Materials and Methods
This cross-sectional study was approved by Ethics Committee on Animal
Use (CEUA) of Universidade Federal Fluminense (UFF) (8405150718) and
licensed by Biodiversity Information and Authorization System
(SISBIO/IBAMA63464-2).
Adult and young free-living opossums were included in this study.
Animals were seized or rescued by environment alauthorities and samples
were collected at wild life rehabilitation facilities of Universidade
Estácio de Sá (22° 58’ 46” S, 43° 27’ 24” W) (eight D. aurita )
and Universidade de Vassouras (22° 24’ 14” S, 43° 39’ 45” W) (sevenD. albiventris ), both located in RJ state, Brazil. Animals were
physically restrained and venipuncture was proceeded from ventral caudal
vein, obtaining up to 1.0mL of blood, which was immediately placed in
ethylenediaminetetraacetic acid (EDTA) tubes.
DNA extraction was performed with the Master PureTMDNA Purification Kit for Blood Version (Epicentre®, Madison, Wisconsin,
USA). In order to avoid false negative results, DNA samples were tested
for presence of amplifiable DNA using the primer set GAPDH-F
(5’-CCTTCATTGACCTCAACTACAT-3’) and GAPDH-R
(5’-CCAAAGTTGTCATGGATGACC-3’), resulting in amplification of a 399 bp
fragment of the housekeeping gapdh gene, encoding
glyceraldehyde-3-phosphate dehydrogenase enzyme, which is present in all
mammals (Birkenheuer et al., 2003).
Subsequently, presence of bacterial DNA was tested by conventional and
quantitative polymerase chain reactions (PCR and qPCR) targeting a 365
bp fragment of Bartonella spp. citrate synthase (gltA )
gene with primers BhCS781p (5’ GGGGACCAGCTCATGGTGG 3’) and BhCS1137n (5’
AATGCAAAAAGAACAGTAAACA 3’), as previously proposed (Norman et al.,
1995). Positive (purified DNA of B. henselae extracted from a
naturally infected cat) and negative (nuclease-free water) controls were
used for each reaction. Amplicons underwent electrophoresis on 1.5%
agarose gel stained with ethidium bromide.