Prevalence of feline coronavirus, feline leukemia virus, and feline immunodeficiency virus in client-owned cats in Croatia

This study aimed to determine prevalences for anti-FCoV antibody, FeLV antigen, FeLV proviral DNA, and anti-FIV antibody among client-owned cats from the cities of Zagreb and Varaždin in Croatia. Subjects included 106 client-owned cats tested at the Faculty of Veterinary Medicine, Vienna, Austria. Blood samples were tested with IFA for anti-FCoV antibody and IFA FCoV antibody titeres, with ELISA for FeLV p27 antigen, with PCR for FeLV proviral DNA, and with RIM for anti-FIV antibody. Prevalence of FCoV and FeLV was 41.51% and 6.60%, respectively. A coinfection with FeLV/FCoV and FIV/FCoV prevalence was 7.55% and 5.66%. No cats were coinfected with FIV and FeLV. All three viruses were detected, confirming their presence in Croatia. The seroepidemiological findings demonstrate that both feline retroviruses and feline coronavirus are important feline pathogens in Croatia.


Introduction
Feline coronavirus (FCoV) belongs to the family of Coronaviridae within the order Nidovirales [1,2,3]. Infection with FCoV is prevalent in pet cats [4]. The prevalence of FCoV in the cat population is very high in multi-cat environments [4]. For example, one study in Israel, Baneth et al., 1999 [5] reported that 83.3% of cats from animal shelters were positive for antibodies against FCoV. In Malaysia, the positive rate of FCoV infection was 84% in cats from catteries [4]. European prevalence is reported to range from 31% to 64% [6,7] when client-owned cats are examined.

Materials and methods
Client-owned cats from the cities of Zagreb and Varaždin were studied, but for the purpose of this study, these cats were treated as a single population. Aseptically, 1 mL of whole venous blood from each of 106 individual client-owned cats was sampled into EDTA-coated tubes. Veterinarians took blood samples from domestic cats in veterinary clinics in Varaždin and Zagreb in their daily work from regular patients during practice. No blood was taken from cats for the purpose of this study. The results of blood tests from domestic cats whose blood samples were taken in the diagnosis process during regular veterinary practice were used for this study. Considering that Article 3 of the Animal Protection Act of the Republic of Croatia (Official Gazette 135/06) stipulates that an experiment is one or more procedures performed on an animal for experimental purposes, which may cause pain, suffering or injury to animals, it is clear that the use of blood test results taken for the purpose of determining the patient's health during regular veterinary practice is not an experiment within the meaning of this Act and therefore no ethical approval was required. Samples were stored at 4°C before transporting on dry ice to the Faculty of Veterinary Medicine, Vienna, Austria. Diagnostic tests were conducted at the Faculty of Veterinary Medicine, Vienna, Austria.

Detection of FeLV antigen and FIV antibodies cat sera
Serum samples were tested with ELISA (ViraCHEK ® /FeLV Synbiotics Corporation, San Diego, CA, USA) for FeLV p27 antigen and with immunomigration test (Witness FIV, Synbiotics Corporation) for anti-FIV antibody. The reported sensitivity and specificity for ViraCHEK ® /FeLV test were 94.9% and 98.4%, and for WITNESS FIV test 94.5% and 99.4% [22]. Interpretation of serological test results was performed according to the manufacturer's instructions.

Test procedure
One well was used for the positive control, one well for the negative control and one well for each sample. One drop or 0.05 ml of Positive Control was added to the first well for positive control and one drop or 0.05 ml of Negative Control to the second well for negative control. 0.05 ml of sample was pipetted into the next well; then one drop or 0.05 ml of Reagent 1 HRP Monoclonal Antibody Conjugate was added to each well. The well holder was tapped (without splashing) for 15 seconds to mix the solutions. After 5 minutes the wells were washed 5 times with deionized/distilled water. After each washing, the holder and blot were firmly inverted onto a paper towel to remove the last drops. Thereafter, two drops or 0.10 ml of Reagent 2, the Chromogenic Substrate Buffer were added to each well. The well holder was then tapped (without splashing) for 15 seconds to mix the solutions. Interpretation of the test results was performed after 5 minutes. The development of a blue color was considered positive for FeLV p27 antigen. A positive test indicates FeLV infection, transient or persistent.

WITNESS FIV test
Test procedure 50 μl of serum was pipetted vertically into the sample well (1). Three drops of buffer were then added to the sample well (1). The test device remained flat during the migration of the reagent complex sample through the reading window. Interpretation of the test results was performed after 10 minutes. The presence of pink bands in the reading windows (2) and (3) indicated that the sample was positive for FIV antibodies. The presence of anti-gp40 antibodies indicates that the cat has been exposed to the virus.

FCoV antibody test
Immunofluorescence for antibodies against group 1 coronaviruses Anti-FCoV antibody and FCoV antibody titeres were determined by a modified, indirect immunofluorescence assay [23]. CrFK monolayers grown in 96 well plates were inoculated with Feline Coronavirus 79-1146 (type 2, ATCC VR 990) until carbapenemase-producing enterobacteriaceae was visible (on day 1 or 2) and fixed with alcohol. For each serum investigated, three dilutions were prepared using phosphate-buffered saline (PBS): 1:10, 1:100, and 1:400. The wells were incubated with 50 μl of the serum dilution for 1 hour at 37°C and washed three times with PBS. 50 μl of a dilution of 1:40 of commercially available fluorescein isothiocyanate (FITC) conjugated goat anti-cat antibodies (Jackson ImmunoResearch) was added to each well. After incubation at 37°C for 1 hour, the wells were washed with PBS and 50 μl of a glycerine-phosphate buffer was added. The plates were evaluated using an inverse UV-microscope. For each sample, the high test dilution with a clear cytoplasmatic fluorescence corresponded to the Coronavirus 1 specific antibody titer. IFA FCoV antibody titeres ranged from 1:10 to 1:400. For FCoV IFA titer <1:10 was indicated as serologically negative, while FCoV IFA titer 1:10 or more as seropositive. An FCoV IFA titer 1:10 was considered low grade positive. A FCoV IFA titer 1:100 was considered medium grade positive, while an FCoV IFA titer ≥1:400 was considered high grade positive.

Real-time PCR for FeLV
Preparation of EDTA blood samples EDTA blood samples were centrifuged at 3400 x g at 4°C for 15 min. The buffy coat was incubated in erythrocyte lysis buffer (buffer EL, Qiagen, Austria) on ice for 10 min and centrifuged at 470 x g at 4°C for 10 min. The supernatant was discarded and the wash step repeated one or two times until the pellet was visibly white. Finally, the pellet was incubated in 180 μl buffer ATL (Qiagen, Austria) and 20 μl Proteinase K at 56°C until complete lysis.
Extraction of viral nucleic acids 140 μl of the leukocyte lysate served as a template for nucleic acid extraction using a commercially available kit (QIAamp ® Viral RNA Kit, Qiagen, Austria) as instructed by the manufacturer. Negative controls consisting of the components of the kit were run together with the samples through all sample preparation and extraction procedures. Extracts (60 μl) were stored at -20°C until PCR analysis.

Real-time PCR assay for FeLV proviral DNA
The real-time PCR method was conducted as described by Tandon et al. (2005) [24]. DNA was amplified in an Applied Biosystems 7300 Real-Time PCR System, Foster City, CA, USA. PCR reactions were prepared with 12.5 μl qPCR TM Mastermix (Eurogentec, Seraing, Belgium), a final concentration of 480 nM of primers (Microsynth, Balgach, Switzerland), 160 nM of fluorogenic probe (Eurogentec) and 5 μl of the extracted nucleic acid samples in a 25 μl total reaction volume. The RT-PCR mixture was prepared using the components of a commercially available kit (SuperScript TM III Platinum ® One-Step qRT-PCR System, Invitrogen) in a reaction volume of 25 μl (2.5 μl template and 22.5 μl PCR mixture). Negative controls (for extraction) and no template controls (for PCR) were run with every assay. Extracts of cell culture supernatants of FeLV (strain FL-237, ATCC VR-721) infected Crandell feline kidney cells served as positive controls. PCR reactions were performed using forward primer FeLV_U3_exo_f: 5 ' -AAC AGC AGA AGT TTC AAG GCC-3 ' , reverse primer FeLV_U3_exo_r: 5 ' -TTA TAG CAG AAA GCG CGC G-3 ' and FeLV_U3_probe 5 ' -CCA GCA GTC TCC AGG CTC CCC A-3 ' as described by Tandon et al. (2005) [24].

Statistical analyses
Statistical analyses were carried out in, Excel, (Microsoft Office 2019 Professional Plus)

Discussion
Since the first serological study of FCoV was conducted in Davis, California, USA Pedersen, 1976; [25], many prevalence studies have been conducted in different countries, some of which are shown in (Table 1). Reports indicate that the prevalence of FCoV varies among countries (Table 1). This variability in FCoV prevalence is caused by the geographical location [5,6], different populations of cats [6], breed [4], and climate [25,26].  The reported prevalence of FCoV in Poland, Turkey and the United States was 38.5%, 45.5%, and 35% [30,29,27] among owned cats. These results are consistent with the results obtained in the current study (41.51%) (Fig. 1).
FCoV positive cats were clinically asymptomatic, similar to the results of Sharif et al., (2009) [4], and Oguzoglu et al., (2010) [1]. However, Oguzoglu et al., 2013 [29] reported that health status was significantly associated with FCoV infection and that clinically ill cats were more frequently positive for FCoV than healthy ones.
Researchers investigated a possible correlation between FCoV antibody titer and fecal virus shedding. Pedersen et al., (2008) [35] confirmed a correlation between the shedding of FCoV and antibody titer. However, some researchers reported contrasting results [36,37]. According to a study by Felten et al., (2020) [37], determination of antibody titers in serum or plasma and FCoV viral RNA in fecal samples provide more reliable and accurate results for FCoV status than each test alone.
Among the FeLV positive cats, only two cats were clinically ill. Except for those cats, 5 of 7 cats (71.43%) were clinically healthy, indicating that these cats were asymptomatic viraemic cats. This finding indicates that it is important to test not only sick cats but also healthy cats to control the spread of FeLV infection among the domestic cat population. However, some other studies reported a significant correlation between infection status and health status. In studies provided by Bande et al., (2012) [9] and Westman et al., (2016) [12] the positivity rates for FeLV infection were significantly higher among sick cats.
Among cats with FeLV and FCoV, six of 8 cats were clinically ill (6/8; 75.00%). This result is inconsistent with the study in Turkey that reported that all cats with FeLV and FCoV were clinically healthy [1].
In the current study, all FIV-infected cats were seropositive for anti-FCoV antibody (5.66%) (Fig. 1) and seroprevalence of coinfection with FCoV and FIV was very similar to results of previous studies [5,2] (Table 4). All cats coinfected with FCoV and FIV were clinically ill in the current study, which is consistent with the study by Tekelioglu et al., (2015) [2].
Based on the data obtained in the current research, the rate of FeLV infection was 14.15% with the inclusion of the FeLV positivity rates in single infection (6.60%) and in coinfection (7.55%) (Figs. 1, and 5). This finding supports observations from a previous Croatian study [21], but not the most recent data from European countries (with the exclusion of prevalence data including stray or shelter cats). FeLV prevalence was higher than observations of studies from Germany [18], Poland [30], Austria [44], Switzerland [48], Italy [52], and Ireland [17].
Additionally, the rate of FCoV infection was 54.72% with the inclusion of the FCoV positivity rates for single infections (41.51%) and in coinfections (13.21%) (Figs. 1, and 5). The seropevalence of antibodies against FCoV in Croatia is similar to those in central European countries. In a study by Moestl et al., (2002) [7] FCoV seroprevalence was 58% in the Czech Republic and 64% in Austria.
The important results from the current study are that the prevalence of FCoV and FeLV is high, FIV is rarer in the Croatian cat population (Fig. 5). The prevalence of FCoV infection was higher than that of FeLV infection (Fig. 5), similar to results of studies of owned cats in Turkey [1,29], and in Poland [30]. In this study, the prevalence of the FeLV infection was higher than that of the FIV infection (Fig. 5), similar to results of studies of owned cats in Germany [18], in Turkey [29], in Poland [30], in Mexico [42], in Hungary [8], and in Italy [52]. However, some other studies in Israel [5], Australia [12], in New Zealand [47], in Cyprus [46], in Brazil [55], and in Ireland [17] reported contrasting results.
Previous study has reported a high rate of retroviral infections in stray cats from the Zagreb metropolitan area in Croatia [21]. The high-density cat population and the large number of stray cats in urban areas increase the chance of contact with other possible FeLV and/or FIV infected stray and free roaming client-owned cats and increase more fighting and aggressive behavior among adult male cats. Therefore, outdoor owned cats are at higher risk of retroviral infections due to more frequent exposure to these viruses. Factors contributing to the high rate of FeLV infection in owned cats and to the FIV infection risk of client-owned cats in the current study may be the high density of cat population and the large number of stray cats living in the urban areas.

Conclusions
This study confirmed the presence of FCoV, FeLV, and FIV infections and coinfections with FeLV/FCoV and FIV/FCoV in the cities of Zagreb and Varaždin. The seroepidemiological findings from this study demonstrate that both feline retroviruses and feline coronavirus are important feline pathogens in the owned cat population in the cities of Zagreb and Varaždin. The results further indicate the importance of preventive testing of cat samples for anti-FCoV antibody, FeLV antigen, FeLV proviral DNA, and anti-FIV antibody to detect the presence of FCoV, FeLV, and FIV infections in the cat population. Preventive measures should include testing, identification of infected cats, segregation and vaccination against FeLV to control these significant infections in owned cats in urban areas in northwestern Croatia. The findings obtained in this study should be helpful for the development of further prophylactic protocols for owned cats in urban areas in Croatia and neighboring countries. However, the results of the current study should be interpreted with caution. The limitations of this research are: a small number of cats and different cat populations (owned, stray, feral cats) from different Croatian areas were not included in this study. Therefore, further more detailed studies with a larger number of owned, stray and feral cats from different urban areas in Croatia are needed to determine the prevalence of FCoV, FeLV, and FIV infections and risk factors for seropositivity among cats in Croatia. The results of this study highlight that data on the prevalence of FCoV, FeLV, and FIV in a larger number of cat samples from different Croatian areas are essential for the development of a prophylactic protocol and for the implementation of effective prevention measures for client-owned, stray and feral cats.

Funding Statement
The author received no financial support for the research or publication of this article.

Conflicts of interest
The author declares that there is no conflict of interest.