Experimental procedures
Tapada de Mafra (TM, 38º57'N, 9º18'W) is an enclosed game area of 819ha of mixed
deciduous woodland, situated in western Portugal and characterised by a predominantly
oceanic climate (Daveau, 1980). For further details on study area description please
refer to Norte et al. (2012). Controlled hunting populations of fallow deer (Cervus
dama), red deer (Cervus elaphus) and wildboar (Sus scrofa) are present in TM, which
contribute to the successful adult tick engorgement and subsequent reproduction (Gray,
1998) and allow the maintenance of tick populations. Questing ticks were collected
from February to June 2013, whereas vertebrate were captured from March to June
2013. Questing ticks were collected by blanket dragging. Birds were captured using
mist nets, small mammals were captured in baited Shermann traps, and lizards were
captured either by hand or using a running noose. Occasional sampling was also made
from one rabbit, which had been run over, and from three wild boars which had been
hunted.
All individuals were identified to species level and marked for recapture
recognition – birds were ringed, small mammals were marked in the ears by collecting
two skin biopsies (one in each ear), and lizards were marked by toe clipping. Biopsies
were only collected at the first capture, but presence of infesting ticks was assessed in
each capture event. Ticks were collected into 70% ethanol tubes. Additionally, two skin
biopsy specimens were collected from each individual bird, lizard and small mammal.
Biopsies were collected from the shin area in birds and from the ears in small mammals
(< 2mm2), and from the tip of the tail from lizards (< 1cm). Ticks were separated by
instar and gender, and identified to species level (Estrada-Peña et al., 2004; Pérez-Eid,
2007). Ticks were washed in 70% ethanol and sterile distilled water, dried on sterile
paper and subsequently boiled in 25% ammonium hydroxide solution, as described
previously by (Schouls et al., 1999) for DNA extraction. Only Ixodes spp. were
analyzed for B. burgdorferi s.l. infection because of established Borrelia vectorial
capacity of several Ixodes spp. (Eisen and Lane, 2002). We tested a maximum number
of 30 ticks of the same species, instar and sex, from the same individual. One of the skin
biopsies of each individual was inoculated in the field into 1ml BSK II medium, which
was then incubated at 34ºC for 15-21 days before examination at the dark field
microscope for the presence of spirochetes, to assess if Borrelia was viable in host
tissues, and subsequent DNA extraction by thermolization (for details see Norte et al.,
2013b). Tissues from two dead A. sylvaticus hearts and bladders were also harvested
and inoculated in BSK II for culture. The other skin biopsy was used for direct DNA
extraction (DNeasy blood & tissue kit, Qiagen). Extracted DNA from ticks, tissues and
their cultures was analysed in a nested PCR targeting the 5S-23S rDNA intergenic
spacer region (IGS; Rijpkema et al., 1995). Amplified products were visualized on 1.5%
agarose gels (Roche Diagnostics GmbH, Mannheim, Germany). A percentage of the
positive samples was further analysed for flaB gene (Johnson et al., 1992). The IGS
positive samples were then tested using a Reverse Line Blot (Gil 2005) using specific
probes described previously (Ripkema et al. 1995, Schouls 1999, Gil 2005, Gern 2010).
The amplicons of those samples showing reactivity to different probes were cloned in
pGEM-T-easy vectors (Promega Biotech Ibérica, SL, Madrid, Spain) following the
manufacturer´s instructions, and the plasmids from 10 colonies were sequenced to
assess whether those positive samples corresponded to mixed infections. Borrelia
genospecies were identified by BLASTn analysis (NCBI), after purifying the amplified
products of nested PCR using the JETquick system (Genomed, Inc.) and sequencing
with the Big-Dye Terminator Cycle Sequencing kit (Applied Biosystems) on an ABI
377 DNA sequencer. Sequencing procedures were performed at the Molecular Biology
Laboratory of National Institute of Health Dr. Ricardo Jorge. The resulting nucleotide
sequences of partial 5S-23S intergenic spacer region were aligned using Multalin
software (Corpet 1988) and compared with other published sequences (from the
positions 254 to 453).
Results are presented including data from recaptures (5 birds and 30 mice).
Birds, lizards and small mammals were captured, marked and skin biopsies were
collected under licence from the ICNF (Licence number: 160/2013/CAPT).
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