene of multiple FeLV strains, which resulted in no cross-amplification with other feline viruses. Under the optimized isothermal amplification conditions at 61 °C for 40 min, the NR-LAMP assay achieved a detection limit of 10 copies/µL. Using a blind clinical test involving 98 samples, the NR-LAMP assay demonstrated perfect agreement with the reference PCR method, providing a sensitivity of 97.3% and a specificity of 100%. This proposed NR-LAMP assay surpasses other related approaches in terms of sensitivity, efficiency, and cost-effectiveness. Consequently, the colorimetric NR-LAMP reaction serves as a robust and convenient diagnostic tool for the inspection of FeLV, offering an alternative molecular method for future clinical applications and commercial utilization.

Prevalence of progressive feline leukaemia virus (FeLV) infection is known to still be high in cats in Europe, especially in Southern Europe, but the prevalence of other outcomes of FeLV infection has not been determined in most countries. The present study aimed to investigate the prevalence of progressive, regressive, abortive, and focal infection in four European countries, two with a high (Italy, Portugal) and two with a low expected prevalence (Germany, France). Blood samples of 934 cats (Italy: 269; Portugal: 240; France: 107; Germany: 318) were evaluated for the p27 antigen, as well as anti-whole virus, anti-SU, and anti-p15E antibodies by enzyme-linked immunosorbent assay (ELISA) in serum and for proviral DNA by quantitative polymerase chain reaction (qPCR) in whole blood. Positive p27 antigen ELISA results were confirmed by reverse transcriptase-qPCR (RT-qPCR) detecting viral RNA in saliva swabs and/or blood. The outcome of FeLV infection was categorised as progressive (antigen-positive, provirus-positive), regressive (antigen-negative, provirus-positive), abortive (antigen- and provirus-negative, antibody-positive), and focal (antigen-positive, provirus-negative) infection. Overall FeLV prevalence was 21.2% in Italy, 20.4% in Portugal, 9.5% in Germany, and 9.3% in France. Prevalence of progressive, regressive, abortive, and focal infection in Italy was 7.8%, 4.5%, 6.3%, and 2.6%; in Portugal 3.8%, 8.3%, 6.7%, and 1.7%; in Germany 1.9%, 1.3%, 3.5%, and 2.8%; in France 1.9%, 3.7%, 2.8%, and 0.9%, respectively. In conclusion, overall FeLV prevalence is still very high, especially in Southern European countries. Therefore, testing, separation of infected cats, and vaccination are still important measures to reduce the risk of FeLV infection.

Feline leukaemia virus (FeLV) is a retrovirus associated with fatal disease in progressively infected cats. While testing/removal and vaccination led to a decreased prevalence of FeLV, recently, this decrease has reportedly stagnated in some countries. This study aimed to prospectively determine the prevalence of FeLV viraemia in cats taken to veterinary facilities in 32 European countries. FeLV viral RNA was semiquantitatively detected in saliva, using RT-qPCR as a measure of viraemia. Risk and protective factors were assessed using an online questionnaire to report geographic, demographic, husbandry, FeLV vaccination, and clinical data. The overall prevalence of FeLV viraemia in cats visiting a veterinary facility, of which 10.4% were shelter and rescue cats, was 2.3% (141/6005; 95% CI: 2.0%–2.8%) with the highest prevalences in Portugal, Hungary, and Italy/Malta (5.7%–8.8%). Using multivariate analysis, seven risk factors (Southern Europe, male intact, 1–6 years of age, indoor and outdoor or outdoor-only living, living in a group of ≥5 cats, illness), and three protective factors (Northern Europe, Western Europe, pedigree cats) were identified. Using classification and regression tree (CART) analysis, the origin of cats in Europe, pedigree, and access to outdoors were important predictors of FeLV status. FeLV-infected sick cats shed more viral RNA than FeLV-infected healthy cats, and they suffered more frequently from anaemia, anorexia, and gingivitis/stomatitis than uninfected sick cats. Most cats had never been FeLV-vaccinated; vaccination rates were indirectly associated with the gross domestic product (GDP) per capita. In conclusion, we identified countries where FeLV was undetectable, demonstrating that the infection can be eradicated and highlighting those regions where awareness and prevention should be increased.

Background Feline leishmaniosis is a vector-borne parasitic disease caused by Leishmania spp. Leishmania infection in dogs is prevalent in the Mediterranean basin, but in other animals, such as cats, it could also play a role in the epidemiology of the disease. Information on the geographical distribution and epidemiological features of L. infantum infection in cats is scarce, particularly in urban stray cats living in regions where canine leishmaniosis is endemic. As diagnosis can be challenging, combining different serological and molecular methods is a useful approach. Our aim was to investigate the prevalence of infection of L. infantum in apparently healthy stray cats in an endemic region of Spain (Zaragoza city) using serological and molecular methods, and to compare the results of the different techniques. Methods The prevalence of Leishmania infection was studied in stray cats captured in urban and peri-urban areas of Zaragoza. Blood was collected from each animal for serology and molecular analysis. Three serological methods, namely the immunofluorescent antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA) and western blot (WB), were used to detect L. infantum antibodies and a real-time PCR (qPCR) assay was used to detect L. infantum DNA. The results were analyzed by Fisher’s exact test and Cohen’s kappa statistic ( κ )  to assess the level of agreement between the diagnostic techniques. Results Serological analysis of blood samples from 180 stray cats revealed 2.2% (4/179) Leishmania infection positivity by IFAT, 2.8% (5/179) by ELISA and 14.5% (26/179) by WB. Leishmania DNA was detected by qPCR in 5.6% (10/179) of the cats. Sixteen cats (8.9%) tested positive by only one serological technique and four tested positive by all three serological methods used. The overall rate of infected cats (calculated as the number of cats seropositive and/or qPCR positive) was 15.6%, and only two cats tested positive by all the diagnostic methods. A significant association was found between male cats and a positive qPCR result. Comparison of the techniques revealed a fair agreement in seropositivity between blood qPCR and IFAT ( κ  = 0.26), blood qPCR and ELISA ( κ  = 0.24), WB and ELISA ( κ  = 0.37) and WB and IFAT ( κ  = 0.40). The highest agreement between seropositive results was between IFAT and ELISA ( κ  = 0.89), and the lowest was between blood qPCR and WB ( κ  = 0.19). The prevalence of the feline leukemia virus antigen was 4.49% (8/178 cats) and that of the feline immunodeficiency virus (FIV) antibody was 6.74% (12/178), while co-infection with both retroviruses was observed in one female cat (1/178). Leishmania ELISA and IFAT seropositivity were statistically associated with FIV status by the chi-square test. Conclusions The results obtained in this study, using serological tests and qPCR, indicate the existence of L. infantum asymptomatic infection in apparently healthy stray cats in the city of Zaragoza, an endemic area in Spain. Graphical Abstract

Feline leukemia virus (FeLV) is one of the most prevalent infectious diseases in domestic cats. Although different commercial vaccines are available, none of them provides full protection. Thus, efforts to design a more efficient vaccine are needed. Our group has successfully engineered HIV-1 Gag-based VLPs that induce a potent and functional immune response against the HIV-1 transmembrane protein gp41. Here, we propose to use this concept to generate FeLV-Gag-based VLPs as a novel vaccine strategy against this retrovirus. By analogy to our HIV-1 platform, a fragment of the FeLV transmembrane p15E protein was exposed on FeLV-Gag-based VLPs. After optimization of Gag sequences, the immunogenicity of the selected candidates was evaluated in C57BL/6 and BALB/c mice, showing strong cellular and humoral responses to Gag but failing to generate anti-p15E antibodies. Altogether, this study not only tests the versatility of the enveloped VLP-based vaccine platform but also sheds light on FeLV vaccine research.

Longitudinal studies of cats naturally infected with feline leukemia virus (FeLV) are important for understanding disease outcomes. Levels of p27 antigen and copy numbers of proviral DNA have been associated with FeLV-infection courses. The purpose of this prospective study was to establish cutoff values for p27 antigen concentration and proviral DNA load that distinguished high positive from low positive groups of cats and to evaluate an association with survival. At enrollment, 254 cats were tested by point-of-care and microtiter plate enzyme-linked immunosorbent assays (ELISAs) for p27 antigen and real-time polymerase chain reaction (PCR) for proviral DNA. The 127 positive cats were retested monthly for six months and monitored for survival over the four-year study. A receiver operating characteristic-based analysis of samples with concordant or discordant qualitative results for p27 antigen and proviral DNA was used to establish cutoff values, and when applied to test results at enrollment for classifying cats as high positive or low positive, a significant difference in survival was observed. High positive cats had a median survival of 1.37 years (95% CI 0.83–2.02) from time of enrollment, while most low positive cats were still alive (93.1% survival). Quantitative results for p27 antigen concentration and proviral DNA load were highly correlated with survival times in FeLV-infected cats.

Feline leukemia virus (FeLV) is a retrovirus distributed worldwide in domestic cats and with different outcomes (progressive, regressive, abortive, focal). The present study reports an epidemiological survey of FeLV frequency and the evaluation of some risk factors and the two main disease outcomes (progressive and regressive) in an urban cat population from Brazil. A total of 366 cats with sociodemographic information and p27 FeLV antigen test performed were included in the study. FeLV DNA (provirus) in the blood samples of all cats was detected via real-time polymerase chain reaction (qPCR). Plasma samples from 109 FeLV-positive and FeLV-negative cats were also submitted to reverse transcription (RT-qPCR) to determine the FeLV viral load. The results demonstrated that 112 (30.6%) cats were positive through the p27 antigen and/or qPCR. A risk factor analysis demonstrated that cats without vaccination against FeLV (OR 9.9, p < 0.001), clinically ill (OR 2.9, p < 0.001), with outdoors access (OR 2.7, p < 0.001), and exhibiting apathetic behavior (OR 3.1, p < 0.001) were more likely to be infected with FeLV. FeLV-infected cats were also more likely to present with anemia (OR 13, p < 0.001) and lymphoma (OR 13.7, p = 0.001). A comparative analysis of the different detection methods in a subset of 109 animals confirmed FeLV infection in 58 cats, including 38 (65.5%) with progressive, 16 (27.6%) with regressive, and 4 (6.9%) with probably focal outcome diseases. In conclusion, this study demonstrates a high prevalence of FeLV in this urban cat population from Brazil and highlights the need to establish more effective prevention strategies (such as viral testing, vaccination programs, specific care for FeLV-positive cats) to reduce diseases associated with this virus in Brazil.

A field study was undertaken to (i) measure the prevalence of feline leukaemia virus (FeLV) exposure and FeLV infection in a cross-section of healthy Australian pet cats; and (ii) investigate the outcomes following natural FeLV exposure in two Australian rescue facilities. Group 1 (n = 440) consisted of healthy client-owned cats with outdoor access, predominantly from eastern Australia. Groups 2 (n = 38) and 3 (n = 51) consisted of a mixture of healthy and sick cats, group-housed in two separate rescue facilities in Sydney, Australia, tested following identification of index cases of FeLV infection in cats sourced from these facilities. Diagnostic testing for FeLV exposure/infection included p27 antigen testing using three different point-of-care FeLV kits and a laboratory-based ELISA, real-time polymerase chain reaction (qPCR) testing to detect FeLV proviral DNA in leukocytes, real-time reverse-transcription PCR (qRT-PCR) testing to detect FeLV RNA in plasma, and neutralising antibody (NAb) testing. Cats were classified as FeLV-uninfected (FeLV-unexposed and presumptively FeLV-abortive infections) or FeLV-infected (presumptively regressive and presumptively progressive infections). In Group 1, 370 FeLV-unexposed cats (370/440, 84%), 47 abortive infections (47/440, 11%), nine regressive infections (9/440, 2%), and two progressive infections (2/440, 0.5%) were identified, and 12 FeLV-uninfected cats (12/440, 3%) were unclassifiable as FeLV-unexposed or abortive infections due to insufficient samples available for NAb testing. In Groups 2 and 3, 31 FeLV-unexposed cats (31/89, 35%), eight abortive infections (8/89, 9%), 22 regressive infections (22/89; 25%), and 19 progressive infections (19/89; 21%) were discovered, and nine FeLV-uninfected cats (9/89; 10%) were unclassifiable due to insufficient samples available for NAb testing. One of the presumptively progressively-infected cats in Group 3 was likely a focal FeLV infection. Two other presumptively progressively-infected cats in Group 3 may have been classified as regressive infections with repeated testing, highlighting the difficulties associated with FeLV diagnosis when sampling cats at a single time point, even with results from a panel of FeLV tests. These results serve as a reminder to Australian veterinarians that the threat of FeLV to the general pet cat population remains high, thus vigilant FeLV testing, separate housing for FeLV-infected cats, and FeLV vaccination of at-risk cats is important, particularly in group-housed cats in shelters and rescue facilities, where outbreaks of FeLV infection can occur.

The surface envelope (SU) protein determines the cell tropism and consequently the pathogenesis of the feline leukemia virus (FeLV) in felids. Recombination of exogenous FeLV (exFeLV) with endogenous retroviruses (enFeLV) allows the emergence of more pathogenic variants. Currently, phenotypic testing through interference assays is the only method to distinguish among subgroups—namely, FeLV-A, -B, -C, -E, and -T. This study proposes a new method for FeLV classification based on molecular analysis of the SU gene. A total of 404 publicly available SU sequences were used to reconstruct a maximum likelihood tree. However, only 63 of these sequences had available information about phenotypic tests or subgroup assignments. Two major clusters were observed: (a) clade FeLV-A, which includes FeLV-A, FeLV-C, FeLV-E, and FeLV-T sequences, and (b) clade enFeLV, which includes FeLV-B and enFeLV strains. We found that FeLV-B, FeLV-C, FeLV-E, and FeLV-T SU sequences share similarities to FeLV-A viruses and most likely arose independently through mutation or recombination from this strain. FeLV-B and FeLV-C arose from recombination between FeLV-A and enFeLV viruses, whereas FeLV-T is a monophyletic subgroup that has probably originated from FeLV-A through combined events of deletions and insertions. Unfortunately, this study could not identify polymorphisms that are specifically linked to the FeLV-E subgroup. We propose that phylogenetic and recombination analysis together can explain the current phenotypic classification of FeLV viruses.