Materials and methods
The zoo is located on the outskirts of Ljubljana (coordinates 46°3′9.25″ N, 14°28′20.08″ E) and covers an area of 6 hectares with a large collection of exotic mammals, birds, and reptiles. Blood samples were collected from the jugular vein of 123 mammals and 65 birds in 2014 and 2015 (Table 1) and the serum was stored at −20 ºC until assay. Tissue samples (brains or liver) of ten animals that died at the Ljubljana Zoo in 2015 were also examined. These included two mute swans (Cygnus olor ), one black stork (Coconia nigra ), two brown rats (Rattus norvegicusvar. alba ), one fat-tailed gerbil (Pachyuromys duprasi ), one alpaca (Vicugna pacos ), two Japanese quail (Coturnix japonica ), and one laboratory mouse (Mus musculus var.alba ) used as food for zoo animals. As a part of the deratization program at the Ljubljana Zoo, sera from 39 wild rodents (n = 39) and brain tissue (n = 11) were collected and used for assays. The rodents included 28 eastern house mice (Mus musculus ), four brown rats (Rattus norvegicus ), four yellow-necked mice (Apodemus flavicollis ), two bank voles (Clethrionomys glareolus ), and one striped field mouse (Apodemus agrarius ). All the samples used in this study were either blood samples collected during surgeries and preventive annual routines where blood was used for other reasons, or tissues from dead animals and rodents collected during the deratization program.
Enzyme Linked Immuno Sorbent Assay (ELISA) was used to detect antibodies to T. gondii , N. caninum , C. burnetii , and C. abortus with the ELISA kit ID Screen T. gondii Indirect Multi-Species, ID Screen N. caninum Indirect Multi-Species, ID Screen Q Fever Indirect Multi-Species, and ID Screen Chlamydia abortus Indirect Multi-Species, IDvet, Grabels, France, respectively. Samples with S/P (%) ≥ 60% and S/P (%) ≥ 50% were classified as positive for C. abortus and for the other infections mentioned above, respectively. Antibodies to E. cuniculi were determined by an indirect immunofluorescent antibody test using MegaScreen Fluoencephalitozoon (Megacor Diagnostic, Hörbranz, Austria) withE. cuniculi antigen and specific conjugates: anti-bovine, anti-goat, and anti-sheep IgG (VMRD, Pullman, Chicago, USA) for Bovidae, anti-camel IgG (VMRD) for Camelidae, anti-deer IgG (KPL Inc. Gaithersburg, Maryland, USA) for Cervidae, anti-horse IgG (VMRD) for Equidae, anti-cat IgG (Sigma Aldrich, St. Louis, USA) for Felidae and Ursidae, anti-dog IgG (Sigma Aldrich) for Canidae, and anti-pig IgG (Sigma Aldrich) for Suidae. Sera were diluted with phosphate-buffered saline twofold starting with 1:50; samples with a titer ≥ 50 were marked as positive.
The DNA was isolated by the QIAamp DNA Mini Kit (Qiagen, Courtaboeuf, France); in the case of E. cuniculi , the tissue was first homogenized using a tissue homogenizer. Detection of T. gondiiwas done by PCR amplification of the TGR1E sequence (Cristina, 1991). The PCR mixture contained 3 µl of DNA, 20 µl of PPP master mix (Top-Bio s.r.o., Prague, Czech Republic), 0.5 µl of each primer (0.1 mM), and 16 µl of PCR-grade H2O. PCR was performed at the following conditions: 94 °C 5 min, 35 cycles of 94 °C 0.5 min, 68 °C 40 s, and 72 °C 40 s and 72 °C 5 min. Genotyping of T. gondii –positive samples was performed with a single multiplex PCR assay with 15 microsatellite markers (Ajzenberg, 2010). Detection of N. caninumwas done with PCR amplification of the Nc-5 region (Muller, 1996). The PCR mixture contained 2 µl of DNA, 12.5 µl of PPP master mix (Top-Bio s.r.o), 0.1 µl of each primer (0.1 mM), and 10.4 µl of PCR-grade H2O. PCR was performed at the following conditions: 94 °C 5 min, 35 cycles of 94 °C 1 min, 63 °C 30 s, and 72 °C 1 min and 72 °C 10 min. Detection of E. cuniculi was done with PCR amplification of a small subunit of rRNA using the primers ECUNF/ECUNR (Valenčáková, 2005). The PCR mixture contained 1 µl of DNA, 12.5 µl of PPP master mix (Top-Bio s.r.o.), 1 µl of each primer, and 9.5 µl of PCR-grade H2O. PCR was performed at these conditions: 95 °C 3 min, 35 cycles of 95 °C 30 s, 60 °C 30 s, and 72 °C 1.5 min and 72 °C 10 min. PCR products were analyzed on 2% agarose gel. The prevalence was statistically analyzed with Pearson’s chi-squared test for independence using STATISTICA Cz 12 (Statsoft, 2013) or with the Monte Carlo method using IBM SPSS Statistics 20. The null hypothesis that seroprevalence in mammals does not differ by orders, families, diet, and sex was tested. The differences were considered statistically significant when the p -value was ≤ 0.05. In the case of a statistically significant difference, the Scheffé multiple comparison method (Statistica Cz 12) was subsequently applied.