The parasite is best known for making infected mice lose their fear of cats, which is good for both cats and the parasite, because the cat gets an easy meal and the parasite gets into the cat’s intestinal track, the only place it can sexually reproduce and continue its cycle of infection.
In humans, it can cause spontaneous abortion in pregnant women and kill those with compromised immune systems. It is also thought that it may be implicated in schizophrenia and a range of human behaviors, including suicide.
Now, new research from the University of California, Berkeley, reveals that the effects may persist long after the parasitic infection is cleared from the body. “Even when the parasite is cleared and it’s no longer in the brains of the animals, some kind of permanent long-term behavior change has occurred, even though we don’t know what the actual mechanism is,” researcher Wendy Ingram said.
Ingram became interested in T. gondii after reading about its behavior-altering effects in rodents and possible implications for its common host, the domesticated cat, and even humans. One-third of people around the world have been infected with T. gondii and probably have dormant cysts in their brains, kept in check by the body’s immune system.
With co-researchers Michael Eisen and Ellen Robey, Ingram set out three years ago to discover how T. gondii affects mice’s hard-wired fear of cats. She tested mice by seeing whether they avoided bobcat urine, which is normal behavior, versus rabbit urine, to which mice don’t react. While earlier studies showed that mice lose their fear of bobcat urine for a few weeks after infection, Ingram showed that the three most common strains of T. gondii make mice less fearful of cats for at least four months.
Using a genetically altered strain of T. gondii that is not able to form cysts and thus is unable to cause chronic infections in the brain, Ingram demonstrated that the effect persisted for four months even after the mice completely cleared the microbe from their bodies. She is now looking at how the mouse immune system attacks the parasite to see whether the host’s response to the infection is the culprit.The findings appear to refute – or at least make less likely – models in which the behavior effects are the result of direct physical action of parasites on specific parts of the brain.
The researchers speculate that the parasite could damage the smell center of the brain so that the odor of cat urine can’t be detected. The parasite could also directly alter neurons involved in memory and learning, in effect, performing its own version of brain surgery.
“The idea that this parasite knows more about our brains than we do, and has the ability to exert desired change in complicated rodent behavior, is absolutely fascinating,” Ingram said. “Toxoplasma has done a phenomenal job of figuring out mammalian brains in order to enhance its transmission through a complicated life cycle.”
The parasitic protozoan Toxoplasma gondii infects about one-third of the population of developed countries. The life-long presence of dormant stages of this parasite in the brain and muscular tissues of infected humans is usually considered asymptomatic from the clinical point of view. In the past 20 years, research performed mostly on military personnel, university students, pregnant women and blood donors has shown that this ‘asymptomatic’ disease has a large influence on various aspects of human life. Toxoplasma-infected subjects differ from uninfected controls in the personality profile estimated with two versions of Cattell’s 16PF, Cloninger’s TCI and Big Five questionnaires. Most of these differences increase with the length of time since the onset of infection, suggesting that Toxoplasma influences human personality rather than human personality influencing the probability of infection. Toxoplasmosis increases the reaction time of infected subjects, which can explain the increased probability of traffic accidents in infected subjects reported in three retrospective and one very large prospective case-control study. Latent toxoplasmosis is associated with immunosuppression, which might explain the increased probability of giving birth to a boy in Toxoplasma-infected women and also the extremely high prevalence of toxoplasmosis in mothers of children with Down syndrome. Toxoplasma-infected male students are about 3 cm taller than Toxoplasma-free subjects and their faces are rated by women as more masculine and dominant. These differences may be caused by an increased concentration of testosterone. Toxoplasma also appears to be involved in the initiation of more severe forms of schizophrenia. At least 40 studies confirmed an increased prevalence of toxoplasmosis among schizophrenic patients. Toxoplasma-infected schizophrenic patients differ from Toxoplasma-free schizophrenic patients by brain anatomy and by a higher intensity of the positive symptoms of the disease. Finally, five independent studies performed in blood donors, pregnant women and military personnel showed that RhD blood group positivity, especially in RhD heterozygotes, protects infected subjects against various effects of latent toxoplasmosis, such as the prolongation of reaction times, an increased risk of traffic accidents and excessive pregnancy weight gain. The modern human is not a natural host of Toxoplasma. Therefore, it can only be speculated which of the observed effects of latent toxoplasmosis are the result of the manipulation activity of the Toxoplasma aimed to increase the probability of its transmission from a natural intermediate to the definitive host by predation, and which are just side effects of chronic infection.
Toxoplasma as the model organism for studying the parasite manipulation hypothesis
Toxoplasma gondii is a parasitic protozoan whose effects on human behaviour, personality and other phenotypic traits have been studied most thoroughly, often in the context of the manipulation theory, the theory suggesting that many parasites change the phenotype of their host to increase their chances of transmission to a new host by, for example, predation. There are various reasons why this particular protozoon has become a favoured model for evolutionary parasitologists, biologists and also psychiatrists.
First of all, Toxoplasma is a very common parasite both in developed and developing countries, and some forms of diseases caused by Toxoplasma infection have very serious impacts on human health; taken together, all forms of toxoplasmosis are a serious socio-economic burdens throughout the world (Pappas et al., 2009; Torgerson and Macpherson, 2011). It is also important to note that the study of the influence of toxoplasmosis on the behaviour of laboratory animals has a very long tradition; this includes a series of about 20 studies that started in the laboratory of William M. Hudtchison in the early 1980s, followed by studies by Joanne P. Webster and Manuel Berdoy in the 1990s, which were succeeded by several other teams (for reviews, see Skallová et al., 2006; Webster, 2007; Webster and McConkey, 2010).
Toxoplasma is an excellent model for studying the manipulation hypothesis as it is trophically transmitted from an intermediate to a definitive host by predation. In contrast to behavioural patterns induced by directly or, more commonly, sexually transmitted parasites, the behavioural patterns induced by a trophically transmitted parasite are relatively easy to recognize (Parker et al., 2009). For example, in a sexually transmitted parasite, the parasite’s and the host’s genes have similar interests: they both ‘try’ to program the host to increase the probability of host reproduction. In contrast, the interests of the host and its trophically transmitted parasite radically differ. The intermediate host, e.g. the mouse, needs to survive (and reproduce) for as long as possible while the parasite, e.g. Toxoplasma, ‘wants’ the definitive host (here, a cat) to kill and eat the infected intermediate host. Some toxoplasmosis-associated behavioural changes, e.g. the prolongation of reaction times in the infected hosts (Hrdá et al., 2000), are rather simple and therefore difficult to recognize from the side effects of the parasitic disease. Other changes, however, are relatively complex and specific, e.g. the fatal attraction phenomenon, i.e. the loss of the fear response to cat odour (and not, for example, to dog odour) in infected rodents (Berdoy et al., 2000). The existence of such complex behavioural patterns suggests that the observed toxoplasmosis-associated changes are the products of the parasite’s manipulative activity rather than side effects of the acute toxoplasmosis (Poulin, 1995). This is further supported by the fact that the intensity of some of the observed behavioural changes increases with the length of time since the onset of infection (Flegr et al., 1996; Havlíček et al., 2001). If the observed behavioural patterns were merely the side effects of the acute form of infection, their intensity would decrease over time from the moment of the infection.
The probable role of Toxoplasma gondii in the origin and progress of some important psychiatric diseases, including schizophrenia, is another reason why this protozoan has become the most important model for studying the influence of a parasite on human behaviour. Schizophrenia afflicts about 0.5–1% of the population in all countries worldwide and its health and socioeconomic impacts are extraordinary (Saha et al., 2005). Since the 1950s it has been known that the prevalence of toxoplasmosis in schizophrenic patients is unusually high (Torrey et al., 2007). This systematic research was initiated and developed by Edwin F. Torrey from the Stanley Research Institute and Robert H. Yolken from the Johns Hopkins University, who showed that the connection between schizophrenia and toxoplasmosis is very strong and that the Toxoplasma infection is most probably a very significant (but not exclusive) cause of schizophrenia (Torrey and Yolken, 1995; Torrey and Yolken, 2005). The effect of latent toxoplasmosis on the risk of schizophrenia is stronger than that of any schizophrenia-associated gene variant identified in genome-wide analyses (The International Schizophrenia Consortium, 2009). A prospective study performed on personnel of the American Army revealed that specific anti-Toxoplasma antibodies show up in the serum of subjects before they contract schizophrenia (Niebuhr et al., 2007). It was shown later that toxoplasmosis increases the concentration of dopamine in the brain of infected hosts, including humans (Flegr et al., 2003), and its genome even contains unique genes for enzymes (tyrosine hydroxylases) that play an important role in the synthesis of dopamine (Gaskell et al., 2009). The increased concentration of dopamine in certain regions of the brain is believed to play a key role in the origin and progress of schizophrenia and the inhibition of dopamine receptors is the basis of the function of all modern drugs used in the treatment of schizophrenia (Tandon et al., 2010). Other studies have shown that the symptom profiles of Toxoplasma-infected and Toxoplasma-free schizophrenia patients differ and the positive symptoms of the disease (hallucinations, delusions) are more severe in Toxoplasma-infected patients (Wang et al., 2006). Modern imaging techniques revealed that the morphology of the brain of schizophrenics differs from that of controls by having a lower density of grey matter (GM) in certain parts of the brain (Shenton et al., 2001). A magnetic resonance imaging (MRI) study published in 2011 showed that these differences (reduction of GM volume bilaterally in the caudate, median cingulate, thalamus and occipital cortex, and in the left cerebellar hemisphere) are observed only in Toxoplasma-infected patients while Toxoplasma-free patients (as well as Toxoplasma-infected controls) have the same brain morphology as Toxoplasma-free controls (Horacek et al., 2012). This observation suggests that toxoplasmosis can induce morphological changes in the brain of genetically predisposed subjects, which, possibly together with a toxoplasmosis-associated imbalance in the levels of dopamine and other neurotransmitters, e.g. serotonin (Henriquez et al., 2009) or nitric oxide (NO) (Kaňková et al., 2010a), can result in schizophrenia.
Changes in the personality profile of humans with latent toxoplasmosis
The personality profile of Toxoplasma-infected subjects was studied using three standard psychological questionnaires, i.e. Cattell’s 16PF, Cloninger’s TCI, NEO-PI-R (Big Five), and one special psychological questionnaire, Toxo94, that searched for specific changes expected to occur in subjects infected by the parasite transmitted from prey to predator (Flegr, 2007). Several studies have shown that infected men exhibited lower scores on Cattell’s factor G – superego strength (they have tendency to disregard rules) and higher scores on Cattell’s factor L – protension (they are more suspicious and jealous). In women, the shift in these two factors is opposite to that of men; they mainly show a positive shift in Cattell’s factor A – affectothymia (they are more warm-hearted, outgoing and easy-going than the more reserved, detached and critical Toxoplasma-free women). With a new version of Cattell’s questionnaire (v. 5), the infected men showed increased, rather than decreased, scores on superego strength [(Flegr, 2010b) for an explanation of discordant results between studies, see chapter 4]. Cloninger’s TCI showed that infected subjects, both men and women, have decreased scores on factor NS – novelty seeking, i.e. a lower tendency to search for new stimuli (Flegr et al., 2003; Skallová et al., 2005). Ethopharmacological studies have shown that lower novelty seeking scores are characteristic for individuals with an increased concentration of dopamine in the brain tissue, which is in an agreement with the increased synthesis of dopamine in tissue cysts of Toxoplasma found in the brain of infected hosts and with results of ethopharmacological studies performed with Toxoplasma-infected mice (Hodková et al., 2007; Skallová et al., 2005). Some studies also suggest that infected subjects have higher scores on Cloninger’s ST – self-transcendence (Novotná et al., 2005; Skallová et al., 2005). The NEO-PI-R questionnaire showed more extraversion in infected subjects, both men and women, and less conscientiousness in comparison with Toxoplasma-free subjects (Lindová et al., 2012).
On the basis of predictions of the manipulation theory and introspection of the Toxoplasma-infected author, a special questionnaire called Toxo94 was constructed (Flegr, 2010b). This questionnaire consisting of only 10 questions was distributed to several groups of subjects tested for toxoplasmosis, such as two large groups of university students and a group of women screened for toxoplasmosis during pregnancy (Flegr, 2010b). The results showed that infected men more often reported that diplomacy is not their strong point, that their instinctive (reflex) behaviour under imminent danger is rather slow and passive, that they believe that some people have the power to impose their will on others with hypnosis or through other means and that when they are attacked, physically or otherwise, or when they should fight for something important, they stop fighting at a certain moment because their own subconsciousness betrays them and they loss the will to fight back. The infected women more often report that diplomacy is not their strong point, that their instinctive (reflex) behaviour under imminent danger is rather slow and passive, that they believe that some people have the power to impose their will on others with hypnosis or otherwise and that they have a weak instinct for self-preservation: in situations where somebody else might be afraid, for example being alone in a forest at night or in an empty house, they remain calm.
Influence of latent toxoplasmosis on human behaviour
Toxoplasma-infected subjects have prolonged reaction times, as measured by a test of simple reaction times (Havlíček et al., 2001). The psychomotor performance gets worse with the level of development of the infection (estimated on the basis of a decrease in the concentration of specific anti-Toxoplasma antibodies). The performance of the subjects in the 3 min simple reaction time test suggests that toxoplasmosis impairs long-term concentration ability rather than maximum performance. The largest performance decrease in the test occurred in RhD negative subjects while the performance of RhD-positive heterozygotes was not influenced by the infection (Flegr et al., 2010; Novotná et al., 2008). The impaired psychomotor performance of infected subjects can explain the higher risk of traffic accidents and work accidents observed in four retrospective studies (Alvarado-Esquivel et al., 2012; Flegr et al., 2002; Kocazeybek et al., 2009; Yereli et al., 2006) and one prospective study (Flegr et al., 2009). The risk of traffic accident is again increased in RhD-negative drivers (Flegr et al., 2009). A double-blind observational study showed that Toxoplasma-infected men scored lower in clothes tidiness than uninfected men, whereas infected women scored higher (but not significantly so) than uninfected women (Lindová et al., 2006). Similarly, infected men scored lower and infected women scored higher in sociability. These outcomes match the results of the personality questionnaires. The infected rural male students scored higher in suspiciousness while infected rural female students scored lower in suspiciousness than their non-infected peers (Lindová et al., 2006), which again agrees with the results obtained with Cattell’s 16PF questionnaire. However, the very opposite was true for students of urban origin – infected male students showed lower and infected female students higher suspiciousness than their Toxoplasma-free peers. Using the method of experimental games, it was shown that both infected men and infected women were less altruistic than Toxoplasma-free subjects in the Dictator game while in the Trust game, the infected men were less altruistic and infected women were more altruistic than Toxoplasma-free men or women (Lindová et al., 2010).
Influence of Toxoplasma on the human phenotype
In addition to its influence on personality profile and behaviour, Toxoplasma is known to affect other phenotypic traits in humans. For example, infected male university students (age 19–22 years) have increased concentrations of testosterone (Flegr et al., 2008a; Flegr et al., 2008b) and, from photographs, their faces are rated as more masculine and dominant by females (Hodková et al., 2007). In contrast, infected female students have decreased levels of testosterone – which corresponds to decreased levels of testosterone in infected male and female mice (Kaňková et al., 2011). Infected male students are 3 cm taller than non-infected male students and both male and female students have a lower index finger to ring finger ratio (Flegr et al., 2005b), which is considered as an indication of being exposed to higher concentrations of testosterone during pregnancy (Manning, 2002). The increased concentration of testosterone was also recently reported in Toxoplasma-infected men, women (Shirbazou et al., 2011) and rats (Vyas, 2013). It should be noted, however, that recent studies performed on two independent populations did not find increased levels of testosterone in infected male soldiers and immunology clinic patients (see Table 1). An alternative explanation for the observed increase in the level of testosterone in males (and expected increase in the level of oestrogen) was suggested by James (James, 2010). He proposed that high testosterone and high oestrogen individuals are more susceptible to any infection, including the Toxoplasma infection. This model can explain the increased concentration of testosterone in men; however, it cannot explain the increased concentration of testosterone in laboratory-infected rodents.
The infected students differ from non-infected students in various morphological traits; however, at least some of the observed differences could be caused by differences between the populations of students coming from towns and from villages (where the prevalence of latent toxoplasmosis is much higher than in Prague) (Kodym et al., 2000). Infected pregnant women have an increased probability of giving birth to a boy; the shift in the sex ratio is especially high in women with relatively recent latent infection. The women with high levels of anti-Toxoplasma IgG antibodies (but with low levels of IgM antibodies) gave birth to 250 boys per 100 girls while the women with low levels of anti-Toxoplasma IgG antibodies gave birth to more girls than boys (Kaňková et al., 2007b). The same effects have been confirmed in mice infected with Toxoplasma in the laboratory (Kaňková et al., 2007a). Pregnant women with toxoplasmosis have increased weight gain: in the subpopulation of RhD-negative Toxoplasma-infected women, the weight gain was nearly twice as high in the 16th week of pregnancy as in other pregnant women (Kaňková et al., 2010b). The rate of early fetal development is lower and the length of pregnancy is about 1.5 days longer in Toxoplasma-infected than in non-infected mothers (Flegr et al., 2005a; Kaňková et al., 2010b). The children of Toxoplasma-infected mothers have lower rates of motor development in the first 18 months of life (Kaňková et al., 2012). Most differences in the reproduction-associated traits between infected and non-infected women can be explained as being a result of immunosuppression and the resulting (expected) decrease in the stringency of embryo quality control (Neuhäuser and Krackow, 2007), which has been observed in both humans (Flegr and Stříž, 2011) and mice (Kaňková et al., 2010a) with latent toxoplasmosis. A large proportion of embryos with various developmental defects, as well as a large percentage of more immunogenic male embryos, are aborted in the early weeks of pregnancy. In immunosuppressed Toxoplasma-infected women, a fraction of such embryos are saved. This phenomenon can explain not only the decreased rates of prenatal and postnatal development of children of infected mothers but also the increased sex ratio in their offspring. The lower stringency of embryo quality control can also explain the observation published in the early 1960s of a dramatically higher prevalence of toxoplasmosis in mothers of children with Down syndrome, 84% versus 32% in controls (Hostomská et al., 1957).
An endocrine hypothesis for the increased sex ratio of recently infected women and decreased sex ratio of women infected for a long time – namely, originally (before the infection) higher oestrogen and testosterone levels in Toxoplasma infection-sensitive subjects and a reduced concentration of these hormones as consequence of long-term infection – has also been suggested (James, 2008; James, 2010). The increased sex ratio of recently infected women can also be explained by Catalano’s stress hypothesis, i.e. selective abortion of male embryos of chronically stressed women (Catalano et al., 2012). It should be noted that the immunological and the endocrine or stress hypotheses are compatible as the increased level of steroids is known to impair the function of the immune system.
An analogous effect to the fatal attraction phenomenon (Berdoy et al., 2000; Kannan et al., 2010; Webster and McConkey, 2010) was observed in Toxoplasma-infected humans. Infected men rated the smell of cat urine as relatively more pleasant while infected women rated it as relatively less pleasant compared with non-infected controls (Flegr et al., 2011). Using urine from four other animal species (tiger, dog, horse, brown hyena), a similar but weaker effect was observed for hyena urine. Like the cat, the hyena is a member of the Feliformia suborder; however, it is not known whether any representatives of this superfamily other than cats (family Felinidae) can be definitive hosts of Toxoplasma. The fatal attraction phenomenon was not observed with tiger urine. This is rather surprising because large cats are definitive hosts of Toxoplasma, and monkeys and apes are a regular component of their prey. It may be that the difference in the effects of the smell of cat and tiger urine on human behaviour is due to the fact that the important pheromone felinine is present in the urine of small cats (Felinae subfamily) but absent in the urine of large cats (Pantherinae subfamily) (Hendriks et al., 1995). It is, however, possible that chance strongly influences which of the urine samples is active in the fatal attraction test. In our study (Flegr et al., 2011), samples of five individuals of each species were used in smell-rating experiments. However, the relative attractiveness of particular samples can still depend on the sample concentration and the time elapsed from sample collection. It has been observed that the effect of toxoplasmosis on olfactory preference follows an inverted-U function – the effect on mice is not observed when using either a high or a very low amount of cat urine (Vyas et al., 2007). Therefore, the results of odour studies partly depend on the dilution of the samples tested. In this context, interesting side results were obtained in one of our evolutionary psychology studies run in parallel with the fatal attraction study. We found that the smell of urine of men and of women in the fertile phase of the menstrual cycle was relatively more pleasant for Toxoplasma-infected male raters (Fig. 1). No significant effect of toxoplasmosis was observed with urine of women in infertile phases of the menstrual cycle. It is possible that the smell of strange male urine might signal a potential danger, which is not avoided to the same extent by infected men – as has been suggested in a similar context by the stress-coping hypothesis (Lindová et al., 2010).
Advantages and disadvantages of the Toxoplasma–human model for studying the manipulation hypothesis
The greatest advantage of the Toxoplasma-human model for studying the manipulation hypothesis is the convenience of obtaining empirical data. Practically any clinical, ethological, anthropological or psychological study could be supplemented with testing the experimental subject for the presence of anamnestic anti-Toxoplasma antibodies and with the comparison of the data from Toxoplasma-infected and Toxoplasma-free subjects. Moreover, all pregnant women are being screened for toxoplasmosis in some countries. Here, we could just ask the women tested to provide informed consent for the use of their clinical data or to complete a special, e.g. psychological, questionnaire.
The human is a long-living animal, especially in contrast with laboratory rodents. This is another very important advantage in manipulation hypothesis studies (but see Webster et al., 2013). Acute toxoplasmosis is usually only a mild disease in humans, a short event in a long human life. The life-long latent toxoplasmosis is mostly considered asymptomatic from the clinical point of view. Therefore, there is little risk of mistaking manifestations of Toxoplasma’s manipulative activity for side effects of the parasitic disease suffered. The possible side effects of acute infection can be identified by searching for a positive or negative correlation between the time elapsed from the infection (which can be derived from the patient medical records or estimated from the concentration of anamnestic antibodies) and the intensity of the observed Toxoplasma-associated phenotypic changes. Of course, the existence of such a positive correlation cannot distinguish whether the observed changes are manifestations of the manipulative activity or only symptoms of the chronic disease. In the case of human parasites, we cannot run a predation study, i.e. we cannot tell whether the manipulation activity objectively increases the efficiency of parasite transmission from intermediate to definitive host by comparing the prevalence of the parasite in intermediate hosts captured and eaten by the definitive host with that in a population of the intermediate host living in the same area. Theoretically, it would be possible to compare the intensity of behavioural manifestations of toxoplasmosis in high-prevalence areas (with high rates of superinfections, i.e. new infections in hosts previously infected with Toxoplasma) with the intensity in low-prevalence areas (where the rates of superinfections are low). The virulence of non-manipulative parasites increases in the high-prevalence areas because in the competition between different genetic lines of the parasite in the body of a superinfected host, the winners are the lines with the highest rates of reproduction and therefore usually those with the highest virulence (Ewald, 1994). In contrast, the virulence of manipulative parasites decreases in the high-prevalence areas, because in the competition within the body of an infected host, the winners are the lines of non-manipulators that, instead of wasting resources in the manipulation activity, invest the maximum resources in reproduction (and leave the manipulation to their competitors). Such studies, however, should be performed in a long-term stable area and it is clear that the human is not a suitable model. Even studies conducted in some suitable model animal in a stable environment would not differentiate between the direct and indirect manipulation activity. It is highly probable that some of the observed effects, for example the shift of the sex ratio in infected humans and mice (Kaňková et al., 2007a; Kaňková et al., 2007b), are only side effects of the manipulative activity of Toxoplasma, primarily aimed at suppressing the activity of the immune system of the infected host and therefore increasing the survival of the parasite in the host organism.
For obvious reasons, a laboratory infection experiment using a Toxoplasma–human model to study the manipulation hypothesis is not feasible. This is an important obstacle to the study of the causal relationship between Toxoplasma infection and various toxoplasmosis-associated traits. For example, the lower NEO PI-R conscientiousness in infected subjects (Lindová et al., 2012) could be an effect of the infection or it may be that there is a higher probability of infection in persons with lower conscientiousness who may have a lower tendency to adhere to hygienic standards. In some cases, the causality direction is quite obvious. It is more probable that toxoplasmosis causes impairment of reaction times than the persons with longer reaction times having a higher probability of infection. Sometimes, a longitudinal study can help; however, a large number of subjects, preferably several thousand, would be needed for such a study when the incidence of the parasitosis is relatively low. Before the presence of genes for dopamine-synthesizing enzymes in the genome of Toxoplasma was revealed (Gaskell et al., 2009) and before an increased dopamine synthesis rate was found in Toxoplasma tissue cysts (Prandovszky et al., 2011), it was not possible to decide whether the positive association between toxoplasmosis and schizophrenia was more probably caused by the effect of Toxoplasma on the brain of predisposed individuals or by a higher risk of Toxoplasma infection in schizophrenics. The results of a prospective longitudinal study performed on US army personnel, however, showed that Toxoplasma infection often precedes the first episode of schizophrenia (Niebuhr et al., 2008).
An important hint concerning the causality can be provided by measurement of the correlation between the duration of Toxoplasma infection and the amount of observed phenotypic change. The existence of a positive correlation suggests that the difference observed between the infected and non-infected subjects is probably the effect of latent infection. A negative correlation suggests that the difference is a fading-out effect of past acute infection and the absence of any correlation is likely to indicate that subjects with a particular phenotype have an increased risk of infection. The published results of similar studies suggest that many statistical associations between latent toxoplasmosis and phenotypic traits are caused by the effect of toxoplasmosis on the host phenotype; however, some associations are probably caused by the effect of a particular trait on the risk of infection and certain associations by parallel effects of some third, known or unknown, factor on the host phenotype and on the risk of Toxoplasma infection.
The last but yet very serious disadvantage of the human model is connected with extreme genetic polymorphism in the human population as well as with extreme heterogeneity of the environmental factors that affect individuals in the study population. Such genetic and non-genetic heterogeneity has a strong negative influence on the observed effect size of any factor studied, including latent toxoplasmosis. In statistics, the effect size is often estimated as the proportion of the total variability of a dependent variable (e.g. of a personality factor) that can be explained by the independent variable studied (e.g. toxoplasmosis). While in studies performed on inbred laboratory animals or on F1 hybrids we can often see factors explaining a high percentage of the total variability of a particular behavioural variable, in ecological and evolutionary studies performed on outbreeding organisms, we mostly see factors explaining 2–7% of variability (Moller and Jennions, 2002); therefore, to find significant effects, we may need to use an order of magnitude larger samples than in inbred animal studies.
A large within-sample and between-samples variability of human populations is also the cause of the fact that various studies performed on different populations often provide different, even opposite, results. In laboratory experiments on inbred animals, we study genetically identical animals that have been exposed to very similar environmental factors during their lives. Therefore, they will probably react identically to the same factor, for example to Toxoplasma infection. In humans, the situation is very different for various reasons. For example, toxoplasmosis influences the human body and mind through several independent pathways. Infected men have a higher concentration of testosterone (Flegr et al., 2008a) and, therefore, are likely to be more competitive, but at the same time they have impaired reaction times (Havlíček et al., 2001). Therefore, if in one study a self-administered simple reaction time test is distributed to groups of 20 draftees during regular military testing while in another study the same test is individually administered by an attractive female PhD student to male university students, it can be expected that in the first study, the negative influence of toxoplasmosis on reaction times will prevail while in the second, the higher competitiveness of the infected students will prevail in the final effect (Flegr et al., 2008c). In both studies, we would find a significant effect of toxoplasmosis on performance in the test; however, in the first study the effect would be negative but in the second it would be positive.
Most physiological processes are regulated on various levels, from the molecular to the psychological. If, for example, Toxoplasma causes an increase in the dopamine concentration in certain regions of the brain, the dopamine-synthesizing cells in other areas of the brain may degenerate. Therefore, at a certain stage of infection, we can detect, paradoxically, a decreased, rather than an increased, level of dopamine in the brain of infected individuals. If toxoplasmosis induces a decrease in superego strength, it could increase the tendency of certain individuals to lie while filling out a questionnaire and therefore we could detect seemingly increased rather than decreased superego strength in these subjects in questionnaire studies. When a subject recognizes some personality change that he/she does not like, for example a toxoplasmosis-associated increase of extraversion, he/she may try, consciously or unconsciously, to mask this change while completing the questionnaire and he/she can even overcompensate for the real personality change by moving from extraversion to introversion.
Some biological theories suggest that a large part of the genetic polymorphism is sustained in a natural outbreeding population as a result of epistatic interactions and frequency-dependent selection (Flegr, 2010a; Mayr, 1963; Templeton, 2008). The particular alleles cannot be fixed or eliminated from the population because they increase a trait positively in the context of one genotype and negatively in the context of another, or because they are advantageous when rare and disadvantageous when common. The population cannot get rid of various mutations by selection and remains polymorphic, and its members react to the same stimuli in different, often even opposite, ways. This affects the results of our ethological and psychological studies. Toxoplasmosis quite often influences the same personality factor in different populations (men and women, rural and urban populations, or RhD-positive and RhD-negative subjects); however, the direction of the effect of a factor may vary between populations. When we study the effect of a factor on a heterogeneous population, we often find a significant increase of variance in certain dependent variables, rather than a significant difference between the population means of particular variables (see Poulin, 2013). For example, comparison of Cattell’s 16PF personality profiles of young women screened for toxoplasmosis during pregnancy showed that infected females scored higher in intelligence and lower in guilt proneness than Toxoplasma-free females. At the same time, they differed in the variance of four other personality factors, namely surgency, protension, shrewdness and self-sentiment integration (Flegr and Havlíček, 1999). In technical articles, tests for equality of variance are commonly used only to check preconditions of the statistical tests. Our experience with real data and the present knowledge of the genetic architecture of phenotypic characters, however, suggest that many genetic and environmental factors influence the variance rather than the mean values of particular characters in polymorphic populations. Therefore, the results of the tests for equality of variance should be published as full-bodied results of such studies.
There are several objective reasons why Toxoplasma gondii is now used as the most important model for studying manipulative activity in humans, which are summarized in the first paragraphs of the present article. However, the most important are subjective reasons and also chance. A large number of parasitic organisms probably exist in helminths, protozoa, fungi, bacteria, archea and viruses that may influence the phenotype of their human host even more than the Toxoplasma. These organisms are, however, still waiting for research teams to engage in a systematic study of their influence on the human host.
Odds Are, You’re Probably Infected With This Brain Parasite And Don’t Even Know It
Have you heard of Toxoplasma gondii, or Toxo for short? Chances are you probably haven’t. Chances are you’re probably infected with it.
Don’t worry, it’s not a deadly disease, but it is insidious in its own way. Toxo is a parasite that’s traditionally found in rats, but breeds inside the stomachs of cats. It does this by subtly rewiring rats’ brains to be attracted to cats. The rat then willingly puts itself in harm’s way for a cat to kill and eat it, thus transferring the parasite to the cat.
Traditional thinking was that while humans can contract Toxo, it’s no real threat to us. In those with impaired immune systems, the parasite can sometimes cause complications. However, for healthy adults, infection results in a short flu-like sickness while the body fights off the parasite. It then proceeds to lay dormant inside the brain.
Well at least that used to be what we thought…
Meet Czech scientist Jaroslav Flegr. Most of what we know about Toxo comes from his research.
It sounds strange, but Flegr began studying Toxo after recognizing that some of his behaviors in many ways mimicked that of other species whose brains were being manipulated by parasites (recklessness, disregard for his own safety). After the fall of Communism, Flegr began to dedicate himself to the of study Toxo and its lifecycle. Eventually, he had himself tested for it, and it turns out he was infected.
It’s estimated that around half the human population is infected with Toxo, it’s actually pretty easy to contact.
In the United States and Europe, many people become infected with Toxo by coming into contact with the litter boxes of outdoor cats. Cats pick up the parasite by killing infected rodents when they hunt. But cat lovers aren’t the only ones who have to worry about contracting Toxo. Because the organism lives in the soil before it’s picked up by rats, you can also contract it by eating unwashed vegetables.
So what does Toxo do exactly?
When rats and other small rodents contract Toxo, essentially the parasite rewires parts of the rats’ brain to make them attractive targets to be killed by cats. It makes the rats bolder and more tolerable of risk. It also makes them attracted to the scent of cat urine. Toxo does this by essentially rerouting dopamine (the reward hormone) in the rat’s brain. Then once the cat kills the rat, Toxo jumps hosts and the cycle begins again.
Effects of Toxoplasma gondii Infection on the Brain
Toxoplasma gondii, an intracellular protozoan parasite, can infect humans in 3 different ways: ingestion of tissue cysts, ingestion of oocysts, or congenital infection with tachyzoites. After proliferation of tachyzoites in various organs during the acute stage, the parasite forms cysts preferentially in the brain and establishes a chronic infection, which is a balance between host immunity and the parasite’s evasion of the immune response. A variety of brain cells, including astrocytes and neurons, can be infected. In vitro studies using non-brain cells have demonstrated profound effects of the infection on gene expression of host cells, including molecules that promote the immune response and those involved in signal transduction pathways, suggesting that similar effects could occur in infected brain cells. Interferon-γ is the essential mediator of the immune response to control T. gondii in the brain and to maintain the latency of chronic infection. Infection also induces the production of a variety of cytokines by microglia, astrocytes, and neurons, which promote or suppress inflammatory responses. The strain (genotype) of T. gondii, genetic factors of the host, and probably the route of infection and the stage (tachyzoite, cyst, or oocyst) of the parasite initiating infection all contribute to the establishment of a balance between the host and the parasite and affect the outcome of the infection.Keywords: toxoplasmosis, toxoplasmic encephalitis, cyst, cell-mediated immunity, genotype, major histocompatibility complex
Toxoplasma gondii is an extremely widespread, and thus successful, protozoan with a complex lifecycle involving felines, in which sexual development occurs, as its definitive host. Humans become infected in one of 3 ways: by ingesting T. gondii tissue cysts (containing bradyzoites) present in the undercooked meat (especially lamb and pork) of infected food animals; by ingesting highly infectious oocysts (containing sporozoites) present in water, garden soil, children’s sandboxes, etc, contaminated by infected cat feces; or through congenital transplacental transmission of rapidly replicating tachyzoites from mothers who become infected during pregnancy (eg, by changing the cat litter) and pass the infection to the fetus.
A possible outcome of congenital infections is severe neurological and ophthalmological disease. The outcome of the other 2 modes of infection is usually a chronic, latent infection that persists for life. This latent infection has been assumed, until recently, to be clinically asymptomatic; as indicated in the accompanying articles, this assumption is being reconsidered.
By definition, latent infections involve a complex interplay between parasite and host, producing some degree of harmony. In humans, T. gondii performs a delicate balancing act that involves, on the one hand, modification of its proximal (and perhaps distal) environment in ways to promote its survival and transmission and, on the other hand, avoidance of overt tissue damage (directly from the parasite or indirectly from the immune response) that would lead to the demise of its host. In the vast majority of T. gondii infections, the parasite: host homeostasis is effectively achieved, resulting in a latent, subclinical infection. A variety of parasite and host factors can influence this balance, however, resulting in effects that can range from subtle to profound. In this review, we discuss the parasite and host determinants that influence the outcome of infection and the effects of these determinants on the brain.
Effects on the Brain
Once it enters the body, T. gondii traverses the intestinal or placental epithelium as a free parasite by paracellular transmigration1 and enters circulating cells such as macrophages2,3 or dendritic cells.3,4 It then appears to use such cells as a “Trojan horse” to gain access to privileged sites such as the brain.
In vitro studies using mouse brain cells have demonstrated that tachyzoites invade microglia,5,6 astrocytes,7,8 and neurons,6,9 and the parasite thereafter forms cysts within these cells.6,8 An in vitro study using human neurons and astrocytes showed that T. gondii also forms cysts in these cells.10 Human cell division autoantigen-1 was recently identified as a key host determinant of bradyzoite development within human fibroblasts.11 Electron microscopy studies on brains of chronically infected mice demonstrated that the majority of cysts are in neurons12,13; the cysts were identified within axons, dendrites, or the cell body of the neurons.13 In mice with congenital toxoplasmosis, cysts were also found within neurons in their brains.14 In humans, proliferating tachyzoites have been detected in glial cells in a patient who had developed toxoplasmic encephalitis.15 In another case of toxoplasmic encephalitis, T. gondii bradyzoites were observed in a Purkinje cell in the cerebellum.16 Toxoplasma gondii cysts have also been reported in astrocytes in humans17; in that study, astrocytes were the only cell type that could be identified due to the poor preservation of the samples. Collectively, these studies demonstrate that T. gondii can infect a variety of brain cells, but additional studies are needed to identify the host cells that preferentially harbor cysts within the brain.
The effects of T. gondii on brain cells can be almost immediate, as shown by the work of Blader et al,18 who used tachyzoites of a type II strain to examine host gene expression profiles in infected human fibroblasts. Within the first 2 hours of infection, although <1% of the 22 000 known human genes examined were upregulated by >2-fold, almost half of the affected genes encoded proteins associated with the immune response. Included among the upregulated genes were those encoding chemokines (GRO1, GRO2, LIF, and MCP1) designed to recruit immune cells, cytokines (IL-1β and IL-6) capable of activating immune responses, and transcription factors (REL-B, NF-κBp105, and I-κBα) that can promote expression of additional immune regulators. Thus, it is clear that the host cell mounts a strong response directed at alerting and activating the immune system to react to the infection.
Twenty-four hours postinfection, by which time the parasite has replicated 2–4 times, a variety of host glycolytic and mevalonate metabolic transcripts are upregulated, presumably, in response to the nutritional drain imparted by the infection. Intracellular tachyzoites are also known to manipulate a variety of signal transduction pathways related to apoptosis,19–21 antimicrobial effector mechanisms,22–25 and immune cell maturation.26 The recent finding of delivery of protein phosphatase 2C released from rhoptries of tachyzoites into the host nucleus27 will likely be a key step forward toward understanding the molecular basis of such transcriptional manipulation. Although similar studies on brain cells have not been reported, it seems likely that T. gondii infection may also influence signaling pathways in the brain.
There is only limited information on manipulation of host cells by bradyzoites. Foudts and Boothroyd28 recently reported that many of the same host genes (eg, cytokines and chemokines) are affected by infection with bradyzoites or tachyzoites in human fibroblasts; however, the number of genes and the magnitude of activation were both lower in bradyzoite infection. Future gene expression studies on tachyzoite and bradyzoite infection of brain cells may reveal cell type–specific changes influencing the secretion of not only cytokines and chemokines but also neurotransmitters, receptors, ion channels, and other central components of brain physiology.
Elevated anti-T. gondii IgG antibody levels have been reported in patients with first-onset schizophrenia,29,30 suggesting an involvement of this parasite in the etiology of schizophrenia. Elevated serum levels of IL-1β have also been detected in individuals with acute schizophrenia, but not chronic schizophrenia,31 and there were no differences in IL-1β or IL-6 serum or cerebro-spinal fluid levels in medicated patients compared with a control group.32 Because tachyzoites induce more pronounced inflammatory cytokine responses in host cells than do bradyzoites, as described above, proliferation of tachyzoites in the brain may be related to the onset of schizophrenia. The lack of elevated IL-1β or IL-6 in medicated patients could be due to the antitoxoplasmic activity of some antipsychotic drugs.33,34 Interestingly, anti-T. gondii IgM antibody, a key indicator of acute acquired infection, is not elevated in the sera of patients with first-onset schizophrenia,29,30 implying that the patients are not in the acute stage of a newly acquired infection. Therefore, a reactivation of chronic infection with the parasite (proliferation of tachyzoites caused by cyst rupture) in the brain might be involved in the onset of the disease. In support of this possibility, expression levels of proinflammatory cytokines, including IL-1β and IL-6, are higher in the brains of a mouse strain in which tachyzoite proliferation occurs in this organ during the later stage of infection compared with the brains of another mouse strain that prevents tachyzoite proliferation during chronic infection.35 It is noteworthy that individuals with congenital T. gondii infection often develop ocular toxoplasmosis later in life,36 and the disease is considered to be due to reactivation of infection. The onset of toxoplasmic chorioretinitis is most frequent during the ages of 20–30,36 correlating well with the age of onset of schizophrenia.37 Therefore, congenital infection with T. gondii may be involved in the etiology of schizophrenia.
Determinants of the Outcome of Infection
A variety of parasite and host factors determine the outcome of infection. When these factors are in balance, a chronic latent infection results. When they are out of balance, active disease may ensue. The most important factors appear to be the mode of infection, parasite strain, host cytokine response, and host genes.
Mode of Infection
It is known that bradyzoites, sporozoites, and tachyzoites show pronounced differences in gene expression, cell invasiveness, replication rate, and migratory proficiency. It thus seems likely that the course of infection and clinical manifestations may be strongly influenced by the mode of the initial infection. Because congenital infections with tachyzoites produce a distinct clinical picture, including chorioretinitis and neurologic disturbance, which can be discovered later in life even when the infection is asymptomatic at birth,36 it is also possible that ingesting tissue cysts containing bradyzoites or ingesting oocysts containing sporozoites may produce different clinical outcomes. The outcomes may also be influenced by the timing of the infection, such as before or after birth.
Strains of T. gondii have been classified into 3 major genotypes (types I, II, and III) based on polymorphisms of their genes.38 Mice infected with type II strains develop toxoplasmic encephalitis after immunosuppressive treatment with anti-interferon-γ (IFN-γ) antibody, whereas animals infected with a type III strain do not.39 Type II is the predominant strain isolated from patients with AIDS, from non-AIDS immunocompromised patients with toxoplasmic encephalitis, and from those with congenital infections.40–42 By contrast, isolates from outbreaks of acute toxoplasmosis, which show a tendency to cause severe ocular disease, are frequently type I.43 Thus, the parasite genotype appears to be an important factor influencing the outcome of clinical illness in humans. If congenital infection with T. gondii is involved in the etiology of schizophrenia, as discussed above, this would implicate type II strains in the etiology of schizophrenia. Because type I strains have a general tendency to grow more aggressively than type II and III strains in host cells, including human fibroblasts in vitro, the aggressiveness of type I tachyzoites might also contribute to the development of schizophrenia. Studies using murine models have demonstrated that the strain (genotype) of the parasite affects the immune responses of infected cells and hosts, the IL-12 response by macrophages following infection in vitro,44 the recognition of infected cells by T cells in vitro,45 and the cytokine response of spleen cells and within the brains of infected mice.46,47
Host Cytokine Response
Among the cytokines produced in response to T. gondii infection, IFN-γ is the most important. The proliferation of tachyzoites during the acute stage of infection is suppressed by IFN-γ–dependent, cell-mediated immune responses48–50 and, to a lesser degree, by humoral immunity.51–53 This leads to the development of chronic infection characterized by T. gondii cysts, primarily in the brain. The immune responses are essential for maintaining the latency of chronic infection. Individuals with immunodeficiencies such as AIDS are at risk for reactivation of infection and the development of life-threatening toxoplasmic encephalitis.54–55 Murine models of the disease have demonstrated that IFN-γ is essential for the prevention of reactivation and development of toxoplasmic encephalitis.56–58 Cyst rupture has also been observed in chronically infected immunocompetent mice, although it is extremely rare.59 The incidence of cyst rupture in the brain may be higher in mice congenitally infected with the parasite.60 In these cases, however, the immune response probably limits proliferation of the parasite.
The main source of IFN-γ are T cells, which infiltrate into the brain following infection.61–65 IFN-γ production by this lymphocyte population is essential for preventing the reactivation of infection.64,65 T cells bearing T-cell receptor Vβ8 are the most numerous population that produces IFN-γ in the brains of infected mice that are genetically resistant to development of toxoplasmic encephalitis.66 Furthermore, adoptive transfer of Vβ8+ T cells alone into infected nude mice (which lack T cells) prevents the development of toxoplasmic encephalitis.66,67 Thus, in murine models, the parasite antigens recognized by this T-cell population appears to play a crucial role in the induction of the protective T-cell responses to prevent reactivation of infection. In addition to T cells, other cells also must produce IFN-γ to prevent reactivation of chronic infection.68 Microglia and blood-derived macrophages are the major non–T-cell populations that produce this cytokine in the brain of infected mice.69
Both human70 and murine microglia5 inhibit intracellular replication of tachyzoites in vitro when activated by IFN-γ plus lipopolysaccharide. Nitric oxide (NO) production by inducible NO synthase is important for the inhibitory effect of activated murine microglia.5 In contrast, NO is not involved in the inhibitory effect of human microglia,70 and the mechanisms of their inhibitory effect are not yet known.
Human astrocytes activated by IFN-γ plus IL-1β inhibit tachyzoite replication in vitro through their production of NO.71 In addition, IFN-γ and TNF-α synergistically induce an expression of indoleamine 2,3-dioxygenease (IDO) in human glioblastoma cell lines and naïve astrocytes, and this IDO activity results in strong toxoplasmostatic effects through the depletion of intracellular pools of tryptophan.72 IFN-γ–activated murine astrocytes also prevent the intracellular multiplication of tachyzoites; their inhibitory effect is not mediated by NO or IDO but by IGTP, a IFN-γ–inducible GTPase of the p47 family.73 More recently, Martens et al74 showed that several p47 GTPases are recruited to the parasite-containing vacuole, where they coordinate membrane vesiculation and destruction of the parasite in murine astrocytes.
In addition to IFN-γ, infection with T. gondii induces a variety of other cytokines by microglia,75–78 astrocytes,75,77 and neurons.75,79 These may promote (eg, IL-1 and TNF-α) or suppress (eg, IL-10 and TGF-β) the inflammatory response. These cytokines appear to play an important role in regulating the resistance of hosts against T. gondii infection in the brain. Although T cells are the predominant lymphocyte population in the brains of infected animals, B cells,80 NK cells,63,69 macrophages,69,80,81 and dendritic cells 3,69,82,83 also infiltrate into the brain after infection.
Susceptibility and resistance to chronic T. gondii infection in the brain is under genetic control in both mice and humans. In mice, the Ld gene in the D region of the major histocompatibility complex (H-2) is important for resistance to development of toxoplasmic encephalitis.84,85 Resistance of mice to the disease is associated with the formation of fewer T. gondii cysts in the brain.84–86 In humans, HLA-DQ3 appears to be a genetic marker of susceptibility to development of cerebral toxoplasmosis in AIDS patients87 and congenitally infected infants,88 whereas DQ1 appears to be a genetic marker of resistance.87 Because the Ld gene in mice and the HLA-DQ genes in humans are part of the major histocompatibility complex that regulates the immune responses, the regulation of the immune responses by these genes appears to be important to determine the resistance/susceptibility of the hosts to the development of toxoplasmic encephalitis.
Conclusions and Future Research
The outcome of T. gondii infection is strongly influenced by both parasite and host determinants. Parasite strains can differ greatly in their aggressiveness during infection and their propensity to form cysts for long-term survival. With respect to the parasite’s ability to influence host gene expression, it is likely that some of these effects are universal, whereas others are cell-type specific. Future research should extend such studies to various types of brain cells and examine differences between bradyzoite and tachyzoite effects on host gene expression. For controlling T. gondii infections, the host critically relies on IFN-γ produced by multiple populations of immune cells, which helps infected cells limit growth of the parasite. Genetic studies also suggest that regulation of the immune response by the major histocompatibility complex probably plays an important role in the susceptibility/resistance to disease. Given the strong influence of both parasite and host on the outcome of infection, it remains to be seen whether specific combinations of parasite and host determinants can uniquely affect brain physiology, as well as psychiatric disorders.
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