Final Year Project - Rebecca Thursfield - The effects of invading bank vole on haemoparasite infections of wood mice in Ireland

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Declaration:
I declare that, with the exception of any statements to the contrary, the contents of this
report/dissertation are my own work, that data presented has been obtained by
experimentation and that no part of the report has been copied from previous
reports/dissertations, books, manuscripts, research papers or the internet.
Signed: _______________________________________________________________________
Print Name: ___________________________________________________________________
Date: _________________________________________________________________________

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Contents Page:
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Table 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4 – 9
8
Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 – 14
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Figure 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Table 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Equation 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Table 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Figure 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Equation 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
15 – 22
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20
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Discussion and Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 – 25
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 – 30
Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 – 38

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Abstract:
Bank voles (Myodes glareolus) have been sited throughout the south-west of Ireland since
1964. It is thought that the rodents were introduced from Germany around 1925-1926 in the
area of the River Shannon and since has begun to spread its range northward. In 2004 a
separate study was conducted throughout Ireland to examine the effects of this invasion on the
Bartonella parasite population in the native wood mice (Apodemus sylvaticus) and discovered
bank voles were acting as dilution hosts.
This study will re-evaluate these dilution effects to determine whether bank voles are still
reducing Bartonella spp. infection in wood mice. Wood mice samples were collected from areas
within the bank vole invasion range (95 samples of which 49.5% were infected) and areas
outside it (82 samples of which 57.3% were infected), and then tested using PCR to determine
whether the samples were Bartonella positive. A total of 179 wood mice were collected,
however, only 177 were usable due to missing area data. A chi-squared test gave a p value
<0.05, meaning any differences seen were not significant and therefore bank voles seem to no
longer be acting as dilution hosts.

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Introduction:
Previous work by Telfer (et al. 2004) which was carried out in Ireland at the start of the
century has suggested that since the bank voles (Myodes glareolus) accidental introduction to
the south of Ireland, there has been a decline in the number of Bartonella spp. infections in the
native wood mouse (Apodemus sylvaticus). Evidence suggested that this invasive species was
acting as a ‘dilution host’ and reducing the parasites prevalence in the wood mouse.
The wood mouse, Apodemus sylvaticus, (also known as the Long-tailed Field Mouse) is a highly
adaptive, common rodent found throughout most of Europe and parts of North Africa. It
occupies a wide variety of habitats ranging from woodland and shrub-land to sand dunes and
wasteland (Schlitter et al. 2008). Being an omnivore, this rodent also has a wide diet, ranging
from plant matter such as seeds, fruit and roots to animal material such as insects
(Montgomery et al. 1990). This species of mouse is the only native small rodent in Ireland
(Telfer et al. 2004) although it now lives in sympatry with other foreign small rodents that have
been introduced, such as the bank (Telfer et al. 2004) and the house mouse (Mus musculus)
(Marnell et al. 2009).
The bank vole, Myodes glareolus (formerly known as Clethrionomys glareolus), is a common
rodent with a wide native range throughout Europe, Asia and Iberia, central Siberia, northern
Scandinavia and the Mediterranean (White et al. 2012). It can populate many different habitats
ranging from dense woodland to river banks and parkland, though heavy cover is preferred
(Amori et al. 2008). Studies have shown that bank voles are extremely common within Europe
with an average density approximately between 6-100 individuals per hectare (Spitzenberg
n.d.). Although it is common throughout Europe and the United Kingdom it was previously
absent from Ireland until the first record of its presence in 1964 (White et al. 2012). However,

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upon analysis of the bank voles mitochondrial DNA it was estimated that the bank voles that
now colonise Ireland are descendants from a small founder group and the true invasion of this
rodent lies somewhere in the late 1920s (White et al. 2012 and Ryan et al. 1996). Work by Ryan
(et al. 1996) shows that there is very little genetic variation between the Irish bank voles,
suggesting that the founding group was very small, possibly even as small as two individuals.
Further investigation suggested that the founding population was originally from Germany and
the year of introduction was around 1925-1926 towards the south-west of Ireland, possibly the
River Shannon, as at this time the Shannon Hydroelectric Scheme was taking place. Large loads
of earth and equipment were being shipping from Germany to this area and this river is the site
at which the first bank vole was captured. (Stuart et al. 2007 and White et al. 2012). By studying
the flea distribution of the bank voles there is more evidence to support that they were
accidentally introduced from Germany with the machinery loads.
A study in 1984 showed that the bank vole was present in over 12,500km2 in the south-west of
Ireland. However, since its introduction (after an initial lag period as the voles established
themselves) it is estimated that this species is expanding its range at a rate of 2-4.5 km x year-1
(Ryan et al. 1996, Telfer et al. 2004 and White et al. 2012), although growth is slower in some
areas due to unsuitable land such as large fields with little cover or thin hedges etc. (Ryan et al.
1996).
Bank voles, along with other rodents, are carriers of disease and parasites, and for the purpose
of this study Bartonella species are of interest. The voles found in Ireland, however, seem to be
immune to infections from Bartonella species despite being host to ticks and fleas that are
themselves infected with Bartonella (Telfer et al. 2004). Studies have shown that bank voles
from other countries can however become infected, such as the population inhabiting a
suburban forest in France which tested positive for Bartonella species in over half of the

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captured voles (Buffet et al. 2012). Studies carried out near Uppsala in Sweden (Holmberg et al.
2003), near the Mazury Lake District region of Poland (Bajer et al. 2000) and in a boreal forest
in the East of Poland (Paziewska et al. 2012) also found many bank voles infected with
Bartonella species. Due to this immunity that the Irish bank voles possess they are acting as a
dilution host for Bartonella, reducing the infection rates in the native wood mouse.
A dilution effect is when the presence of another host species, in this case the bank vole, of a
feeding vector (arthropods such as ticks and fleas) that has a low infection capacity or immunity
to any parasites and diseases the vectors may transfer, causes a lower infection rate in the
other vector host species (wood mouse), therefore diluting and reducing the disease risk the
vectors possess (Schmidt et al. 2001). The ticks and fleas feed on the vole but do not become
infected with Bartonella since the vole is immune and so is not infected. If these arthropods
then feed on a wood mouse they will not pass on any Bartonella parasites. Over time the
infection rates of Bartonella in the wood mice has dropped because of this dilution (Telfer et al.
2004).
Research conducted by Telfer (et al. 2004) stated that the most plausible explanation for this
immunity is that these Irish bank voles may just have a natural resistance to Bartonella or they
are not susceptible to the native strains of Bartonella found in Ireland. The strains found here
may differ from those found in other parts of the world and may be unable to infect bank voles.
An explanation for the natural resistance in all Irish bank voles may be due to an original
immunity found in the small founding colony. If this small group of voles were originally
resistant to Bartonella then they may have passed it on to all their offspring, although further
studies would be needed to confirm this theory.
When a previous investigation was conducted by Telfer (et al. 2004) three flea species were
found; Amalaraeus penicilliger Dale, Ctenophthalmus nobilis Rothschild and Hystrichopsylla

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talpae Curtis. All species of flea are ectoparasites that live off the blood of other organisms and
undergo four different life stages; egg, larva, pupa and adult. Only the adult stage feeds on
blood, and therefore if the only stage that can transmit Bartonella, and can survive long periods
of time without a blood meal. Eggs can also lay dormant for great periods of time until the ideal
hatching conditions are met. Work by Chomel (2011) states that vertical transmission from
adult fleas to their eggs does not occur and therefore newly hatched adults can only become
infected via horizontal transmission. Morick (et al. 2011) confirms this with work conducted on
Xenopsylla ramesis fleas.
Fleas thrive in warm temperatures and are most active in the spring and summer months.
However, they can survive all year and, since wood mice and bank voles don’t hibernate, can
continue transmitting parasites and diseases throughout the rodent populations.
Bartonella is a genus of parasitic bacteria classed as being microparasites due to their small
size, along with other viruses and bacteria. They are gram-negative, facultative intracellular
bacteria that are characterised by their fastidious and pleomorphic aerobic coccobacillary or
bacillary rods being around 0.3 μm x 1 μm in size (Saisongkorh et al. 2009). The bacteria are
transmitted by blood-sucking arthropod vectors, such as ticks and fleas, when the vector feeds
on the host where they enter the bloodstream and infect cells. Due to this they are classed as
intracellular haemoparasites, however, they can be differentiated from other closely related
organisms by their hemotropic lifestyle, consisting of a long-lasting infection of the hosts
erythrocyte cells (red blood cells), lasting the remaining life of the red blood cell which can be
several weeks. Upon infection of the host the bacteria target and infect endothelial cells using a
unique method of cellular invasion which involves the activation of a pro-inflammatory
phenotype (Kosys et al. 2012). Approximately five days after infection the Bartonella are
released and go on to invade mature erythrocyte cells where they multiply and can remain for

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several weeks. After this initial release from the endothelial cells the cycle continues, releasing
new Bartonella every three to six days which themselves go on to infect new cells and multiply
(Dehio, 2001). Once the bacteria have multiplied they wait for another arthropod vector to
ingest them in a blood meal and then go on to infect another host.
There is now estimated to be between 30–40 species of Bartonella (Kosoy et al. 2012), at least
thirteen of which are known to cause zoonotic infections in humans (Chomel et al. 2010).
Bartonella
species
First cultivation Area Reservoir Human disease
(s)
B. birtlesii Mouse (Apodemus
spp.)
Bodensee,
Germany
Rat
B. taylorii Woodland mouse
(Apodemus spp.)
United Kingdom Rat
B. grahamii Woodland mammal
(Clethrionomys
glareolus)
United Kingdom Rat,
insectivores
Neuroretinitis
Table 1 The Bartonella species which can infect Apodemus spp. and M. glareolus, along with the area
found, the reservoir host and any diseases it may cause to humans are indicated (Saisongkorh et al.
2009).
Table 1 indicates that M. glareolus can become infected with the species B. grahamii which
causes Neuroretinitis in humans, affecting the eyes. However, research from Telfer (et al. 2004)
suggested that this species of Bartonella is absent from Ireland as only B. taylorii and B. birtlesii
were recovered from mice and voles. Also, these species of Bartonella appear to be unable to
infect humans and so there is no risk of zoonosis occurring.
However, disease in humans due to Bartonella is quite common, one of the most frequent
being cat scratch disease which can be caused by Bartonella henselae, Bartonella clarridgeiae
and Bartonella koehlerae (Chomel et al. 2012), although other more serious diseases are
associated with these species, such as Leishmaniasis, anthrax and bubonic plague (Schmidt,
2001 and Swaddle, 2008). By studying the effects of dilution hosts it may be possible to
eventually apply these same effects to other host species in an attempt to reduce not only the

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frequency of potentially harmful Bartonella species but also of other pathogenic parasites. This
may not only help to save many human lives but also those of domesticated animals,
potentially saving farmers millions of dollars in losses each year.

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Materials and Methods:
Wood mice were trapped in three areas throughout Ireland and DNA samples extracted from
each by W. Ian Montgomery (Montgomery, 2012). These samples were then donated to the
University of Salford for various uses by students.
The method used to identify if the rodents samples contain any strains of Bartonella was by a
semi-nested PCR reaction. A semi-nested PCR was required to improve the likelihood of
detecting any Bartonella present as they may pass unnoticed in a regular PCR. This semi-nested
PCR tests allow for greater specificity as it uses three different primers (BigF, BogR, BigR) over
the course of the reaction rather than just two that would be used in a standard PCR, and so
can detect product where the standard would not. The first PCR works to amplify a specific DNA
sequence for Bartonella and the second round amplifies the first PCR product further to
produce a more specific product.
Materials for the PCR:
 Various sizes of micropipettes and tips
 Eppendorf tubes
 Eppendorf tube rack
 Polystyrene box
 Ice
 2x 1µl BigF primer per sample
 1µl BigR primer per sample
 1µl BogR primer per sample
 12µl Taq 2X colourless master mix (Bioline, London) per sample
 12µl Taq 2X red master mix (Bioline, London) per sample
 8.5µl distilled water per sample
 1µl DNA per sample
 1µl positive control DNA per PCR batch
 PCR thermocycler

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Materials for the gel electrophoresis:
 Measuring cylinder
 Conical flask
 Weighing boat
 Spatula
 Scientific scales
 Microwave
 Transparent gel casting tray
 Casting gates
 Gel caster/clamp
 Gel electrophoresis apparatus
 Agarose powder
 TBE buffer solution
 Gel red
 Hyperladder II
 Transilluminator
The desired wood mouse DNA samples, along with the positive control DNA sample, Taq 2X
clear master mix (Bioline, London) and the BigF and BogR primers were removed from the
freezer and allowed to defrost. Meanwhile ice was collected and stored in a polystyrene box
and an eppendorf tube tray placed on top. An appropriate number of eppendorf tubes were
then paced into the tray in preparation. For each sample 12.5µl of clear master mix, 8.5µl of
distilled water, 1µ BigF and 1µl BogR is needed. To save time the total amounts needed were
calculated and transferred to a large eppendorf tube. To account for pipetting errors an extra
37.5µl of clear master mix, 25.5µl distilled water, 3µl BigF and 3µl BogR was also added to the
calculated amount (e.g. for seven rodent samples, two negative controls and one positive
control (10 tubes) 125µl + 37.5µl of clear master mix, 85µl + 25.5µl of distilled water, 10µl + 3µl
of BigF and 10µl + 3µl BogR would be measured and pipetted into the large tube). 23µl of this
solution was then transferred to each eppendorf tube and kept on ice. The primers, clear
master mix and any remaining solution were returned to the freezer so they may be used again
in later experiments. In each tube 2µl of different wood mouse DNA was added to make up a

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25µl solution in each tube. For every four tubes containing a DNA sample one tube was left
untouched with no DNA added to it to act as a negative control. If any of these negative
controls showed positive results at the end it would be known that contamination had occurred
and the results would be unreliable. A positive control DNA sample was also used to test for
contamination. This sample contained a strain of human Bartonella (Bartonella bacilliformis)
that is not be found in rodents and so if any sample tested positive with a band size the same as
this it would be known contamination had occurred. At this stage it was important to keep the
samples on ice to ensure non-specific binding did not occur. The eppendorfs were marked on
the top of their lids with a permanent marker to indicate which tube contained which DNA
sample, placed into the PCR thermocycler, set to BIGBART and left to run.
During this time more eppendorf tube were set up as done previously in preparation for the
second round. The Taq 2X red master mix (Bioline, London) and the primers BigR (used in place
of BogR for the second round) and BigF were taken out of the freezer and allowed to defrost.
The total volume required was then calculated (12.5µl of red master mix, 1µl of BigF, 1µl of
BigR and 9.5µl of distilled water was pipetted per sample, along with an extra 37.5µl of red
master mix, 3µl of BigR and BigF, and 28.5µl of distilled water to account for pipetting errors)
and pipetted into a large eppendorf tube. 24µl of this was then pipetted into each of the
smaller eppendorf tubes and left on ice.
Once the thermocycler was finished 1µl of the first round PCR product was transferred to a
different eppendorf tube for each product, making the total amount in each tube to 25µl. These
were then kept on ice to prevent non-specific binding and their lids labelled appropriately. The
tubes were then returned to the thermocycler and set to run on the BIGBART cycle once more.
Meanwhile the agarose gel was prepared. The gel is made at a 1.5% concentration and so for
an electrophoresis tank that requires a 50ml gel 0.75g of agarose powder is needed along with

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50ml of TBE buffer solution. The TBE buffer was measured to 50ml using a measuring cylinder
and transferred to a conical flask. A weighing boat was then place on a set of scientific scaled
and a spatula used to collect the agarose powder and carefully transfer the powder into the
boat. Once 0.75g was weighed out the agarose powder was transferred to the conical flask with
the TBE buffer and swirled. The conical flask was then placed in a microwave and heated at full
power for around 30 seconds or until the solution came to a boil and the liquid was clear. The
conical flask was left on the side and allowed to cool slightly.
During this time the gel casting tray was set up. An appropriately sizes transparent gel tray was
collected along with a clamp and two casting gates, each large enough to create 15 wells
(creating a total of 30 wells in the finished gel). The gel tray was placed into the clamp and the
clamp tightened to prevent liquid from escaping. The casting gates were then put in place.
Once the TBE-agarose solution had cooled slightly 50µl of Gel red was added and mixed in via
swirling. This solution was then carefully poured into the casting tray and left to cool
completely and set. After 15 to 30 minutes the gel was set and the casting gates were carefully
removed to reveal the wells in the gel. The clamp was released and the casting tray, along with
the gel, was transferred to a gel electrophoresis apparatus where TBE buffer solution was
carefully poured over the top to completely cover the gel.
Once the second PCR round in the thermocycler had finished 10 µl of each PCR product was
pipetted into a well in the gel. For every PCR product containing a DNA sample, a blank PCR
product was loaded. 10µl of hyperladder II was also pipetted into two wells, one made by each
casting gate, which served to indicate band sizes once viewed under a transilluminator. The
electrophoresis apparatus was then turned on and set to run at 100V for 30 minutes.
When the electrophoresis apparatus was complete the machine was turned off, the gel
removed and transferred to a transilluminator where it could be viewed under UV light allowing

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the DNA fragments to be seen. Samples testing positive showed a white band while those
testing negative showed no bands. A picture was taken and the gel discarded appropriately.
This PCR technique allows for the recognition of inter-Bartonella species hypervariability and
therefore samples testing positive produce different band sizes depending on the Bartonella
species.

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Results:
Wood mice were trapped and collected in a separate study across Ireland by W. Ian
Montgomery (et al. 2012) and their DNA samples shared with the University of Salford for use
by the students. The trapping was undertaken in late autumn and winter of 2010/2011 and was
restricted to field boundaries as it was the most prevalent habitat for the different rodent
species. These samples were collected from three different areas; Zone 1 in Northern Ireland
was an area free of bank voles, Zone 2 was towards the middle of Ireland and was an area just
over the bank vole invasion range and was free of bank voles, and Zone 3 was towards the
south of Ireland in an area where bank voles were present.
From across these zones, three different batches of samples were collected. Batch 1 included
93 wood mouse samples and was collected from both Zone2 and Zone 3 in order to get a
mixture of wood mice samples from areas where bank voles were present and areas where
bank voles had yet to invade. Batch 2 included 33 wood mice samples and was taken from Zone
3 to ensure bank vole presence. Batch 3 included 53 samples and was taken from Northern
Ireland in Zone 1 to ensure bank vole absence. It was necessary to collect from various areas to
be able to determine the effects of the bank vole invasion on Bartonella spp. infection in wood
mice.
The positive results gained from the electrophoresis gels suggest that more than one
Bartonella spp. was present in the wood mice samples as several different band sizes occurred,
as illustrated by Figure 1. However, due to time restraints the samples were unable to be
sequences and therefore specific Bartonella species were not identified.

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Figure 1 Electrophoresis gel of wood mice samples 33 to 48 from Batch 1, including blanks and
hyperladder. Hyperladder II was loaded into the sixth well on the top row and the fourth well on the
bottom row. Samples were loaded into the first four wells and then a blank in the fifth. This method was
repeated along the gel, with a blank loaded into a well for every four samples. A positive control sample
was loaded into the final well on the bottom row.
Samples from each batch were compiled and sorted into two groups; ‘bank voles present’ and
‘bank voles absent’.
Bank voles present Bank voles absent
Number of wood
mice
95 Number of wood
mice
82
Number of
individuals infected
with Bartonella (%)
49.5
Number of
individuals infected
with Bartonella (%)
57.3
Table 2 A total of 179 wood mice individuals were collected from across Ireland. However, due to
missing area data for samples 30 and 31 from Batch 1, only 177 samples could be used and sorted into
Table 2. Out of the total 177 usable samples collected 94 gave a positive infection from Bartonella spp.
The data in Table 2 seems to suggest that wood mice living in bank vole areas have a lower
infection rate than those in bank vole-free areas. However, a chi-squared test is required to

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determine whether there is a significant difference between the two data sets (Fowler, 1998).
The null hypothesis is the presence of bank voles does not have a significant effect on the
frequency of Bartonella infected wood mice.
An adjustment is required to the chi-squared formula due to the data being a 2x2 table. Yates's
correction for continuity is needed, the formula being where ‘|’ is the
absolute value.
Infected
Bank Vole Presence Expected frequency (E) Observed frequency (O)
[|O - E| - 0.5]2 /E
Bank vole area 50.4545 47 0.173
Bank vole-free area 43.5502 47 0.1998
Uninfected
Bank Vole Presence Expected frequency (E) Observed frequency (O)
[|O - E| - 0.5]2 /E
Bank vole area 44.5455 48 0.196
Bank vole-free area 38.4498 35 0.2263
Equation 1 Chi-squared test to determine whether bank voles have a significant effect on the frequency
of Bartonella infections in wood mice.
X2 is the sum of all ‘[|O - E| - 0.5]2 /E’ values.
x2 0.7951
Bank Vole presence Total Number of
Wood mice
Total Infected Total Uninfected
Bank Vole area 95 47 48
Bank vole-free area 82 47 35
Total 177 94 83
Infected Formula Answer
Overall Prevalence 94/177 0.5311
The expected number for Bank vole 0.5311x95 50.4545
The expected number for No Bank vole 0.5311x82 43.5502
Uninfected Formula Answer
The expected number for Bank vole 0.4689x95 44.5455
The expected number for No Bank vole 0.4689x82 38.4498

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By referring to a chi-squared table using the x2 value and 1 degree of freedom, the tabulated
critical value at p=0.05 can be found to be 3.84. The x2 value of 0.7951 is less than this critical
value and so means there is not a significant difference between the two data sets (p = <0.05)
and the null hypothesis can be accepted.
Wood mouse samples from Batch 1 (Zone 2 and 3) also had their weight and sex recorded.
However, data for some samples was incomplete due to rats taking all but the head and ears of
certain wood mouse individuals. Out of the 93 samples collected only 85 were usable for
mathematical tests relating to sex and weight. The weight of an individual may be used as an
indicator of age, with heavier wood mice generally being older.
To determine whether the host weight/age is related to infection a spearman's rank
coefficient test can be used. The null hypothesis is the age/weight of the wood mouse
individual does not have a significant effect on infection from Bartonella spp.
Weight
Class (g)
Number of
Wood mice
Total
Infected
Percentage
Infected
Weight
Rank
Infection
Rank
D D2
13-14.9 5 1 20% 1 1 0 0
15-16.9 7 5 71.42% 2 8 6 36
17-18.9 28 12 42.86% 3 4 1 1
19-20.9 14 9 64.29% 4 6 2 4
21-22.9 17 12 70.59% 5 7 2 4
23-24.9 8 3 37.50% 6 3 -3 9
25-26.9 4 1 25% 7 2 -5 25
27+ 2 1 50% 8 5 -3 9
Total 85 44 51.76% Total 88
Table 3 Shows the calculated weight and infection data for all suitable Batch 1 samples. The smallest
weight recorded was 13.3g and the largest 28.5g. At least seven categories are required for a
Spearman’s rank coefficient test and so weights were divided into eight equal groups. Weight ranks
were allocated smallest to highest in terms of weight class. Infection ranks were allocated in the same
manner to the ‘percentage infected’. The column labelled ‘D’ is the difference between the weight rank
and infection rank.

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The information contained in Table 3 can be input into the spearman’s rank coefficient
equation, rs = 1 - [6total / (n3 - n)], where 6 is a constant peculiar to the formula and n is the
number of units in the sample.
rs = 1 – [(6x88) / (512-8)]
rs = 1 – [528 / 504]
rs = -0.0476
By checking the critical values in a spearman’s rank correlation coefficient table for n=8 (for the
purpose of this equation the minus sign is ignored when checking the critical value) a tabulated
critical value of 0.881 for p=0.01 and 0.643 for p=0.1 can be found. The critical value for p=0.1
shows the lowest level of significance, while the critical value for p=0.01 shows the highest level
of significance. Since the calculated rs value was 0.0476, and is therefore lower than the critical
values, the null hypothesis is accepted.

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Sex may also influence Bartonella infection rate throughout bank vole and bank vole-free
areas.
Figure 2 Indicates the percentage prevalence of Bartonella spp. infecting male and female wood mice of
Batch 1 (Zone 2 and 3) from areas where bank voles are present and areas where bank voles are yet to
invade. From Batch 1; 32 male and 24 females were trapped in a bank vole area, and 15 males and 14
females were trapped in a bank vole-free area.
To study this theory a chi-squared test was calculated. The following test explores the
relationship between the sex of an individual and the Bartonella spp. infection rates in wood
mice. The null hypothesis is the sex of a wood mouse individual does not have a significant
effect on infection from Bartonella spp.
50%
73.33%
45.83% 42.86%
0
10
20
30
40
50
60
70
80
Bank Voles Present Bank Voles Absent
Prevalence(%)
Bartonella spp. Prevalence in Male and Female
Wood mice
Males
Females

21
Infected
Sex Expected frequency (E) Observed frequency (O)
[|O - E| - 0.5]2 /E
Males 24.3272 27 0.1941
Females 19.6688 17 0.2391
Uninfected
Sex Expected frequency (E) Observed frequency (O)
[|O - E| - 0.5]2 /E
Males 22.6828 20 0.2101
Females 18.3312 21 0.2566
Equation 2 Chi-squared test to determine whether sex of an individual has a significant effect on the
frequency of Bartonella spp. infections in wood mice. Yate’s correction for continuity has been taken
into account when calculating the x2
value.
x2 0.8999
Since the x2 value is less than the tabulated critical value of 3.841 for 1 degree of freedom, the
p value is greater than 0.05 and therefore there is no significance between the two data sets
and the null hypothesis can be accepted.
The results gained from the statistical tests suggest that, although bank voles once acted as a
dilution host (Telfer et al. 2004), this is no longer the case. Wood mice from bank vole areas are
just as likely to become infected with Bartonella as the wood mice from bank vole-free areas.
Sex Total Number of
Wood mice
Total Infected Total Uninfected
Males 47 27 20
Females 38 17 21
Total 85 44 41
Infected Formula Answer
The expected number of infected males 0.5176x47 24.3272
The expected number of infected females 0.5176x38 19.6688
Uninfected Formula Answer
The expected number of uninfected males 0.4824x47 22.6828
The expected number of uninfected females 0.4824x38 18.3312

22
Infection from Bartonella spp. also seems to be randomly distributed among males and
females, and old and young wood mice are equally likely to become. Out of the entire 179
wood mice that were sampled, a total of 94 (52.5%) were infected with Bartonella spp. making
about half the population Bartonella positive.

23
Discussion and Conclusion:
The importance of dilution effects is widely known throughout the scientific community, with
zoonosis contributing to many serious diseases in humans today (Swaddle, 2008). Interactions
between humans and wildlife, as well as domestic animals, have led to the spread of diseases
such as Leishmaniasis, Chagas' disease, avian influenza, West Nile virus, anthrax and bubonic
plague (Schmidt, 2001 and Swaddle, 2008) to name just a few. However, the most well-known
and well studies zoonoses is Lyme disease, being the most common vector-borne disease in
North America. This disease is transmitted by ticks belonging to the Ixodidae family, which live
primarily on the ubiquitous white-footed mouse (Peromyscus leucopus) (Schmidt, 2001 and
Swaddle, 2008). By introducing other hosts with a low reservoir competence such as squirrels
and shrews the results gained were encouraging (LoGiudice, 2003).
It seems that a dilution effect is increased by the diversity of different host species available.
However, until the invasion by bank voles, rats seemed to be the only other reservoir host for
B.birtlesii and B.taylorii (the Bartonella spp. found in the native wood mouse)(Saisongkorh et al.
2009) and no dilution effect had previously been seen. Once bank voles had been introduced,
and has sufficient time to become established, a dilution effect began to occur as they
appeared to possess a natural immunity to the Bartonella parasites (et al. Telfer, 2004).
However, from the data gained above this no longer seems to be the case as the tests showed
no significant different in the prevalence of Bartonella spp. infection from wood mice in bank
vole invasion areas and those from bank vole free areas. Telfer (et al. 2004) suggested that
some Irish bank voles may have infections from Bartonella but at such low prevalence (3% or
less) that it may have been missed. A study by Butterworth (2013) has found that certain Irish
bank vole samples tested positive for Bartonella, with a prevalence of about 10%, which seems

24
to support Telfer’s theory. This Bartonella prevalence in bank voles may have risen to such a
level where they can no longer act as competent dilution hosts and so the prevalence in wood
mice throughout the country, in both bank vole and bank vole-free areas, is starting to level off
to equal amounts.
There is evidence that some strains of Bartonella associated with rodents seem to be host-
specific (Telfer et al. 2004) which may explain the ‘immunity’ to Bartonella the bank voles
seemed to possess. However, if the bank vole invasion started in 1925-1926 with the River
Shannon project (Stuart et al. 2007 and White et al. 2012) this 90 year interaction between the
two species (and the tick and flea species they share) may have allowed sufficient time for the
wood mouse Bartonella strains to adapt to infect bank voles, and the effects may just becoming
apparent in recent years.
Additional information relating to sex and weight were recorded for the wood mice individuals
from Batch 1, which stretched both sides of the bank vole invasion boarder. The statistical tests
conducted suggest that sex is not an influential factor for Bartonella infections, and neither is
the age of an individual. The Telfer study (et al. 2004), as well as a study conducted by Colton
(2011), both showed similar results relating to sex and age. All wood mice trapped were of
breeding age and so not correlation could be made in terms of maturity, although Telfer (et al.
2004) found no relationship between maturity and Bartonella infection.
However, younger mice may be more susceptible to infection due to underdeveloped
immune systems. It may be possible for younger mice and nursing pups to contract Bartonella
from the fleas deposited in the nest by their parents, although this was not explored. A study
with rats suggested that the spleen plays a vital role in the immune response for Bartonella spp.
by acting to ‘strain’ the bacteremia and allow them to be more easily phagocytised. By
removing the spleen by splenectomy, a clear increase in the mortality rate for those with

25
endemic Bartonella infections was seen (Haller, 1966). However, the spleens of newly born
wood mouse pups may not be strong enough to fight off the invading bacteremia.
Fleas are more active in the spring and summer months and thrive in warm environments. A
study my Janecek (et al. 2012) also stated that Bartonella spp. infection rates were recorded to
be higher in May which seems to coincide with the flea season. Trapping was conducted in the
late autumn/winter when flea numbers and Bartonella spp. infection rates are lower and so
additional trapping could be conducted in the spring/summer time to compare data and
determine whether season may have an effect on the prevalence of Bartonella spp. throughout
wood mice from both bank vole and bank vole-free areas.
Infections from Bartonella spp. may last for several months to years (Schülein et al. 2001) and,
since wood mice usually live one to two years, an individual that has contracted the bacteremia
can potentially be infected its entire life. During this time it can also pass on the Bartonella
parasite to other ticks and fleas which in turn infect other wood mice individuals, and also now
bank voles it would seem.
Although at the start of the century there was encouraging results suggesting the prevalence
of the Bartonella parasite was being reduced significantly due to bank voles acting as dilution
hosts (Telfer et al. 2004) this no longer seems to hold true, and without one or more
competent dilution hosts present in Ireland Bartonella spp. will continue to be transmitted
throughout the wood mouse population. If bank voles are losing their immunity to Bartonella
spp. then the prevalence within this rodent species is also predicted to increase to similar levels
seen in wood mice over the coming years.

26
Acknowledgements:
Thank you to Dr Kevin Bown, my project supervisor, for invaluable help and advice throughout
the project.
Ian W. Montgomery and his Masters students for supplying the wood mouse samples and data
collected from Ireland.
The University of Salford Manchester for funding my project.
Technicians at The University of Salford Manchester for providing equipment and general help.

27
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31
Appendix:
Appendix 1 Lists all positive and negative results gained along with sample number what Batch the sample was from (Batch 1 =
Zone 2 and 3, Batch 2 = Zone 3, Batch 3 = Zone 1).
With Bank Voles Without Bank Voles
Batchnumber
Samplenumber
Positive=1
Negative=0
Batchnumber
Samplenumber
Positive=1
Negative=0
1 2 0 1 1 0
1 5 0 1 3 1
1 8 1 1 4 1
1 9 0 1 12 1
1 10 0 1 18 0
1 11 0 1 27 1
1 13 0 1 28 1
1 14 0 1 32 0
1 15 1 1 34 1
1 16 1 1 36 1
1 17 1 1 37 1
1 21 0 1 38 0
1 22 0 1 44 1
1 23 1 1 45 1
1 24 1 1 48 1
1 25 1 1 49 1
1 26 1 1 51 0
1 29 1 1 52 1
1 33 0 1 53 1
1 35 0 1 56 0
1 39 0 1 60 0
1 42 1 1 64 1
1 43 1 1 65 1
1 46 1 1 69 0
1 47 1 1 71 0
1 50 0 1 73 0
1 54 0 1 83 1
1 55 1 1 89 0
1 58 0 1 92 0
1 59 1 3 1 0
1 61 1 3 2 1
1 62 0 3 3 0
1 63 1 3 4 0
1 67 0 3 5 1
1 72 1 3 6 0
1 74 0 3 7 0

32
1 76 1 3 8 0
1 77 1 3 9 0
1 78 0 3 10 1
1 79 1 3 11 1
1 80 1 3 12 1
1 81 0 3 13 0
1 82 1 3 14 0
1 84 1 3 15 0
1 86 0 3 16 0
1 87 0 3 17 1
1 88 0 3 18 1
1 90 1 3 19 0
1 91 0 3 20 1
1 93 0 3 21 1
2 6 0 3 22 0
2 7 0 3 23 1
2 19 1 3 24 1
2 20 1 3 25 1
2 40 0 3 26 1
2 41 1 3 27 0
2 57 1 3 28 1
2 66 1 3 29 1
2 68 0 3 30 0
2 70 1 3 31 1
2 75 0 3 32 1
2 85 0 3 33 1
2 1 0 3 34 0
2 2 0 3 35 1
2 3 0 3 36 1
2 4 1 3 37 1
2 5 1 3 38 0
2 6 0 3 39 1
2 7 1 3 40 1
2 8 1 3 41 1
2 9 0 3 42 0
2 10 0 3 43 1
2 11 0 3 44 1
2 12 0 3 45 0
2 13 0 3 46 0
2 14 0 3 47 0
2 15 0 3 48 0
2 16 1 3 49 1
2 17 0 3 50 0
2 18 1 3 51 1
2 19 0 3 52 1
2 20 1 3 53 1

33
2 21 1
2 22 0
2 23 1
2 24 1
2 25 1
2 26 1
2 27 1
2 28 0
2 29 0
2 30 1
2 31 1
2 32 0
2 33 1
Appendix 2 Table listing all data for the Batch 1 wood mice
Invasion
Range
SampleID
IDElodie
Reproductive
Condition
Weight(g)
Sex
Batch
number
Zone
Sample
number
Positive=1
Negative=0
No bank voles S7181-9 S7181-9 NP 17.2 Female 1 2 and 3 1 0
No bank voles N4130-7 N4130-7 NP 19.3 Female 1 2 and 3 3 1
No bank voles N7000-7 N7000-7 TWD 19.4 Male 1 2 and 3 4 1
No bank voles S6060-8 S6060-8 TWD 22.2 Male 1 2 and 3 12 1

34
No bank voles S7505-3A S7505-3_1 TWD 16.4 Male 1 2 and 3 64 1
No bank voles N2040-1A N2040-1_1 TWD 25.5 Male 1 2 and 3 69 0
No bank voles R5180-8 R5180-8 TWD 22.5 Male 1 2 and 3 89 0
Bank voles R5050-8 R5050-8 NP 22.9 Female 1 2 and 3 10 0
Bank voles R8030-10A R8030-10_1 NP 19.7 Female 1 2 and 3 21 0
Bank voles N2110-8 N2110-8 NP 20.7 Female 1 2 and 3 25 1
Bank voles M9021-8 M9021-8 NP 25.4 Female 1 2 and 3 26 1
Bank voles S4061-3 S4061-3 NP 13.3 Female 1 2 and 3 33 0
Bank voles R8000-4B R8000-4_2 NP 16.6 Female 1 2 and 3 46 1
Bank voles M8020-10 M8020-10 NP 16.5 Female 1 2 and 3 50 0
Bank voles M8020-2 M8020-2 TWD 17.8 Male 1 2 and 3 5 0
Bank voles N2000-8 N2000-8 TWD 22.0 Male 1 2 and 3 8 1
Bank voles S0080-5 S0080-5 TWD 28.5 Male 1 2 and 3 9 0
Bank voles R8030-6 R8030-6 TWD 22.2 Male 1 2 and 3 35 0
Bank voles M9040-6A M9040-6_1 TWD 18.9 Male 1 2 and 3 54 0
Bank voles S2090-10A S2090-10_1 TWD 21.3 Male 1 2 and 3 79 1

35
Bank voles R5040-10 R5040-10 unknown unknown unknown 1 2 and 3 2 0
Bank voles S4000-7 S4000-7 unknown unknown unknown 1 2 and 3 24 1
Bank voles R5020-8 R5020-8 unknown unknown unknown 1 2 and 3 29 1
Bank voles R8290 R8290 unknown unknown unknown 1 2 and 3 76 1
Bank voles S3050-10 S3050-10 unknown unknown unknown 1 2 and 3 81 0
Bank voles S4010 S4010 unknown unknown unknown 1 2 and 3 90 1
Unknown S3090-5 S3090-5 unknown unknown unknown 1 2 and 3 30 0
Unknown N0030-6 N0030-6 unknown unknown unknown 1 2 and 3 31 0
Appendix 3 Gel photograph of batch 1 - samples 1 to 32

36
Appendix 5 Gel photograph of batch 1 - samples 49 to 76. To the left shows a darkened image to highlight the bands. To the
right shows a brightened image to highlight the hyperladder.

37

38

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