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Theses & Dissertations

Boston University Theses & Dissertations

2013

Toxoplasma gondii and behavioral modification in hosts Carey, Robert Francis IV Boston University http://hdl.handle.net/2144/12065 Boston University

BOSTON UNIVERSITY SCHOOL OF MEDICINE

Thesis

TOXOPLASMA GONDII AND BEHAVIORAL MODIFICATION IN HOSTS

by

ROBERT FRANCIS CAREY IV B.S., Duke University, 2006

Submitted in partial fulfillment of the requirements for the degree of Master of Arts 2013

Approved by

First Reader Stephanie M. Oberhaus, Ph.D. Assistant Professor of Microbiology

Second Reader Dr. Carol Sulis, M.D. Associate Professor of Medicine

ACKNOWLEDGEMENTS I’d like to sincerely thank both of my readers, Dr. Stephanie Oberhaus and Dr. Carol Sulis, for their generosity in agreeing to participate, their patience during the process, and their constructive feedback that went into the finished product. I especially want to thank Dr. Oberhaus for helping me to complete this thesis remotely by helping me with submission.

I’d also like to thank my advisor at BU GMS, Dr. Simon Levy, for all of his guidance and encouragement during the past two years.

Finally, I’d like to thank the administrators, especially Dr. Gwynneth Offner, for all their help with completing the MAMS program and enabling me to continue my education at Boston University.

iii

TOXOPLASMA GONDII AND BEHAVIORAL MODIFICATION IN HOSTS

ROBERT FRANCIS CAREY IV Boston University School of Medicine, 2013 Major Professor: Stephanie M. Oberhaus, Ph.D., Assistant Professor of Microbiology ABSTRACT Toxoplasma gondii is a heteroxenous protozoan parasite that is found in nearly every species of mammal and billions of latently infected humans worldwide. The symptoms and morbidities associated with acute, congenital, and AIDS-associated toxoplasmosis are familiar to many, while those associated with latent toxoplasmosis are not nearly as well known. Behavioral manipulation is a common strategy of parasite and parasitoid species, and recent research into T. gondii has revealed that T. gondii infection alters the way rodents respond to the odor of the urine of its feline predators, which are also the definitive hosts of T. gondii. Humans have been found to be potentially affected by T. gondii as well: associations have been identified between latent T. gondii infection and psychiatric diseases (including schizophrenia), personality changes, and traffic accidents. This review investigates the state of current scientific knowledge related to Toxoplasma gondii, analyzes recent developments, and examines the implications on public health. We also provide critical analysis of the published literature and make suggestions for future research. iv

TABLE OF CONTENTS

Title

i

Reader’s Approval Page

ii

Acknowledgements

iii

Abstract

iv

Table of Contents

v

List of Tables

vi

List of Figures

vii

List of Abbreviations

viii

Introduction

1

Objectives

4

Review of Published Data

6

Discussion and Proposals for Future Research

45

Conclusions

56

Figures

57

Tables

63

References

66

Vita

87

v

LIST OF TABLES

Table 1

Title A Selection of Some Known Parasitic Effects on Host

Page 63

Phenotypes 2

Cattell’s 16 Personality Factors

64

3

Cloninger’s Temperament and Character Inventory (TCI)

65

vi

LIST OF FIGURES

Figure 1

Title Electron-Micrograph Composites of a Tachyzoite and

Page 58

Bradyzoite 2

Major Routes of Transmission of T. gondii

59

3

Relative Importance of Meat-Producing and Game

60

Animals in the Transmission of T. gondii to Humans 4

Preference or Avoidance of Feline Odor by Subject and

61

Control Rats 5

Schematic Model of Toxoplasma-Induced Changes to Host Limbic System

vii

62

LIST OF ABBREVIATIONS

AIDS

Acquired Immune Deficiency Syndrome

ELISA

Enzyme-linked immunosorbent assay

HAL

Haloperidol

IgG

Immunoglobulin G

IgM

Immunoglobulin M

NMDA

N-methyl-D-Aspartate

OCD

Obsessive-compulsive disorder

PD

Pyrimethamine + Dapsone treatment

QALY

Quality-Adjusted Life Years

RhD

Rhesus factor D

T. gondii

Toxoplasma gondii

VA

Valproic Acid

WHO

World Health Organization

viii

INTRODUCTION

Toxoplasma gondii is a heteroxerous protozoan parasite that is found commonly in cats, birds, rats, mice, livestock, and humans (Dubey 2009). Transmission of the parasite varies from species to species, but there are three primary mechanisms: consumption of fecal oocysts, consumption of tissue cysts, and vertical transmission from parent to fetus (Figure 1) (Tenter et al., 2000). Cats are the definitive host for T. gondii and are the only known source for fecal transmission, while all other intermediate hosts can serve as a reservoir for infective tissue cysts (Elmore et al., 2010). Post-natal infection in humans with T. gondii is typically asymptomatic or quite mild (Dubey & Jones, 2008), but latent infection in immunocompromised individuals (i.e. those with acquired immune deficiency disease (AIDS) or taking corticosteroids for other diseases) or congenital infection often leads to symptoms with a variety of manifestations (Dubey & Jones, 2008; Montoya & Liesenfeld, 2004). A meta-analysis by Torrey et al. (2007) found that “individuals with schizophrenia have an increased prevalence of antibodies to T. gondii.” Exposure to maternal blood sera with elevated levels of Immunoglobulin G (IgG) antibody to T. gondii is also associated with later development of schizophrenia (Mortenson et al., 2007; Brown et al., 2005). In the past ten years much research has been done on hypothesized behavioral effects of T. gondii on humans and the societal and public health implications of those effects. 1

Flegr and Webster have led recent research efforts to investigate the effect of T. gondii on the behavior of infected hosts. In a 2002 study of traffic accidents in Prague, Flegr found that “subjects with latent toxoplasmosis have significantly increased risk of traffic accidents than the non-infected subjects.” Another study in Turkey also showed a statistically significant increased risk for traffic accidents in drivers with latent toxoplasmosis (Yereli et al., 2006). This followed a study in 2001 that found a decrease in reaction time in humans infected with latent toxoplasmosis (Havlicek et al., 2001). Two earlier studies (Flegr & Hrdy, 1994; Flegr et al., 2000) found a gender-specific personality effect of latent toxoplasmosis, summarized by Flegr (2007) as men being “more likely to disregard rules and…more expedient, suspicious, jealous, and dogmatic” and females being “more warm hearted, outgoing, conscientious, persistent, and moralistic.” The extent of those personality changes seen in women was found to correlate to the duration of latent T. gondii infection (Flegr et al., 2000), suggesting that the parasite caused those changes. These human studies have inherent limitations, most prominently the lack of a true control, and therefore can be used to infer correlation but not causation. Mice and rats, intermediate hosts of T. gondii and frequent prey of its definitive host, also display significant behavioral changes in response to T. gondii infection, including increased activity levels (Webster 1994) and reduced fear of novel stimuli (Hay et al., 1983; Webster et al., 1994). More remarkably, latent infection with T. gondii was shown to dramatically alter rats’ response to 2

the scent of cat urine; rats are usually highly averse to areas treated with cat urine, but toxoplasmosis-affected rats actually prefer those areas to other neutral-scented areas (Berdoy et al., 2000). The manipulation hypothesis, which posits that parasites modify the behavior of their intermediate hosts to increase the likelihood of that parasite returning to its definitive host, offers an explanation for some of these changes in rodents. By subtly altering a rodent’s behavior to increase its chance of being eaten by a cat (known as the fatal attraction phenomenon) and passing the tissue cysts back into the digestive system of the cat, the parasite would increase its reproductive capability. Webster summarized the many ways T. gondii was known to affect non-human animal behavior in a review in 2007 for the Schizophrenia Bulletin (Webster, 2007). More recently, it has been found that humans with latent toxoplasmosis also have different responses to cat urine odor than uninfected individuals, with men finding the odor significantly more pleasant and women finding the odor significantly less pleasant (Flegr et al., 2011). Combined with studies linking T. gondii to schizophrenia and a recent study showing a correlation between the prevalence of T. gondii and the national suicide rates of 20 European countries (Lester 2010), this result suggests the ability of T. gondii to directly alter human behavior in ways that were not previously apparent. More research is needed to determine the extent and nature of these effects, ways to prevent further infections, and what medical and psychiatric interventions are possible and advisable for those with latent toxoplasmosis. 3

Objectives The scientific research related to T. gondii is entering a new phase; having established the plausibility of parasitic manipulation of human behavior, researchers are trying to determine the precise mechanisms of these modifications, the nature and scope of the resulting pathologies and public health hazards, and the proper way to diagnose and treat toxoplasma-associated disorders. Some skepticism of the causality of T. gondii for these observed changes persists, and increased scrutiny of these claims will accompany the increasing publicity and awareness. Thus, the goal of this study is to provide a comprehensive review and critical analysis of the state of current scientific knowledge related to Toxoplasma gondii, analyze recent developments, examine the implications on public health, and suggest directions for future research. Specifically: (1)

Review what is known about the physiology, life cycle, and transmission mechanisms of T. gondii and determine areas that require further study.

(2)

Review the known behavioral effects of T. gondii on its hosts and their mechanisms, with particular attention paid to the effects on humans.

(3)

Summarize and discuss the recent finding that latent toxoplasmosis is associated with significantly increased risk profiles for some 4

psychiatric illnesses and serious automobile-related accidents, and how this is changing the public and scientific attitude towards both psychiatric illness and parasites generally believed to be latent or harmless. (4)

Discuss the public health implications of these findings and determine areas of potential future research.

In the course of this study, the nature of parasite-host interaction and the ways parasites manipulate their host’s behavior will be examined.

5

REVIEW OF PUBLISHED DATA

TOXOPLASMA GONDII

Toxoplasma gondii is a heteroxenous protozoan parasite that is found most commonly in cats, birds, rats, mice, livestock, and humans (Dubey 2009), but is known to infect almost all mammalian species. T. gondii is an intestinal coccidium in the Apicomplexa phylum (other members include the malarial Plasmodium and Babesia), defined as being parasitic of animals, unicellular, and spore-forming (Levine, 1971). Like most apicomplexa, T. gondii has a distinctive organelle called the apicoplast (Figure 1) – a plastid external to the nucleus containing approximately 35kB of DNA encased by four membranes, a revealing feature shared by chloroplasts in plants and algae (Levine 1988; Ralph et al., 2004). T. gondii also has an apical complex, a group of polar structures that are essential for invasion and proliferation (Morrissette & Sibley, 2002). During invasion, the apical complex coordinates the mechanical attachment and penetration (Mondragon & Frixione, 1996), and regulates autocrine and paracrine signaling related to attachment, invasion, and subsequent parasitiphorous vacuole formation (Hu et al, 2006; Sibley et al., 1995). Life Cycle T. gondii has three distinct infectious stages: tachyzoite, bradyzoite, and sporozoite. Asexual reproduction occurs in intermediate hosts in two stages. 6

The first, rapid phase entails quick endodyogeny of tachyzoites in many tissues and cells. Next is the initiation of tissue cysts, containing slowly multiplying bradyzoites (also by endodyogeny). These cysts have a high affinity for skeletal and cardiac muscle and the central nervous system (CNS) (Dubey & Beattie, 1988). They are immediately infectious and periodically revert to tachyzoites and repeat the process. Tachyzoites are usually broken down by the stomach, while bradyzoites are much more resilient, requiring extreme temperatures (less than 12° or greater than 67° Celsius) or salinity/radiation for inactivation (Dubey & Beattie, 1988). In general, when a host consumes and is infected by T. gondii in any phase, the parasite penetrates the intestinal enterocytes, enters the lamina propria, and converts to the tachyzoite phase. Tachyzoites disperse and, via phagocytosis, actively penetrate host cells in almost all organs and tissues, though there is a tropism toward skeletal and cardiac muscle and CNS cells. Subsequently, a parasitiphorous vacuole is generated, derived from both parasite and the host cell components. After rapidly multiplying into many clonal tachyzoites via endodyogeny (production of two cells that consume the parent cell), these tachyzoites then convert into bradyzoites, encased in tissue cysts (Dubey et al., 1998). Interconversion between tachyzoite and bradyzoite phase is an area of intense research. These distinct stages of the life cycle are defined by the expression of a distinct subset of genes; the control of this expression and the 7

triggering stimuli are still not fully elucidated, though cellular stress, fever, and various host immune response factors are thought to contribute (Sullivan et al., 2009). Bohne et al. (1994) and Jerome et al. (1998) have also found that a slowed replication rate is associated with development of tachyzoites into bradyzoites. One major difference between tachyzoites and bradyzoites is the development of a cyst wall. Bradyzoites, from the time they enter a host cell, begin creating the cyst wall. Within the tissue cysts, they also reproduce via endodyogeny, albeit at a much more gradual pace (Dubey et al., 1998). The cyst is advantageous to long-term survival of T. gondii in several ways: it is preserved even within an immunologically capable host cell and helps to defend the parasite against pharmacological or immune attacks (Weiss & Kim, 2000). A cyst can survive ingestion in order to reach the stomach and enter small intestinal tissue (Jacobs et al., 1960). Within the cysts, bradyzoites remain infectious, allowing transmission to a predator as it consumes infected prey. Additionally, the cyst provides a barrier between the bradyzoite and the host internal environment, enabling benign persistence by T. gondii that is not lethal to the host (Sullivan et al. 2009). Bradyzoites, from the time they enter a host cell, begin creating the cyst wall. This wall is preserved even within an immunologically capable host cell, and helps to defend the parasite against pharmacological or immune attacks

8

(Weiss & Kim, 2000). Within the tissue cysts, they also reproduce via endodyogeny, albeit at a much more gradual pace (Dubey et al., 1998). Sexual reproduction, involving the sporozoite phase, only occurs in the epithelia of the felid small intestine. Tachyzoites, bradyzoites, or oocysts are ingested by felids and invade the epithelia. The primary mode of sexual reproduction is via ingestion of tissue cysts containing bradyzoites. Proteolytic enzymes in the stomach and small intestine break down the cyst wall. The newly released bradyzoites enter the small intestinal epithelial cells and begin asexually reproducing. Several generations later, after approximately two days, the sexual cycle begins. Male microgametes penetrate and fertilize mature females by propelling themselves with their flagella, forming zygotes (Dubey et al., 1998). A five-layered oocyst wall is formed (Ferguson et al., 1975), and the oocysts are discharged into the lumen when the small intestinal cells rupture (Dubey et al., 1998). The oocysts will ultimately be expelled with the feces into the environment. It is rare that different strains of T. gondii infect the same feline in the short timeframe that would allow sexual reproduction. It is also possible for T. gondii to undergo “selfing”, a type of sexual reproduction in which a single genotype forms gametes of both genders, which then fuse to yield nearly identical offspring (Wendte et al., 2010). This type of reproduction may explain the predominance of a handful of genotypes in the wild (Wendte et al., 2011).

9

These oocysts, once passed into the environment with the feces, are nearly indestructible; highly resistant to cold, disinfectants, dryness; they can live for up to 18 months outside a host (Frenkel et al., 1975; Dumetre et al., 2008; Wainwright et al., 2007). Transmission Transmission of the parasite varies from species to species, but there are three primary mechanisms: consumption of fecal oocysts, consumption of tissue cysts, or vertical transmission from parent to fetus (Figure 2) (Tenter et al., 2000).

Horizontal transmission can occur via several vectors: ingesting

undercooked meat containing tissue cysts is by far the most common in humans, but oocyst-contaminated food and accidental environmental ingestion of oocysts are also possible (Dubey and Jones, 2008; Kijlstra and Jongert, 2008). Livestock and omnivorous game can become infected by consuming feline fecal matter while grazing in areas with a high density of feral cats. Animals that eat other animals infected with tissue cysts also become carriers. Felines are the definitive host of T. gondii and are the only known source for fecal transmission, while all other intermediate hosts can serve as a reservoir for infective tissue cysts (Elmore et al., 2010). Post-natal infection in humans with T. gondii has long been thought to be typically

asymptomatic

or

quite

mild,

while

latent

infection

in

immunocompromised individuals (i.e. those with AIDS or taking corticosteroids for other diseases) or congenital infection often leads to symptoms with a variety 10

of manifestations (Dubey & Jones, 2008; Montoya & Liesenfeld, 2004).

A

pregnant woman who is newly infected (or who has a reactivated strain of toxoplasmosis as a result of immunosuppression) is at significant risk of transmitting T. gondii transplacentally to her fetus (Montoya & Remington, 2008). Risk of transmission is 10-15% during the first trimester, but is accompanied by the most severe associated resulting defects.

In the third trimester, risk of

transmission is 50-70%, but with more mild associated defects (Stillwaggon et al., 2011; Chatterton 1992; Remington & Desmonts, 1990). Potential symptoms of congenital toxoplasmosis acquisition include spontaneous abortion, stillbirth, hydrocephaly, microcephaly, cerebral calcifications, retinochordoiditis, failure to thrive, CNS issues, and learning disabilities.

Early diagnosis and treatment

dramatically improves outcomes for children with congenital toxoplasmosis (McLeod et al., 2006). It has also been discovered that, in canines, rabbits, and brown rats, T. gondii can be sexually transmitted from males to females (Arantes et al., 2009, Liu et al., 2006, Dass et al., 2011). Further work on sexual transmission of T. gondii in other species is needed. Prevalence Latent toxoplasmosis, long thought of as an asymptomatic and insignificant infection as compared with the highly deleterious congenital or immunosuppression-associated disease, is critically important in large part because of its prevalence. Effects that seem mild or appear in only a small 11

percentage of cases take on much greater public health significance when the cohort is approaching three quarters of the population in some areas. Prevalence of latent toxoplasmosis in humans varies dramatically among countries, geographical regions, and ethnic groups (Tenter et al., 2000). Due to frequent blood testing of military recruits in many countries, as well as prenatal screening for toxoplasmosis and other diseases, there is an abundance of data related to T. gondii prevalence. Studies in various parts of the world have found prevalence ranging from 0 to 100% (Chatterton 1992; Remington & Desmonts, 1990; Dubey & Beattie, 1988). Tenter et al. (2000) have done an exhaustive review of prevalence data worldwide, and estimate that approximately one-third of childbearing adults is infected with latent toxoplasmosis. In the 1990s alone, they found that results from prevalence studies ranged between 37-58% in Central Europe, between 51-72% in Latin America, between 54-77% in West Africa, between 4-39% in Eastern Asia, and between 11-28% in Scandinavia. Three studies in the United States found prevalence ranging from 10.8-22.5% but decreasing from 1988 to 2004 (Jones et al., 2001; Jones et al., 2003; Jones et al., 2007). Most mammals are capable of infection by T. gondii. Due to the potential for horizontal transmission by the consumption of undercooked meat, prevalence among livestock and game has been widely studied. Documented carriers and hosts of T. gondii include domestic and feral cats, pigs, cattle, chickens, sheep, dogs, deer, bears, raccoons, foxes, otters, dolphins, seals, bobcats, cougars, 12

lynx, panthers, new world monkeys, and marsupials. Prevalence tends to be highest among sheep, goats, pigs, free- ranging poultry, and game (Figure 3). T. gondii exhibits significant phenotypic and genotypic variation globally (Su et al., 2012). Until fairly recently, T. gondii was thought to consist of four genotypes: Type I, II, III, and X (Type X is sometimes referred to as Type 12). (Wendte et al.) Type II and III clonal lineages exist in people on all continents and are dominant in North America, Africa, and Europe (Lehman et al., 2006). Type I, limited to North America and Europe, is less common, but more virulent (Howe & Sibley, 1995).

Among wildlife, the recently discovered Type 12

genotype is dominant in North America, with Types II and III also persisting (Dubey et al., 2011); two distinct strains predominate among wildlife in Africa (Mercier et al., 2010). A recent study of 956 T. gondii specimens by Su et al. (2012) that combined restriction fragment length polymorphism, microsatellite marker, and sequence-based marker genotyping techniques found 138 unique genotypes that can be divided into 15 haplogroups and six major clades. This study provides the most precise and comprehensive look at the genetic variation among T. gondii populations, and the old “type” nomenclature will likely be replaced by the clades or haplogroups identified therein. Interestingly, Central and South America has been found to have significantly greater genetic diversity. There is no dominant strain but there are approximately 42 strains that are relatively localized, most of which are not found anywhere else in the world (Rajendran et al., 2011). Lehmann et al., (2006) 13

hypothesize that T. gondii originated in South America and spread to the rest of the world by means of just a few events: either rare transatlantic migration of infected birds or transport of infected rodents in naval trading vessels. Thus, T. gondii would have had a long evolutionary history in South America; the several genotypes that were exported to the rest of the world have had far less time to recombine and, accordingly, show much greater genetic homogeneity. The nearly universal host range of T. gondii makes it one of the most successful infectious agents on the planet.

As discussed earlier, it can

reproduce both asexually and sexually, can be transmitted horizontally and vertically, and has multiple vectors of host-host transmission. Boothroyd (2009) even suggested that in light of recent investigation of new strains and ecological niches, “perhaps it is time to revisit the ‘felines-only’ dogma.” Aside from raising the possibility that certain strains of T. gondii may have evolved the ability to sexually reproduce outside of feline species, Boothroyd eloquently highlights the truly expansive milieu of T. gondii and the role its adaptability has played in its staggering evolutionary success, and identifies four traits of T. gondii that have allowed it to achieve such success. First, the definitive host has a far-reaching habitat, with felines of various species existing in forests, jungles, tundra, mountains, and cities. Second, the large number (>100,000,000) of resilient and infectious oocysts shed by a host into the environment is a way to infect large numbers of omnivorous or herbivorous foraging species. Third, T. gondii has evolved what Boothroyd calls a “magic molecular formula,” just basic and 14

adaptable enough to infect almost any intermediate host while still remaining infectious to a feline that consumes that host. Fourth, and most importantly, is the infectivity of intermediate hosts to other intermediate hosts. As T. gondii evolved, it likely transitioned from a strictly feline parasite to one that could infect other animals in a dead-end manner and finally arrived at its current heteroxenous identity. This capacity to infect multiple hosts via multiple vectors, combined with the abilities to remain quiescent and non-lethal once in the host and reproduce both sexually and asexually has been the formula for its great success as a parasite.

PARASITIC BEHAVIORAL MANIPULATION

Our planet is home to countless organisms linked in an almost infinitely complex web of competition, predation, symbiosis, and coexistence. In such a crowded space with a finite amount of resources, natural selection has produced almost every adaptation imaginable as species (unwittingly) jockey between and amongst themselves in a perpetual struggle to develop even the slightest edge. Species have carved out evolutionary niches and developed various strategies to survive and pass on their genes. Parasitism is one such strategy that entails dependence on a host organism for nutrients, protection, distribution, growth and reproductive environment, or all of the above. It may be the case that there are more parasitic species on our planet than non-parasites (Rohde, 1982), and 15

some estimates of the percentage of parasitic species range as high as 80% (Zimmer, 2000).

Especially when compared to vertebrates, the relative

preponderance of parasites is staggering. Helminthes that prey upon vertebrates (i.e. parasitic worms including tapeworms, flukes, and roundworms) alone may make up more than 300,000 distinct species; there are about 45,000 known species of vertebrates (Dobson et al., 2008). There is no accurate way to estimate the number of parasitic species of protozoa, bacteria, viruses, or fungi, but it is certainly on the order of millions of species (Dobson et al. 2008). The obvious implication is that parasitism is a highly successful evolutionary adaptation.

As Zimmer said in his book Parasite Rex (Zimmer

2000), “parasites have been a dominant force, perhaps the dominant force, in the evolution of life.

Or perhaps I should say in the minority of life that is not

parasitic.” In order to understand the way T. gondii affects its hosts, it’s important to first understand parasitism and the way that the species involved interact. Moore (2013) explained that parasites experience selective pressure to gain access to a host and then influence the host to act in a way that benefits the parasites’ survival and reproduction.

Hosts, on the other hand, experience

selective pressure to avoid being a host to parasites. If they do become a host, they are pressured to minimize the negative consequences of this parasitism on their fitness. Parasites have evolved a myriad of ways of gaining entry into another the body of its host organism.

Host organisms have responded by

evolving a number of defense mechanisms, including specialized features of the 16

immune system. In turn, parasites have evolved ways to avoid detection, disable the host immune system, or use components of the host organism against itself. Certain parasites, including T. gondii, have developed ways of interacting with the central nervous system of their hosts, resulting in changes in behavior, coordination, and movement of the host (Table 1) (Moore, 2002). Many of these changes in host behavior increase the parasite’s reproductive success and result in transmission to another host (Thomas et al., 2005; Adamo, 2013).

This

concept of behavioral manipulation, often sensationalized in popular media as “zombieism” or “brain control”, is rooted in basic biology; cells within the brain and nervous system communicate via relatively simple neurotransmitters, and collectively direct the behavior of the organism. The resulting altered behaviors of these organisms are examples of the concept of an “extended phenotype” pioneered by Richard Dawkins (1982). The behavior of the host organism can be considered a physical extension or manifestation of the parasite’s genome, since that altered behavior is actually controlled and induced by genes of the parasite. Further, that behavior benefits and maximizes the survival of those genes, in the same way that a peacock’s tail or a giraffe’s long neck is encoded by certain genes and functions to increase the odds of passing on those genes. Several comprehensively studied examples of parasitic behavioral manipulation in other species can greatly contribute to the understanding of T. gondii’s effects on its host and the mechanisms of its actions. No two parasite17

host interspecies interactions are identical, but there are a few general categories of strategies whereby the parasite takes control and enhances its own transmission. There is the “zombie” or “enslaver” strategy, in which the parasite takes complete possession of the host’s motor function and induces it carry out specific tasks or engage in patterns of behavior that are completely abnormal and beneficial only to the parasite. The host typically dies shortly thereafter. Perhaps the most dramatic example of this strategy is that of the fungus, Ophiocordyceps unilateralis, a parasite of ants. After infection, an ant proceeds through several tightly choreographed steps: first is a phase of erratic wandering, ensuring that the ant is outside of its colony when it dies; second is a phase of climbing to an elevated location, ensuring the dispersal of the spores, which are too large to be dispersed by the wind; finally the ant uses its large mandibles to clamp tightly to the leaf or bark, allowing it to stay in place while the necessary reproductive organ of the fungus sprouts (Andersen et al, 2009). The ant then convulses and dies, the biting action persists, and a large stalk, which eventually forcefully releases the spores of the fungus’s next generation, grows out of the back of the ant’s head (Pontoppidan et al., 2009).

Each of these altered behaviors is

essential for the completion of the fungal life cycle, but provide no benefit to the ant (Hughes et al., 2011). The precision and extent of control exhibited by this fungus is remarkable. In a similar form of manipulation, bees infected by conopid fly larva are induced to dig into the soil before they die, resulting in larger, heavier 18

flies (Muller 1993). Parasitic wasps, of which there are an estimated 5,00010,000 species (Smith et al., 2008), are masters of the enslaver strategy. One type of parasitic wasp can induce its host spider to weave a special cocoon-like structure to protect the wasp pupae against heavy rain (Eberhard, 2000), while another paralyzes a cockroach by injecting venom (containing dopamine) into it then bringing it back to its nest, where it remains paralyzed for eight days before the wasp offspring consume it alive (Gal et al., 2005). Other parasites are known to manipulate host habitat choice in order to complete their complex life cycle. Thomas et al. (2002) witnessed nine species of crickets commit suicide by jumping into a river or creek, induced by Paragordius tricuspidatus, an aquatic hairworm that requires an arthropod host for juvenile development. Another species of hairworm, Spinochordodes tellinii, was found to exert a similar influence on grasshoppers.

Biron et al. (2005)

concluded that hairworms can manipulate the central nervous system (CNS) functions of a grasshopper through molecular mimicry: S. tellinii can produce molecules from the Wnt family that directly influence CNS development, and grasshoppers were found to have differential protein expression in pathways linked to neurotransmitter secretion and release. Mimicry of neurotransmitters or other integral proteins is an effective mechanism of manipulation that appears in many parasites; the role of dopamine in T. gondii’s manipulation of rodents and humans will be discussed later in this review. 19

While these examples demonstrate that parasites, in order to proceed to another phase in their life cycle, are capable of steering their hosts into environments conducive to that transformation, there are also parasites that induce behavioral changes that lead to predation of their host. The parasites are also consumed and are transmitted to their definitive hosts as a result. Infection of Gammarus pulex, a crustacean, with the acanthocephalan Pomphorhynchus minutus causes G. pulex to swim closer to the surface, where they become easier prey for waterbirds, the definitive host of P. minutus (Tain et al., 2006). Another species, P. laevis results in a loss of typical light avoidance by G. pulex (Tain et al., 2006). This results in increased predation by fish, the definitive host of P. laevis. These parasites were later shown to bring about this effect by altering the production of serotonin, another common essential neurotransmitter (Tain et al., 2007). The trematode, Euhaplorchis califorensis, is also known to alter its host’s behavior by manipulating the dopamine and serotonin pathways. E. califorensis has a three-stage life cycle involving horn snails, killifish, and shorebirds. After the trematode leaves the snail, it is eaten by the killifish and forms cysts in its brain. Killifish, an intermediate host, then displays conspicuous swimming behaviors and is more frequently caught by its avian predators and the final host in the trematode life cycle (Shaw et al., 2009). One final example of parasite manipulation is particularly relevant to this review of T. gondii. A baculovirus that infects the gypsy moth caterpillar has been found to carry DNA that encodes for two proteins – EGT, ecdysteroid UDP20

glucosyl transferase, which influences climbing behavior, and PTP, protein tyrosine phosphatase, which has been repurposed as a viral structural protein – that are nearly identical to proteins found in the gypsy moth. This virus, upon entering the caterpillar’s brain, produces these proteins and releases them to dramatically alter brain function and cause the caterpillar to climb high up in trees. After the virus has replicated, the caterpillar then liquefies as a result of the combination of viral-encoded chitinase and cathepsin proteins (Hawtin et al., 1997), and the virus is dispersed over a wide area, increasing its chance to infect another host (Katsuma et al., 2012). T. gondii also possesses in its genome a copy of an important host protein, tyrosine hydroxylase. Just like the baculovirus, it uses this “pirated” gene to synthesize proteins (normally produced and recognized by its host to effect physiologic behavior) that may result in behavioral manipulations.

T. GONDII MANIPULATION OF RODENT BEHAVIOR

Behavioral manipulation is a widespread phenomenon among parasites and an effective strategy that exists in potentially tens of thousands of parasitic species (Dobson et al., 2008). This manipulation is typically highly specific and can often manifest in behavior that is directly harmful to the host species. T. gondii causes a similarly significant and harmful behavioral change in its hosts, which are mammals. 21

Although there are many parasites that effect changes on mammalian hosts, including humans, these effects are not direct behavior alteration. For example, the malarial parasite Plasmodium falciparum, also a member of the apicomplexa phylum, causes the blood of infected humans to become more attractive to mosquitoes, increasing the transmission of this parasite to new hosts (Lacroix et al., 2005). While this is certainly manipulation of a trait of the host, it cannot be described as behavioral. Likewise, there are many viral, bacterial, and protozoan species, e.g. norovirus, that infect mammalian hosts and result in vomiting, diarrhea, sneezing, and other actions that almost certainly increase transmission because of the propulsive and difficult-to-repress ejection of bodily fluids that contain many copies of the infectious agent (Johnston et al., 2007; Wikswo & Hall, 2012). But attributing the cause of these transmission-assisting symptoms to behavioral modification is difficult. It is more likely that this is an example of a pathogen evolving to take advantage of the physiological response of mammalian hosts to vomit or produce diarrhea when infected with these organisms rather than pure behavioral manipulation (Nesse & Williams, 1996). The behavior is a physioglogical response of the host from which the parasite has evolved to benefit. The definitive hosts of T. gondii are felines. Similar to other parasites, T. gondii has evolved to manipulate the behavior of the prey species of this definitive host: rodents (Hutchinson et al., 1980). Upon exposure to cat odor or feline urine, uninfected rodents display stereotypical behavior: they hide, they 22

avoid unfamiliar and open areas, and they display a strong aversion to areas where the odor is present (Dielenberg et al., 2001).

Several different brain

regions influence these innate responses, but the strongest associations have been found with a circuit made up of the medial hypothalamus (Canteras et al., 1997) and inputs from the ventral hippocampus and the basolateral and medial amygdala (Dielenerg et al., 2001; Blanchard et al., 2005).

Rats showed a

statistically significant increase in expression of Fos, a protein known to be associated with fear in rodents (Milanovic et al., 1998), in those regions (Dielenberg et al., 2001). In addition, experimentally produced lesions in those regions significantly reduced the production of Fos and the fear response (Canteras et al., 1997; Blanchard et al., 2005). Rodents that are infected with T. gondii have been shown to have increased activity levels as compared with uninfected rodents, or rodents infected with a exclusively murine parasite, Syphacia muris (Webster 1994). T. gondii-infected rodents have also been found to have reduced aversion to novel stimuli.

Barnett et al. (1976) tracked the response of wild and

laboratory-bred rodents in a plus-maze to novel stimuli. They concluded that wild rats, which display great caution or complete avoidance in response to these new spaces or objects (in contrast to the curiosity and exploratory responses demonstrated by domesticated rodents), have highly neophobic innate tendencies. Berdoy et al. (1995), in a study of 36 wild and hybrid (half wild, half laboratory-bred) rats in a naturalistic outdoor enclosure (100 m2), found that 23

those with T. gondii infection displayed reduced neophobia, measured by exploration of novel stimuli in the enclosure, as compared to uninfected rats. The most striking and specific behavioral alteration in infected rodents is what is known as the “fatal feline attraction” phenomenon: the loss of the aversion to cat urine odor.

Berdoy et al. (2000) observed the nocturnal

exploratory behavior of 23 infected and 32 control rats in a square enclosure. Each corner was treated with an odor: the rat’s own scent, a neutral scent (water), rabbit urine, or cat urine. Infected rats displayed statistically significant reduced aversion to the area with cat urine, but no statistically significant difference in response to the other three areas or overall activity, indicating that the effect of T. gondii infection is not a gross impairment of smell or a dramatic change in movement level but a precise alteration of the response to feline urine odor. Figure 4 is a visualization of the difference in exploratory behavior among the most active rats in each experimental group (seven infected, seven control). Lamberton et al. (2008) performed a similar experiment, using a Y-shaped maze design and replacing rabbit urine with mink urine (a rodent predator but not a definitive host of T. gondii). They also found that rats and mice, upon infection with T. gondii, do not display avoidance of (and actually develop a mild attraction to) feline urine odor compared with their own odor or a predator that is not a definitive host of T. gondii. As mentioned above, this avoidance is typically mediated by specific limbic brain regions (e.g. the medial hypothalamus along with hippocampal and 24

amygdalar inputs). A parallel pathway has been described by Choi et al. (2005) as mediating sexual reproductive behaviors. These pathways are distinct, but have significant anatomical overlap, both following a path through the medial amygdala and hypothalamus. T. gondii seems to disrupt the functioning of the avoidance pathway while stimulating the parallel sexual pathway. House et al. (2011) placed 36 rats, 18 infected and 18 uninfected, in a cage with either feline odor or an inaccessible estrous female rat. They then sacrificed the rats, and measured the levels of c-Fos, a protein rapidly produced in response to certain cellular stimuli, as a proxy for neural activation in the two pathways. They found that infected rats exposed to urine had significant disturbance of the defensive avoidance pathway and an increase in the activity in the reproductive pathway. The level of activity in the sexual pathway of infected rats exposed to urine matched the level of activity in the same pathway in uninfected rats exposed to an estrous female. They concluded that an attraction to the cat urine, mediated by the sexual limbic pathway, competes with and mitigates the nearby parallel defensive pathway (Figure 5). It is important to note that these effects are not part of a generalized pathology in response to T. gondii infection. In their comparison, Vyas et al. (2007) saw no change in growth rates, body weight, food consumption, nonaversive learning, or olfaction between infected and infected rats. No negative impact on mating success or social status has been discovered, behaviors that

25

are mediated by the limbic pathways and are known to be energetically costly and indicative of general fitness (Berdoy et al., 1995). Generally, the infection with a parasite can be detected by female rodents in the urine of the males and is used to discriminate against these infected males in mate selection.

Willis & Poulin (2000) showed that female rats can

differentiate between the urine of a parasitized rat and an uninfected rat by measuring the amount of time the female investigated the respective urine samples of rats with or without the tapeworm Hymenolepis diminuta. Kavaliers et al. (1998) demonstrated that female mice have a preference for male mice that are uninfected with two common parasites over infected mice. They exposed the females to urine samples of males infected with a nematode parasite, Heligmosomoides polygyrus, or a protozoan parasite, Eimeria vermiformis, and uninfected mice and measured their subsequent analgesic response.

When

exposed to urine of an infected mouse, females showed increased analgesic response, a defense reaction that is thought to prepare a female mouse for an encounter with an infected male and results in reduced interest in or avoidance of that male. In addition to using urine as a cue for parasitism status, female rats can also detect relative testosterone levels in male urine and show a strong preference for males with higher levels, as measured by female urine marking responses to the urine of castrated or intact males (Taylor et al., 1982). In contrast to other parasites, there is evidence that T. gondii infection enhances the sexual attractiveness of males to uninfected females (Dass et al., 26

2011). In a study of 72 estrous female rats’ behavior in response to urine of T. gondii-infected and uninfected males, female rats showed a statistically significant preference for areas marked with urine from infected males. Females also allowed greater mating opportunities to infected males when placed in a space with one infected and one uninfected male. It is unsurprising, then, that Lim et al. (2012) found that T. gondii infection increased expression of genes mediating testosterone synthesis and resulted in greater testicular testosterone production in male rats. Collectively, these studies indicate that T. gondii is able to manipulate the response of rodents to their feline predators, yet has no effect on overall health, and even improves reproductive success. Mechanisms How does a simple protozoan parasite effect these very specific and complex behavioral and physiologic changes? T. gondii cysts tend to localize to the CNS and muscle tissue – this ability to cross the mammalian blood-brain barrier could allow T. gondii to interfere with neurotransmitter signaling. Stibbs first detected differences in brain chemistry between acutely infected and uninfected mice in 1985; he also noticed elevated dopamine levels in chronically infected mice. Since then, a number of studies have set out to discover the mechanism by which T. gondii influences behavior. Adamec et al. (1999) discovered that blockade by dizocilpine, a noncompetitive antagonist of N-methyl-D-aspartate (NMDA) receptors, which 27

typically mediate the fear response to predators, in the amygdala of rodents led to similar behavior as those infected with T. gondii.

Rats given dizocilpine

approached felines at a greater rate than controls, and did not display the induction of anxiety-like behavior, lasting several weeks, that the researchers found was typical of an untreated rat after exposure to a feline. One prediction, based on these findings, is that high concentrations of T. gondii cysts would localize in areas known to be specifically involved in the response to detection of predator urine in infected animals: the medial hypothalamus (Canteras et al., 1997) and inputs from the ventral hippocampus and the basolateral and medial amygdala (Dielenerg et al., 2001; Blanchard et al., 2005). Several studies have tested this hypothesis by infecting rodents, sacrificing them at various intervals, and investigating the distribution of cysts in brain and spinal cord sections with detection by immunocytochemistry and/or bioluminescence; however, only one found more cysts in the amygdala than in other parts of the CNS (Vyas et al., 2007). Other studies reported highly variable results (Ferguson et al., 1991; Dellcasa-Lindberg et al., 2007; Kittas et al., 1984; Gonzalez et al., 2007). Afonso et al. (2012), using similar detection methods as the previous studies, found that although the distribution of cysts was not random as compared to a Lorentz curve, there was no particular tropism of cysts for one region. They did find, however, that certain patterns of cyst distribution were associated with certain behavioral phenotypes. They hypothesized that there is selective pressure on T. gondii to alter neuronal circuits, and that this may be accomplished by cyst 28

accumulation in the different parts of that circuit rather than one specific area. These results suggest that while T. gondii cysts localize in a variety of places in the CNS, they may be able to exert an effect in the precise locations that modulate the target behaviors. One possible way to exert this effect would be alteration of the level of key neurotransmitters. In fact, Gaskell et al. (2009) found two genes within the T. gondii genome that encode tyrosine hydroxylase, an enzyme that produces dopamine precursor L-DOPA and is rate-limiting in the production of dopamine. Prandovszky et al. (2011) infected PC12 cells, a commonly used in vitro model of dopaminergic neurons, with T. gondii and, after a five-day incubation period, used high-performance liquid chromatography with electrochemical detection to compare the amount of dopamine produced in infected vs. uninfected PC12 cells.

Infected cells showed a greater than three-fold increase in dopamine

production. In the same study, brain tissue sections of mice infected with T. gondii 6-8 weeks prior were probed with an antibody to dopamine and stained. Intense localization of staining was noted in cyst-containing neural cells; within those cells, staining was found to localize to the tissue cysts themselves rather than the host cell.

These data show that T. gondii induces an increase in

dopamine production in infected cells. Prandovskzy et al. (2011) acknowledged that some of these data are puzzling: T. gondii cysts contain tyrosine hydroxylase, but an additional enzyme not encoded in the toxoplasma genome, DOPA decarboxylase, is required to 29

make dopamine. McConkey et al. (2013) hypothesized that the effect exerted by T. gondii could be explained by the localization of this enzyme to the targeted pathways rather than the localization of tissue cysts themselves. That is, T. gondii cysts could infiltrate cells in the CNS without a clear localization to the pathways that it seems to target, and the different concentrations of DOPA decarboxylase in different areas of the brain would result in the effect of the cysts being localized. Prandovszky et al.’s study would have been strengthened by differentiating between L-DOPA and dopamine localization (which is admittedly very difficult because of the structural similarities) and by comparing staining for dopamine in infected neurons in tissue sections of the brain known to have different levels of DOPA decarboxylase. While the connection between T. gondii-mediated production of L-DOPA and the ultimate increase in dopamine is an area that requires further research, the ability of T. gondii tissue cysts to disrupt the dopamine metabolism of neuronal cells remains a plausible explanation for the source of the fatal feline attraction phenomenon in rodents. The effect of certain antipsychotic drugs on the fatal attraction behavior and the level of T. gondii supports this hypothesis of dopaminergic disruption.

Jones-Brando et al. (2003) treated human foreskin

fibroblast cells with twelve antipsychotic or mood-stabilizing compounds, and then exposed them to T. gondii that had been genetically modified to permit visualization of replication by spectrophotometric analysis. Haloperidol (HAL), a dopamine antagonist, and valproic acid (VA), a GABA inhibitor, were found to 30

inhibit replication of T. gondii; Goodwin et al. (2011), using a similar experimental system, found similar results for three antischizophrenic agents, fluphenazine, trifluoperazine, and thioridazine, but found no effect for HAL. It is unclear why their results differed from Jones-Brando et al. (2003). . Webster et al. (2006) found that HAL, VA, and the standard anti-T. gondii chemotherapeutic cocktail pyrimethamine plus dapsone (PD) ameliorated the feline attraction behavior in infected rats. They infected 46 rats with T. gondii, then, after 14 days, treated them with HAL, VA, PD, or water for 14 days. The same procedure was followed with 39 control rats, except that they were treated with isotonic saline. The timing of this treatment was aimed to inhibit replication of tachyzoites prior to the development of tissue cysts and bradyzoites, which are less susceptible to drug treatment. Six to eight weeks after the infection and treatment protocol, the rats were observed in a manner similar to the Berdoy et al. (2000) study described above. Infected untreated rats displayed the feline attraction behavior as seen in other studies, spending more time in areas treated with feline urine and staying in those areas longer after entrance. Infected rats treated with HAL and PD spent significantly less time in the areas treated with feline urine (VA showed a similar but not statistically significant effect) and demonstrated a reduction in total number of visits to the feline-treated area that approached significance. Skallova et al. (2006) observed a comparable effect in a similar experiment using dopamine selective uptake inhibitor GBR 12909 instead of HAL, VA, or DP. 31

Interestingly, Webster et al. (2006) found that uninfected rats treated with all three drugs displayed a statistically significant increased feline attraction behavior and were statistically significantly more active than the uninfected untreated cohort, though this increase fell short of those seen in infected untreated individuals. The authors hypothesized that this behavior is a result of the desired anti-anxiolytic effect of such drugs. These results have interesting implications on the possible mechanisms by which these drugs achieve their effects in human psychiatric patients.

T. GONDII MANIPULATION OF HUMAN BEHAVIOR

Toxoplasma gondii has been identified as a human parasite for over 100 years, but for much of that time latent toxoplasmosis has been regarded as asymptomatic and insignificant (Webster et al., 2013). The initial interest in T. gondii came from its appearance in an infant with encephalomyelitis (Wolf et al., 1939), and we now know that T. gondii can cause significant birth defects or abortion if the mother becomes infected during pregnancy (Jones et al., 2003). With the advent of AIDS in the 1970s and 1980s, it was found that T. gondii was reactivated as a result of immunosuppression and could cause an opportunistic infection that was one of the more common causes of death in untreated AIDS patients (Luft et al., 2004; Dubey & Jones, 2008). In contrast, post-natal infection in humans with T. gondii is typically asymptomatic or quite mild (Dubey & Jones, 32

2008). For this reason, and because of the difficulty in eradicating T. gondii cysts due to broad dispersal in the body and resistance to pharmacological therapy (Samuel et al., 2003), there has been relatively little attention paid to the effect of latent toxoplasmosis on human health and behavior. But as evidence mounted that rodents’ behavior was manipulated in association with T. gondii infection, some scientists began to question whether this organism had any effect on immunocompetent humans. It was previously shown that human behavior can be modified by infectious agents: Reiber et al. (2010) found an increase in social behavior in the 48 hours following flu vaccination (despite the use of inactivated virus, the body still recognizes the vaccine as an antigenic threat and mounts an immune response), and Novotna et al. (2005) found a decrease in novelty-seeking in patients infected with Cytomegalovirus. A majority of the research on T. gondii’s possible effects on human behavior has been published after 1999 (Torrey & Yolken, 2003). Results have shown evidence of human behavioral modification and involvement in the etiology of certain mental disorders; for the purpose of this review these findings will be divided into three general categories: schizophrenia and other mental disorders, personality changes, and traffic accidents. Before discussion of the data on the possible effects of T. gondii on humans, it is important to note that the determination the infection status of a human for T. gondii is done indirectly. Direct detection of T. gondii cysts by biopsy is possible but not typically performed, as cysts are usually located in the 33

CNS or deep muscle tissue and the precise distribution within these locations varies. The most commonly used method is enzyme-linked immunosorbent assay (ELISA), which was established in the Bulletin of the World Health Organization (WHO) to be the most consistent and reliable test for seropositivity when compared on ability to correctly determine seropositivity status of serum samples; other methods tested included the Sabin-Feldman dye test, complement-fixation test, indirect hemagglutination, and immunofluorescence detection (Carlier et al., 1980). Lin et al. (1980) outlined a standardized ELISA assay protocol, and since 2004 ELISA values have been compared to an international reference reagent, containing IgG antibodies to Toxoplasma gondii, as established by the WHO (Rigsby et al., 2004). The level of IgG anti-T. gondii antibodies in a sample is compared to the standard in order to infer the likelihood of infection. In general, the studies of T. gondii separate IgG levels into several groups (usually negative, moderate, and high) based on these relative antibody levels. The presence of anti-T. gondii antibodies is not absolutely indicative of infection, as uninfected individuals have been found to have low levels (Potasman et al., 1986). In the literature reviewed, “infected” and “uninfected” patients were diagnosed as such by indirect and non-definitive methods unless otherwise noted. Schizophrenia and other psychiatric disorders A review of case reports and studies on AIDS-related toxoplasmic encephalitis found that in up to 60% of AIDS patients with reactivated 34

toxoplasmosis symptoms that included altered mental status, hallucinations, thought disorders, and delusions may occur (Israelski & Remington, 1988). T. gondii has the capacity to cross the blood-brain barrier and has been shown to have a preference for infecting CNS neural cells and muscle tissue. Therefore, early efforts were made to determine whether latent toxoplasmosis could impact brain function. In 2001, Yolken et al. tested the serum of 38 patients having primary schizophrenic episodes for elevated levels of antibodies to T. gondii.

As

compared to 27 healthy individuals, matched by sex, socioeconomic status, and age to the case patients, they found statistically significant increased levels in the patients having schizophrenic episodes. Positive T. gondii infection status was determined based on reactivity to 2 separate IgG peptides along with an ELISA reactivity level above a minimum predetermined optical density relative to a previously-established WHO standard. Torrey & Yolken (2003) found that several studies had been carried out on prevalence of T. gondii in psychiatric patients between 1953 and 1979, mostly in Eastern Europe and Latin America. Twelve of the thirteen studies found higher levels of T. gondii antibodies in study patients compared to controls, with eight of those studies attaining significance.

A later meta-analysis by Torrey et al.

(2007), using some of the same studies, found that individuals with schizophrenia have higher levels of T. gondii antibodies than the non-schizophrenic patients. The odds ratio of association was found to be 2.73, higher than any other known 35

genetic or environmental factor associated with schizophrenia. Another metaanalysis by Arias et al. (2012) found the odds ratio to be 2.70.

Maternal

exposure to T. gondii and presence of IgG T. gondii antibodies in maternal sera have also been found to be risk factors for later development of schizophrenia. Mortensen et al. (2007), using a nested case-control model comparing sera taken from 71 Danish schizophrenic patients and 684 controls several days after their birth and stored until the study, found an increased risk (odds ratio 1.79) of having IgG antibodies in the tested sera for schizophrenics. Brown et al. (2005) also found an increased risk (odds ratio 2.61) of IgG antibodies in a similar experimental study with 63 schizophrenic patients and 123 controls, the only difference being that maternal sera were tested. In both studies, the IgG would have been maternal in origin due to its passage through the placental barrier and the delay of T. gondii IgG production until approximately 3 months after birth in infected infants (Wilson & McAuley, 1999). One study in Turkey, using ELISA detection of anti-T. gondii IgG antibodies to determine seropositivity status of 100 schizophrenic patients matched by gender, age, socioeconomic status, and dietary habits as compared with 50 “depressive” patients and 50 healthy volunteers, found that 66% of schizophrenic patients had evidence of latent toxoplasmosis as compared to 24% of patients with “depressive disorder” and 22% of healthy controls (Cetinkaya et al., 2007). Another study compared 180 soldiers discharged from the United States military with a diagnosis of schizophrenia to 532 healthy 36

controls matched for age, enlistment date, sex, race, and branch of military service. Sera samples taken before and after the diagnosis of schizophrenia were significantly more likely than those of controls to be positive for T. gondii, as determined by anti-T. gondii IgG antibody levels measured by ELISA (Niebuhr et al., 2008). There are other studies that also report an association between T. gondii and schizophrenia (Alvarado-Esquivel et al., 2011; Bachmann et al., 2005; Dogruman-al et al., 2009). One of the most accepted current hypotheses (Howes & Kapur, 2009) of the mechanism of schizophrenia posits that multiple genetic and environmental risk factors predispose individuals to presynaptic striatal hyperdopamingergia – as Howes and Kapur put it, “schizophrenia is thus dopamine dysregulation in the context of a compromised brain.”

T. gondii is known to carry tyrosine

hydroxylase, an enzyme necessary for dopamine production (Prandovszky et al., 2011) and cysts are widespread throughout the brain (Dubey & Beattie, 1988); these findings suggest that T. gondii may play a role in some cases of schizophrenia. Schizophrenia is not the only psychiatric illness linked to both dopamine dysregulation and T. gondii. Miman et al. (2010a) found a statistically significant increase in seropositivity for anti-T. gondii IgG antibodies measured by ELISA in 42 patients with obsessive-compulsive disorder (OCD) compared to 100 healthy controls. Using experimental methods similar to that study, Miman et al. (2010b) also found a statistically significant association of T. gondii with Parkinson’s 37

disease. There are also anecdotal reports of improvement of OCD (Brynska et al., 2001; Smadja et al. 1995) and Parkinsonian (Murakami et al., 2000; Carranzana et al., 1989) symptoms in patients positive for T. gondii upon antitoxoplasma therapy. Additionally, associations with autism (Prandota, 2010) and Tourette’s syndrome (Krause et al., 2010) have been postulated. As pointed out in a review by McConkey et al. (2013), it is very notable that all of these disorders are at least associated with a disruption in the production, uptake, or metabolism of dopamine. There is no evidence of direct causation, since neither all patients with a disorder are positive for T. gondii nor are all people seropositive for T. gondii possessed of an accompanying psychiatric disorder. But the number of diseases that have been shown to have an association with T. gondii combined with the potential contribution of T. gondii to dopamine dysregulation does suggest that it may be a contributing factor.

McConkey et al. (2013)

hypothesized that a number of variables in T. gondii infection could influence whether psychiatric illness ensues, such as age and severity of initial infection, the genotype of the infective strain, and the number and geographic distribution of cysts in the brain. Suicide attempts have also been associated with T. gondii infection. Okusaga et al. (2011) found in a study of 950 schizophrenics that, for those schizophrenic patients under the median age of 38, there was a significant positive association between a history of suicide attempts and T. gondii infection at the time of the study (as determined by ELISA measurement of IgG antibodies 38

to T. gondii). Pederson et al. (2012) performed a prospective study carried out from 1992-2006 on a cohort of over 45000 women in Denmark, 26.8% of whom were find to be seropositive for T. gondii at the time of their pregnancy. They measured the IgG antibodies to T. gondii in blood samples taken from their newborn children (whose IgG levels would be entirely attributable to maternal seropositivity status as described by Wilson & McAuley (1999)) by ELISA and compared the levels to the WHO international standard serum (Rigsby et al., 2004); levels of IgG greater than 24% of this standard were treated as seropositive. Pederson et al. (2012) found statistically significant associations between T. gondii infection and self-directed violence, violent suicide attempts, and suicide; T. gondii-infected mothers, as compared with mothers not infected with T. gondii, were 53% more likely to have self-directed violence, 81% more likely to attempt a violent suicide, and 105% more likely to commit suicide. Interestingly, higher anti-T. gondii IgG levels were associated with increased relative risk.

Higher levels of IgG antibodies have been associated with

increased number of tachyzoites, as measured by amounts of mRNA for tachyzoite-specific surface antigen 1, in the brains of infected mice (Singh et al., 2010), but no association has been shown between parasite burden and IgG levels in humans.

More research must be done to determine whether IgG

antibody levels correlate with concentration of T. gondii cysts or if differences in antibody levels reflect some other variable, such as the different immune responses of individuals to the same stimuli, infection by different strains of T. 39

gondii, or some other unknown factor.

In another study, Ling et al. (2011)

compared the national rates of suicide reported to the WHO and the prevalence of T. gondii infection, adapted from a review of available national prevalence studies (Tenter et al., 2000), in women of 20 European countries. An association was found between levels of T. gondii and suicide for women aged 45-74, but this study does have significant limitations such as not testing individual correlations and not correcting for population size. Finally, in a comparison of the 7440 responses to the National Health and Nutrition Survey, bipolar disorder was also shown to be associated with high levels of IgG anti-T.gondii antibodies in ELISA measurement (Pearce et al., 2012). Personality Changes T. gondii has been correlated with subtle personality changes in infected individuals. Flegr has overseen several large studies of personality traits using Cattell’s Personality Factors (Table 2) (Cattell, 1957) and Cloninger’s Temperament and Character Inventory (Table 3) (Cloninger et al., 1994). Flegr and Hrdy (1994), in a study of 338 people, found a statistically significant correlation between latent toxoplasmosis, as determined by a delayed skin hypersensitivity test (a measurement of activated T-cell response to an antigen; a positive response to an antigen indicates likely prior exposure), and two Cattell factors: low rule-consciousness (G) and high vigilance (L). A similar study by Flegr et al. (1996) expanded upon these findings by examining dichotomous 40

gender personality differences in infected subjects, also determined by a delayed skin hypersensitivity test. They administered Cattell’s questionnaire to 394 staff and students of Prague’s Charles University, and then determined T. gondii infection status using the same skin hypersensitivity test. As compared to uninfected controls, infected male subjects showed lower rule-consciousness (G) and higher vigilance (L), while infected female subjects showed higher warmth (A) and higher rule-consciousness (G). Both male and female infected subjects showed higher apprehension (O) than uninfected controls. In 2003, Flegr et al. looked for associations in 857 Czech military conscripts between performance on Cloninger’s Temperament and Character Inventory questionnaire and T. gondii status, using both complement fixation tests and ELISA to determine positivity. They found a statistically significant reduction in novelty-seeking, shown to be associated with increased levels of dopamine in humans (Hansenne et al., 2002), in infected individuals as compared to uninfected controls (Flegr et al, 2003). It is possible that these differences between infected and uninfected subjects are not caused by T. gondii infection, but represent a correlation between certain personality traits and an increased likelihood of acquiring T. gondii infection. One could hypothesize that different personality traits could lead to differential likelihood of exposure to oocysts due to possession of a pet cat or increased likelihood of eating undercooked meat. To try to determine whether T. gondii was causing these changes rather than certain personality traits altering an individual’s likelihood of acquiring T. gondii, a number of studies focused on 41

the correlation of duration of infection and intensity of personality change, reasoning that if these personality differences between infected and control subjects intensified with increased duration of infection, it could be reasonably inferred that T. gondii was contributing to the difference rather than vice versa. Flegr et al. (1996) tested 190 men who had been clinically diagnosed with acute toxoplasmosis and confirmed by testing for IgG and IgM anti-T. gondii antibodies at some point in the preceding 13 years. The results of Cattell’s personality factor questionnaire were compared with the time passed since the diagnosis of acute toxoplasmosis, and there was a statistically significant association between longer duration of infection and lower rule-consciousness (G). Flegr et al. (2000) repeated this experimental model with 230 women who had been diagnosed with acute toxoplasmosis in the preceding 14 years, and found a statistically significant association between longer duration of infection and higher ruleconsciousness (G). Both studies controlled for age in their statistical analyses. These studies provide some evidence that these personality changes were amplified over time, but much more research must be conducted on this topic before causality can be confirmed.

These studies did not measure

personality changes in the same subjects before and after infection with T. gondii but compared cohorts of already-infected patients with healthy controls.

A

longitudinal study measuring changes in personality factors over time of a large cohort, some of whom become infected during the study, would provide much

42

stronger evidence for the hypothesis that latent toxoplasmosis causes increasing personality changes over time. Traffic accidents T. gondii has been shown to decrease reaction times in rodents (Hrda et al., 2000). Havlicek et al. (2001) performed a double-blind study of reaction times and T. gondii infection status on 116 human subjects. Reaction time was tested using a computerized program that measured response to the appearance of a white square in the center of a black computer screen by the pressing of a key on a special keyboard. This test was administered for one and three minutes to each subject. T. gondii status was determined by ELISA and complement fixation testing.

The results of the infected and uninfected groups, after

correction for age, showed a statistically significant increase in reaction times in the infected subjects. This finding led to several studies of T. gondii’s effect on driving performance.

A retrospective study in Prague found a significantly higher

seropositivity for anti-T. gondii antibodies in drivers who had been involved in major traffic accidents (only those who were deemed to be at least partially at fault in the accident report were included, and no driver whose car was struck on the side or rear was included) as compared to seronegative controls (Flegr et al. 2002). The 145 car accident drivers were drawn from a Prague hospital surgery unit after their accidents and were matched to 446 controls also from Prague, and seropositivity status was determined using ELISA and complement fixation 43

testing. The authors concluded that the value of the odds ratio between T. gondii infection and involvement in a major car accident in their study means that those with latent toxoplasmosis have an increase in 165% to their risk of getting into a major accident (Flegr et al., 2002). Yereli et al. (2006) matched 185 residents of Izmir or Manisa, Turkey, all between 21-40 years old, who had been involved in auto accidents in the preceding six-month period to 185 control subjects who had not been involved in auto accidents of the same age from the same cities.

They found that

seropositivity for antibodies to T. gondii, as determined by ELISA testing, was almost four times higher in the accident group compared to the control group. Another group in Turkey found seropositivity for anti-T. gondii antibody in 53.5% of subjects who had been in an accident vs. in 28.3% of those in the control group (Kocazeybek et al., 2009). Interestingly, several recent studies have shown that presence of Rhesus factor D (RhD), a blood antigen, eliminates these effects on reaction times in those infected with T. gondii (Novotná et al., 2008; Flegr et al., 2009). RhD is a blood antigen that appears on the surface of red blood cells in those with the corresponding gene. The factor is well known as the cause of erythroblastosis fetalis, a hemolytic disease that usually affects newborns who are positive for the factor and are born to mothers who are negative to the factor but have previously immunized by giving birth to a RhD-positive child (Avent & Reid, 2000). The prevalence varies among different ethnicities, but RhD-positive genotypes have 44

been reported to exist in 85% of Caucasians, 92% of Blacks, and 99% of Asians (Reid & Lomas-Francis, 2004).

The continued existence of variation in RhD

phenotype among populations has been perplexing to evolutionary biologists (Perry et al., 2012).

Novotná et al. (2008) performed a reaction-time study,

similar to that used by Havlicek et al. (2001), and found that subjects infected with T. gondii and heterozygous for the RhD factor did not show the same delay in reaction times as compared to uninfected controls. RhD-negative infected subjects were shown to have longer reaction times than both RhD-positive infected subjects and uninfected controls.

Flegr et al. (2009) performed a

prospective study looking at the T. gondii seropositivity status, RhD status, and traffic accident history of 3890 military drivers in the Czech Republic from 20002003. They tested each draftee for RhD status using a standard Rh blood test and for T. gondii status using ELISA and complement fixation.

They then

determined the accident status of each draftee after their service based on military police records. They found that in RhD-negative subjects, those with T. gondii showed a statistically significant increase in traffic accidents compared with uninfected drivers, while no difference was observed between RhD-positive drivers based on T. gondii status. Similarly, among T. gondii-infected drivers, RhD-negative subjects showed a statistically significant increase in traffic accidents compared with RhD positive subjects.

45

DISCUSSION AND PROPOSALS FOR FUTURE RESEARCH

For most people unfamiliar with the research on T. gondii, their initial response upon hearing that it can affect its hosts is disbelief that a tiny protozoan could possibly influence the behavior of humans.

It is, admittedly, quite

unsettling – absurd, even – to think about a parasite controlling our behavior, even in the subtle ways outlined here. Nonetheless, there are data from a variety of studies suggesting this is possible, e.g. T. gondii parasitizes cats and can only sexually reproduce in feline intestinal systems; T. gondii also parasitizes rodents, which are a major prey of felines, as intermediate hosts; T. gondii has genes that encode for tyrosine hydroxylase, uses those genes to increase dopamine levels, and that dopamine increase may alter its host’s response to cat urine odor and increase its chances of completing its life cycle; T. gondii has evolved the ability to infect nearly every mammalian species, all of whom use dopamine as an important neurotransmitter; T. gondii’s potential ability to manipulate dopamine activity, though not evolutionarily targeted at other species, has been correlated with changes in behavioral phenotype in other mammals, including humans. Clearly the neural complexity and interconnectedness of circuitry in rodent and human brains are not identical, but as Adamo (2013) said, “If the mind is a machine, then anything can control it – anything, that is, that understands the code and has access to the machinery.” Webster et al. (2013) went further and claimed that it would be costly for a parasite to have the mechanisms to 46

distinguish

non-definitive

hosts

and

discriminately

manipulate

behavior

depending on whether it finds itself in a definitive or non-definitive host. There would be no selective benefit for this trait, they claim, because it would only be manifested in parasites that are already in “dead-end” hosts and will not reproduce. Applying this to T. gondii, once it is established that it can manipulate the behavior of rodents, the presence of the capability to manipulate the behavior of other mammals, though perhaps not in such direct and logical ways as inducing attraction to the scent of a predator, is actually more likely than a lack of such capability. Some of these “by-product” effects of infection are quite serious and are beginning to be aggressively researched, as is the case with psychiatric illness, suicide, and traffic accidents.

But, as interest in latent toxoplasmosis has

increased, other significant and sometimes bizarre effects of T. gondii have been found.

T. gondii retains its ability to alter the response to cat urine across

species, as infected humans have different responses to cat urine odor than uninfected individuals, with infected men finding the odor statistically significantly more pleasant and infected women finding the odor statistically significantly less pleasant (Flegr et al., 2011). Despite the well-known negative effects of acute toxoplasmosis in a pregnant woman on her fetus, latent toxoplasmosis has recently been found to also have notable, though not nearly as significant, effects. The sex ratio of offspring of mothers with latent toxoplasmosis have been found to be different as compared with uninfected mothers: women with 47

latent toxoplasmosis give birth to more boys, and women with the highest anti–T. gondii IgG antibody titers gave birth to 260 male babies for every 100 female babies (Kaňková et al., 2007); the normal sex ratio is 104 males per 100 females in most populations. In addition, infected, RhD-negative pregnant mothers gain statistically significantly more weight during gestation (Kaňková et al., 2010) and their children are shown to have lower rates of motor development during their first 18 months (Kaňková et al., 2012). Mothers with toxoplasmosis are much more likely to have children with Down syndrome (Hostomská 1957), T. gondiiinfected males are typically about 3 cm taller than others (Flegr et al., 2005), men infected with T. gondii are rated as more masculine and attractive by women (Hodkova et al., 2007) – the list of changes in humans goes on and on. Outside of humans, otters with toxoplasmic encephalitis behave and swim erratically, resulting in dramatically increased predation by sharks and posing a threat to the recovery of this already endangered species (Kreuder et al., 2003) – a behavior pattern quite similar to that of gammarids, the crustacean that is parasitically induced to swim in well-lit areas close to the water’s surface and is subsequently preyed upon by fish and birds. Given the number and variety of behavioral alterations that may result from latent toxoplasmosis as well as the prevalence of infection, thought to be at least one billion people and more likely between two and three billion (Tenter et al., 2000), T. gondii poses a significant public health risk.

As Adamo and

Webster (2013) summarized in a recent article, the public health significance of 48

latent toxoplasmosis on etiology, treatment regimens, and morbidity associated with many diseases, especially psychiatric diseases, is not well understood. Toxoplasmosis was, and often still is, described as presenting with initial mild

flu-like

symptoms

and

then

an

asymptomatic

infection

(http://www.cdc.gov/parasites/toxoplasmosis/disease.html). As recently as 2009, awareness of toxoplasmosis was low enough that it was labeled an orphan disease, or diseases that “attract disproportionately low scientific and public health attention for the impact that they can have,” in the UK (Halsby et al., 2013).

Even in studies explicitly about toxoplasmosis such as “Cats and

Toxoplasmosis: Implication for Public Health”, the effects of latent toxoplasmosis are minimalized: “One such pathogen, the protozoan parasite Toxoplasma gondii, rarely causes clinical manifestations in cats or immunocompetent humans.” (Dabritz & Conrad 2010) Within the past several years, there has been a noticeable change in the amount of attention paid to latent toxoplasmosis as well as the tone and scope of that discussion within the scientific research, medical, and psychiatric communities.

T. gondii has been named one of five “neglected parasitic

infections” in the United States and “targeted by CDC as priorities for public health action, based on the number of people infected, severity of the illnesses, ability to prevent and treat them” (http://www.cdc.gov/parasites/npi.html). There have been calls for prospective studies on whether schizophrenic patients with T. gondii respond differently from other schizophrenic patients to several first-line 49

antipsychotic drugs, the implication being that patients with toxoplasmosis may be sufficiently divergent as to require distinct treatment protocols or even diagnoses (Fond et al., 2013). Work on a T. gondii vaccine has accelerated in recent years (Kur et al., 2009; Meng et al., 2012). There is an entire chapter dedicated to the influence of toxoplasmosis on psychiatric illness in the recent textbook Frontiers in Neuroscience (“Toxoplasma gondii, the Immune System, and Suicidal Behavior”, Okusaga & Postolache, 2012). The number of scientific papers published on T. gondii, particularly on its connections to mental illness, have surged as well: a search of PubMed for papers matching the search terms “toxoplasma” and “schizophrenia” resulted in 58 publications matching these terms since 2009, more than the prior 30 years. A similar search for “latent toxoplasmosis” showed that the last decade has seen almost as many papers published on the topic than in the 90 preceding years. Despite the amount of research done on T. gondii, it is not clear what its actual impact on public health is. There have been exhaustive studies done on the potential impact on livestock (Dubey 2009).

In a study on foodborne

pathogens in the United States – which did not include chronic or congenital conditions – toxoplasmosis was linked to 4,428 hospitalizations and 327 deaths per year (Batz et al., 2012). The same study found that the disease burden costs $3 billion per year and 11,000 in Quality-Adjusted Life Years (QALY).

Only

Salmonella causes more deaths annually than toxoplasmosis (Scallan et al., 2011). Flegr has been particularly outspoken in describing the potential public 50

health significance, declaring that, due to its high prevalence in much of the world and corresponding high attributable mortality, latent toxoplasmosis might represent a far more significant economic and public health issue than was previously thought (Flegr, 2013). In an interview with The Atlantic (McArdle, 2012) he suggested that T. gondii might contribute to the deaths of as many people as malaria every year. T. gondii is a serious problem, and warrants more attention and research. In order to make that case more effectively, scientists researching this parasite ought to acknowledge and address several weaknesses in the models that are used to study it. In this review, no studies that looked at the changes in an individual rodent or human subject after infection with T. gondii were found. All studies cited in this paper compared the behavior of an infected group to a control group; with rodents, it may seem reasonable to assume that there is very little difference between the behavior of the control group and the way the infected subjects would have had acted had they not been infected, but this assumption needs to be tested. Similarly, all the studies of T. gondii’s impact on human behavior compares the behavior of infected cohorts to uninfected controls. It is possible that the risks for contracting T. gondii infection are not evenly distributed, and that some of the effects that are attributed to the parasite may, in fact, be preexisting factors that contribute to future infection status. Additionally, the tests that are used to determine seropositivity for T. gondii antibodies ought to be refined and standardized to a greater degree, and work 51

must be done to determine the precise significance of higher antibody titers in some individuals. In many studies, the association of T. gondii infection and a hypothesized effect are strongest in those infected subjects with the highest IgG antibody titers, but no explanation or evidence is provided as to why that is the case or what these higher titers tell us about parasite burden or disease state. Much work has been done on detection technique, but studies that correlated findings of T. gondii infection with autopsy reports of brain cysts were not found. Such a finding would buttress the claim that presence of anti-T. gondii antibodies are sufficient evidence for infection. Finally, the studies that address personality changes in humans with T. gondii infection suffer from the imprecise and scientifically unverifiable measurements that are used. There is no way to rectify this problem, as the evaluation and enumeration of someone’s personality is particularly difficult to quantify in objective and scientific measurements. Studies that produce results consisting of objectively verifiable or scientifically measurable data, such as neurotransmitter or antibody levels, suicides, or traffic accidents, are superior to studies that are inherently subjective. In addition to greater scientific rigor in the research on T. gondii and its effect on humans, better public health studies, with scientifically calculated estimates of actual impact, ought to be a priority. Here are several other recommendations for future studies and strategies that will help resolve unsettled questions about T. gondii, clarify how it impacts our world, and, hopefully, set a more serious tone in the discussion on how to address it. 52

First, large-scale prospective studies must become the rule rather than exception. Because of the relative ease and reduced costs, retrospective studies make up a majority of the research on toxoplasmosis. Studies on rodent models are also instructive, but, in addition to the qualification that accompanies all animal research that animal and human models are very different, the vastly longer lifespan of humans means that many of the significant symptoms and side effects of T. gondii on humans will be beyond the scope of the those experiments. Prospective human studies along the model (but not necessarily the scale) of the Framingham Heart Study would be a landmark accomplishment allowing scientists to monitor seroprevalence in a population over time, make new connections between T. gondii and diseases, and gain further insight into the relationship between toxoplasmosis and a host of psychiatric disease. Second, there must be increased testing for T. gondii seropositivity among psychiatric patients. The association with schizophrenia is the most significant finding related to toxoplasmosis, and must be exhaustively explicated.

All

patients newly diagnosed with a psychiatric disorder should be tested for toxoplasmosis status, and differences in disease progression and reaction to therapy

between

T.

gondii-positive

and

T.

gondii-negative

cohorts

of

schizophrenic patients ought to be carefully monitored. The differential efficacy of HAL, VA, PD, and other standard dopaminergic, anti-schizophrenic, or moodstabilizing drugs on these cohorts should be studied, as it is possible that the positive results observed in these drugs on schizophrenia or other mood 53

disorders is mediated or augmented in large part by their effects on T. gondii (Webster & McConkey, 2010).

Webster et al. (2006) found that uninfected

rodents treated with HAL or VA showed induction of the fatal feline attraction that is symptomatic of T. gondii; that is, treatment with antipsychotics in the T. gondiinegative cohort induced the same effect that was ameliorated in the T. gondiipositive cohort.

A recent study by Horacek et al. (2012) found statistically

significantly greater reduction in grey matter among T. gondii-positive schizophrenic subjects as compared to uninfected schizophrenic subjects, while little difference was found between uninfected schizophrenic subjects and controls. These findings raise the possibility that T. gondii-positive schizophrenia constitutes a distinct pathology and ought to have distinct treatment protocols. Additionally, future work should also include testing second-generation or “atypical” antipsychotic drugs for the effect demonstrated by HAL and VA on rodent response to cat urine odor and on T. gondii growth, as discovery of other psychiatric drugs that attack T. gondii would expand treatment options for practitioners and broaden the understanding of both schizophrenia and toxoplasmosis. Third, it would be helpful to determine the exact differences in driving tendencies between infected and uninfected cohorts.

If a clear difference

emerged that was shown to contribute to the dramatically higher accident rate in infected people, steps could be taken to address the difference; e.g. a finding of reduction in attentiveness or dramatic decline in reaction time after several 54

consecutive hours of driving could result in a campaign to encourage taking short breaks or in-car timers. In countries with high prevalence of dashboard video cameras, studies could be done on different driving habits in those with and without toxoplasmosis, and accidents could be studied to determine if there are patterns in the accidents occurring in the different groups.

Car insurance

companies could contribute to the research in this area by requiring a T. gondii screen for any customers with more than one accident. As more effective and inexpensive treatments become available, it may be prudent to require either a negative screen for T. gondii or proof of current treatment for latent toxoplasmosis before acquiring a driver’s license. With estimates of potential driving deaths caused by T. gondii in the hundreds of thousands (Flegr 2013), even moderate progress in this area could have a dramatic impact on lives saved. Fourth, the differences in virulence and prevalence of the different strains of T. gondii must be elucidated. As Webster et al. (2013) discussed, there have been differences noted in the way these strains affect rodents and wildlife; similar differences could also be observed in clinical and behavioral outcomes in humans. Type II and III strains were found to express tyrosine hydroxylase at a higher level than Type I (Gaskell et al., 2009). Progress has been made recently in the development of more effective knockout strains (Fox et al., 2011), which will allow researchers to see the affects of specific genes on the various effects of T. gondii. 55

Fifth, more work remains to be done on the life cycle. T. gondii is found at high levels in the Arctic region (Prestrud et al., 2010) and other areas with little or no feline presence, leading some to declare that “perhaps it is time to revisit the ‘felines-only’ dogma” (Boothroyd 2009).

Recently, there has been increased

attention on the possibility that sexual transmission may be a more common mode of infection than was previously thought (Vyas 2013).

Combined with

studies showing that T. gondii manipulates testosterone levels (Kaňková et al., 2011) and that infected males have greater reproductive success (Vyas 2013), these data raise the interesting question of whether testosterone has a more important role in behavioral manipulation and T. gondii’s widespread success than was suspected. Finally, a study establishing how many cases of human toxoplasmosis comes from cats versus tainted meat would help to dispel some of the myths that detract from the public health awareness of toxoplasmosis. The media frequently propagates the stereotype of the “crazy cat lady”, and the misconception that most human toxoplasmosis comes from domestic cats distracts from the real problem of raw or undercooked meat. While pet cats certainly contribute to the prevalence of T. gondii, it is much more practical to try to reduce transmission via raw meat than to attempt to curb or eliminate cat ownership.

56

CONCLUSION

Toxoplasma gondii is a widespread parasite that has enormous consequences on global public health, even in its so-called latent state. Behavioral manipulation is a common strategy among parasites, and its use by parasites on human hosts ought not to be surprising. Research in the last several years has refined our knowledge of the dramatic ways that T. gondii may modify mammalian, specifically human, behavior by interfering with dopaminergic pathways. The repercussions of this modification may be associated with increased psychiatric illness, suicide, and traffic accidents. This thesis summarizes the current literature on T. gondii and its potential manipulation of human behavior, and outlines the recent changes in attitude towards T. gondii, suggests areas that warrant increased focus, and recommends several future studies.

.

57

FIGURES

Figure 1. Electron-Micrograph Composites of a Tachyzoite (left) and Bradyzoite (right). You can see the apicoplast near the Golgi complex in both images. Notice the differences in the apical complex between the tachyzoite and bradyzoite. (Figure taken from Dubey et al., 1998).

58

Figure 2. Major Routes of Transmission of T. gondii. T. gondii can be transmitted between species in a variety of ways. Horizontal transmission via oocysts, represented by blue lines above, occurs when a human or animal ingests feline fecal material. Horizontal transmission via tissue cysts, represented by the red lines above, occurs when a human or animal eats the raw or undercooked flesh of another animal. Vertical transmission, represented by green lines above, occurs when a mother passes the infection on to her offspring. Sexual transmission is also known to occur in several species (not 59

humans), but is a thought to be a minor contributor to overall transmission. (Figure taken from Tenter et al., 2000).

Figure 3. Relative Importance of Meat-Producing and Game Animals in the Transmission of T. gondii to Humans. Prevalence in different animal species varies dramatically. Pigs, sheep, and goats have the highest prevalence among animals commonly eaten by humans. This figure is a generalization of typical prevalence; certain game animals, such as bears or deer, can have even higher prevalence than that of pigs sheep or goats. Notice that free-raised animals almost always have higher T. gondii prevalence than commercially raised animals. (Figure taken from Dubey et al., 1998, adapted from the Report on the WHO Workshop on Public Health Aspects on Toxoplasmosis, Meeting of the Working Groups “Husbandry, Household, and Environment” and “ Food Hygiene”, Bilthoven, The Netherlands, 23-24th October, 1989)

60

Figure 4. Preference or Avoidance of Feline Odor by Subject and Control Rats. The results represent the difference in cumulative visits to cells with rabbit or feline odor by rats during a night of activity. Sorties are defined as active periods separated by at least 1 minute of sheltered inactivity. Infected rats showed a gradually increasing preference for feline odor, while non-infected rats showed a strong increasing aversion to feline odor and a preference for the rabbit odor. (Figure taken from Berdoy et al., 2000)

61

Figure 5. Schematic Model of Toxoplasma-Induced Changes to Host Limbic System. This figure illustrates the brain pathways activated by A) a general odor B) the odor of a female rat C) cat urine odor and D) cat urine odor in a T. gondiiinfected rat. In B) the female odor activates an inhibitor (MEApd) of an inhibitor (VMHvl) of approach behavior, resulting in approach. In C) cat odor activates an activator (MEApv) of the inhibitor (VMHvl) of approach as well as an activator (VMHdm) of avoid behavior, resulting in avoidance. In D) cat odor activates the inhibitor (MEApd) and activator (MEApv) of the inhibitor (VMHvl) of approach behavior, with inhibition of the inhibitor dominating and resulting in approach. (Figure taken from House et al., 2011) AOB, accessory olfactory bulb; MEA, medial amygdala; VMH, ventromedial hypothalamus.

62

TABLES

Table 1. A Selection of Some Known Parasitic Effects on Host Phenotypes. Parasites exhibit a wide range of effects on their hosts. This table some of the Focus more thoroughly researched and well-understood examples of prototypical 1999). canthocephalan cysta- manipulation. (Table taken from Poulin & Thomas, Table 1. Some documented effects of parasites on host phenotypesa

lso good examples of nimals displaying alterh behaviour and body

ortant to note that in d examples of parasitenges in host phenodence rests on compariaturally infected hosts. is possible that some notypes are a cause of er than its consequence; mental infections can lutely that the differn healthy and infected re the result of parasitic

Hostb

Parasite

Phenotypic effect

Effects on motor activity Copepod (E) Cestode Cockroach (E) Acanthocephalan Smelt and eel Various helminths Mouse (E) Protozoan Mouse (E) Nematode

Increased swimming activity Reduced running speed Reduced swimming speed Impaired motor performance Impaired motor performance

Effects on behaviour Snail Trematode Isopod Acanthocephalan Mayfly Nematode Cockroach (E) Acanthocephalan Beetle (E) Cestode Sockeye salmon Various helminths Bully (fish) Trematode Stickleback Cestode Killifish Trematode Mouse (E) Nematode Mouse (E) Nematode Humans (E) Nematode

Altered microhabitat choice Altered microhabitat choice Female behaviour in males Altered microhabitat choice Altered tendency to migrate Impaired orientation during migration Impaired predator evasion Altered vertical distribution Decreased tendency to school Loss of dominance Altered microhabitat choice Impaired memory

Refs 16 37 38 39 40 20, 41 14, 15 30 37 42 43 44 11 45 46 40 47

ns for these changes e categories, each diffiguish from the others10. induced changes in host ight represent adaptive Effects on morphology Snail (E) Trematode Increased body size 48, 49 the host, aimed at elimiIsopod Acanthocephalan Altered body colouration 14 asting the parasite, or at Midge Nematode Males resembling females 29 s effects on host fitness. Mayfly Nematode Males resembling females 30 ges in host phenotype Stickleback Cestode Altered body colouration 13 expression of parasite Rat (E) Cestode Increased body size 50 g the host phenotype in nefit the parasite, for ex- a This list is not comprehensive. easing its probability of b Studies using experimental manipulations of parasite levels are denoted by (E). Third, parasite-induced the host phenotype might be mere pathoin studies of behavioural modification of hosts by paraquences of infection, not adaptive to either sites: for most behavioural measures, the range of values ite. Each of the examples listed in Table 1 obtained for parasitized hosts overlaps with that obt of one or more of these three scenarios. tained for unparasitized hosts, but causes the overall eir evolutionary origin, changes in host mean value to shift towards one extreme15,16. Lauckner, an influence the immediate and long-term studying shell sizes in marine gastropods, described ural selection on hosts. The examples conthe effects of larval trematodes as a ‘grotesque distorw assume that parasites alter host phenotion’ of the frequency distribution of shell sizes17. The ut suppressing host reproduction comresulting overall population distribution was either the case with many examples in Table 1). highly skewed or multimodal, depending on locality, whereas that of the unparasitized component of the the distribution of host phenotypes gastropod population was unimodal and normal. ffect many continuous phenotypic variIf the shift in phenotype caused by parasites is large, , such as body size or behavioural traits. and if prevalence is less than 100%, the distribution of ected hosts, the frequency distribution of trait values is likely to become bimodal, with paraten follows a normal distribution that resitized and unparasitized individuals forming distinct ic differences combined with environmengroups (Fig. 1b). Whether the distribution of trait valasitic infection can shift the mean value of ues becomes skewed or bimodal, it no longer reflects 63 trait one way or the other, and increase the distribution of trait values expected from host n the overall host population (Fig. 1). Of genotypes, and selection on that trait can be disrupted mportance of this effect for the host popuby parasites in many ways. ds on the abundance of the parasite, or the Strong environmental effects on phenotypes can r of parasites per host. When the host is render selection ‘myopic’, ie. capable of seeing and act-

Table 2. Cattell’s 16 Personality Factors. These are Raymond Cattell’s 16 personality factors with descriptions of the characteristics that determine low or high score for each factor (Table adapted from Cattell, 1957).

Low Score

Factor

High Score

A

Cool, detached

Warmth

Warm, outgoing

B

Lower problem-solving ability, les intelligent

Reasoning

Higher problem-solving ability, more intelligent

C

Less Stable

Emotional Stability

More Stable

E

Deferential, accommodating, obedient

Dominance

Dominant, assertive

F

Restrained

Liveliness

Lively, expressive

G

Non-conforming

RuleConsciousness /Superego

Conforming, rule-conscious

H

Shy, timid

Social Boldness

Uninhibited, bold

I

Unsentimental, Objective

Sensitivity

Sentimental, subjective

L

Unsuspecting, trusting

Vigilance

Suspicious, distrustful

M

Grounded, conventional, practical

Abstractedness

Imaginative, abstract

N

Guileless, forthright

Guardedness

Private, guarded

O

Secure, self-assured

Apprehension

Apprehensive, insecure

Q1

Traditional, not as open to change

Openness to Change

Open to change, flexible

Q2

Group-oriented

Self-Reliance

Self-reliant, solitary

Q3

Unexacting, lax

Perfectionism

Perfectionistic, compulsive

Q4

Relaxed, patient

Tension

Tense, impatient

64

Table 3. Cloninger’s Temperament and Character Inventory (TCI). These are C. Robert Cloninger’s temperaments and characters (Table adapted from Cloninger, 1994). Cloninger’s Temperament and Character Inventory Temperaments Characters Novelty Seeking

Self-Directedness

Harm Avoidance

Cooperativeness

Reward Dependence

Self-Transcendence

Persistence

65

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VITA ROBERT F. CAREY (847) 962-4943 [email protected] 2426 Ontario Rd. NW #303, Washington, DC, 20009 Born 1984 EDUCATION Boston University School of Medicine, Boston MA, Class of 2017 • Matriculating August 2013 Boston University, Boston MA, Class of 2013 • Candidate for Master of Arts of Medical Science, May, 2013 • Coursework: Biochemistry and Cell Biology, Medical Histology, Medical Pathology, Advanced Human Physiology, Biostatistics • GPA: 4.0 Duke University, Durham NC, Class of 2006 • Major: Biology (BS) with a Neuroscience Concentration • Minor: Chemistry New York University in Florence, Italy. Fall 2004 WORK EXPERIENCE 2012 - 2013 – Princeton Review – SAT and MCAT Biology Instructor • Teach SAT and MCAT prep classes 2006 - 11 – St. Jude Medical– Pacemaker Clinical Specialist, Merlin.net Remote Monitoring Supervisor • Spearheaded efforts to implement new remote monitoring technology compatible with electronic medical records in area clinics • Assisted in pacemaker and defibrillator implants • Ran follow-up clinics for patients with implanted pacemakers and defibrillators • Taught continuing education classes for RNs and MAs about ICDs • Received award for Sales Team of the Year and Region of the Year, 2007

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2005 - Sangamo Biosciences, Inc., Richmond, CA – Research Assistant • Performed lab based experiments resulting in the discovery and generation of zinc finger proteins implicated in neuropathic pain. • Assisted in discovering zinc-finger proteins that promote deactivation of nerve growth hormone expression. 2003 - InStadium, LLC, Chicago, IL - Assistant Manager of Operations • Sales representative and assistant to the director of operations for a start-up company focused on sports advertising. • Performed demographic research. • Traveled to stadiums across the country to install product. RESEARCH ACTIVITIES 2012 - 2013 – NIH, National Cancer Institute, HIV/AIDS Malignancy Branch, lab of Dr. Robert Yarchoan • Special Volunteer: Perform research on KSHV-infected B cells exploring the function of Hypoxia-Inducible Factors 1 and 2 and the influence of localized hypoxia on the viral life cycle. 2011 - UCSF Cardiology and Electrophysiology Department, lab of Dr. Jeffrey Olgin • Research Assistant: Perform, prepare, assist and observe electrical mapping and genetic experiments with a focus on causes of Atrial Arrhythmias, particularly AF. 2011 - UCSF Pediatric Multiple Sclerosis Center, office of Drs. Emmanuelle Waubant and Ellen Mowry • Research Assistant: Accumulate, edit and organize data for metaanalyses and studies related to causes and predictive factors of MS. • Second author on two papers relating to MS-predictive genotypes (in review stage). VOLUNTEER ACTIVITES 2012- current– AIDS Healthcare Foundation: Work with the Mobile Testing Unit and the STD clinic. 2012 - Wizards: Set up and ran fun science experiments for children ages 4-10 at an after-school center at the local YMCA – January 2012 – June 2012. 88

2011 - Pacemaker Mission Trip with Solidarity Bridge to Tarija, Bolivia: Lead Pacemaker Technician – May 2011 2011 - Medical Mission Trip with Family Health Ministries to Port-auPrince, Haiti: Triage and General Medical Services Assistant – May 2011 LEADERSHIP/ACTIVITIES 2002-2005 - Duke University Club Football - President, Coach, Captain • Organize schedule, budget and recruit for a football team which competes against other university club and Division III teams. • Defensive all-league twice, league defensive MVP 2003. 2003-2006 - Sigma Nu Fraternity - Class President, Pledge Marshal, Head of Philanthropy

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