Infection
To survey gastrointestinal helminth infection status, the stomach,
caecum, small intestines and large intestines were dissected under a
microscope and all parasites observed were visually identified and
recorded, including both larval and adult forms. Two species of helminth
worms were commonly found in the gastrointestinal tracts of the
dissected animals: the whipworm Trichuris muris and the pinwormSyphacia obvelata . Also recorded was the tapewormHymenolepsis sp. , though this was rarely recorded (5 instances
over 366 recorded mice) and was not included in further analysis.
To measure the level of infection by the hepatic helminth Calodium
hepaticum (also known as ÂCapillaria hepaticum ), the
liver was visually assessed during initial dissection and scored based
on the level of decolouration (from 0 = no decolouration, to 5 =
extensive discoloration).
To assess ectoparasite infections, following dissection, the fur of each
mouse was examined under a dissecting microscope with all parasites
identified using a visual key (key created at University of Nottingham,
from photos by Laura Myhill), and total number of each species counted.
As fleas rapidly move to and from the body, including after capture and
after death, numbers are highly dynamic, and therefore only prevalence
was recorded.
To record microparasite prevalence, DNA was extracted from the frozen
blood clots using a 2-step extraction kit (Extracta DNA Prep, Quanta Bio
Massachussetts, USA), and the resulting DNA used for PCR. The
microparasites selected for detection were the apicomplexan protozoanBabesia microti (although in fact another closely related
apicomplexan was detected instead – see below), the flagellate
protozoan Trypanosoma sp. and the bacteria Anaplasma
phagocytilum and Bartonella spp. (see Supplementary Materials).
These parasites were chosen based on species and genera which are
commonly detected across wild rodent populations (Duh, Petrovec, Trilar,
& Avsic-Zupanc, 2003; Healing, 1981; Taylor et al., 2018).
Abundance measures of these parasites could not be reliably recorded,
and so only presence/absence data from these species are used in
analyses. Amplified DNA was run on a 2% agarose gel for 30 minutes at
100V, and visualised under ultraviolet light to confirm amplification.
Identity of successfully amplified samples was confirmed through
sequencing at an external company (Source Bioscience, Nottingham, UK)
and comparison against reference sequences on BLAST (National Center for
Biotechnology Information). Successfully amplified samples fromB.microti primers were found to actually be from infections by
another apicomplexan parasite, Sarcocystis dispersa . The identity
of this parasite was further confirmed through PCR of an additional gene
with Sarocycstis -specific primers, sequencing of PCR product as
described above.
Infection by C.hepaticum was not recorded for the first 150 mice
caught, and blood clot material for these mice was lost, so no C.
hepaticum or microparasite data was available for 150 of 366 mice.