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ARTICLE
Parasitic infection increases risk-taking in a social,
intermediate host carnivore
Connor J. Meyer
1,2,3
✉
, Kira A. Cassidy
1,3
, Erin E. Stahler
1
, Ellen E. Brandell
1
, Colby B. Anton
1
,
Daniel R. Stahler
1
& Douglas W. Smith
1
Toxoplasma gondii is a protozoan parasite capable of infecting any warm-blooded species and
can increase risk-taking in intermediate hosts. Despite extensive laboratory research on the
effects of T. gondii infection on behaviour, little is understood about the effects of tox-
oplasmosis on wild intermediate host behavior. Yellowstone National Park, Wyoming, USA,
has a diverse carnivore community including gray wolves (Canis lupus) and cougars (Puma
concolor), intermediate and definitive hosts of T. gondii, respectively. Here, we used 26 years
of wolf behavioural, spatial, and serological data to show that wolf territory overlap with areas
of high cougar density was an important predictor of infection. In addition, seropositive
wolves were more likely to make high-risk decisions such as dispersing and becoming a pack
leader, both factors critical to individual fitness and wolf vital rates. Due to the social hier-
archy within a wolf pack, we hypothesize that the behavioural effects of toxoplasmosis may
create a feedback loop that increases spatial overlap and disease transmission between
wolves and cougars. These findings demonstrate that parasites have important implications
for intermediate hosts, beyond acute infections, through behavioural impacts. Particularly in a
social species, these impacts can surge beyond individuals to affect groups, populations, and
even ecosystem processes.
https://doi.org/10.1038/s42003-022-04122-0
OPEN
1
Yellowstone Wolf Project, Yellowstone Center for Resources, P.O. Box 168 Yellowstone National Park, WY 82190, USA.
2
Wildlife Biology Program,
Department of Ecosystem and Conservation Sciences, W. A. Franke College of Forestry and Conservation, University of Montana, Missoula, MT 59812, USA.
3
These authors contributed equally: Connor J. Meyer, Kira A. Cassidy.
✉
email: Connor.meyer@umontana.edu
COMMUNICATIONS BIOLOGY | (2022) 5:1180 | https://doi.org/10.1038/s42003-022-04122-0 | www.nature.com/commsbio 1
1234567890():,;
T
oxoplasma gondii is a ubiquitous multihost protozoan
parasite capable of infecting any warm-blooded species
and requires a felid definitive host to sexually reproduce
1
.
Infection spreads either through the ingestion of oocysts shed in
the environment by a definitive host (e.g., environmentally
mediated transmission via water or vegetation), the ingestion of
infected tissue cysts in definitive or intermediate hosts
1,2
, or,
if the fetus survives infection, vertically through congenital
transmission
2
.
Once an intermediate host is exposed, the infection spreads from
the intestinal lining to form cysts in the brain and muscle tissue and
acute toxoplasmosis occurs
2
. If acute infection occurs during
pregnancy it can lead to birthing complications, spontaneous
abortions, and stillbirths; and in young or immunosuppressed
individuals can cause fatal encephalitis
3,4
. Immunocompetent
individuals generally exhibit no clinical symptoms but will have a
chronic lifetime infection due to the presence of cysts
2
.Experi-
mental studies have shown that chronic infections, even in healthy
individuals, can lead to increased dopamine
5,6
and testosterone
production
7,8
. These hormone changes can cause increased
aggression
9,10
and risk-taking behaviour such as increased hyper-
active movement, failure to avoid olfactory predator cues (i.e.,
seeking out instead of avoiding felid urine), and decreased
neophobia
7,11–13
.
Considering the effects that T. gondii infection can have on
intermediate host reproduction and behaviour, T. gondii’s role in
wild ecosystem processes are understudied. One of the few studies
focused on infection impacts on behavior in a wild mammal,
Gering et al. (2021) found that toxoplasmosis was associated with
increased boldness in hyena (Crocuta crocuta) cubs and that
seropositive hyenas of all ages were more likely to be killed by
African lions (Panthera leo)
14
. That study demonstrated a
mechanistic link between toxoplasmosis and an individual’s fit-
ness through behaviour and decision-making.
Gray wolves (Canis lupus) in Yellowstone National Park (YNP)
have been the subject of extensive research over several decades,
primarily focused on predator-prey dynamics, population
dynamics, genetics, behaviour, and canine pathogens
15
. YNP is a
complex multi-carnivore system, where wolves and a definitive T.
gondii host, cougars (Puma concolor), overlap spatially due to
high landscape heterogeneity and prey movements
16
. Thus,
similar multispecies T. gondii transmission pathways as those
found between spotted hyenas and lions could be present between
wolves and cougars in North American systems, where wolves
that spatially overlap with cougars may have increased T. gondii
transmission risk via direct or indirect contact with cougars. T.
gondii has been documented in the YNP gray wolf
17
and we seek
to understand T. gondii’s role in this social, intermediate
host carnivore using 26 years of gray wolf serological and
observational data.
Our first aim was to determine which demographic and eco-
logical factors affect T. gondii infection in wolves in YNP. We
tested individual demographic factors, including age, sex, social
status at the time of capture, and coat color due to their potential
variation in disease susceptibility. Previous research has found the
risk of T. gondii infection increases with age due to accumulating
risk of exposure with time
17,18
. The other three wolf demographic
factors were included because of their links to certain hormones,
which may influence an animal’s susceptibility to infection
19
. Sex
hormones play a role in infection risk and, once infected, hor-
mone production may be altered
19
; however, other studies found
no link between T. gondii seroprevalence and sex
14,17,18
. Due to
natural variations in hormone levels (testosterone, progesterone,
estrogen, etc.) between the sexes
20
, there may be differing risks
and subsequent behavioral responses to infection. Previous
research has found social status (e.g., pack leaders)
21
and coat
color (gray coat color wolves have higher cortisol levels and
increased behavioral aggression)
22
linked to varying hormone
levels and immune defense
23,24
. To determine if seroprevalence is
affected by the amount of spatial overlap with a T. gondii defi-
nitive host (i.e., cougars), we included an overlap index for each
wolf and areas of high cougar density.
Our second aim was to determine if T. gondii infection influ-
ences wolf behavior. We identified three wolf behaviours asso-
ciated with greater risk-taking: (1) dispersing from a pack, (2)
achieving dominant social status (referred to as becoming a lea-
der), (3) approaching people or vehicles (referred to as habitua-
tion), and two causes of death associated with increased risk:(a)
intraspecific mortality (i.e., death by other wolves through
interpack fights), or (b) anthropogenic mortality (i.e., death by
humans due to decreased proximity to humans or human
structures). As behavior can be influenced by many factors, we
controlled for certain variables in each of the behavior models:
sex can influence behaviors such as dispersal, and age can
influence the probability of a certain behavior occurring
25
.
Northern YNP has very high wolf density, the roads are open
year-round, the elevation is lower and provides winter range for
ungulates and opportunities for wolf hunters just outside the park
boundary. All these factors may affect wolf behavior as the wolves
there may have increased opportunities to disperse, to die, and
may be more susceptible to habituation. Therefore, we controlled
for YNP system (northern or not) as well. In controlling for these
factors that may influence wolf behavior, we aim to isolate the
influence of T. gondii infection on behavior. We tested if ser-
ostatus influenced the odds of a wolf performing these behaviors
or dying of one of these causes. We discuss the findings from both
of our aims, factors influencing T. gondii seroprevalence and
determining if toxoplasmosis affects wolf behavior, with respect
to interspecific disease dynamics and how behavioural changes
can impact gray wolves at multiple scales.
Here we found that T. gondii infection in wolves was predicted
by pack overlap with a definitive host, cougars, and that wolves
seropositive for T. gondii changed their behaviour to take greater
risks—being more likely to disperse and to become pack leaders
than seronegative wolves. Due to a wolf pack’s social structure,
these behaviour changes may cause a feedback loop that leads to
pack-level increases in risk-taking with important implications
for further disease transmission, interspecific competition with
cougars, and wolf survival.
Results
Serology. Of the 62 cougars tested for T. gondii, 51.6% were
seropositive. Seroprevalence in cougars increased from 45%
during the first sampling time (n = 47, 1999 to 2004) to 73%
during the second sampling time (n = 15, 2016 to 2020). This test
confirmed the presence of T. gondii in YNP’s most-abundant
definitive host.
Between 1995 and 2020, an average of 11.8 sera samples were
collected each year (range = 4–22) to test for T. gondii antibodies.
All 50 tests from 1995 through 1999 were negative, then three
wolves tested seropositive in 2000. Thereafter, between one and
eight wolves were seropositive each year. Seventeen equivocal
samples were detected using the ELISA and were then rerun using
the MAT assay, which allowed us to distinguish eleven
seropositive and six seronegative samples. The pooled seropre-
valence was 0.0% from 1995 to 2000, 24.5% from 2000 to 2004,
18.7% from 2005 to 2009, 42.9% from 2010 to 2014, and 36.5%
from 2015 to 2020. Using samples collected from 2000 to 2020,
we ran 273 tests on 256 samples. Prevalence was 27.1% (n = 74)
with 61.9% negative (
n = 169) and 11.0% equivocal (n = 30).
Twenty-five individuals were tested more than once throughout
ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-022-04122-0
2 COMMUNICATIONS BIOLOGY | (2022) 5:1180 | https://doi.org/10.1038/s42003-022-04122-0 | www.nature.com/commsbio
their life due to multiple captures, and therefore have multiple
samples, spaced at least eleven months apart. Eight males were
tested twice, 15 females were tested twice, and two females were
tested three times. Accounting for multiple tests, 229 individuals
were tested: 116 males, 112 females, and one hermaphrodite.
Females (31.25%) had slightly higher seroprevalence than males
(25.00%), but these proportions were not different (z-score =
−1.05, p = 0.15).
Wolf age was recorded both as a continuous variable and
categorical variable with 100 pups, 53 yearlings, 88 adults (aged
2.0–5.9), and 15 old adults (aged 6.0 and older). Seroprevalence
was similar between the three younger categories (pup = 29.00%,
yearling = 28.30%, adult=26.14%) and only increased with old
adults (46.67%). A test comparing pup seroprevalence to all other
age categories was not significant (z-score=0.346, p = 0.36). The
biggest difference was between old adults and all other age
categories pooled (z-score = −1.40, p = 0.08). We also tested for
differences in T. gondii exposure between gray and black coat
colors and found no difference: gray wolves (n = 115) had 25.22%
and black wolves (n = 114) had 31.58% seropositivity (z-score =
−1.07, p = 0.14). Similarly, we tested social status at the time of
sampling and found no difference: subordinates (n = 197) had
30.45% and leaders (n = 59) had 23.73% seropositivity.
Wolves that had at least 42.1% overlap with cougar density
≥1.8/100 km
2
(HCO) had a higher proportion of seropositive
tests than wolves with MCO (5.1–42.0%), which was higher than
wolves with LCO (0 to 5% overlap). Twelve wolves (n = 83,
14.46%) with LCO were positive, 30 wolves (n = 83, 36.14%) with
MCO were positive, and 31 wolves (n = 84, 36.90%) with HCO
were positive. The proportion of seropositive wolves in a pooled
MCO and HCO was greater than wolves with LCO (z-score =
−3.65, p = <0.001). To visualize cougar density and overlap with
different wolf pack territories we pooled seropositive tests in nine
general wolf use areas and plotted them on a map of YNP with
high cougar density highlighted (Fig. 1).
Demography analysis results. The full model testing the prob-
ability of seropositivity, with a w
i
= 0.99, included SEX, AGE IN
YEARS, SOCIAL STATUS, COAT COLOR, and COUGAR
OVERLAP INDEX (Table 1). The NULL model performed
poorly, with a w
i
= 0.01.
The COUGAR OVERLAP index (β = 1.089, 95% CIs:
0.176–2.003) was an important factor in the odds a wolf was
seropositive for T. gondii. An increase from LCO to MCO to
HCO was associated with a higher likelihood of testing positive.
The odds ratio of COUGAR OVERLAP was 2.97 (exp[1.089]),
meaning the odds an MCO wolf was seropositive was nearly three
times higher than an LCO wolf. The odds an HCO wolf was
seropositive was almost 9 times higher odds of being seropositive
than a wolf in LCO. Predicted probabilities for seropositivity
(Fig. 2), based on the full model, showed that seropositivity
increased non-linearly with cougar overlap: wolves living in areas
with LCO had a predicted 4.7% prevalence, whereas wolves living
in MCO had a predicted 12.5% prevalence, and wolves in HCO
had a predicted 28.4% prevalence.
Unexpectedly, AGE did not have an effect on T. gondii
infection (β = 0.296, 95% CIs: −0.158–0.751) and the 95%
confidence intervals overlapped zero. The full model included
SEX, but this variable was nonsignificant and the confidence
interval overlapped zero (β = 0.769, 95% CIs:−0.4984–2.022).
SOCIAL STATUS at the time of testing was non-significant
(β = −0.836, 95% CIs:−1.982–0.311) as was COAT COLOR
(β = 0.516, 95% CIs:−0.726–1.757).
Behaviour analysis results. Wolves classified as dispersers had
nearly double the T. gondii seroprevalence of non-dispersers:
36.26% for dispersers and 18.42% for non-dispersers (z-
score=3.11 p < 0.001). The model (DISP
1
) that included TOXO
performed better (w
i
= 0.92; Table 2) than the model without
TOXO (DISP
2
). All four variables were significant with p values <
0.05 and none of the confidence intervals overlapped zero. Males
were more likely to disperse than females, wolves living in
northern YNP were more likely to disperse than wolves in the
interior of YNP, wolves were more likely to disperse with
increasing time monitored, and seropositive wolves were more
likely to disperse than seronegative wolves (β = 2.459, 95% CIs:
0.298–4.620). The odds ratio for TOXO was 11.69 (exp[2.459]),
meaning the odds a seropositive wolf disperses was 11 times
higher than the odds a seronegative wolf disperses.
Fig. 1 Map of cougar density and T. gondii seroprevalence in wolves in Yellowstone National Park (YNP). a Map of cougar density and T. gondii
seroprevalence in wolves in Yellowstone National Park (YNP). Yellow indicates cougar density <1.8/100 km
2
and purple indicates cougar density ≥1.8/
100 km
2
. Pie charts show the T. gondii seroprevalence (seropositive=black; seronegative=white/transparent) from wolves living in nine general areas
throughout YNP, pooled across years 2000–2020. b A sample year (2015) of wolf pack territory minimum convex polygons in YNP along with each pack’s
cougar overlap index level (LCO, MCO, or HCO) based on percentage of overlap with cougar density ≥1.8/100 km
2
(purple).
COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-022-04122-0 ARTICLE
COMMUNICATIONS BIOLOGY | (2022) 5:1180 | https://doi.org/10.1038/s42003-022-04122-0 | www.nature.com/commsbio 3