1. Kendra McAlister
Senior Practicum
1 March 2016
The behavioral impact of Toxoplasma gondii infection on the Owens Valley vole (Microtus
californicus vallicola)
Kendra McAlister1
, Janet Foley2
, Amanda Poulsen2
1
Department of Animal Biology, University of California, Davis
2
Department of Veterinary Medicine and Epidemiology, University of California, Davis
Abstract:
The protozoan parasite Toxoplasma gondii infects a broad range of intermediate warm-blooded
hosts, notably small mammals such as mice and rabbits. In order to sexually reproduce the
parasite requires access to definitive felid hosts which prey on wild rodents. The parasite appears
to block the innate aversion of some rodents to cat odors, thus promoting an attraction that may
increase the likelihood of transmission between intermediate and definitive hosts. Little is known
about the behavioral impact of T. gondii infection on rodents such as the Owens Valley vole,
which likely has contact with the parasite in its natural habit. The aim of this study was to
evaluate the potential for T. gondii to promote an anxious emotional state in the Owens Valley
vole which might favor its transmission in the environment. Results revealed that chronic
infection had no significant impact on the performance of fear based behaviors such as self-
grooming, wall-seeking and freezing in Owens Valley voles.
Introduction:
T. gondii is an obligate protozoan parasite that completes its sexual life cycle within the
intestines of its definitive host, a member of the cat family (Haroon et al. 2012). Following
sexual reproduction oocysts are shed with cat feces, often to be consumed by grazing warm-
blooded intermediate hosts such as mice and rabbits (Gatkowska et al. 2012). Voles are among
the known intermediate hosts prone to T. gondii infection by ingestion of shed oocysts (Dabritz
et al. 2007). After contact with the oral cavity T. gondii replicates and spreads throughout the
body of its host, infecting various muscles and major organs, including the central nervous
system (Haroon et al. 2012). Acute T. gondii infection in rodents elicits strong behavioral
alterations such that the rodents’ natural aversion toward feline smell diminishes (Gatkowska et
al. 2012). This lack of avoidance of cat predator odor may facilitate the parasite’s transmission
from intermediate to definitive host, allowing it to complete its life cycle and produce more
oocysts (McConkey et al. 2012). While this phenomenon has been well documented in captive
mice and rats little is known whether wild rodents such as voles exhibit the same infection
outcomes.
One subspecies of California vole, the Owens Valley vole (Microtus californicus vallicola),
represents an interesting model of study for the behavioral impact of T. gondii infection.
Although little is known about the Owens Valley vole, potential exposure to T. gondii exists
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2. based on its similarity to a phylogenetically close and endangered California vole subspecies, the
Amargosa vole (Microtus californicus scirpensis); the Amargosa vole has significant contact
with T. gondii in its natural habitat (Ellsworth et al. 2015; Ott-Conn et al. 2013). Given the lack
of state records on the Owens Valley vole, investigating the effect of T. gondii on its behavior
and emotional state would help demystify the nature of the subspecies and the impact T. gondii
has on other species of rodent. Under the “behavioral manipulation” hypothesis, T. gondii likely
reduces feline aversion in rodents to better its chances of transmitting to a felid host (Vyas et al.
2007). While reduced feline avoidance may be the likely outcome for infected voles compared to
healthy voles perhaps there are other behavioral patterns vulnerable to parasitic influence.
Assuming infected Owens Valley voles display a preference for cat pheromones, for the benefit
of parasite transmission, will infected voles engage in other behavior patterns for the benefit of
the parasitic host? The aim of this study is to determine whether T. gondii has an effect on the
performance of other potentially maladaptive behaviors such as self-grooming, freezing
(signified by a sustained crouching position) and wall-seeking behavior (moving along the
boundary and walls of spaces), which are generally associated with fearful emotional states in
rodents. Infected Owens Valley voles will be compared to uninfected controls to determine if
both groups exhibit differing levels of fear-based behaviors. To answer this question we initially
infected six Owens Valley voles with T. gondii while allowing an equal number to remain
uninfected for control purposes. One month following inoculation with oocysts, we performed
scent tests with one type involving bobcat urine and another type involving domestic cat urine.
Voles were placed in cages with four quadrants and allowed to explore the arena for 24-minutes.
Initially these trials were analyzed for vole response to the scented tiles placed in three out of
four quadrants. From the total number of trials a few were selected for analysis of fear behavior
parameters measured at one-minute intervals. McConkey et al. (2012) determined that rodents
prefer non-predator pheromone, either their own or a sympatric species, under healthy
conditions; however, infected rats exhibited an attraction toward feline pheromone compared to
non-predator pheromones. Following this conclusion, we selected trials based on infected vole
preference for bobcat and domestic cat quadrants and non-infected preference for home
quadrant. We also noted the amount of time spent in theoretically nonfavored quadrants (home
for infected voles and bobcat and domestic cat for uninfected voles). In addition, we wanted to
further analyze trial videos based on the number of entries to the quartered and central parts of
the arena, i.e. spontaneous locomotor activity. Locomotor activity was our measure of
opportunity for the voles to respond to the trial conditions.
Methods:
Animals
A sample size of 12 Owens Valley voles (six infected, six uninfected) was used for this study.
The groups, consisting of female and male individuals, were housed in an indoor rodent facility
located in Davis, CA. The infected group was treated with an inoculum which administered 100
T. gondii oocysts per vole. Special protocols were followed to prevent infection in the control
group and exchange of contaminants between the room designated for this experiment and other
areas of the rodent facility. Experimental trials were conducted one month after primary infection
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3. to allow time for chronic invasion of tissue in the central nervous system (Vyas et al. 2007). All
experimental procedures were approved by the California Department of Fish and Wildlife and
an animal use permit issued by the US federal government. The voles were euthanized at the end
of the experimental procedure.
Trial Types and Arena
Two kinds of trials were conducted: Type 1, a test for vole response to bobcat scent, and Type 2,
a test for vole response to domestic cat scent. Trials took place in a square arena divided into
four quadrants with wooden planks. The quartered sections were designated “home”, “control”
(mouse odor), “bobcat/cat” and “blank”. Ceramic tiles were placed in the center of each, except
for the blank quadrant.
• Home quadrant: tile kept in vole’s cage for two weeks prior to trials, serving as familiar
scent.
• Control quadrant: ceramic tile infused with urine from house mice, which are a sympatric
species in the Owens Valley vole habitat.
• Bobcat/cat odor quadrant: a ceramic infused with commercially purchased bobcat urine
for trial Type 1, and domestic cat urine for Type 2.
• Blank: no tile.
Trial Procedure and Replicates
Voles were placed into the center of the arena facing the blank quadrant at the start of each trial.
Their movements were recorded on video for 24 minutes. “Bobcat/cat” and “control odor”
quadrants were always positioned adjacent to the “home” quadrant in each trial. However, the
position of “home” quadrant was randomized for each individual trial to control for location
preferences (i.e. “home” placed across from “blank” to avoid bias toward or against “bobcat/cat”
quadrant). The 12 voles (six infected, six uninfected) experienced three rounds of each trial type
(Type 1: bobcat, Type 2: domestic cat) to control for behavior associated with the vole being
unfamiliar with a new setting. With 12 voles and three replicates of each trial type the planned
number of individual trials was 72.
Scan Sampling
Six 24-minute trials were sampled at one-minute intervals involving a sample size of three
infected and three uninfected voles from the scented tile tests. Three uninfected trials were
selected based on home quadrant preference (50%+ time spent in quadrant during trials) while
three infected trials were selected due to time spent in either the Trial Type 1 or Trial Type 2 cat
quadrant (50%+ time spent in quadrant during trials). The amount of time spent in home and
bobcat and cat quadrants was noted for all six trials regardless of preference. Locomotor activity
measures were analyzed to determine whether the vole groups had equal opportunity to explore
the arena. Three anxiety behavior parameters were measured during the scan samples: (a) wall-
seeking behavior, (b) freezing behavior and (c) self-grooming.
Analysis
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4. Quadrant preference data are shown as the mean value ± standard error of the mean (SE). This
data was analyzed by t-test. All other behavior data was analyzed by a chi-square goodness of fit
test. The two experimental groups and the scan sampling data collected from the select trials
were used to determine the main effects of T. gondii on Owens Valley vole behavior.
Results:
Scan Sample Selection Criteria
Obtained results revealed that the number of entries to the quadrant and central areas of the arena
did not differ based on infection or control status of the voles, c2
(1, N = 6) = 0.03, p = .8575.
These results indicate that both vole groups had the same level of opportunity to explore the
arena during scented trial tests (Table 1). Further, as presented in Fig. 1A, uninfected voles
chosen for scan sampling spent significantly more time in the home quadrant compared to
infected voles demonstrating a strong preference for home in the healthy control group (p <
0.006). Conversely, infected voles spent significantly more time in the Type 1 and Type 2 cat
quadrant compared to uninfected voles (Fig. 1B) suggesting strong influence by the T. gondii
challenge (p < 0.0001). The quadrant preference results stand in good agreement with McConkey
et al.’s (2012) model for non-predator and predator scent preference among infected and non-
infected rodents.
Scan Sample Behavior Parameters
Although Table 2 shows a slightly higher freezing behavior count for infected voles compared to
uninfected voles, the difference was not significant, c2
(1, N = 6) = 0.13, p = 0.7194. Additionally,
compared to non-infected voles no significant difference in the wall-seeking behavior count was
evident for infected voles, c2
(1, N = 6) = 0.13, p = 0.7194. Both groups preferred being away
from the center of quartered parts of the arena (Table 3). Finally, Table 4 shows similar self-
grooming behavior counts between infected and non-infected voles with no significant difference
between the two groups, c2
(1, N = 6) = 0.93, p = 0.3359. The obtained results revealed that T.
gondii invasion does not significantly influence the normal fearful behavior of Owens Valley
voles suggesting fearful emotional states do not promote parasite transmission between voles and
cats.
Table. 1 Spontaneous Locomotor Activity
Category Observed # Expected #
Uninfected Voles 61 62
Infected Voles 63 62
Total 124 124
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5. Fig. 1 Time spent in home (A) and Type 1 and Type 2 cat (B) quadrants during 24-minute trial
observations.
Table. 2 Freezing Behavior
Category Observed # Expected #
Uninfected Voles 36 34.5
Infected Voles 33 34.5
Total 69 69
Table. 3 Wall-Seeking Behavior
Category Observed # Expected #
Uninfected Voles 60 62
Infected Voles 64 62
Total 124 124
Table. 4 Self-Grooming Behavior
Category Observed # Expected #
Uninfected Voles 11 13.5
Infected Voles 16 13.5
Total 27 27
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6. Discussion:
Vyas et al. (2007) postulated that under the “behavioral manipulation” hypothesis a parasite
enhances its transmission efficiency by altering the innate defensive behaviors of its host. In
doing so, the parasite increases its chances of reproducing and inheriting favorable genetic traits
in the next generation. Our study replicated this finding by determining a strong level of
attraction to feline odors in infected Owens Valley voles compared to healthy controls. Utilizing
these results, and near equal locomotor activity measures, we discovered that the impact of
infection on aversion to cat pheromones was the only notable maladaptive trait because infection
did not impact fear and anxiety-like behaviors in voles. Although behaviors such as self-
grooming, wall-seeking and freezing are related to the innate fear of feline predators there were
no reported differences in how frequently healthy and infected voles engaged in those behaviors
across scan samples.
While one could argue that predator scent preference in voles may have less to do with parasite
invasion and may more accurately reflect general sickness, the manipulation hypothesis provides
basis for further investigation of parasite strategies for improving transmission efficiency.
Whether the weakened fear of feline predator results in immediate transmission from vole to cat
or movements through multiple indefinite hosts, the parasite has ample opportunity to exploit its
host in order to sexually reproduce (Vyas et al. 2007). However, data comparing predation rates
between control and infected rodents could help provide a more direct experimental observation
in support of the manipulation hypothesis. As it stands, predator-prey system models help to
demonstrate that even a small selection force favoring vulnerability of infected prey animals
would suffice to increase parasitic load in predator populations (Vyas et al. 2007).
Perhaps we were unable to replicate the findings in reference to fear based behaviors due to very
small sample size and unintended factors impacting the experimental procedure. For instance,
not all animals received equal handling treatment as some were released at later times than others
into the arena after being trapped in their home cages. In addition, we may have neglected
environmental distractions that could influence vole behavior. For instance, vole urine creates a
visible UV trail that may have stained the wooden planks used to quarter the arena. Whereas all
other parts of the arena were sanitized between trials we never treated the wooden planks. There
existed the possibility of voles being exposed to scent residues from infected and non-infected
neighbors.
In addition, some infected voles were considerably older and further along in their infection
status. Wasting syndrome contributed to the euthanasia of one infected individual resulting in 11
individuals toward the end of the experimental procedure. Specific to the scan sampling
measures, voles would occasionally be hidden from view leading to lesser values for self-
grooming and freezing behavior. Were there multiple cameras observing the trials from various
angles the sampling behavior values may have resulted in more significant results relative to the
“behavioral manipulation” hypothesis.
In short, data presented in this study supports evidence of blocked aversion toward cat odors in
voles, though the effects of infection may be just specific to olfaction and not fear based
behaviors. Studies involving acute states of infection in voles, in addition to other types of
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7. behavior such as rearing and climbing, will provide more information regarding the impact of T.
gondii infection on Owens Valley vole behavior.
Acknowledgements:
I would like to express my great appreciation to my faculty mentor, Prof. J. Foley, for providing
me an opportunity to work with endangered animals within permit guidelines and for her
valuable and constructive assistance during the planning and development of this research work.
Her patience and willingness to give her time so generously has been very much appreciated.
I would also like to offer special thanks to my research supervisor, A. Poulsen, who let me
experience research in disease ecology and offered valuable lessons in collaboration,
commitment to study designs and maintaining compliance with protocols established by persons
in charge.
I am also grateful to Elvira Hack, academic advisor for the Animal Biology undergraduate
program. I am extremely thankful and indebted to her continuous support, guidance and
encouragement throughout my undergraduate career.
I take this opportunity to express gratitude to Kim Mahoney of the College of Agricultural and
Environmental Sciences Dean’s Office for her patience and professionalism. I also thank my
family and friends for their unceasing encouragement and support throughout this venture.
I also place on record, my sense of gratitude to one and all, who directly or indirectly, have lent
their hand in this endeavor.
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8. References:
Dabritz, H., M. Miller, E. Atwill, I. Gardner, C. Leutenegger, A. Melli, and P. Conrad. 2007.
Detection of Toxoplasma gondii-like oocysts in cat feces and estimates of the
environmental oocyst burden. J. Am. Vet. Med. Assoc. 231:1676-1684.
Ellsworth, A., Parmenter, S. and Parmenter B. Owens valley vole research. 2015. California
Department of Fish and Wildlife.
Gatkowska, J., M. Wieczorek, B. Dziadek, K. Dzitko and H. Dlugonska. 2012. Behavioral
changes in mice caused by Toxoplasma gondii invasion of the brain. Parasitology
Research 111:53-58.
Haroon, F., U. Hädel, F. Angenstein, J. Goldschmidt, P. Kreutzmann, H. Lison, K-D. Fischer, H.
Scheich, W. Wetzel, D. Schülter and E. Budinger. Toxoplasma gondii actively inhibits
neuronal function in chronically infected mice. 2012. PLoS ONE 7(4).
McConkey, G. A., H. L. Martin, G. C. Bristow and J. P. Webster. 2012. Toxoplasma gondii
infection and behavior – location, location, location? The Journal of Experimental
Biology 216:113-119.
Ott-Conn, C., D. Clifford, T. Branston, R. Klinger, and J. Foley. 2013. Survey of pathogen
exposure and ectoparasites in the federally endangered Amargosa vole (Microtus
californicus scirpensis).
Vyas, A., S-K. Kim, N. Giacomini, J. C. Boothroyd, and R. M. Sapolsky. Behavioral changes
induced by Toxoplasma infection of rodents are highly specific to aversion of cat odors.
2007. PNAS 104(15).
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