Toxoplasma: Molecular and Cellular Biology
Preface
The direction and progress of any research discipline is
determined by the community’s collective ability to navigate, through
observation and experimentation, in the pursuit of illumination. From the
outset, the route to understanding Toxoplasma gondii as a parasitic
protozoan was convoluted. In hindsight, this can be largely attributed to it
possessing acomparatively complex, elusive life cycle, remaining a parasite of
“doubtful nature” decades after its discovery (Wenyon, 1926; Lapage, 1968). More
recently, a migration of researchers working with a core of established
Toxoplasma investigators has led to something of a renaissance in the field.
At this point, our collective knowledge is sufficiently great, so, as one of the
contributors to this volume amusingly suggested, it is time to “demystify”
Toxoplasma.
T. gondii was a relative latecomer in its discovery as a parasitic
disease agent, and even the current name was not established until a couple of
years after its initial description by Charles Nicolle at the Institut
Pasteur, Tunis, in 1907 (Nicolle, 1907). Although Nicolle was better known for
his work on typhus, for which he was awarded the Nobel prize, he was a scientist
with the “Pastorian mission” to eradicate infectious disease (Pelis, 2006).
Thus, he retained a broad interest in infectious disease agents, including
enigmatic intracellular parasites of rodents. Based on similarities to the
pattern of replication of piroplasms, the parasite taken from the desert rodent
Ctenodactylus gundi (or gondi as referred to by Nicolle) was tentatively
classifed as Piroplasma quadrigeminum (Nicolle, 1907). Nicolle, wishing to share
his finding, kindly lent a blood smear from the infected rodent to the then
world’s expert on piroplasms, the Cambridge parasitologist George Nuttall.
Nuttall, a native Californian and head of the Molteno Institute for
Parasitology, concluded that it did not belong to the Piroplasma and suggested a
new genus, classifying the parasite as Nicollia quadrigemina (Nuttall,
1913). This name and information was presumably offered to Nicolle in a personal
communication, but was never formally published by
Nuttall¹. After further
observation, in 1908, Nicolle and his colleague L. Manceaux made another
proposal based on “grandes analogies entre lui et les Leishmania” and
suggested a new name Leishmania gondii (Nicolle and Manceaux, 1908). This
speculation was based on several criteria, but two are notable. First, in liver
preparations:
Les éléments parasités sont toujours des mononucléaires; on ne rencontre jamais de protozoaires dans les globules rouges, dans les polynucléaires ni dans les cellules du foie.
Second:
…la présence fréquente de deux corps chromatiques: grand et petit karyosomes.
The first observation that the parasite resides in monocytes and not red blood cells argued against the parasite being a piroplasm, and the apparent absence in liver cells reinforced the leishmania paradigm of monocyte occupation. The second and frequent observation of a small chromatin body in addition to the nucleus is certainly reminiscent of a kinetoplast, and it is ironic that its true nature as the apicoplast would not be definitively revealed for another 90 years. Concurrent with this analysis in 1908, A. Splendore in Brazil published the first in a series of three papers that described effectively the same parasite isolated from a rabbit. He also tentatively identified it as a leishmania (Splendore, 1908). Finally in 1909, after extensive microscopic analysis of several tissues and experimental infection studies, Nicolle and Manceaux endowed the parasite with its current name (Nicolle and Manceaux, 1909):
…et nous proposons, pour désigner ce genre nouveau, le nom
de Toxoplasma (de τοξον,
arc). Le nom du parasite du Gondi sera donc T. gondii.
Notably, they finish this seminal publication by reporting on a friendly collaboration with A. Splendore, concluding that the parasite he isolated from the rabbit was “morphologiquement identique” to their newly named T. gondii. Splendore, apparently in agreement with this assessment, classified his parasite as T. cuniculi and published these results in the third paper of his series the following year (Splendore, 1910). This was the first of many such papers describing the isolation of Toxoplasma from a variety of host species.
Over the next 50 years, Toxoplasma (including species now known to be gondii) was isolated from a wide variety of animal species including humans. In this time, the parasite’s current status as a model organism came to the fore as the acute disease form could be easily cultured and lent itself to microscopic study. EM studies began in the early 1950s and the ultrastructure of T. gondii, descriptions of the apical complex, and secretory organelles such as the rhoptries became a paradigm for the invasive form of apicomplexan parasites (see, for example, Gustafson et al., 1954; Scholtyseck and Melhorn, 1970). Ironically, the morphology of the parasite was well described, but the nature and extent of T. gondii infection remained a mystery because only the acute disease form of the parasite was readily observed as the parasite was primarily seen post-mortem. This situation changed dramatically in 1948 with the advent of the serologically based dye test developed by Albert Sabin and Harry Feldman (Sabin and Feldman, 1948). The test not only allowed diagnosis of human toxoplasmosis but also provided a way to gather epidemiological data from which the close relationship between T. gondii isolates and the solidification of the single species concept emerged. Although T. gondii had been isolated from many mammalian and bird species, the natural route of transmission remained unknown. Since the large majority cases of human toxoplasmosis were via congenital transmission, the rarity of the condition could not explain the widespread infection in both man and animals.
Like many infectious agents, epidemiological data provided the first clues. In the 1950s and early 60s, evidence began to accrue that the consumption of undercooked meat may be an important route of T. gondii infection (Weinman and Chandler 1954; Jacobs et al., 1960). Through observation of children in a Paris sanitorium in the mid-1960s, Desmonts and co-workers followed children before and after admission to the sanitorium and assessed the rate of seroconversion as a measure of infection (Desmonts et al., 1965). From a baseline 10% seroconversion per year, the figure rose to 50% with the daily consumption of undercooked beef or horse meat and to nearly 100% with rare lamb. A study of the adult population in Paris at this time showed that over 80% were positive for antibodies against T. gondii, consistent with the rate of acquired infection observed in the sanitorium. Curiously, a survey of strict vegetarians in India showed that the prevalence of infection was similar to non-vegetarians and herbivorous animals (Rawal, 1959). This and similar investigations prompted the search for other routes of infection.
As current Toxoplasma researchers, the coccidian nature of the parasite is such an integral part of our thinking that we tend to forget the difficulties in understanding the parasite in the absence of this knowledge (D. Ferguson, personal communication). In the mid-1950s, L. Jacobs and co-workers first tested fecal transmission of T. gondii in dogs, albeit with negative results ( Jacobs et al., 1955). Unfortunately between their lack of success and the similarity to other known coccidian parasites of cats and dogs, T. gondii transmission remained enigmatic for another 10 years. William Hutchison, working at the University of Strathclyde in the mid-1960s, revisited the fecal transmission route hypothesis. Although he wanted to follow Jacobs and use dogs for his experiments, these could not be maintained at his facility so he used a cat instead (J.P. Dubey, personal communication). Hutchison showed that it was possible to isolate from cat feces, 2 weeks after infection with tissue cysts, an unknown form of the parasite that could transmit infection to mice (Hutchison, 1965). Importantly, this form was very stable and could survive for up to 12 months stored in water. However, since the feces-derived inoculum potentially contained pathogens other than T. cati ova and an unknown form of T. gondii (specified pathogen-free cats were not available at the time), the only way Hutchison could control the infection was by a microscopic examination of the inoculum, and he noted:
The only living material observed, apart from bacteria, were the oocysts of Isospora and the ova of T. cati.
In retrospect, it probable that the Isospora-type oocysts were in fact the unrecognized form of T. gondii, highlighting how unthinkable it was to consider Toxoplasma as a coccidian at the time (D. Ferguson, personal communication). Thus, the prime hypothesis was that the unknown infectious form could be protected within the nematode eggs, but Hutchison cautiously concluded:
At present it is impossible to say whether the infection induced by this material was transmitted by helminth ova or by some other means.
This finding motivated Hutchison and other researchers, notably J. Frenkel, J.P. Dubey, H.G. Sheffield and M.L. Melton to address this question. Despite some data to the contrary, the collective results clearly showed that T. gondii transmission was not dependent on nematode ova (Frenkel et al., 1969; Sheffield and Melton, 1969). Finally, with the discovery of the sexual cycle in the cat intestine, these researchers and their colleagues revealed the true coccidian nature of T. gondii and better defined the unique asexual transmission between secondary hosts, culminating in the elucidation of the complete life cycle (Frenkel et al., 1970; Huchison et al., 1970, 1971). Following this painstaking microscopy by Dubey, D. Ferguson and co-workers described gametogenesis and the sexual cycle in great detail within the cat intestine (Dubey and Frenkel, 1972; Ferguson et al., 1974, 1975, 1979a, 1979b; Speer and Dubey, 2005). For asexual reproduction, Frenkel coined the now commonly used terms “tachyzoite” to describe the rapidly dividing form seen in the acute infection and “bradyzoite” to describe the slowly dividing form seen in the chronic infection (Frenkel, 1973). Through characterizations of the bradyzoite infection and infective fecal forms, Dubey and co-workers clarified a confusing situation in the literature by defining the bradyzoite containing mass as “tissue cysts” (Dubey and Beattie 1988) compared to the fecal shed “oocysts” (Dubey and Frenkel 1976).
With knowledge of the life cycle in hand, in the late 1970s and early 80s, Elmer Pfefferkorn and co-workers at Dartmouth initiated a series of studies to establish the transmission genetics of T. gondii. In an elegant set of crosses using drug resistance and auxotrophic markers they had previously developed (Pfefferkorn et al., 1977, 1978), Pfefferkorn confirmed Mendelian inheritance and the haploid nature of the parasite (Pfefferkorn and Pfefferkorn, 1980). Moreover, they and others showed that a single cloned parasite could give rise to the complete sexual cycle in the cat, demonstrating that the sexual cycle did not involve fixed mating types (Pfefferkorn et al., 1977; Cornelissen and Overdulve, 1985).
These results were consistent with microscopic/cellular observations of gametogenesis in the cat intestine, where both macro- and microgametocytes appeared to be produced from a single organism. Thus, in mixed infections, both selfing and outcrossing occur in about the same frequency (Pfefferkorn and Pfefferkorn, 1980). These studies laid the foundation for what would become the molecular genetics and genomics of T. gondii.
Compared to other model organisms, the early path to understanding T. gondii ran a circuitous route, but from the beginning, the community of investigators has been intellectually incisive and generous with sharing information, techniques and authorship on publications. The current community is a testament to this tradition.
We would like to thank J.P. Dubey and D. Ferguson for historical insights and, most importantly, thanks to all of the contributors to this book and publisher who made it possible.
James W. Ajioka, Cambridge, UK
Dominique Soldati, Genéve, Suisse
July, 2007
¹ This is inferred from a section describing Nicolle’s parasite in Nuttall’s 1913 review of Piroplasms [ voltar ].
