Main 2
Main 2
Main 2
identification, especially in cases (like ours) of very similar oocyst 2.2. Flotation and microscopical analysis of oocyst
morphology in different species (Duszynski and Wilber, 1997; Long and
Joyner, 1984). For that reason, tools based on DNA amplification and Fecal samples were washed with tap water to eliminate potassium
sequencing have been included as routine strategy not only for detec- dichromate and homogenized. After oocyst were flotated using a satu-
tion, but also for taxonomic assessment (Fernandez et al., 2003; Hnida rated salt solution (specific gravity = 1.18–1.20), they were collected
and Duszynski, 1999; Kawahara et al., 2010; Morris et al., 2007; by centrifugation (3234×g/room temperature/10 min), washed with
Schnitzler et al., 1999; Su et al., 2003; Vrba et al., 2010). distilled water (1800×g/room temperature/10 min). The flotations
Up to 16 species of Eimeria have been described from house mice were screened for the presence of oocyst using a Leica® DM750 M light
(Levine and Ivens, 1965) and some of them use different niches in the microscope under the 10X objective. To estimate the intensity of in-
intestine. The reasons for this diversity are still elusive (Zhao and fection, flotated oocysts were counted using a Neubauer chamber and
Duszynski, 2001a) and artificial splitting of morphologically plastic the results were expressed as oocyst per gram (OPG) of faeces. Samples
forms of the same species (in the same of different hosts) might con- were then preserved in a fresh solution of potassium dichromate 2.5%
tribute to this. (w/v) and sporulated in a water bath at 30 °C for 10–12 days for further
Eimeria species described from house mice include E. falciformis, the characterisation.
first coccidia described in house mice (Eimer, 1870), which has some- Eimeria isolates, corresponding to different phylogenetic groups (see
times been regarded as the most prevalent species in mice (Becker, below), were selected to take photomicrographs of sporulated oocysts
1934; Owen, 1976). This species (and especially the BayerHa- using a Carl-Zeiss microscope at 100x magnification. Measurements
berkorn1970 isolate) are the most commonly studied coccidia model in were made on ~30 oocysts and ~30 sporocysts, using Adobe Photoshop
laboratory mice. Life cycle progression (Haberkorn, 1970) and host CC v14.2.1 (3778 pixels = 100, 0000 μm). Length and width were
response (Mesfin et al., 1978; Schelzke et al., 2009; Schmid et al., 2012) measured and reported in micrometers. The Length/Width (L/W) ratio
are relatively well studied and the whole genome of this species has was calculated for both oocysts and sporocysts including means, stan-
been sequenced and annotated in detail (Heitlinger et al., 2014). dard deviation and variation coefficients. Additionally, main morpho-
E. vermiformis was first described in 1971 (Ernst et al., 1971) but logical traits (oocyst wall, oocyst residuum, micropyle, polar granule,
since then, to our knowledge, not reported in wild house mice. Similar sporocyst residuum, refractile bodies and Stieda body) were described,
to E. falciformis, most of the information on this species comes from according to the protocol of Duszynski and Wilber (1997).
laboratory infection experiments (Figueiredo-Campos et al., 2018; Rose
et al., 1990; Rose and Millard, 1985; Todd Jr and Lepp, 1971), making 2.3. DNA extraction
the timing of life cycle progression and its effect on the host relatively
well studied. DNA from colon content was extracted using the NucleoSpin® Soil
E. ferrisi was originally described from M. m. domesticus from North kit (MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany) according
America (Ankrom et al., 1975; Levine and Ivens, 1965). Laboratory to the manufacturer's recommendations, adding a mechanical lysis
infections with this parasite have confirmed its shorter life cycle, process in a Mill Benchtop Mixer MM 2000 (Retsch GmbH, Haan,
compared to E. vermiformis or E. falciformis (Schito et al., 1996). Germany). DNA from cecum and ileum tissues was isolated using the
To the best of our knowledge, just few investigation of prevalences innuPREP DNA Mini Kit (Analytik Jena AG, Jena, Germany) following
and intensities of coccidia has been conducted in free-living populations the instructions of the manufacturer after disruption of the tissue with
of M. musculus (Ball and Lewis, 1984; Ernst et al., 1971; Golemanski, liquid nitrogen in a mortar. Quality and quantity of isolated DNA was
1979; Owen, 1976; Parker et al., 2009; Yakimoff and Gousseff, 1938). measured spectrophotometrically in a NanoDrop 2000c (Thermo
In the present work we studied the prevalence of Eimeria in house mice Scientific, Waltham, USA).
from a transect of the well-studied European house mice hybrid zone
(HMHZ) (Boursot et al., 2003; Ďureje et al., 2012; Phifer-Rixey and 2.4. PCR amplification for detection (ap tRNA) and identification (nu 18S
Nachman, 2015). We established methods for detection, species iden- rDNA and mt COI)
tification and quantification of Eimeria in these wild commensal po-
pulations of house mice. For detection of Eimeria, amplification of a conserved tRNA region
of the apicoplast genome (Ap5) was used. Primers Ap5_Fwd (YAAAG-
GAATTTGAATCCTCGTTT) and Ap5_Rev (YAGAATTGATGCCTGAGYG-
2. Material and methods GTC) were designed based on the complete apicoplast genomes se-
quences available in the GenBank from Eimeria tenella (NC_004823.1),
2.1. Collection of samples E. falciformis (CM008276.1) and E. nieschulzi (JRZD00000000.1).
For all samples with oocysts detected during flotation or successful
Between 2015 and 2017, 378 house mice (Mus musculus) were amplification of Ap5, genotyping PCRs were performed to confirmation
captured in 96 farms and private properties in a transect 152.27 km of detection and further identification of parasite species. A fragment of
long and 114.48 km wide, within the German federal state of nuclear small subunit ribosomal RNA (~1500 bp) and mitochondrial
Brandenburg (capture permit No. 2347/35/2014) (Fig. 1A, Supple- cytochrome C oxidase subunit I (~800 bp) were amplified using pri-
mentary data 1). On average 20 traps were set overnight per locality. mers 18S_EF and 18S_ER (Kvičerová et al., 2008) and Cocci_COI_For/
Mice were house individually in cages over night and euthanised by Rev (Ogedengbe et al., 2011), respectively. An alternative pair of pri-
cervical dislocation. All mice were dissected within 24 h after capture. mers was used in case of failure to amplify COI: Eim_COI_M_F (ATGT
Faeces for microscopical diagnosis of Eimeria spp. were preserved in CACTNTCTCCAACCTCAGT) and Eim_COI_M_R (GAGCAACATCAANAG
potassium dichromate (K2Cr2O7) 2.5% (w/v) and stored at 4 °C until CAGTGT). These primers were designed based on the mitochondrial
further processing, colon content was preserved in liquid nitrogen and genome of E. falciformis (CM008276.1) (Heitlinger et al., 2014) and
stored at −80 °C. For a subset of 163 mice (from Brandenburg in 2016) amplify a ~700 bp fragment of COI gene.
tissue samples from cecum and ileum were collected for DNA extraction PCR reactions were carried out in a Labcycler (SensoQuest GmbH,
and molecular identification of Eimeria spp. All samples were kept in Göttingen, Germany) using 0.025 U/μL of DreamTaqTM DNA
liquid nitrogen during transportation and maintained at −80 °C until Polymerase (Thermo Scientific, Waltham, USA), 1X DreamTaq Buffer,
processing. 0.5 mM dNTP Mix, 0.25 μM from each primer and 1–20 ng/μL of DNA
template in 25 μL reaction. A concentration of 0.25 mM dNTP Mix and a
supplementation with 2 mM MgCl2 was used for the amplification of
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