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.