Foamy viruses (FVs), also known as spumaviruses, belong to the
In many hosts where FVs are prevalent, serum antibodies as well as neutralising antibodies against viral proteins have been associated with FV infection (10, 11); therefore, the diagnosis is mainly based on serological tests, while molecular methods are used rather for phylogenetic studies, especially those on interspecies transmission of simian foamy viruses to different primate species and humans (4, 31, 34). Serological methods like neutralising tests and immunoblots were explored in FFVfca research in the past, while currently the ELISA method described by Romen
Our study aimed to investigate the seroreactivity of domestic cats from Poland for FFVfca antigens using sera collected from three distinct regions of the country. Additionally, we tried to investigate the association of FFVfca seroreactivity with selected demographic variables.
Cut-off values were calculated from the netOD450 Gag values of all 223 samples as 2 × (meanGag + 3 SD). Positive outliers were excluded and the procedure was repeated until the calculated cut-off value no longer changed after their exclusion. The Bet and Env cut-off was calculated from the group of Gag-negative sera as 2 × (meanBetorEnv + 3 SD) (33).
The distribution of the individual reactivities showed that no clear distinction of FFV Gag-positive and negative reactivity was apparent (Fig. 1), as was also previously reported (3). Therefore the cut-off value for Gag was newly calculated as described in the previous section. To confirm the calculated cut-off values for Gag (netOD450 = 0.199), selected sera were used for immunoblot analysis. In total, 22 cat sera were tested by immunoblotting with cellular antigen of CRFK cells infected with FFVfca. The tested samples were those displaying Gag reactivity around and above the statistically calculated Gag cut-off value as well as clearly FFVfca-positive and -negative sera as determined by ELISA. Uninfected CRFK cell lysate was used as a control. Cat sera clearly positive in ELISA were also positive in immunoblot, since the FFVfca Gag proteins (52 kDa precursor and 48 kDa processed Gag) were detected (Fig. 2); similarly ELISA-negative sera did not react with FFVfca antigen in immunoblot.
Distribution of feline foamy virus Gag, Bet and Env antigens seroreactivity in 223 domestic cats from Poland. Dashed red lines indicate determined cut-off values
Detection of FFVfca-specific antibodies by immunoblotting assays with a cellular antigen in representative feline serum samples. M – prestained protein ladder; A – lane with Crandell-Rees feline kidney cells (CRFK)/FFVfca cells lysate as antigen; B – lane with uninfected CRFK cells lysate as antigen; P – FFVfca positive control serum; N – FFVfca negative control serum; 34/64, 34/79, 34/15 – representative samples with their ELISA reactivity to FFVfca Gag antigen (OD Gag)
However, there were 10 serum samples which had been positive in ELISA (net OD450 from 0.211 to 0.421) but which showed no reactivity with FFVfca Gag proteins in immunoblot. Sera with netOD450 equal or higher than 0.539 were also positive in immunoblot assay. Such re-evaluation resulted in a Gag cut-off value of netOD450 = 0.539 with sera at or above this value being Gag positive and sera below this value being scored Gag negative (Fig. 1). This new cut-off clearly distinguished a group of low-level Gag-reactive sera that were negative in immunoblotting from those that were Gag positive in both tests (Fig. 1). Previous reports confirmed that Bet is not consistently detectable in cat sera by immunoblot assays (1, 3), and therefore we could not use the same procedure as for Gag antigen to experimentally determine the Bet cut-off value. Thus, we calculated the cut-off for Bet from Gag-negative sera as netOD450 = 0.517. The cut-off for Env antigen was calculated in the same way and was established as netOD450 = 0.654 (Fig. 1).
Using the determined cut-off values, 100 out of the 223 domestic cat sera showed the presence of FFVfca antibodies, of which 99 reacted to Gag and 80 to Bet antigens, while only 56 did so with Env. Moreover, 80% of Gag-positive samples also reacted to Bet antigen, while only 55.5% with Env antigen. The scatter plot analysis confirmed a stronger correlation between the reactivities to Gag and Bet than between those to Gag and Env antigens (Fig. 3). Interestingly, only 51 out of 223 tested samples reacted to all three antigens. Only one serum sample showed reactivity exclusively to the Env antigen. These results seem to be concordant with those of previously reported studies, which showed that Gag was the antigen of choice for serological surveys of FV infections.
Scatter plot of ELISA results showing relationship between (A) feline foamy virus Gag and Bet and (B) Gag and Env antigens; correlation coefficients are indicated on both graphs. Circles indicate Gag ELISA negative and squares Gag ELISA positive samples
In summary, 44% of the examined domestic cat sera from Poland were positive for FFVfca. Among them, 35% represented cats from the Warsaw, 45% from the Gdańsk and 74% from the Kraków agglomerations (Fig. 4).
Distribution of the cat serum samples’ reactivity to feline foamy virus Gag antigen by animal origin. Black circles indicate Gag ELISA-negative and grey squares Gag ELISA-positive samples; A – Warsaw; B– Gdańsk; C– Kraków agglomerations
Since demographic information was available for 113 cats from Gdańsk and Kraków (Table 1, Fig. 5) a chi-squared test was used to analyse the association between FFVfca infection and variables such as age, gender and contact with other cats. A statistically significant association between the prevalence of FFVfca infection and the age of the cat was observed (χ2 = 10.30, P = 0.0061). Only 30% of domestic cats under the age of 1 year tested positive for FFVfca. The seroprevalence of FFVfca was clearly higher in the two remaining age groups: among adult cats (1–10 years) 67% of individuals were assessed FFVfca positive and in the group of the oldest cats (over 10 years) 55% showed seroreactivity to FFVfca Gag. In domestic cats, no significant association was found between FFVfca infection and gender or between infection and contact with other cats (Table 1).
Box-plot distribution of feline foamy virus Gag-seropositive and -seronegative cat samples from the Gdańsk and Kraków agglomerations analysed by variables
Serological prevalence of FFVfca in domestic cat populations from the Gdańsk and Kraków agglomerations aligned with demographic variables
Variable | Number of cats | ||
---|---|---|---|
FFVfca seropositive | Total | ||
Gender | Female | 22 | 51 |
Male | 37 | 61 | |
Age | Young (≤1 year) | 10 | 22 |
Adult (1–10 years) | 33 | 49 | |
Old (>10years) | 16 | 29 | |
Contact with other cats | Yes | 48 | 86 |
No | 10 | 23 |
Serum samples from 223 domestic cats originating from three agglomerations were tested for the presence of FFVfca Gag-, Bet- and Env-specific antibodies using GST-capture ELISAs for the first time in Poland. In serological studies on FVs, Gag protein was acknowledged as the antigen of choice in the detection of infection with such viruses. Gag protein is known to be highly immunogenic and contains sequences which are conserved among different isolates of FFVfca, including two FFVfca serotypes identified due to differential neutralisation (7, 37). It has also been demonstrated that Gag protein elicited a strong and long-lasting immune response in all species infected by FVs even through interspecies transmission (1, 12, 177, 22, 377). Therefore, in our study FFVfca seroprevalence was determined based only on the seroreactivity of cat sera to Gag antigen. Such seroprevalence as determined, 44%, is in accordance with that found in previous studies in domestic cats in Australia, Vietnam, Germany, Switzerland and US showing 30–80% FFVfca-positive individuals depending on age, gender and geographic region (3, 8, 13, 24, 26, 33, 388). In a further similarity to previous studies (13), the rate of FFVfca-positive animals varied by location.
Similarly to the study reported by Bleiholder
Additionally to Gag, we also used Bet and Env antigens to test all cat sera. As previously observed (3), we also confirmed the stronger correlation between the reactivities to Gag and Bet than Gag and Env, however, both antigens reacted with clearly lower number of sera than the Gag antigen. Similar results were noted previously not only for FFVfca but also for other FVs (9, 23) and can be explained by the cessation of production of Bet protein after the productive phase of FVs infection and its diminution in the persistent one lessening its diagnostic value (33). The assay using the Env antigen was primarily developed in order to find an alternative to serotype-specific PCR and neutralisation assays (3, 377, 41) for distinguishing between FFVfca serotypes. Unfortunately, this antigen did not detect serotype-specific antibodies, probably due to its C-terminus fusion to the GST moiety, which alters the quaternary structure of the resulting fusion protein and limits the access of antibodies to the specific epitopes. This fact, of course, disqualifies Env fusion protein as the main diagnostic antigen but does not negate its supportive value, similarly to Bet antigen (18, 19).
In order to investigate the risk factors favouring FFVfca infection, we performed statistical analysis based on serological data and some demographic information available for most of the cats from the Gdańsk and Kraków agglomerations. Significant association of FFVfca infection with cat age was observed, which is consistent with the results of previous studies and supportive of the hypothesis that FFVfca infections are preferentially accumulated in cat populations through horizontal transmission (27, 38). In this context, the fact that no association was found between FFVfca infection and outdoor contact with other cats is quite surprising, especially since social contacts are considered to be the main route of FV transmission (15). However, it can be simply explained by the unequal size of the tested groups of cat sera having led to biased results. Additionally, in concurrence with previous reports from Australia, we also noted that FFVfca prevalence did not vary between genders (388). Interestingly, recent reports from the USA suggested that male cats are at higher risk of FFVfca infection; however, since this observation was made in cat shelters in Colorado, regional variation or spurious association can be considered (13).
This study is the first report confirming FFVfca infections among cats in Poland. Using a GST-capture ELISA, we detected FFVfca-specific seroreactivity in 44% of cat serum samples. Additionally, we found a significant association between seroreactivity to FFVfca antigen and the age of cats, suggesting that adult animals have a higher probability of being infected with FFVfca. Since FFVfca has been associated with a higher risk of other retroviral infections, further epidemiological and clinical studies should be conducted to investigate the potential influence of infection with this virus on the health status of domestic cats.