History

Investigators on two continents first described T. gondii in 1908. Nicolle and Manceaux, at the Institut Pasteur in Tunis, identified and named the parasite in a cricitine rodent, the North African gondi (Ctenodactylus gundi), native to the mountains of southern Tunisia, and maintained in their laboratory (Nicolle and Manceaux, 1908, 1909). Splendore, in Brazil, noted identical forms in a laboratory rabbit (Splendore, 1908, 1909). Appreciation of the spectrum of disease that the parasite can cause came slowly. Wolf and Cowen (1937) at Columbia University identified the parasite in central nervous system lesions in infants that had been diagnosed with meningoencephalitis. Understanding of the role of chronic infection came with the identification by Wilder (1952) of Toxoplasma in necrotic lesions of the retina of eyes previously thought to have been involved with tuberculosis or syphilis. The high prevalence of the infection in various populations was first shown by the serological test developed by Sabin and Feldman (1948), which relied on the ability of human serum to induce leakage of

Principles and Practice of Clinical Parasitology

Edited by Stephen Gillespie and Richard D. Pearson © 2001 John Wiley & Sons Ltd extracellular dye into live tachyzoites in the presence of complement. The recognition of congenital toxoplasmosis in infants came before either generalized disease in adults or the lymphadenitis of primary Toxoplasma infections in adults was appreciated (Wolf and Cowen, 1937). The role of reactivation of latent infections in the production of disease in immunosup-pressed adults was recognized at the outset of solid organ transplantation (Ruskin and Remington, 1976). In the early 1980s, central nervous system reactivation with multifocal encephalitis became a major presentation of disease in patients with AIDS (Luft et al., 1983a).

DESCRIPTION OF THE ORGANISM Classification

The parasite is a member of the phylum Apicomplexa, class Sporozoa, subclass Coccidia, order Eucoccidia and suborder Eimeria (Levine et al., 1980). It is therefore related to malaria and a large number of coccidians that generally infect birds and mammals. The parasite was recognized as a coccidian only in 1969, when four laboratories independently established the sexual cycle (Frenkel, 1970; Frenkel et al., 1970). Traditional classification schemes have relied on morphological comparisons of the various life stages, most importantly the sexual stages. By these criteria, T. gondii closely resembles Isospora spp. and Sarcocystis spp. and, although it has been argued that the organism name should be changed, it continues to be validly named as T. gondii. More recently, molecular genetic techniques have shown that T. gondii is a single species related to Isospora, Sarcocystis, Frenkelia and Hammon-dia but most closely related to Neospora caninum (Guo and Johnson, 1995).

Molecular analysis of genes of T. gondii indicates that some genetic elements of the parasite may derive from a member of the green algae (Fichera and Roos, 1997; Stokker-mans et al., 1996). This situation may have arisen by an incorporation of algal DNA by endosym-biosis, and may be of importance in development of novel drug targets that take advantage of differences between 'plant-like' and mammalian gene characteristics. A membrane-bound, plastid-

like structure, the apicoplast, contains a 35 kb circular genome and can be specifically inhibited by ciprofloxacin, clindamycin and macrolide antibiotics, which block parasite replication in a peculiar delayed fashion. The target of these drugs is likely protein synthesis in the apicoplast. Plastid replication is immediately affected, but overall parasite growth is maintained until the second or third parasite replication cycle (Fichera and Roos, 1997). Many of the plastid genes have been transferred to the nucleus and may explain the plant-like character of T. gondii structural proteins such as tubulin (Stokkermans et al., 1996). Other evidence that T. gondii has plantlike characteristics conveyed by the apicoplast is that the parasite expresses enzymes of the shikimate pathway, which is essential for the synthesis of folate, ubiquinone and aromatic amino acids in algae and plants (Roberts et al., 1998). A well-characterized inhibitor of the shikimate pathway, the herbicide glyphosate, also inhibits T. gondii. Four enzymes of the shikimate pathway have been detected in T. gondii, and the pathway is also present in the apicomplexan parasites Plasmodium falciparum and Cryptosporidium parvum.

Life-cycle

The asexual stages of T. gondii can cause disease in humans and most animals (Figure 5.1). There are two asexual forms. The first form, called the tachyzoite, can invade all types of cells and divides rapidly, leading to cell death (Figure 5.2). The second form, called the bradyzoite, divides slowly and forms cysts, most prominently in muscle and brain (Figure 5.3). Tachyzoite replication causes acute disease, while encysted bradyzoites are long-lived, with slow turnover, and are responsible for maintaining the latent infection. Cysts in tissue elicit no inflammation, and presumably have little effect on surrounding cellular function until they break down and release the bradyzoites, which can convert to tachyzoites and cause necrosis and inflammation. Reactivation of bradyzoites from cysts is responsible for most disease in immunosuppressed hosts. The infection is maintained in nature in numerous animals, both wild and domesticated.

Acute Infection

Bradyzoítes

Bradyzoítes

Carnivores

Bradyzoites in Meat Ingested by Humans

Congenital Reactivation Transmission Encephalitis

Fig. 5.1 Life-cycle of T. gondii. The cat is the definitive host, in which the sexual cycle is completed. Oocysts shed in cat feces can infect birds, rodents and grazing animals or humans. The cysts found in the muscle of food animals may infect humans eating insufficiently cooked meat. Human disease takes many forms, but congenital infection and encephalitis from reactivation of latent infection in the brains of immunosuppressed persons are the most important manifestations of disease

Rodents and birds ingested by cats keep the sexual cycle going in the wild. Human food animals, especially sheep, pigs and goats, may harbor cysts in muscle, which are infectious for people and other carnivores when ingested in raw or undercooked meat (Dubey, 1990, 1992; Dubey et al., 1995). The sexual cycle takes place in the superficial epithelium of the small intestine of both wild and domestic members of the cat family (Figure 5.4). Oocysts, which are shed in feces of recently infected cats, are resistant to desiccation and heat (Dubey, 1995; Jacobs et al., 1960). Oocysts are less dense than water and remain in the upper soil horizon, where they may contaminate skin and may be ingested, either directly by hand-to-mouth transmission or on raw vegetables (Frenkel et al., 1970). Oocysts require exposure to air, after cat feces are deposited in soil, for at least 12 hours but up to several days in order to complete sporulation, after which they are infectious by mouth (Frenkel et al., 1975). This information is useful in the management of cat litter boxes, which have a lower chance of harboring infectious oocysts if the feces are removed daily.

Population Genetics

Strains of T. gondii from all continents have been compared genetically and shown to be a homogeneous single species with less than 5% sequence variation between isolates from any area of the world (Boothroyd, 1993). The species sorts genetically into three major clonal lineages (designated as I, II and III), with little evidence of recombination (Howe and Sibley, 1995). Demonstration of the sexual cycle in the cat intestine indicates that sexual recombination is possible, and it can be shown to occur in

Fig. 5.2 T. gondii infection of cultured human fibroblasts, demonstrating 'rosettes' of tachyzoites (tg) within parasitophorous vacuoles in the cytoplasm of the host cells. The parasites are orientated with their posterior poles to the inside of the ring, n, host nucleus. Bar=10 ^m
Fig. 5.3 Bradyzoite cyst in the retina, stained with PAS. No inflammation is evident adjacent to the intact cyst. Individual bradyzoites cannot be distinguished. Bar = 50 ^m

experimental infections (Pfefferkorn et al., 1977; Pfefferkorn and Pfefferkorn, 1980). This must be relatively infrequent in nature, however, probably because it would require a cat to ingest two separate strains of T. gondii in close temporal proximity, so that the initial intestinal infection produced gametes that could cross-fertilize. Virulence differences between three defined genotypes can be demonstrated in experimental infections of inbred mouse strains. There is evidence, from analysis of a collection of 109 isolates from around the world, that the type II genotype, as defined by Sibley, is over-

Fig. 5.4 Sexual stages of T. gondii in epithelial cells of cat small intestine. (A) Early sexual stages (types B and C), 40 hours after infection, stained by PAS. (B) Later stages (types D, E and gamonts) in the periphery of intestinal epithelial cells after 8 days, stained with haematoxylin and eosin. Bars=75 ^m. Histological preparations courtesy of Dr Jack Frenkel, University of Kansas Medical Center

Fig. 5.4 Sexual stages of T. gondii in epithelial cells of cat small intestine. (A) Early sexual stages (types B and C), 40 hours after infection, stained by PAS. (B) Later stages (types D, E and gamonts) in the periphery of intestinal epithelial cells after 8 days, stained with haematoxylin and eosin. Bars=75 ^m. Histological preparations courtesy of Dr Jack Frenkel, University of Kansas Medical Center represented in human disease, and that type III is more frequent in animals (Howe and Sibley, 1995; Sibley and Howe, 1996). Type I, which is most virulent in the mouse model, may be more frequent in human congenital disease (Sibley and Howe, 1996). Genetic tools, including genetic crosses (Pfefferkorn and Pfefferkorn, 1980), transfection and homologous recombination to produce knockout phenotypes (Roos et al., 1994) a preliminary genetic map (Sibley and Boot-hroyd, 1992; Sibley et al., 1992) and an expressed-sequence-tag library (Ajioka et al., 1998), have been developed to aid the genetic analysis of the parasite.

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