Integrative and Comparative Biology
Integrative and Comparative Biology, volume 54, number 2, pp. 118–128
doi:10.1093/icb/icu060
Society for Integrative and Comparative Biology
SYMPOSIUM
What Makes a Feline Fatal in Toxoplasma gondii’s Fatal Feline
Attraction? Infected Rats Choose Wild Cats
M. Kaushik, S. C. L. Knowles and J. P. Webster1
Department of Infectious Disease Epidemiology, School of Public Health, Imperial College Faculty of Medicine, St Mary’s
Campus, London W2 1PG, UK
From the symposium ‘‘Parasitic Manipulation of Host Phenotype, or How to Make a Zombie’’ presented at the annual
meeting of the Society for Integrative and Comparative Biology, January 3–7, 2014 at Austin, Texas.
1
E-mail: [email protected]
Synopsis Toxoplasma gondii is an indirectly transmitted protozoan parasite, of which members of the cat family
(Felidae) are the only definitive hosts and small mammals such as rats serve as intermediate hosts. The innate aversion
of rodents to cat odor provides an obstacle for the parasite against successful predation by the feline definitive host.
Previous research has demonstrated that T. gondii appears to alter a rat’s perception of the risk of being preyed upon by
cats. Although uninfected rats display normal aversion to cat odor, infected rats show no avoidance and in some cases
even show attraction to cat odor, which we originally termed the ‘‘Fatal Feline Attraction.’’ In this study, we tested for the
first time whether the ‘‘Fatal Feline Attraction’’ of T. gondii-infected rats differed according to the type of feline odor
used, specifically whether it came from domestic cats (Felis silvestris catus) or wild cats—cheetahs (Acinonyx jubatus) or
pumas (Felis concolor). In two-choice odor trials, where wild and domestic cat odors were competed against one another,
consistent with previous findings we demonstrated that infected rats spent more time in feline odor zones compared with
uninfected rats. However, we further demonstrated that all cat odors are not equal: infected rats had a stronger preference
for wild cat odor over that of domestic cats, an effect that did not differ significantly according to the type of wild cat
odor used (cheetah or puma). We discuss these results in terms of the potential mechanism of action and their implications for the current and evolutionary role of wild, in addition to domestic, cats in transmission of T. gondii.
Introduction
Toxoplasma gondii is a protozoan parasite with an
indirect lifecycle, of which the only known definitive
hosts are members of the cat family (Felidae) (Dubey
2010). The sexual cycle of the parasite occurs within
the feline intestine, after which oocysts are shed in
the feces. Although transmission was traditionally attributed to the domestic cat Felis silvestris catus, several feline species are now thought to be definitive
hosts of T. gondii. Although prevalence of infections
are variable, shedding of oocysts has been demonstrated both experimentally and naturally in several
wild species, including, but not exclusive to, the
European wildcat F. silvestris silvestris, the African
wildcat Felis lybica, the bobcat (Lynx rufus), the leopard cat Felis bengalensis euptilurus, the puma Felis
concolor (also referred to as cougars or American
mountain lions), and the cheetah Acinonyx jubatus
(Lukesova and Literak 1998; Aramini et al. 1999;
Miller et al. 2008; Dubey 2010).
Toxoplasma gondii has a wide range of intermediate hosts, including small birds and mammals, such
as the rat (Rattus norvegicus) (Webster and
Macdonald 1995). In the wild, R. norvegicus are subject to predation by a number of species, including
cats, foxes, and mink. Rodents, both rats and mice,
have an innate aversion to the odor of these predators, which should decrease their risk of predation
(Blanchard et al. 1990; Dielenberg and McGregor
2001). Laboratory rats with no exposure to cats for
hundreds of generations still show significant behavioral and physiological aversion to the odor of cats’
urine (Zangrossi and File 1992). For instance, exposure to such odor has been shown to increase anxiogenic responses in social interaction and generalized
anxiety tests, which can be detected for up to an
Advanced Access publication June 6, 2014
ß The Author 2014. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved.
For permissions please email: [email protected].
119
Toxoplasma gondii’s wild cat versus domestic cat attraction
hour after exposure to the odor (Zangrossi and File
1992). The physiological changes in rats when exposed to cats’ odor have been demonstrated to include raised corticosterone levels and greater c-Fos
expression in many regions of the brain, particularly
in the medial amygdala, medial hypothalamus, and
periaqueductal gray (File et al. 1994).
The innate aversion of rodents to cat odor presents an obstacle for parasite transmission, which requires predation by the feline definitive host. Earlier
investigations into the effect of T. gondii infection on
a rat’s response to feline odor had surprising results
(Berdoy et al. 2000). Toxoplasma gondii appeared to
alter a rats’ perception of feline predation risk: uninfected rats displayed normal aversion to the odor of
cat urine, whereas infected rats showed no aversion
and in some cases even attraction, which we originally termed the ‘‘Fatal Feline Attraction’’ (Berdoy
et al. 2000). This behavior has been consistently
demonstrated over several studies (Berdoy et al.
2000; Webster et al. 2006; Vyas et al. 2007a; House
et al. 2011), utilizing a range of methods including
either four-choice or two-choice assays (Webster
et al. 2006; Lamberton et al. 2008), and odors from
either domestic cats (Berdoy et al. 2000; Webster
et al. 2006; Lamberton et al. 2008) or bobcats
(Vyas et al. 2007). Such altered behavioral responses
have been shown to be specific to felids, and not to
non-predatory mammalian such as rabbits (Berdoy
et al. 2000; Webster et al. 2006) or to predators
that are not definitive hosts, such as mink
(Lamberton et al. 2008), dogs (Kannan et al. 2010),
or foxes (Dielenberg et al. 2001; McGregor et al.
2002). This adds weight to the parasite-manipulation
hypothesis, which suggests that the parasite may specifically alter the rat’s behavior in ways that increase
its chances of entering the feline definitive host, and
thereby completing its life cycle (Webster 2001). This
subtle ability to distinguish between the odors of a
definitive and a non-definitive host of T. gondii indicates that this is a specific alteration in the cognitive perception of the predation risk by the rat,
rather than a more general destruction of a behavioral trait or of olfaction (Webster and McConkey
2010).
Here we test, for the first time, whether there is
any evidence for differential aversion or ‘‘Fatal Feline
Attraction’’ to the odor of feline urine in relation to
whether the source is domestic cats versus wild cats.
By comparing feline species known to shed T. gondii
oocysts under experimental and natural conditions
(Lukesova and Literak 1998; Elmore et al. 2010),
that of the cheetah (A. jubatus) and the puma
(F. concolor), in comparison to domestic cats’
(F. silvestris catus), we test the species specificity of
T. gondii-altered response to feline odor. In accordance with the manipulation hypothesis, we predicted that T. gondii infection in rats would cause
decreased aversion, or even the ‘‘Fatal Feline
Attraction’’ preference, in response to both wild
and domestic definitive feline hosts. However, it
could also be argued that a lack of recent co-evolution between the rat intermediate host and wild
feline species might reduce any parasite manipulation
of host response to their urine odor, when compared
with domestic cats. Alternatively, if the odor signal(s)
responsible for predator attraction are stronger in
wild cat compared with domestic cat urine, one
might observe greater preference for wild cat compared with domestic cat odor among infected rats.
Materials and methods
Strains of host and parasite
Male (n ¼ 15) and female (n ¼ 15) Lister-hooded rats
were obtained at 3 weeks of age from Harlan UK
Ltd, and in order to minimize any generalized
non-specific stress/anxiety level, were accustomed to
handling until adulthood. Rats were housed in
groups of 4, until male rats reached 500 g in
weight, after which males were housed in pairs.
Female rats did not reach this weight. All rats were
housed in aerosol-controlled plastic cages with solid
bottoms, with wood shavings, and woodchips used
as bedding. Bedding was changed two or three times
weekly, and the cage sterilized weekly. Rats were
fed and watered ad libitum on a pelleted rodent
diet and sipper tubes. Environmental enrichment
was provided through addition of items such as
cardboard tunnels, wooden chew-sticks, and shredded paper.
Two ‘‘wildtype’’ Pru (type II) lines of T. gondii
were used for experimental infections, chosen because these are avirulent cyst-forming strains (Grigg
et al. 2001). All parasites were maintained in human
foreskin fibroblasts (HFFs) and obtained from the
University of Leeds. Toxoplasma gondii parasites
were released from HFF cells, counted, and then
transported to London in Dulbecco’s modified
Eagle’s medium (DMEM) solution. The parasites
were syringe-lysed using a 27-gauge needle to release
tachyzoites, then centrifuged. The DMEM was removed, and the parasite material re-suspended in
sterile phosphate-buffered saline (PBS) and densities
estimated. Infection was conducted via interperitoneal (IP) injection with 0.2 ml per rat containing
1 106 tachyzoites in sterile PBS. This amount
was chosen based on a previous study which also
120
used IP inoculation of Pru strain tachyzoites in rats
(Vyas et al. 2007), and did not result in any morbidity, mortality, or complications. Uninfected sham
control rats were IP injected with 0.2 ml of sterile
PBS. Rats were infected at 3 months of age.
Sample sizes were calculated using Mead’s resource
equation, E ¼ N – T (Mead 1990).
Urine of cheetahs (A. jubatus), pumas (F. concolor), and
domestic cats (Felis domesticus)
Urine of domestic cats was obtained from Archway
Veterinary Surgery, Wiltshire, and the Royal
Veterinary College (RVC), Hertfordshire. Cheetah
and puma urine was also obtained from the RVC.
Urine samples from several individuals of each
species were pooled into three species-specific
mixes for use in trials, in order to maintain consistency across trials and minimize any effects due to
individual variation among cats. The pooled urine
samples were stored in the freezer and aliquoted
out for individual trials in order to further maintain
consistency.
Four studies of T. gondii in cheetahs reported
seroprevalences ranging from 27.3% to 77.7%,
whereas 14 studies in pumas indicated a range of
7% to 100% (Dubey 2010). Many studies in domestic cats have indicated seroprevalence ranging from
5.4% to 84.9% (Dubey 2010). Therefore, there is no
obvious difference in natural seroprevalence levels
between the three feline species examined here, and
as with all species infected with the parasite, prevalence of T. gondii is likely to vary widely of the basis
of geographical location and sampling distributions
(Dubey 2010).
M. Kaushik et al.
from one of the two wild cats considered here—
cheetah or puma. This design allowed us to elucidate
how feline species-specific the level of aversion or
attraction to, feline odor is (Webster et al. 2013).
The two-choice test has previously been used to
compare responses of infected and uninfected rats
to the odors of definitive and non-definitive hosts
(Lamberton et al. 2008). In this study, we aimed to
test whether rats infected by T. gondii respond differently to the odors of different feline species.
The two-choice apparatus consisted of two arms
separated from an entrance arena (neutral zone) by a
sliding door, with the floor covered by a thin layer of
woodchips (Fig. 1A). In one arm, 0.2 g of tissue
paper soaked in 0.7 ml of domestic cat’s urine was
positioned, whereas in the other arm, 0.2 g of tissue
paper soaked in 0.7 ml of the urine either of cheetah
or puma was positioned. At the start of each behavioral trial, individual rats were placed in the arena’s
‘‘neutral zone’’ facing away from the feline odor
Behavioral assay: the two-choice feline species odor
test
Assays of rats’ response to feline urine odor were
conducted at 6–8 months post exposure to T.
gondii, and hence at a time when chronic T. gondii
cysts would be established in the Central Nervous
System. Behavioral tracking was conducted with
Ethovision XT (Noldus, Wageningen, Netherlands).
This was used to record data such as ID of the rat,
date, and coded condition (unexposed uninfected
and exposed infected). Automated tracking also enabled accurate recording of the movement velocity,
and thereby activity, of individual rats.
The two-choice test of response to feline odors
enables measurement of the number of entrances
into, and the time spent in, zones with different
feline odors. In each trial, one odor was always
from domestic cat urine, whereas the other came
Fig. 1 Two-choice behavioral assay of odor. Arms/zones were
separated from an entrance ‘‘neutral’’ arena by a sliding door.
Within each arm, a small layer of woodchips was placed. In one
arm, 0.2 g of tissue paper soaked in 0.7 ml of domestic cat’s urine
was positioned, whereas in the other arm, 0.2 g of tissue paper
soaked in 0.7 ml of the urine either of cheetah or puma was
positioned. Rats were placed in the entrance facing away from
the choice arms. The sliding door was lifted, and the rats were
allowed to freely explore the maze for 5 min. Automated behavioral tracking was conducted with Ethovision XT (Noldus,
Waginen, Netherlands) with no human observer present during
the trials.
121
Toxoplasma gondii’s wild cat versus domestic cat attraction
arms (feline zones). The sliding door was lifted and
the rats were allowed to freely explore for 5 min. As
both odours are potentially aversive, a short test time
was used to minimize stress for the animal. Likewise,
rats could avoid entering the feline odor zones and
remain in the neutral zone. The location of odors
within the two arms was randomized to avoid positional bias and woodchips were changed and all apparatus wiped down with Trigene to remove odors
before and after each test. Experiments were performed in two experimental blocks. Twelve rats
were given both a cheetah and a puma trial in the
first experimental round, 13 rats were given both
trials in the second experimental round, and 5 rats
were given both trials in both rounds. Parameters
calculated from behavioral data collected in each
trial included: duration of time in each zone (neutral, domestic cat zone, and wild cat zone), velocity
of the rat both overall and in each zone separately
(as previous studies suggested that infection by
T. gondii increases activity in rats and mice
(Webster 1994, 2007).
At the end of the study, all experimental rats were
euthanized by rising concentrations of isofluorane
gas and cervical dislocation. The presence of T. gondii’s antibodies was confirmed by the IgG and IgM
indirect latex agglutination test (Toxoreagent; Mast
Group Ltd). Titres more than 1:16 were considered
positive (Webster 1994; Webster et al. 1994), which
showed that all rats exposed to T. gondii sustained
infection. The work was performed under Home
Office project license and all procedures were classed
as ‘‘mild.’’
Calculation of attraction and discrimination ratios
We first assessed each rat’s preference/aversion for
spending time in any feline zone when compared
with the neutral zone, via an ‘‘Attraction ratio,’’ calculated as duration in any feline zone/total time.
Subsequently, for those rats that entered a feline
odor zone at least once, we calculated a ‘‘Feline discrimination ratio,’’ to assess that individual’s relative
‘‘preference’’ for domestic cat or wild cat odor. This
was calculated as duration in domestic cat zone/total
time in feline odor zones. If there was no difference
between the rats’ response to domestic cats’ odor and
wild cats’ odor, a feline discrimination ratio not significantly different from 0.5 would be expected.
Statistical analyses
All statistical analyses were performed in R version
2.15.2. Since individual rats were subjected to multiple trials (at least one cheetah and one puma trial
per rat, sometimes two trials of each type), to make
full use of the data we used mixed models in analysis, implemented in the lmer package, including rat
identity as a random intercept term. Models were
simplified by backwards stepwise simplification
using likelihood ratio tests (on models fit via maximum likelihood) to obtain a final, minimal model
with all terms significant at the 5% level. Final estimates of parameters were obtained using models
fitted using restricted maximum likelihood. For all
models, assumptions were checked graphically by inspecting histograms of residuals for normality, and
plots of residuals against fitted values to assess homoscedasticity. The attraction ratio (proportion of
total time spent in feline zones) was arcsine
square-root transformed before analysis, to ensure
model assumptions were met. Feline discrimination
ratio and movement speed were approximately normally distributed, and no transformation was required for model assumptions to be upheld. The
number of entries into the wild cat zone (a count
ranging between 0 and 11) was modeled using a
Poisson mixed model with a log link. No evidence
for overdispersion was found in this model, or in an
equivalent standard Poisson GLM with the randomintercept term omitted. The following predictors
were tested in all models: infection status (two-level
factor: infected/uninfected), trial-type (two-level
factor: cheetah or puma as the wild cat), rat’s sex,
and experimental round. An interaction term between trial-type and infection was included in all
models to test whether the influence of infection
on the response variable varied between cheetah
and puma trials. To assess whether the two
T. gondii strains used may have induced different
effects on rats, all models were repeated including
parasite strain as a three-level factor (uninfected,
strain 1, and strain 2) instead of the binary variable
‘‘infection status.’’ No significant differences were
found between strains in any model; thus, only results from models including the simpler ‘‘infection
status’’ term are reported.
Results
In total, 70 two-choice feline odor trials (35 cheetah
versus domestic cat and 35 puma versus domestic
cat) were performed, including 30 individual rats.
During cheetah trials, all rats entered at least one
of the feline odor zones at some point, although
two uninfected rats never entered the cheetah zone.
In the puma trials, five rats (two uninfected and
three infected) did not enter either arm and remained within the odor-neutral zone, whereas one
122
uninfected rat did not enter the wild cat zone at any
point. In all other trials, rats entered both feline odor
zones at some point.
M. Kaushik et al.
cat zone, with infected rats entering that zone more
frequently than did uninfected rats (Table 1C; predicted entries into the wild cat zone for uninfected
rats was 2.8, compared with 4.5 for infected rats).
Feline attraction
Attraction ratio was most strongly associated with
trial-type, with rats spending much less time in any
feline zone during puma trials than in cheetah trials
(Table 1A). A significant effect of infection status was
also found, with infected rats spending more time in
any feline zone compared with uninfected rats (mean
predicted attraction ratio: infected rats 0.31, 95% CI:
0.24–0.38; uninfected rats 0.18, 95% CI: 0.10–0.28;
back-transformed values from a model including infection status only). Under a null hypothesis of no
discrimination between the three zones, a simple expectation would be an ‘‘attraction ratio’’ of 0.66.
Thus, in this experiment both infected and uninfected rats show avoidance of the feline odor arms,
but infected rats to a significantly lower degree.
Female rats also spent slightly less time in feline
odor zones than male rats (Table 1A). In the minimal model, 29% of variance in the attraction ratio
was explained by the rat’s individual identity, indicating that rats showed within-individual consistency
(or persistent inter-individual differences), in the
proportion of time they spent in the feline odor
zones across trials, over and above the effects of infection status or gender.
Discrimination of feline odor
Among rats that entered a feline zone at least once,
rats infected by T. gondii spent relatively more time
in the wild cat zone compared with the domestic cat
zone, an effect that did not differ significantly according to the type of wild cat odor being considered
(Table 1B and Fig. 2A). Uninfected rats showed no
significant preference for one feline odor over the
other, spending nearly 60% of their total time in
feline odor zones within the domestic cat zone
(mean model-predicted feline discrimination
ratio ¼ 0.578, 95% CI: 0.491–0.664). On the other
hand, rats infected with T. gondii showed a significant preference for wild cat odor compared with domestic cat odor, with 540% of their total time in
feline odor zones being spent in the domestic cat
zone (mean model-predicted feline discrimination
ratio was: 0.386, 95% CI: 0.326–0.445). The discrimination ratio showed less within-individual consistency than the attraction ratio, with individual
identity explaining only 6% of the variance in the
minimal model. Infection with T. gondii also predicted the number of times a rat entered the wild
Speed of movement (velocity)
In models of overall rat movement speed during
trials, a significant interaction between type and infection status was found, whereby rats infected with
T. gondii moved faster than uninfected rats during
trials involving the odor of pumas, but this was not
the case in trials with the odor of cheetahs (Table 1D
and Fig. 2B). However, among trials in which rats
entered both feline odor zones, rats infected with
T. gondii moved more slowly in the wild cat zone
compared with the domestic cat zone (Table 1E).
Thus, while overall speed during the trial was
higher among infected rats, particularly in the
puma trials, they appears to slow down once in the
wildcat zone (Fig. 2C).
Discussion
Wide interest in the evolutionary ecological literature
on parasite–host interactions has focused on parasite-induced manipulation of the host. For trophically-transmitted parasites, an ability to manipulate
the host’s phenotype in a way that increases susceptibility to predation by definitive hosts may be favored by selection (Holmes and Bethel 1972; Moore
et al. 2005). The specificity and intensity of the response of prey to different types of predators remain,
however, little studied (Grostal and Dicke 1999),
even before consideration of the effect parasites
may have on such behavior. Previous findings have
suggested that T. gondii may enhance transmission to
a feline definitive host by causing infected rats (the
intermediate host) to lose their innate avoidance of,
and in some cases even become attracted to cat odor,
while having no impact on a rat’s response to the
odor of non-predators (Berdoy et al. 2000; Webster
et al. 2006; Lamberton et al. 2008) or of mammalian
predator species that are not definitive hosts for
T. gondii (Lamberton et al. 2008). The results of
the current study show that, given a choice of only
wild cat odor versus domestic cat odor, infected rats
displayed a significant ‘‘preference’’ for cheetah and
puma odors, whereas unexposed control rats displayed a lack of discrimination or an equal aversion
both to domestic and wild cat odor. Our results also
augment previous data revealing that rats infected by
T. gondii are more active than are their uninfected
counterparts overall (Webster 1994, 2001, 2007).
However, the automated behavioral tracking assays
123
Toxoplasma gondii’s wild cat versus domestic cat attraction
Table 1 Results from mixed models assessing the impact of Toxoplasma gondii infection on rats’ behavior in a two-choice test of
responses to feline urine odor
Variable
df
Parameter estimate
SE
Likelihood ratio 2
P
(A) Attraction ratioa
Infection (infected)
1
0.146
0.066
4.958
0.0260
*
Trial-type (Puma)
1
0.181
0.042
15.509
0.0001
***
Infection trial-type
1
0.091
0.089
1.102
0.2938
Rat’s sex (male)
1
0.146
0.059
6.053
0.0139
Experiment round (second)
1
0.017
0.051
0.107
0.7431
Infection (infected)
1
0.192
0.054
11.357
0.0008
Trial-type (Puma)
1
0.010
0.047
0.054
0.8162
*
(B) Discrimination ratiob
Infection trial-type
1
0.048
0.101
0.225
0.6356
Rat’s sex (male)
1
0.016
0.051
0.127
0.7215
Experiment round (second)
1
0.004
0.049
0.012
0.9143
***
(C) Number of wildcat zone entriesc
Infection (infected)
1
0.399
0.188
4.221
0.0399
Trial-type (Puma)
1
0.141
0.120
1.410
0.2351
Infection trial-type
1
0.326
0.276
1.427
0.2323
Rat’a sex (male)
1
0.441
0.163
6.796
0.0091
Experiment round (second)
1
0.042
0.145
0.084
0.7724
*
**
(D) Overall speedb
Infection (infected)
1
0.172
0.375
Trial-type (Puma)
1
0.647
0.333
Infection trial-type
1
1.123
0.410
7.201
0.0073
**
Rat’s sex (male)
1
0.887
0.281
9.506
0.0020
**
Experiment round (second)
1
0.125
0.240
0.335
0.5630
Infection (infected)
1
0.842
0.423
4.034
0.0446
Trial-type (Puma)
1
0.352
0.398
0.842
0.3588
(E) Speed difference wildcat versus domesticb
Infection trial-type
1
0.403
0.852
0.245
0.6205
Rat’s sex (male)
1
0.390
0.399
1.007
0.3156
Experiment round (second)
1
0.810
0.398
4.208
0.0402
*
*
Notes: For significant terms (in bold), statistics are reported from the minimal model, while for non-significant terms statistics are reported for
the time a variable was removed from the model. For two-level factors (infection, trial-type, sex and experiment round), parameters show the
difference in that parameter between the factor level indicated in brackets compared to the other level.
a
Results from a linear mixed model, with arcsine square root values of attraction ratio as the response.
b
Results from linear mixed models, using an untransformed response variable.
c
Results from a Poisson mixed model with a log link.
*significant at p50.05 **significant at p50.01 ***significant at p50.001.
used here also revealed that, while infected rats
spent relatively more of their time in wild cat
zones compared with domestic cat zones, they
also moved more slowly when in the wild cat zone.
Thus, rather than ‘‘freezing’’ in a fear response, infected rats appear to maximize their time exploring
the wild cat zones, a behavior that under natural
conditions would likely increase their risk of
predation.
The findings of these tests raise a number of questions regarding the specificity and evolution of
T. gondii’s ‘‘Fatal Feline Attraction.’’ These results
may imply, for instance, that wild cats have greater
capacity to be definitive hosts for T. gondii relative to
domestic cats, and/or that they have a longer/stronger co-evolution with the parasite. Although data are
limited, the shedding of oocysts by T. gondii has
been demonstrated in all three feline species
124
Uninfected
2.0
Infected
Uninfected
0.0
1.0
Puma
Cheetah
●
●
−2.0
●
●
−1.0
●
Speed difference (cm/s)
4.0
5.0
6.0
C
3.0
0.4
0.3
●
Overall speed (cm/s)
●
0.5
0.6
0.7
0.8
B
0.2
Feline discrimination ratio
A
2.0
M. Kaushik et al.
Infected
Uninfected
Infected
Fig. 2 Effects of Toxoplasma gondii on rats’ (A) feline discrimination ratio, (B) overall speed, and (C) speed difference (average speed
when in the wild cat zone—average speed when in the domestic cat zone), during two-choice trials of response to feline odor.
Predicted means and standard errors are plotted from mixed models (see the ‘‘statistical analyses’’ section) that include only an
infection by trial interaction.
examined here (Lukesova and Literak 1998; Elmore
et al. 2010). There is, however, some suggestion that
wild cats may be superior definitive hosts for
T. gondii. Although infections in domestic cats are
typically characterized by initial intensive shedding of
oocysts after exposure, followed by long-lasting (several years) immunity to subsequent homologous or
heterologous T. gondii exposure (Dubey 1995, 2010),
data from wild cats indicate that T. gondii oocysts
may continue to be shed intermittently, and thereby
continue to contaminate the environment and maximize transmission over extended periods (Ruiz and
Frenkel 1980).
Likewise, although domestic cats are often implicated in toxoplasmosis outbreaks, improvements in
parasite genotyping, combined with broader sampling designs, have revealed significant roles of wild
cats in several outbreaks. These include, for instance,
the role of pumas, as well as domestic cats, in the
1995 toxoplasmosis outbreak among humans in
British Columbia, Canada (Aramini et al. 1999).
There are also examples of outbreaks of toxoplasmosis in which very high morbidity and mortality of
animals occurred. In coastal California, over 72%
of southern sea otters (Enhydra lutris nereis) were
found to be infected with a non-archetypical
T. gondii strain, designated Type X T. gondii
(Conrad et al. 2005). Recent testing of potential
sources of transmission in coastal watersheds adjacent to these sea otter habitats found that three wild
felids (two pumas and a bobcat), but no domestic
cats, tested positive for Type X. If Type X strains are
detected more commonly from wild felids in subsequent studies, this could suggest that these wild cats
are more important land-based sources of T. gondii
for marine wildlife than domestic cats (Miller et al.
2008). Even if the relative preferences of infected
intermediate hosts for wild cats over domestic cats
observed here are relatively subtle, mathematical
models describing another predator–prey–parasite
system demonstrate that even a small increase in
susceptibility of an infected prey population to a
specific predatory host would be sufficient to cause
a significant increase in parasite load within the definitive host and therefore in transmission of the
parasite (Vervaeke et al. 2006).
The results presented here also leads to speculation about the evolutionary role of wild cats in the
life-cycle of T. gondii. Large wild cats may have
played a significant role in the historical transmission
of T. gondii by preying on both small and large intermediate hosts, potentially including humans.
Molecular genotyping indicates that the apicomplexan radiation, and the emergence of coccidians,
occurred well before the evolution of mammals, or
perhaps even of chordates (Escalante and Ayala
1995). Toxoplasma gondii nowadays infects any
warm-blooded animal, including about one-third of
humans worldwide (Tenter et al. 2000). Many of the
same subtle behavioral alterations observed in rats
infected by T. gondii are also observed in humans
infected with T. gondii (Flegr 2007). This includes
a recent demonstration of the apparent ‘‘Feline
Attraction’’ in humans testing seropositive for T.
gondii, who found the odor of cat urine more attractive than seronegative individuals did (Flegr et al.
2011). Before humans were hunters, the Felidae
were the most successful, powerful predators across
most of the world. The giant cheetah (Acinonyx pardinensis), for example, at about 120–150 kg, coexisted with early humans and may have hunted
them. At a 1.8-million-year-old site at Dmanisi in
Toxoplasma gondii’s wild cat versus domestic cat attraction
the Republic of Georgia, one of the oldest known
sites for ancient human species outside of Africa,
scientists recently found fossils of a dirk-toothed
cat (Megantereon cultridens) and a similar scimitar
cat (Homotherium crenatidens) (Hemmer et al.
2011). Large wild cats have been, and still are, significant predators of non-human primates, such as
chimpanzees (Tsukahara 1993) and baboons (Condit
and Smith 1994). Indeed 10% (Mills and Bigg 1993)
to 24% (Hoppe-Dominik 1984) of leopards’
(Panthera pardus) diets has shown to consist of predated terrestrial primates. If large primates like chimpanzees are subject to predation by felids today, it is
plausible that predation by large felids could have
been a strong selective force on early hominids
(Isbell 1994). Thus, although T. gondii-associated behavioral alterations observed in latently infected
humans are often viewed as by-products of selection
for behavioral changes in intermediate hosts such as
rats and mice, and hence ‘‘parasite constraint’’ in
secondary hosts rather than ‘‘parasite manipulation’’
per se (Webster 2001; Webster et al. 2013), the results
presented here lead us to consider the hypothesis
that humans too may have served as intermediate
hosts of T. gondii in our evolutionary past.
In terms of potential mechanisms of aversion/attraction to feline odor, several neuromodulatory and
endocrine mechanisms have been suggested to be
involved (Webster and McConkey 2010; Kaushik
et al. 2012; McConkey et al. 2013; Webster et al.
2013). One of the most topical hypotheses is that
T. gondii may directly increase levels both of hostand/or parasite-derived dopamine in the host’s brain,
which subsequently influences behavior (Webster
and McConkey 2010; Prandovszky et al. 2011). It
has also been reported that exposure to feline odor
increases turnover rates of dopamine in the hypothalamus and striatum (Belzung et al. 2001). The
role of dopamine in the reward process was classically associated with the ability to experience pleasure and the emotion of obtaining a reward.
However, recent data suggest a more motivational
role, such as the emotion of craving a reward
(Bechara et al. 1992; Dichiara and North 1992;
Bressan and Crippa 2005).
There is also the possibility that the infection may
be linked to a less-specific effect, such as a reduced
aversion to a more pungent or intense feline odor.
One could postulate that domestic cats may have
been selected to be ‘‘less smelly’’ compared with
their wild counterparts, although domestic tomcats’
urine is proposed to be much smellier than the urine
of either cheetahs or pumas (Apps et al. 2014). It is,
however, plausible that the observed differential
125
responses to different feline species in relation to
the status of infection with T. gondii could be explained, at least in part, by different levels of a particular urinary compound or olfactory cue that could
elicit the rat’s avoidance/attraction response. Many
animals use chemicals as pheromones to communicate between individuals of the same species, for example to influence mate choice or to assert
dominance. Pheromonal communication is an open
broadcast system that can be intercepted by unintended receivers, both predator and prey. Although
data are limited and occasionally conflicting, particularly from free-ranging wild cats, components of
urine do differ between domestic and wild cats (Asa
1993; McLean et al. 2007; Apps et al. 2014). Neither
cheetah nor puma urine contains lipids, although
lipids are present in the urine of domestic cats
(Asa 1993). 3-Mercapto-3-methylbutanol is the intensely odorous component of the urine of male domestic cats. It is produced by hydrolysis of the felidspecific amino acid felinine, whose production is catalyzed by cauxin, a carboxylesterase enzyme that is
unusually abundant in domestic cats’ urine.
Although presence of 3-mercapto-3-methylbutanol
in (intact male) domestic cats is high, this component appears to be only rarely detected in large wild
cats, such as leopards, and not at all in pumas or
cheetahs (Apps et al. 2014). Presence and levels of
felinine, a putative pheromone important in attracting female cats and marking territories, have also
been shown to differ among species of felid. It has
been reported by some to be present only in the
urine of domestic cats but absent in the majority
of large cats, with the potential exception of leopards
and the Indian Leopard cat (Prionailurus bengalensis)
(Hendriks et al. 1995a). Cauxin is also reportedly
either absent or at lower levels in big wild cats relative to domestic cats (McLean et al. 2007). There
are no reports of cauxin being detected in cheetahs’
urine and likewise it is reportedly absent from
pumas’ urine (Miyazaki et al. 2006). Since infected
rats showed a stronger response to wild cat compared with domestic cat urine, neither 3-mercapto3-methylbutanol, felinine, or cauxin appear to be
clear candidates for any apparent ‘‘Fatal Feline
Attraction.’’
Further narrowing of candidate compounds could
come from considering the sex, age, and other attributes of the cat involved, since the components of
feline urine differ according to sex, age, health
status, and whether the cat is intact or castrated.
Felinine is higher in male cats than in female cats,
and also in intact compared with castrated males
(Hendriks et al. 1995a). If felinine is involved in
126
T. gondii-associated behavioral alterations, one might
hypothesize that male cats would evoke a stronger
‘‘Fatal Feline Attraction’’ than female cats. Indeed,
some evidence from field studies suggests males
might play a more important role in the transmission of T. gondii: studies of feral domestic cats
(Afonso et al. 2007), free-ranging pumas (Kikuchi
et al. 2004), and bobcats (Oertley and Walls 1980)
have all found higher prevalence of T. gondii in
males than females. Thus, future experimental studies would certainly benefit from explicitly testing
whether the sex of the donor influences the response
of infected and uninfected rats. Age and health status
of the cat may also have an impact on the active
components within its urine (Miyazaki et al. 2006),
as, for example, geriatric domestic cats at risk of
azotemia display an elevated cauxin-to-creatinine
ratio (Jepson et al. 2010). The dose and strength of
urine odor has been demonstrated to influence species-specific responses of rats to predators (Vyas
et al. 2007). Thus, although volume of the urine
sample and the mass of tissue both were standardized in this study, and a consistent source of pooled
urine was used for each feline species so as to minimize any potential confounding effects due to variation among the donors of urine, it will be of
interest for future studies to investigate how individual variation among predators affect the response of
infected rats to felines. This could also include, for
instance, testing the prediction of an enhanced ‘‘Fatal
Feline Attraction’’ of rats to uninfected cats compared with infected (and/or immune) cats.
To conclude, the current study has revealed that
while uninfected rats show an aversion to both domestic and wildcat treated zones, infected rats show a
significant preference for wild-cat-treated zones over
domestic-cat-treated zones. These results raise further questions about the mechanism of action and
mode of discrimination in ‘‘Fatal Feline Attraction’’
as well as about the role of wild cats in the transmission of T. gondii both today and in our evolutionary past.
Acknowledgments
The authors are very grateful to Kelly Weinersmith
and Zen Faulkes for organizing the symposium. They
are also very grateful to Dr Simon Wolfensohn from
Archway Veterinary Surgery, Wiltshire, and Prof.
Alan Wilson from the RVC, Hertfordshire, for supplying the urine samples of domestic and wild cats,
and to Dr Robert Deacon from the Department of
Experimental Psychology, University of Oxford, for
supplying the two-choice maze, and to Dr Glenn
M. Kaushik et al.
McConkey at the University of Leeds for supplying
the parasite strains. A final thank you goes to Prof.
Janice Moore, Dr Peter Apps, and Prof. Harold
Heatwole for comments on the text. J.P.W. and
M.K.: designed the study; M.K. and J.P.W.: collected
the data; S.K., J.P.W. and M.K.: analyzed the data;
J.P.W., S.K. and M.K.: wrote the paper; and J.P.W.:
presented the data at the symposium.
Funding
Support for participation in this symposium was
provided by the Society for Integrative and
Comparative Biology (Division of Invertebrate
Biology, Division of Animal Behavior, and Division
of Neurobiology), The American Microscopical
Society, and the National Science Foundation [IOS
1338574]
(to
J.P.W.)
and
the
Medical
Research Council and the Stanley Medical Research
Institute.
References
Afonso E, Thulliez P, Pontier D, Gilot-Fromont E. 2007.
Toxoplasmosis in prey species and consequences for prevalence in feral cats: not all prey species are equal.
Parasitology 134(Pt. 14):1963–71.
Apps P, Mmualefe L, Jordan NR, Golabek KA, McNutt JW.
2014. The ‘‘tomcat compound’’ 3-mercapto-3-methylbutanol occurs in the urine of free-ranging leopards but not in
African lions or cheetahs. Biochem Syst Ecol 53:17–9.
Aramini JJ, Stephen C, Dubey JP, Engelstoft C, Schwantje H,
Ribble CS. 1999. Potential contamination of drinking water
with Toxoplasma gondii oocysts. Epidemiol Infect
122:205–315.
Asa CS. 1993. Relative contributions of urine and anal-sac
secretions in scent marks of large felids. Am Zool
33:167–72.
Bechara A, Harrington F, Nader K, Vanderkooy D. 1992.
Neurobiology of motivation—double dissociation of 2 motivational mechanisms mediating opiate reward in drugnaive versus drug-dependent animals. Behav Neurosci
106:798–807.
Belzung C, El Hage W, Moindrot N, Griebel G. 2001.
Behavioral and neurochemical changes following predatory
stress in mice. Neuropharmacology 41:400–8.
Berdoy M, Webster JP, Macdonald DW. 2000. Fatal attraction
in Toxoplasma-infected rats: a case of parasite manipulation
of its mammalian host. Proc R Soc Lond Ser B 267:267.
Blanchard RJ, Blanchard DC, Rodgers J, Weiss SM. 1990. The
characterization and modelling of antipredator defensive
behavior. Neurosci Biobehav Rev 14:463–72.
Bressan RA, Crippa JA. 2005. The role of dopamine in reward
and pleasure behaviour—review of data from preclinical
research. Acta Psychiatr Scand Suppl:14–21.
Condit VK, Smith EO. 1994. Predation on a yellow baboon
(Papio cynocephalus cynocephalus) by a lioness in the Tana
River National Primate Reserve, Kenya. Am J Primatol
33:S7–64.
Toxoplasma gondii’s wild cat versus domestic cat attraction
Conrad PA, Miller MA, Kreuder C, James ER, Mazet J,
Dabritz H, Jessup DA, Gulland F, Grigg ME. 2005.
Transmission of Toxoplasma: clues from the study of sea
otters as sentinels of Toxoplasma gondii flow into the
marine environment. Int J Parasitol 35:1155–68.
Dichiara G, North RA. 1992. Neurobiology of opiate abuse.
Trends Pharmacol Sci 13:185–93.
Dielenberg RA, Hunt GE, McGregor IS. 2001. ‘When a rat
smells a cat:’ the distribution of Fos immunoreactivity in
rat brain following exposure to a predatory odor.
Neuroscience 104:1085–97.
Dielenberg RA, McGregor IS. 2001. Defensive behavior in rats
towards predatory odors: a review. Neurosci Biobehav Rev
25:597–609.
Dubey JP. 1995. Duration of immunity to shedding of
Toxoplasma gondii oocysts by cats. J Parasitol 81:410–5.
Dubey JP. 2010. Toxoplasmosis of animals and humans,
Second Edition. Boca Raton (FL): CRC Press, Taylor and
Francis group.
Elmore SA, Jones JL, Conrad PA, Patton S, Lindsay DS,
Dubey JP. 2010. Toxoplasma gondii: epidemiology,
feline clinical aspects, and prevention. Trends Parasitol
26:190–6.
Escalante AA, Ayala FJ. 1995. Evolutionary origin of
Plasmodium and other Apicomplexa based on rRNA
genes. Proc Natl Acad Sci USA 92:5793–7.
File SE, Zangrossi H Jr, Sanders FL, Mabbutt PS. 1994. Raised
corticosterone in the rat after exposure to the elevated plusmaze. Psychopharmacology (Berl) 113:543–6.
Flegr J. 2007. Effects of Toxoplasma gondii on human behaviour. Schizophr Bull 33:757–60.
Flegr J, Lenochova P, Hodny Z, Vondrova M. 2011. Fatal
attraction phenomenon in humans: cat odour attractiveness
increased for Toxoplasma-infected men while decreased for
infected women. PLoS Negl Trop Dis 5:e1389.
Grigg ME, Bonnefoy S, Hehl AB, Suzuki Y, Boothroyd JC.
2001. Success and virulence in Toxoplasma as the result of
sexual recombination between two distinct ancestries.
Science 294:161–5.
Grostal P, Dicke M. 1999. Direct and indirect cues of predation risk influence behavior and reproduction of prey: a
case for acarine interactions. Behav Ecol 10:422–7.
Hemmer H, Kahlke R-D, Vekua AK. 2011. The cheetah
Acinonyx pardinensis (Croizet et Jobert, 1828) at the hominid site of Dmanisi (Georgia): a potential prime meat supplier in Early Pleistocene ecosystems. Q Sci Rev
30:2703e2714.
Hendriks WH, Moughan PJ, Tarttelin MF, Woolhouse AD.
1995a. Felinine: a urinary amino acid of Felidae. Comp
Biochem Physiol B Biochem Mol Biol 112:581–8.
Hendriks WH, Tarttelin MF, Moughan PJ. 1995b. Twentyfour hour felinine excretion patterns in entire and castrated
cats. Physiol Behav 58:467–9.
Holmes JC, Bethel WM. 1972. Modification of intermediate
behaviour by parasites. In: Canning EU, Wright CA, editors. Behavioural aspects of parasite transmission. New
York: Academic Press. p. 123–49.
Hoppe-Dominik B. 1984. Etude du spectre des proies de la
panthkre, Panthera pardus, dans le Parc National de Tai en
Cote d’Ivoire. Mammalia 48:477–87.
127
House PK, Vyas A, Sapolsky R. 2011. Predator cat odors activate sexual arousal pathways in brains of Toxoplasma
gondii infected rats. PLoS One 6:e23277.
Isbell LA. 1994. Predation on primates: ecological patterns and evolutionary consequences. Evol Anthropol
61:61–71.
Jepson RE, Syme HM, Markwell P, Miyazaki M, Yamashita T,
Elliott J. 2010. Measurement of urinary cauxin in geriatric
cats with variable plasma creatinine concentrations and
proteinuria and evaluation of urine cauxin-to-creatinine
concentration ratio as a predictor of developing azotemia.
Am J Vet Res 71:982–7.
Kannan G, Moldovan K, Xiao J-C, Yolken RH, JonesBrando L, Pletnikov MV. 2010. Parasite strain-dependent
effects of Toxoplasma gondii on mouse behaviour. Folia
Parasitol 57:151–5.
Kaushik M, Lamberton PH, Webster JP. 2012. The role of
parasites and pathogens in influencing generalized anxiety
and predation-related fear in the mammalian central nervous system. Horm Behav 62:191–201.
Kikuchi Y, Chomel BB, Kasten RW, Martenson JS, Swift PK,
O’Brien SJ. 2004. Seroprevalence of Toxoplasma gondii in
American free-ranging or captive pumas (Felis concolor)
and bobcats (Lynx rufus). Vet Parasitol 120:1–9.
Lamberton PH, Donnelly CA, Webster JP. 2008. Specificity of
the Toxoplasma gondii-altered behaviour to definitive versus
non-definitive
host
predation
risk.
Parasitology
135:1143–50.
Lukesova D, Literak I. 1998. Shedding of Toxoplasma gondii
oocysts by Felidae in zoos in the Czech Republic. Vet
Parasitol 74:1–7.
McConkey GA, Gaskell E, Bristow GC, Webster JP. 2013.
Toxoplasma gondii brain infection and behaviour: location,
location, location? J Exp Biol 216:113–9.
McGregor IS, Schrama L, Ambermoon P, Dielenberg RA.
2002. Not all ‘predator odours’ are equal: cat odour but
not 2,4,5 trimethylthiazoline (TMT; fox odour) elicits specific defensive behaviours in rats. Behav Brain Res
129:1–16.
McLean L, Hurst JL, Gaskell CJ, Lewis JC, Beynon RJ. 2007.
Characterization of cauxin in the urine of domestic and big
cats. J Chem Ecol 33:1997–2009.
Mead R. 1990. The design of experiments: statistical principles for practical applications. Cambridge UK:
Cambridge University Press (First paperback edition,
with corrections).
Miller MA, Miller WA, Conrad PA, James ER, Melli AC,
Leutenegger CM, Dabritz HA, Packham AE, Paradies D,
Harris M, et al. 2008. Type X Toxoplasma gondii in a
wild mussel and terrestrial carnivores from coastal
California: new linkages between terrestrial mammals,
runoff and toxoplasmosis of sea otters. Int J Parasitol
38:1319–28.
Mills MGL, Bigg SHC. 1993. Prey apportionment and related
ecological relationships between large carnivores in Kruger
National Park. In: Dunstone N, Gorman ML, editors.
Mammals as predators. Oxford: Clarendon Press.
p. 253–68.
Miyazaki M, Yamashita T, Hosokawa M, Taira H, Suzuki A.
2006. Species-, sex-, and age-dependent urinary excretion
128
of cauxin, a mammalian carboxylesterase. Comp Biochem
Physiol B Biochem Mol Biol 145:270–7.
Moore J, Adamo S, Thomas F. 2005. Manipulation: expansion
of the paradigm. Behav Process 68:283–7.
Oertley KD, Walls KW. 1980. Prevalence of antibodies to
Toxoplasma gondii among bobcats of West Virginia and
Georgia. J Am Vet Med Assoc 177:852–3.
Prandovszky E, Gaskell E, Dubey JP, Webster JP, McConkey GA.
2011. The neurotropic parasite Toxoplasma gondii increases
dopamine metabolism. PLoS One 6:e23866.
Ruiz A, Frenkel JK. 1980. Toxoplasma gondii in Costa Rican
cats. Am J Trop Med Hyg 29:1150–60.
Tenter AM, Heckeroth AR, Weiss LM. 2000. Toxoplasma
gondii: from animals to humans. Int J Parasitol 30:
1217–58.
Tsukahara T. 1993. Lions eat chimpanzees: the first evidence
of predation by lions on wild chimpanzees. Am J Primatol
29:1–11.
Vervaeke M, Davis S, Leirs H, Verhagen R. 2006. Implications
of increased susceptibility to predation for managing the
sylvatic cycle of Echinococcus multilocularis. Parasitology
132:893–901.
Vyas A, Kim SK, Giacomini N, Boothroyd JC, Sapolsky RM.
2007a. Behavioral changes induced by Toxoplasma infection
of rodents are highly specific to aversion of cat odors. Proc
Natl Acad Sci USA 104:6442–7.
Vyas A, Kim SK, Sapolsky RM. 2007b. The effects of
Toxoplasma infection on rodent behavior are dependent
on dose of the stimulus. Neuroscience 148:342–8.
M. Kaushik et al.
Webster JP. 1994. The effect of Toxoplasma gondii and other
parasites on activity levels in wild and hybrid Rattus norvegicus. Parasitology 109:583–9.
Webster JP. 2001. Rats, cats, people and parasites: the impact
of latent toxoplasmosis on behaviour. Microb Infect
3:1037–45.
Webster JP. 2007. The impact of Toxoplasma gondii on animal
behaviour: playing cat and mouse. Schizophr Bull 33:752–6.
Webster JP, Brunton CFA, Macdonald DW. 1994.
Effect of Toxoplasma gondii on neophobic behaviour in wild
brown rats, Rattus norvegicus. Parasitology 109:37–43.
Webster JP, Kaushik M, Bristow GC, McConkey GA. 2013.
Toxoplasma gondii infection, from predation to schizophrenia: can animal behaviour help us understand human behaviour? J Exp Biol 216:99–112.
Webster JP, Lamberton PHL, Donnelly CA, Torrey EF. 2006.
Parasites as causative agents of human affective disorders?
The impact of anti-psychotic and anti-protozoan medication
on Toxoplasma gondii’s ability to alter host behaviour. Proc R
Soc Lond Ser B 273:1023–30.
Webster JP, Macdonald DW. 1995. Parasites of wild brown
rats (Rattus norvegicus) on UK farms. Parasitology 111(Pt
3):247–55.
Webster JP, McConkey GA. 2010. Toxoplasma gondii-altered
host behaviour: clues as to mechanism of action. Folia
Parasitol 57:95–104.
Zangrossi H Jr, File SE. 1992. Behavioral consequences in
animal tests of anxiety and exploration of exposure to cat
odor. Brain Res Bull 29:381–8.