Discussion
Our survey of T. gondii in NPs across Costa Rica documented
widespread infection and species-specific risks of infection. The
seroprevalence obtained for captive NPs is similar to other studies in
South America, such as 30.8% and 76% using MAT in captive Cebus
apella, (Leite et al., 2008; Pires et al., 2012), and 79% inCebus sp. and 57% in Ateles sp. from zoos in São Paulo,
Brazil (Bouer et al., 2010). The high prevalence of NPs in captivity may
be due to management practices, including improperly washed fruits or
vegetables, and raw or undercooked meat (Pires et al., 2012; Valentini
et al., 2004), proximity of wild or domestic cats, and invasion of
enclosures by infected birds and rodents that might be ingested by NPs
(Pires et al., 2012; Valentini et al., 2004). These sources of infection
would be similar for male and female NPs in captivity, consistent with
our findings.
The 11.6% seroprevalence in wild Costa Rican NPs was lower than the
26.6% (n = 60) reported by Garcia et al (2005) using MAT in Brazil, and
the prevalence we found for A. palliata (6.6%) is lower than
documented for Alouatta caraya by Garcia et al (2005) in Paraná
(17%, 3/17) and Molina et al (2014) in São Paulo, Brazil (75%, 15/20).
However, it is very similar to findings by de Thoisy et al (2001), who
found only 4% (2/50) seropositive Alouatta senilicus in French
Guiana, and similar to this study had a higher proportion of positive
females. Our data did not indicate a significant difference between
sexes for any of the species, although this should be studied further
given the sex bias observed in French Guiana and among domestic cats
(Afonso et al., 2007).
In the case of C. imitator , the prevalence is equal to that
reported by Garcia et al. (2005) in Cebus spp. with 30.2%
(13/43). In fact, several studies have identified that carnivorous diet
is a risk factor for T. gondii infection (Cabezón et al., 2011;
Hejlicek et al., 1997). On the other hand, the prevalence found inA. geoffroyi (40%, 2/5) appears to be high, which is surprising
considering its arboreal and herbivorous (frugivorous) behavior,
characteristics that minimize its exposure to the parasite. No
prevalence reports were found for wild Ateles sp . to compare this
finding to. However, sample sizes for both A. geoffroyi andS. oerstedii were very small, so these findings should be
interpreted with caution. The difference in seropositivity observed
between howler (A. palliata ) and capuchin monkeys (C.
imitator ) coincides with Garcia et al. (2005) and corresponds to
behavioral characteristics between species. Howlers are predominantly
folivorous, supplementing their diet with fruits, flowers and seeds, and
obtaining most of the water they need from their food (reviewed by
Wainwright, 2007). However, they can drink from water accumulated in
branches, trunks or bromeliads (Glander, 1978; Gilbert & Stouffer,
1989), or search for it on the ground (Almeida-Silva et al., 2005). The
source of infection for howlers would be water bodies infected with
oocysts. Meanwhile, capuchins are the most omnivorous NPs, feeding on
various sources like fruits, insects and small vertebrates such as
birds, rodents, squirrels, coatis, bats, frogs and lizards (reviewed by
Wainwright 2007 and Catão-Dias et al., 2013). They frequently go down to
the undergrowth and ground while foraging and traveling (Feagle, 1999).
In addition, Cebus spp. drink water directly from puddles
(Fragaszy et al., 2004). These characteristics give capuchins greater
exposure to oocysts in the soil / water or in invertebrates (transport
hosts) and to tissue cysts present in infected vertebrates.
The low antibody titers obtained for the wild NPs (16 and 32) coincide
with those reported by Garcia et al. (2005), who found mostly 16 and 32
for a single individual. While A. palliata had low antibody
titers, two A. geoffroyi and two C. imitator had very high
antibody titers (262,144 - 1,048,576). Molina et al (2014) reported
values of 25 for Callithrix penicillata (a very susceptible
species) and higher titers (up to 1600) for A. caraya , arguing
that differences in prevalence and titers could respond to differences
in host susceptibility, contact rates or post-exposure time. Indeed,Alouatta ’s susceptibility in comparison to Cebus could be
reflected in serological differences, since the probability of
post-infection survival and, therefore, developing an immune response is
naturally lower for howlers. Furthermore, if these individuals die, they
would be excluded from the population and the sample, reducing the
number of individuals captured with high titers. In contrast, titers of
captive NPs were high in our study. Leite et al. (2008) reported
antibody titers of 8000 by MAT for Cebus sp . in captivity, while
Ekanayake et al. (2004) reported antibody titers >256 (up
to 4096) in 3 of 21 positive free-ranging but urban macaques in Sri
Lanka. Management factors along with the increased life expectancy forCebus sp. and Ateles sp. in captivity could explain the
chance of infection and high titers observed in this group.
It is worth highlighting the large number of positive samples from the
Gulf of Nicoya (Figure 1.A.). Tempisque River is one of the most
important basins in the country, draining 10.6% of the territory and
flowing into the Gulf of Nicoya (Gutiérrez et al 1985). Areas close to
bodies of water could represent sources of infection for NP, because
water can be contaminated at any point, transport oocysts long
distances, and favor their survival (Lindsay & Dubey, 2009). Samples
from protected areas with high levels of human contact also showed high
prevalence and high titers in the two wild C. imitator mentioned
before. In certain national parks and private reserves, feeding wild
monkeys is a common practice, and capuchin and squirrel monkeys
sometimes exhibit agonistic behaviors that include taking food directly
from humans and coming down to the ground. Such opportunistic behavior
(Campbell, 2013) may increase exposure to infectious agents (Wolfe et
al., 1998; Ekanayake et al., 2004).
Besides identifying specific areas where there could be elevated risk of
infection, our data indicates that environmental variables such as
forest cover and precipitation could be associated with exposure risk.
Seropositivity in A. palliata was higher when there was a higher
percentage of forest cover and less annual precipitation. Forest cover
can protect oocysts from sunlight, allowing them to remain viable where
protected for 1-1.5 years (Ruiz et al., 1973; Frenkel et al., 1975).
Smith & Frenkel (1995) and Almería et al. (2004) found greater
seroprevalence in hares and other mammals sampled in forested areas
versus more arid grasslands, arguing that shadow and relative humidity
provided by forest cover act on oocyst conservation by decreasing the
evaporation rate and desiccation of oocysts.
Wet seasons tend to increase oocyst survival (Frenkel et al., 1975). In
fact, T. gondii seroprevalence in cats (Afonso et al., 2013) and
humans (Hubálek 2005) has been associated with rainy and warm episodes
(North Atlantic Oscillation), and in wild ruminants (Gamarra et al.,
2008) with humid areas. Contrary to expectations, in this study
precipitation was inversely related with seropositivity in both NP
species. Costa Rica is a tropical country with high relative humidity
and stable temperature overall. It is possible that increased
precipitation in Costa Rica results in greater runoff, transporting
oocysts towards the coasts and away from the animals. In recent past
years, low rainfall due to the El Niño phenomenon has generated severe
droughts and forest fires in some areas of Costa Rica. Among many other
animals, A. palliata were severely affected by water and food
shortages, with high mortality due to dehydration and starvation, as
well as injuries due to troops fighting for food. Because behavior
change driven by droughts can increase exposure to parasites present in
the scarce sources of water, the risk of disease increases especially
for animals weakened by starvation and dehydration.
In the present study, the source of exposure of wild NP could be wild
and not domestic cats, which might explain the low effect of human
population density on seropositivity. Contact with humans has been
associated with high seropositivity in macaques (Ekanayake et al.,
2004), which can become infected by ingesting human food from the ground
in areas frequented by domestic cats (Tenter et al., 2000). Domestic cat
population estimates as well as sampling in areas where humans feed and
interact with wildlife should be included in future studies.
Additionally, Toxoplasma genotypes produce different degrees of
virulence in humans and mice (Xiao & Yolken, 2015), and given the high
diversity recently found in Central and South America (Ajzenberg et al.,
2004; Lehmann et al., 2004; Rajendran et al., 2012; Vitaliano et al.,
2014; Rego et al., 2018; Vethencourt et al., 2019) the observed
differences in seroprevalence between wild and captive NP might be due
to different genotypes. Little is known about the genotypes circulating
in wildlife, and associations between strain type, lesion patterns and
clinical outcome have not been reported in wildlife frequently. Thus,
future studies that focus on genotyping and virulence of T.
gondii isolates in wildlife and domestic animals from wild and
anthropized environments would be of great value.
In the present study, MAT was used as it is simple and fast, doesn´t
require specific modifications, can be used for large sample sizes and
diverse host species, and can be run on serum and plasma (Desmonts &
Remington, 1980; Shaapan et al., 2008; Dubey, 2010). An antibody titer
of 1/25 is often considered as evidence of exposure to T. gondiiin many mammals (Dubey et al., 1995) and 1/5 in birds (Dubey et al.,
2016). However, there is no antibody titer that is considered specific
for primates and our analysis required that we be able to maximize
sensitivity of detection given the high susceptibility and low
seroprevalence in some NP species, thus we reported all antibody titers
of 1/16 or higher (Garcia et al., 2005).
Among the reagents used for MAT is mercaptoethanol which destroys
immunoglobulin M (IgM) and therefore MAT only detects IgG (Seefeldt et
al., 1989). Because IgG are maintained for life (Dubey, 2010), their
detection indicates exposure to T. gondii at some point during
the animal´s life; inability to discriminate recent infections precludes
assessing longitudinal shifts in seropositivity. However, IgG presence
can be used to detect the spatial influences of environmental factors.
Further analyses through longitudinal sampling and detection of IgM are
recommended.
In conclusion, our study documented widespread T. gondiiinfection in NP and species-specific risks of infection for the first
time in Costa Rica. The high seroprevalence and titers found in captive
capuchin and spider monkeys may be due to management practices, the
proximity of cats or intermediate hosts, and the increased life
expectancy in captivity for these species. The low seroprevalence and
titers in wild NP varied between species. The difference in
seropositivity observed between wild howler (A. palliata ) and
capuchin monkeys (C. imitator ) in this study agrees with
behavioral and dietary characteristics, in which capuchins are more
exposed to oocysts while foraging on the ground, and by ingesting
invertebrates (transport hosts) and vertebrates (tissue cysts). However,Alouatta ’s susceptibility compared to Cebus might also
explain the observed serological differences, due to decreased survival.
Our data indicated that specific areas could represent an elevated risk
of infection (i.e. water runoff and human interaction), and
environmental variables such as abundant forest cover and low
precipitation could be associated with higher exposure risk in wild NP.
Surveillance of T. gondii in NP is required to better understand
the infection status, genotypes and drivers involved in wild and captive
NP, including individuals in the process of reintroduction, so that
biosecurity measures are improved, avoiding the release of infected
individuals, and develop novel strategies to protect wild populations.