INTRODUCTION Zoonoses are infectious diseases common to humans and certain animal species that are naturally transmitted from animals to humans and vice versa. Today, more than 200 zoonoses are known to require different epidemiological approaches in disease detection and control. A large number of zoonoses are transmitted indirectly through intermediaries - vectors, most often ticks or mosquitoes. For a large number of vector-borne zoonoses, there is no systematic data on the prevalence in wildlife that are often reservoirs of these zoonoses, so such species pose a higher risk due to the possibility of more frequent contact with domestic animals and humans. From an epidemiological point of view, it is important to know which and to what extent the causative agents of zoonoses are carried by wild animals, that can be transmitted by vectors to domestic animals or humans. One of the wild species of canids that represents a reservoir of vector-borne diseases is the Red Fox (Vulpes vulpes L.), which is present in large numbers in rural and suburban areas. The Red Fox (Vulpes vulpes L.) is one of the adaptable species of animals whose numbers are increasing in human proximity, and increasing the risk of transmitting the disease to domestic animals and humans. It is known from the available literature that foxes can be reservoirs of various zoonoses that pose a public health problem because they can be transmitted not only to domestic animals, including pets, (especially dogs) but also to humans, which have not yet been studied in Croatia. This group of diseases includes anaplasmosis, Lyme borreliosis, ehrlichiosis and heartworm disease. MATERIAL AND METHODS This research was conducted on 179 shot carcasses of red fox (Vulpes vulpes L.) from the area of 9 counties, including 127 habitats (hunting grounds), foxes were of both sexes and aged from several months to seven years. Blood and an ear were sampled from each fox. Fromchain reaction (PCR) for amplification of the target part of DNA Dirofilaria immitis, nested and seminested polymerase chain reaction (nested PCR) for amplification of regions within the 16S rRNA and GroEL section of the Anaplasma phagocytophilum genome. From the collected ear samples, nested PCR method was performed for amplification the target part of Borrelia burgdorferi DNA. Molecular studies were performed in order to compare with the results of the SNAP test. Genomic DNA isolation was performed using the commercial kit ReliaPrep ™ Blood gDNA Miniprep System (Promega, USA). To determine the nucleotide sequences of the target gene 5.8S-ITS2-28S rDNA, two primers were used: DIDR-F1 and DIDR-R1 for the PCR reaction of D. immitis amplification (Sigma Aldrich, USA). For the PCR amplification reaction of D. immitis from samples that were positive on the SNAP® 4Dx® Plus Test, a commercial GoTaq® Green Master Mix kit (Promega, USA) was used, which contains all the necessary optimized reagents. Specific primers (Sigma Aldrich, USA) were used to amplification of regions within the 16S rRNA and GroEL section of the A. phagocytophilum genome. For both methods, the nested PCR in which the region within the 16S rRNA section of the genome A. phagocytophilum and the seminested PCR in which the region within the GroEL section of the genome A. phagocytophilum was amplified, two sets of primers were used. In the nested PCR reaction for amplification of regions within the 16S rRNA and GroEL section of the A. phagocytophilum genome a commercial ALLinTM Red Taq Mastermix, 2x kit (highQu) was used. Two pairs of primers were used to amplify of regions within the 16S rRNA section of the A. phagocytophilum genome; ge3a/g10r and ge9f/g2. Two pairs of primers were used to amplify regions within the GroEL section of the A. phagocytophilum genome; EphplgroEL(569) F/EphplgroEL(1193)R and EphplgroE(569)F/EphplgroEL(1142)R. The obtained PCR reaction products were checked by electrophoresis in 1,5% agarose gel. After electrophoresis for 1 hours at room temperature at 90 V and 9 mA, the obtained results were visualized using a UV transilluminator (Vilber Lourmat, Torcy, France). The purified PCR products were sent for sequencing to Macrogen Inc. (Amsterdam, The Netherlands). To determine the nucleotide sequence, the resulting chromatograms were analyzed using Sequencher 5.4.6 (http://www.genecodes.com, Genes Codes Corporation). The following programs and software packages were used in the computer analysis of the obtained sequences: ClustalX, version 1.83 (THOMPSON et al., 1997), for multiple sequence alignment in order to find changes/mutations in the studied nucleotide sequences, and to prepare data for phylogenetic analysis; BioEdit, version 7.0.5.3 (HALL, 1999), for editing aligned sequences; the collected blood samples, testing was performed by SNAP® 4Dx® Plus Test, polymerase chain reaction (PCR) for amplification of the target part of DNA Dirofilaria immitis, nested and seminested polymerase chain reaction (nested PCR) for amplification of regions within the 16S rRNA and GroEL section of the Anaplasma phagocytophilum genome. From the collected ear samples, nested PCR method was performed for amplification the target part of Borrelia burgdorferi DNA. Molecular studies were performed in order to compare with the results of the SNAP test. Genomic DNA isolation was performed using the commercial kit ReliaPrep ™ Blood gDNA Miniprep System (Promega, USA). To determine the nucleotide sequences of the target gene 5.8S-ITS2-28S rDNA, two primers were used: DIDR-F1 and DIDR-R1 for the PCR reaction of D. immitis amplification (Sigma Aldrich, USA). For the PCR amplification reaction of D. immitis from samples that were positive on the SNAP® 4Dx® Plus Test, a commercial GoTaq® Green Master Mix kit (Promega, USA) was used, which contains all the necessary optimized reagents. Specific primers (Sigma Aldrich, USA) were used to amplification of regions within the 16S rRNA and GroEL section of the A. phagocytophilum genome. For both methods, the nested PCR in which the region within the 16S rRNA section of the genome A. phagocytophilum and the seminested PCR in which the region within the GroEL section of the genome A. phagocytophilum was amplified, two sets of primers were used. In the nested PCR reaction for amplification of regions within the 16S rRNA and GroEL section of the A. phagocytophilum genome a commercial ALLinTM Red Taq Mastermix, 2x kit (highQu) was used. Two pairs of primers were used to amplify of regions within the 16S rRNA section of the A. phagocytophilum genome; ge3a/g10r and ge9f/g2. Two pairs of primers were used to amplify regions within the GroEL section of the A. phagocytophilum genome; EphplgroEL(569) F/EphplgroEL(1193)R and EphplgroE(569)F/EphplgroEL(1142)R. The obtained PCR reaction products were checked by electrophoresis in 1,5% agarose gel. After electrophoresis for 1 hours at room temperature at 90 V and 9 mA, the obtained results were visualized using a UV transilluminator (Vilber Lourmat, Torcy, France). The purified PCR products were sent for sequencing to Macrogen Inc. (Amsterdam, The Netherlands). To determine the nucleotide sequence, the resulting chromatograms were analyzed using Sequencher 5.4.6 (http://www.genecodes.com, Genes Codes Corporation). The following programs and software packages were used in the computer analysis of the obtained sequences: ClustalX, version 1.83 (THOMPSON et al., 1997), for multiple sequence alignment in order to find changes/mutations in the studied nucleotide sequences, and to prepare data for phylogenetic analysis; BioEdit, version 7.0.5.3 (HALL, 1999), for editing aligned sequences; chain reaction (PCR) for amplification of the target part of DNA Dirofilaria immitis, nested and seminested polymerase chain reaction (nested PCR) for amplification of regions within the 16S rRNA and GroEL section of the Anaplasma phagocytophilum genome. From the collected ear samples, nested PCR method was performed for amplification the target part of Borrelia burgdorferi DNA. Molecular studies were performed in order to compare with the results of the SNAP test. Genomic DNA isolation was performed using the commercial kit ReliaPrep ™ Blood gDNA Miniprep System (Promega, USA). To determine the nucleotide sequences of the target gene 5.8S-ITS2-28S rDNA, two primers were used: DIDR-F1 and DIDR-R1 for the PCR reaction of D. immitis amplification (Sigma Aldrich, USA). For the PCR amplification reaction of D. immitis from samples that were positive on the SNAP® 4Dx® Plus Test, a commercial GoTaq® Green Master Mix kit (Promega, USA) was used, which contains all the necessary optimized reagents. Specific primers (Sigma Aldrich, USA) were used to amplification of regions within the 16S rRNA and GroEL section of the A. phagocytophilum genome. For both methods, the nested PCR in which the region within the 16S rRNA section of the genome A. phagocytophilum and the seminested PCR in which the region within the GroEL section of the genome A. phagocytophilum was amplified, two sets of primers were used. In the nested PCR reaction for amplification of regions within the 16S rRNA and GroEL section of the A. phagocytophilum genome a commercial ALLinTM Red Taq Mastermix, 2x kit (highQu) was used. Two pairs of primers were used to amplify of regions within the 16S rRNA section of the A. phagocytophilum genome; ge3a/g10r and ge9f/g2. Two pairs of primers were used to amplify regions within the GroEL section of the A. phagocytophilum genome; EphplgroEL(569) F/EphplgroEL(1193)R and EphplgroE(569)F/EphplgroEL(1142)R. The obtained PCR reaction products were checked by electrophoresis in 1,5% agarose gel. After electrophoresis for 1 hours at room temperature at 90 V and 9 mA, the obtained results were visualized using a UV transilluminator (Vilber Lourmat, Torcy, France). The purified PCR products were sent for sequencing to Macrogen Inc. (Amsterdam, The Netherlands). To determine the nucleotide sequence, the resulting chromatograms were analyzed using Sequencher 5.4.6 (http://www.genecodes.com, Genes Codes Corporation). The following programs and software packages were used in the computer analysis of the obtained sequences: ClustalX, version 1.83 (THOMPSON et al., 1997), for multiple sequence alignment in order to find changes/mutations in the studied nucleotide sequences, and to prepare data for phylogenetic analysis; BioEdit, version 7.0.5.3 (HALL, 1999), for editing aligned sequences; MEGA, version 5.0. (TAMURA et al., 2011), for phylogenetic and molecular evolutionary analyzes using different methods and procedures and BLAST (ALTSCHUL et al., 1990), to search databases (http://www.ncbi.nlm.nih.gov). The commercial NucleoSpin® Tissue Kit (Macherey-Nagel GmbH & Co. KG, Germany) was used for PCR to amplify the target part of DNA B. burgdorferi. Evidence of the presence of B. burgdorferi by nested PCR is carried out in two parts using two different pairs of primers in two separate reactions. In an external PCR reaction, a specific DNA fragment encoding the OspA protein of B. burgdorferi is amplified. The obtained PCR product serves as a mold for the internal PCR reaction in which a small part of the PCR products of the external PCR reaction is multiplied, thus increasing the sensitivity and specificity of the PCR method itself. Twenty randomly selected ear samples were screened using the nested PCR method. Statistical analysis included descriptive statistics, frequency distribution, significance tests, and nonparametric tests: the Mann-Whitney test when two groups were tested and the Kruskal-Wallis analysis for multiple analysis. For binary and categorical variables, statistically significant differences in seroprevalence between groups were estimated using hi-square statistics for more than the two groups included and Fisher’s exact test for two-way comparisons only. All seroprevalence of the sample was estimated for the whole population by calculating the standard error (SE) and the 95% confidence interval (CI 95%). To assess the strength of the association between infection and risk factors, including geographical location (counties), breed, age and sex, the prevalence ratio (PR) was calculated as well as the Odds ratio (OR) and the corresponding 95% confidence intervals. Logistic regression analysis was performed to provide valid estimates of interconnection strength between groups. Significance among the groups was taken at the 5% level (p <0.05) for the two-way test. All statistical and epidemiological analyzes were performed using the software STATISTICA 12 and WinEpiscope ver. 2. RESULTS The rapid diagnostic SNAP® 4Dx® Plus test used to detect Dirofilaria immitis antigen, Borrelia burgdorferi antibody, Anaplasma phagocytophilum antibody and Ehrlichia canis / Ehrlichia ewingii, was positive in 30 or 16.76% (CI 95% 11.29) fox. The highest percentage of positive foxes obtained by the SNAP test was found in Karlovac County (38.45%, CI 95% 19.75 - 57.15%), and the lowest in Varaždin County (7.70%, CI 95% -6.79 - 22, 19%) (P ˂ 0.05). Gender was found in 175 individuals, of which 108 or 59.78% were males and 67 or 37.43% were females, and in four individuals the sex was unknown. Of the 108 male animals searched, a total of 12.96% (N = 14, CI 95% 6.63 - 19.29%) were positive, and 22.39% of female foxes were found (N = 15, CI 95% 12), 41 - 32.38%) positive. Regarding age, a positive SNAP test was in 24 or 18.05% of foxes. The highest share of infected foxes was found in foxes of younger age groups up to one year (20.00%, CI 95% -0.24 - 40.24%) and at the age of 1-2 years (21.43%, CI 95% 10), 69 - 32.18%), while a slightly lower percentage of foxes were found in the age group of 2-3 (16.13%, CI 95% 3.18 - 29.08%) and 3-4 years (12, 50%, CI 95% -3.71 - 28.71%). Depending on the individual causative agent, 12 or 6.70% (CI 95% 3.20 - 10.19%) positive samples for Dirofilaria immitis antigen were found in 18 or 10.05% (CI 95% 5.8 - 14) of the individual test was positive for antibodies to Anaplasma phagocytophilum / A. platys, indicating a 1.63-fold higher exposure of foxes to anaplasma compared to heartworm, but this difference was not statistically significant (P = 0.2526, χ2 = 1,309). For other vector-borne diseases tested by this test, namely Borrelia burgdorferi and Ehrlichia canis/Ehrlichia ewingii antibodies were not detected. In Zagreb County, a statistically significantly lower number of positive foxes for D. immitis antigen was found (1.89%, CI 95% -1.78 - 5.56%) compared to other counties (P <0.05). However, in most counties this percentage was equal and ranged from 10.53% (CI 95% -3.27 - 24.33%) (Međimurje County) to 15.39% (CI 95% 1.52 - 29, 06%) (Karlovac County) (P> 0.05). D. immitis was not found in Varaždin and Bjelovar-Bilogora counties. Antibodies to A. phagocytophilum have not been established in Krapina-Zagorje County and the City of Zagreb. In other counties, the proportion of antibodies was found to be from 4.55% (CI 95% -4.16 - 13.76%) (Požega-Slavonia) to 23.08% (CI 05% 6.88 -39.28%) Karlovac County. According to the age structure of the studied fox population in relation to the positive test result according to the causative agent, the prevalence was not statistically significant (P> 0.05). A positive result for antigen on D. immitis was found only in foxes of younger age groups at the age of 1-2 years (14.29%, CI 95%, 6.34 -25.50%) and 2-3 years 3.23%, CI 95%, 2.99 -9.45%) (P> 0.05). Positive result for antibodies to A. phagocytophilum / A. Platys are also in the highest percentage found in younger foxes up to four years of age. In order to test age as a risk factor, the animals were divided into three age groups: (1) young juveniles up to 1 year or until puberty, (2) then as young adults from 1 to 3 years and (3) at population of older adult foxes aged 3 to 7 years. The results of a regression analysis showing the possible correlation of the obtained prevalence of A. phagocytophilum / A. platys and D. immitis with potential risk factors. The results show that there are differences in the exposure of foxes to risk factors such as geographical areas, but sex and age has not established a statistically significant association. A standard PCR method to display the target gene 5.8S-ITS2-28S rDNA filaria was performed on eight fox samples that were positive and eight fox samples that were negative for D. immitis antigen using the SNAP® 4Dx® Plus Test. Analysis of PCR products amplified from the target DNA of foxes that were positive for D. immitis antigenic SNAP® 4Dx® Plus test indicates that none of the eight samples were positive, which means that in none of the eight samples was the amplification of the investigated fragment for D .immitis. Also, the presence of D. repens and A. reconditum was not determined by standard PCR method. as well as the presence of possible coinfection. Molecular analysis confirmed that samples that were negative using the SNAP® 4Dx® Plus test were also negative using the standard PCR method. The PCR method for the display of the 16S rRNA and GroEL section of the A. phagocytophilum genome was performed on nine fox samples that were positiv for A. phagocytophilum antibodies using the SNAP® 4Dx® Plus test. Analysis of PCR products amplified from DNA foxes that were been SNAP® 4Dx® Plus test positives for A. phagocytophilum antibodies, four of the nine samples showed a positive result, which means that in four of the nine samples was the amplification of the studied fragment for A. phagocytophilum. One of the significant vector-borne diseases in domestic and wild animals is Borrelia burgdorferi. The rapid SNAP® 4Dx® Plus diagnostic test found no antibodies to this bacterium. In order to verify the negative results of the rapid diagnostic test, and to further assess the possible infection of wild foxes with this bacterium, a sample of all foxes was collected to prove the presence of Borrelia burgdorferi using the nested PCR method. The presence of Borrelia burgdorferi was not detected in any of the twenty randomly selected ear samples. DISCUSSION Conducting an epidemiological study of the prevalence of vector-borne diseases in foxes that can be detected by rapid screening SNAP® 4Dx® Plus Test used for detection of Dirofilaria immitis antigen, Borrelia burgdorferi antibody, Anaplasma phagocytophilum / Anaplasma platys and Ehrlichia canis /Ehrlichia ewingii In Croatia, a total of 16.76% of positive foxes were found, of which 10.05% of foxes tested positive for antibodies to Anaplasma phagocytophilum / Anaplasma platys, and 6.70% of foxes were positive for Dirofilaria immitis antigen. This means that of the total positive foxes (N = 30), the ratio of anaplasma-positive to heartworm-positive was 6:4, in 1.63 times higher exposure of foxes to anaplasma compared to heartworm, but this difference was not statistically significant (P = 0.2526, χ2 = 1.309). Although this paper does not prove a direct connection between the fox as a reservoir of anaplasmosis and heartworm for dogs, it cannot be ruled out, especially that in areas of northwestern Croatia, mutual contact is possible, foxes with dogs and other domestic animals, because habitats (hunting grounds) positive foxes, are areas near the settlement or are even within the populated area itself. However, comparing the differences in the number of positive foxes by pathogen in individual counties, the found number of positive foxes for antibodies to Anaplasma spp. Species between counties was not significant (P ˃ 0.05), while only statistically significantly lower prevalence of D. immitis was found in Zagreb County (1.89%) compared to other counties (P <0.05). The differences obtained can be explained by the different density of foxes in a habitat. With regard to gender, although 1.73 times more total positive female foxes were found compared to male foxes, this difference was not statistically significant. Epidemiological analysis also revealed a slightly higher general exposure to the causative agents of vector-borne diseases in female animals. The prevalence of D. immitis was equal in male and female animals, but a slightly higher exposure of female animals compared to males was also found, but this difference was also not statistically significant (P = 0.1746). Approximately the same exposure was found for anaplasma, although 2.3 times more were positive for A. phagocytophilum / A. platys antibodies in female animals than in males (P = 0.0673). Comparing the general prevalence as well as the prevalence by pathogens according to age structure, it is evident that the largest number of positive foxes was up to four years of age. The general prevalence ranged from 12.50% in individuals 3-4 years to 21.43% in foxes aged 1-2 years. It should also be noted that the largest number of foxes examined was at the age of 1-2 years (N = 31). Testing of the differences in proportions did not reveal a significant difference (P> 0.05). D. immitis was 4,23 times more established at the age of 1-2 years compared to foxes at the age of 2-3 years (3,23%) but this difference was not statistically significant (P = 0.1068). Unlike D. immitis, antibodies to A. phagocytophilum / A. platys are established in all age groups up to four years and between the ages of five and six. The lowest number of serologically positive foxes was found at the age of one to two years 4.14%, and the highest in juvenile foxes up to one year 20.00%. Comparing the results obtained by rapid screening SNAP®4Dx® Plus test with the results of applied molecular methods, standard PCR and nested PCR method it is clear that SNAP®4Dx® Plus is highly specific because all negative results obtained by screening were confirmed as negative by molecular methods, which indicates a specificity of 100%. The positive results of antibodies to A. phagocytophilum and A. platys indicate an infection of the fox that resulted in the development of antibodies, although by the nested PCR method it was found in the circulation in only four positive animals out of a total of nine. This can be explained by the fact that the SNAP®4Dx® Plus test detects antibodies and is therefore useful at a later stage of infection when PCR, due to low bacteremia or after the causative agent is no longer detectable in the blood and may give negative results, which is confirmed by other authors. According to their research, SNAP®4Dx® Plus test shows high sensitivity for detection of D. immitis antigen by 94.1%. Compared to the autopsy findings in dogs, also confirmed the high sensitivity of the test for D. immits from 94.26% to 99.18%, while the specificity was lower and ranged from 83.45% to 98.75 %. Negative PCR results of positive foxes by screening test for D. immitis can be explained by insufficient sample to detect the required DNA fragments in the blood and thus a small number of microfilariae. Some authors also confirm that by applying the standard PCR method, false-negative PCR test results can be obtained because they confirmed the presence of the causative agent of DNA by applying more sensitive nested PCR in the same samples. This research represents a contribution to the research of vectors of communicable diseases of wild animals in Croatia and the results confirm the circulation of vector-borne diseases in the fox population in the researched areas. Detecting the prevalence of the disease, as well as understanding the risk factors, allow surveillance planning and are an essential prerequisite for the implementation of control programs. Therefore, these results can contribute to a better understanding of the researched diseases in Croatia and can help in the implementation of further epidemiological research and surveillance, as well as the control program, depending on the prevalence rate and risk factors.