Toxocara canis

Toxocara canis is a remarkable nematode parasite, commonly found in dogs but able to infect a wide range of other hosts including humans (Glickman and Schantz, 1981; Lewis and Maizels, 1993; Hotez & Wilkins 2009; Smith et al 2009). Among its many striking features are: a tissue-dwelling phase which can endure many years; the ability to cross the placenta to infect unborn pups; a tropism for neurological tissue in paratenic hosts (such as humans); survival in vitro for many months in serum-free medium; the secretion of a set of biologically active glycoproteins in vivo and in vitro; and the possession of a surface glycocalyx which is jettisoned under immune attack. For all these reasons, T.canis presents an attractive model system for parasitic nematodes (Maizels et al 2000). In addition, toxocariasis causes a significant pathology in humans (Gillespie, 1987; Gillespie, 1993) as well as dogs (Lloyd, 1993), and elimination of this infection would be a highly desirable goal (Hotez & Wilkins 2009).

Developmental Biology of Toxocara

T.canis infection begins on ingestion of embryonated eggs into the stomach. The resistant eggshell breaks down and infective larvae emerge, and penetrate the intesinal mucosa. Invasion occurs in all mammalian species, but only in canids do larvae follow a full developmental pathway by migrating through the lungs, trachea and oesophagus back to the gastrointestinal tract. In other species, the larvae remain in the tissue-migratory phase without developing (Sprent, 1952); arrest at the larval stage is only released if the paratenic host is carnivorously consumed by canid species (Warren, 1969). In dogs, there is a further fascinating property: many invading larvae are restrained from development, arrrested in the tissues until reactivated during the third trimester of pregnancy; then, trans-placental invasion of the gestating pups can occur, and subsequently additional larvae pass into the colostrum to cause post-natal infection (Sprent, 1958; Griesemer et al, 1963; Scothorn, Koutz and Groves, 1965; Burke and Roberson, 1985). Thus female dogs contain a reservoir of infection, producing waves of larvae for each new litter of offspring, and the majority of pups are infected at birth with T. canis.

Dormant tissue larvae have been recorded as long as 9 years after infection in a mammalian host (Beaver, 1962; Beaver, 1966), while incubation in vitro permits larvae to survive for up to 18 months (de Savigny, 1975). In neither context is any morphological development observed. However, the parasites maintain a high metabolic rate, being dependent (in vitro, at least) on a regular supply of glucose and amino acids. Cultured larvae secrete copious quantities of glycoprotein antigens, which are described as T. canis Excreted-Secreted (TES) products (Sugane and Oshima 1983; Maizels et al 1984; Badley et al 1987; Meghji and Maizels 1987).

Immune Evasion

The ability of arrested-stage larval parasites to survive in the tissues for many years must depend on potent immune-evasive and anti-inflammatory mechanisms operated by the parasite (Ghafoor et al., 1984; Smith, 1991). Secreted macromolecules are the primary candidates for immune evasion mediators, and indeed larvae are found to release large quantities of glycoproteins in vitro. Antibodies to secreted TES glycoproteins also detect antigens in vivo which appear to have been released from parasites. For this reason, we have analysed the secreted proteins in detail, as set out below. Several routes have been adopted: the direct, biochemical approach of protein analysis; an immunological path of identifying antigenic determinants; and most recently a molecular biological strategy. In the latter respect, we have undertaken a broader survey of the major products encoded by this parasite, expecting that it must devote a large part of its energy in evading the immune response (Tetteh et al., 1999). A range of fascinating molecules have emerged, some of which can be predicted from their primary sequence structure to fulfill certain functions : mucins, proteases, enzyme inhibitors, etc (Maizels et al, 2000)

The Surface Coat

The external surface of the T. canis larva is covered by a carbohydrate-rich glycocalyx termed the surface coat (Maizels and Page, 1990; Page et al., 1992a), a fuzzy envelope 10 nm in thickness and detached by a similar distance from the epicuticle. The surface coat appears to play a primary role in immune evasion, as it is shed when the parasite is bound by granulocytes (Fattah et al., 1986) and or antibodies (Smith et al., 1981; Page et al., 1992a). Surface coats are a common feature of nematode organisms, both parasitic and free-living (Blaxter et al., 1992), so that this very direct means of immune evasion may be a simple adaptation to parasitism.

Biochemical analysis of the surface coat, coupled with electron microscopy, showed that its principal constituent is TES-120, the set of secreted mucins (Gems and Maizels 1996; Loukas et al 2000). Other TES glycoproteins, such as TES-32 and TES-70, are not found in the surface coat, but remain associated with the nematode body cuticle. The surface coat carries a strong negative charge, binding to cationic stains such as ruthenium red and cationized ferritin (Page et al., 1992a), but the identity of this charge group has yet to be determined.

The coat mucins are thought to be secreted from two major glands in the larval body, the oesophageal and secretory glands (Page et al 1992b). The latter, previously termed the excretory cell, is directly connected to the cuticle by a duct opening at the secretory pore (Nichols, 1956). It is not certain whether the panel of secreted mucins are all represented in the coat, or whether there are important differences between the two compartments.

Carbohydrate Moieties

Ascarid nematodes were shown more than 70 years ago to contain high levels of carbohydrate antigens (Campbell, 1936). Analyses of whole TES products revealed a surprisingly high level of glycosylation, mostly accounted for by galactose and N-acetylgalactosamine (Meghji and Maizels, 1986). As the core sugars for asparagine (N)-linked oligosaccharides are mannose and N-acetylglucosamine, this composition indicated a predominance of O-linked glycosylation. Analysis by mass spectrometry (Khoo et al., 1991; Khoo et al., 1993) defined two major O-linked glycans, both variants of a trisaccharide containing N-acetylgalactosamine, galactose and fucose . The fucose is invariably O-methylated, and the galactose is methylated in 50% of the molecules. In T.cati the galactose is fully methylated, so that the trisaccharide containing a single methylation site on fucose alone represents a T.canis-specific structure. The N-linked oligosaccharide structure which predominates has also been determined to be a biantennary trimannosylated structure linked to a chitobiose-asparagine core, resembling products found in insects (Khoo et al., 1993).

Carbohydrate specificities were also pre-eminent in a panel of monoclonal antibodies generated to TES (Maizels et al., 1987). MAbs Tcn-2 and Tcn-8 recognise a spectrum of TES glycoproteins, including TES-400, -120, -70, -55 and -32, through a periodate-sensitive determinant presumed to be conjugated to various peptide backbones. There is a striking contrast between the two antibodies, however, in that Tcn-2 reacts only to T.canis glycoproteins, while Tcn-8 cross-reacts fully with products from T.cati. We have recently shown, with synthetic glycans, that Tcn-2 binds to the mono-methylated form (Schabussova et al 2007).

The T.canis trisaccharide can be considered to be a mammalian blood group antigen H (or type O) to which has been added additional methyl groups which represent a substantial modification. There is however, some evidence for minor unmodified bloodgroup-like oligosaccharides (Khoo et al., 1993). Moreover, the presence of tetrasaccharides containing N-acetylgalactosamine linked to the galactose is indicated by the blood group A-like reactivity of TES in antibody and lectin binding analyses (Smith et al 1983; Meghji and Maizels, 1986).

 Links

NHS Information pages on Toxocariasis

Scottish Parasite Diagnostic Laboratory Toxocariasis test

Toxocariasis in dogs

Conclusions

Toxocara has an exceptional ability to withstand attack by the immune system, most probably due to the specific glycoproteins which constitute the surface and secreted compartments of the parasite. Tissue-dwelling larvae appear to devote a large part of their metabolic energy into production of the TES proteins. We have discovered that two of the major products released by the infective larvae are similar at both structural and functional levels to host C-type lectins, a family of proteins which are pivotal in the immune system (Loukas et al 1999, 2000). These sugar-binding proteins mediate key events in the immune response to infection, particularly in inflammation when cells and harmful molecules are focussed on the site of invasion. Host lectins are required to bind to carbohydrate 'danger' signals in order to initiate inflammatory influx around the parasite, and the release of a parasite lectin may block this process.

The molecular characterisation of the TES proteins now provides an opportunity for immunological investigations at a much finer level of definition. Significant contrasts have been observed between infected patients with different syndromes, with respect to surface binding, proportions of anti-peptide and -carbohydrate specificities, and isotype (Smith, 1993), and it may now be possible to relate these patterns to recognition of individual antigens. The T cell response to TES is also known to be remarkably skewed towards the Th2 phenotype (Del Prete et al., 1991) and large quantities of TES given to mice induces eosinophilia (Sugane and Oshima, 1984). These biological activities may soon be attributed to identified molecular products of the parasite.

There are practical potentials for much of this work. As the parasite is essentially a successful tissue transplant, residing in muscle or other soft tissue without rejection, we believe that parasite products could prove useful in enhancing tissue graft success in clinical medicine. On a more immediate basis, we have isolated a range of parasite proteins which can be synthesised in the laboratory, promising a much better diagnostic reagent for determining parasite infection whether in dogs, the natural host, or on the occasions when human infection occurs. Finally, and perhaps most importantly, the isolation of genes encoding the major larval antigens paves the way for development of a vaccine against canine toxocariasis, which may in future lead to the eradication of this threat to both human and veterinary health.

References

Badley, J. E., Grieve, R. B., Bowman, D. D., Glickman, L. T. and Rockey, J. H. (1987). Analysis of Toxocara canis larval excretory-secretory antigens: physicochemical characterization and antibody recognition. Journal of Parasitology 73, 593-600

Beaver, P. C. (1962). Toxocarosis (visceral larva migrans) in relation to tropical eosinophilia. Bulletin de la Société de Pathologie Exotique 55, 555-576

Beaver, P. C. (1966). Zoonoses, with particular reference to parasites of veterinary importance. Biology of Parasites (ed. Soulsby, E. J. L.) pp 215-227. Academic Press, New York. 

Blaxter, M. L., Page, A. P., Rudin, W. and Maizels, R. M. (1992). Nematode surface coats: actively evading immunity. Parasitology Today 8, 243-247

Burke, T. M. and Roberson, E. L. (1985). Prenatal and lactational transmission of Toxocara canis and Ancylostoma caninum : experimental infection of the bitch before pregnancy. International Journal for Parasitology 15, 71-75

Campbell, D. H. (1936). An antigenic polysaccharide fraction of Ascaris lumbricoides (from hog). Journal of Infectious Diseases 59, 266-280

de Savigny, D. H. (1975). In vitro maintenance of Toxocara canis larvae and a simple method for the production of Toxocara ES antigen for use in serodiagnosis test for visceral larva migrans. Journal of Parasitology 61, 781-782

Del Prete, G., De, C. M., Mastromauro, C., Biagiotti, R., Macchia, D., Falagiani, P., Ricci, M. and Romagnani, S. (1991). Purified protein derivative of Mycobacterium tuberculosis and excretory-secretory antigen(s) of Toxocara canis expand human T cells with stable and opposite (type 1 T helper or type 2 T helper) profile of cytokine production. Journal of Clinical Investigation 88, 346-350

Fattah, D. I., Maizels, R. M., McLaren, D. J. and Spry, C. J. F. (1986). Toxocara canis: interaction of human eosinophils with infective larvae in vitro. Experimental Parasitology 61, 421-433

Gems, D. H. and Maizels, R. M. (1996). An abundantly expressed mucin-like protein from Toxocara canis infective larvae: the precursor of the larval surface coat glycoproteins. Proceedings of the National Academy of Sciences USA 93, 1665-1670

Ghafoor, S. Y. A., Smith, H. V., Lee, W. R., Quinn, R. and Girdwood, R. W. A. (1984). Experimental ocular toxocariasis: a mouse model. British Journal of Ophthalmology 68, 89-96

Gillespie, S. H. (1987). Human toxocariasis. Journal of Applied Bacteriology 63, 473-479

Gillespie, S. H. (1993). The clinical spectrum of human toxocariasis. Toxocara and Toxocariasis: Clinical, Epidemiological and Molecular Perspectives (ed. Lewis, J. W. and Maizels, R. M.) pp 55-61. Institute of Biology, London.-

Glickman, L. T. and Schantz, P. M. (1981). Epidemiology and pathogenesis of zoonotic toxocariasis. Epidemiologic Reviews 3, 230-250

Griesemer, R. A., Gibson, J. P. and Elsasser, D. S. (1963). Congenital ascariasis in gnotobiotic dogs. Journal of the American Veterinary Medical Association 143, 962-964

Hotez, P.J., Wilkins, P.P. (2009) Toxocariasis: America's most common neglected infection of poverty and a helminthiasis of global importance? PLoS Negl Trop Dis 3, e400.

Khoo, K.-H., Maizels, R. M., Page, A. P., Taylor, G. W., Rendell, N. and Dell, A. (1991). Characterisation of nematode glycoproteins: the major O-glycans of Toxocara excretory secretory antigens are methylated trisaccharides. Glycobiology 1, 163-171

Khoo, K.-H., Morris, H. R. and Dell, A. (1993). Structural characterisation of the major glycans of Toxocara canis ES antigens. Toxocara and Toxocariasis: Clinical, Epidemiological and Molecular Perspectives (ed. Lewis, J. and Maizels, R. M.) pp 133-140. Institute of Biology, London.

Lloyd, S. (1993). Toxocara canis : the dog. Toxocara and Toxocariasis: Clinical, Epidemiological and Molecular Perspectives. (ed. Lewis, J. W. and Maizels, R. M.) pp 11-24. Institute of Biology, London.-

Loukas, A.C., Hintz, M., Tetteh, K.K.A., Mullin, N.P., Maizels, R.M. (2000) A family of secreted mucins from the parasitic nematode Toxocara canis bear diverse mucin domains but share similar flanking six-cysteine (SXC) repeat motifs. Journal of Biological Chemistry 275, 39600-39607.

Loukas, A. C., Mullin, N. P., Tetteh, K. K. A., Moens, L. and Maizels, R. M. (1999). A novel C-type lectin secreted by a tissue-dwelling parasitic nematode. Current Biology 9, 825-828

Loukas, A. C., Doedens, A., Hintz, M. and Maizels, R. M. (2000). Identification of a new C-type lectin, TES-70, secreted by infective larvae of Toxocara canis , which binds to host ligands. Parasitology 121: 545-554. 

Maizels, R. M., de Savigny, D. and Ogilvie, B. M. (1984). Characterization of surface and excretory- secretory antigens of Toxocara canis infective larvae. Parasite Immunology 6, 23-37

Maizels, R. M., Kennedy, M. W., Meghji, M., Robertson, B. D. and Smith, H. V. (1987). Shared carbohydrate epitopes on distinct surface and secreted antigens of the parasitic nematode Toxocara canis. Journal of Immunology 139, 207-214

Maizels, R. M. and Page, A. P. (1990). Surface associated glycoproteins from Toxocara canis L2 parasites. Acta Tropica 47, 355-364

Maizels, R. M., Tetteh, K. K. A. and Loukas, A. C. (2000) Toxocara canis : genes expressed by the arrested infective larval stage of a parasitic nematode. International Journal of Parasitology 2000, 30:495-508.

Meghji, M. and Maizels, R. M. (1986). Biochemical properties of larval excretory- secretory (ES) glycoproteins of the parasitic nematode Toxocara canis . Molecular and Biochemical Parasitology 18, 155-170

Nichols, R. L. (1956). The etiology of visceral larva migrans. I. Diagnostic morphology of infective second-stage Toxocara larvae. Journal of Parasitology 42, 349-362

Page, A. P., Rudin, W., Fluri, E., Blaxter, M. L. and Maizels, R. M. (1992a). Toxocara canis : a labile antigenic coat overlying the epicuticle of infective larvae. Experimental Parasitology 75, 72-86

Page, A. P., Hamilton, A. J. and Maizels, R. M. (1992b). Toxocara canis: monoclonal antibodies to carbohydrate epitopes of secreted (TES) antigens localize to different secretion-related structures in infective larvae. Experimental Parasitology 75, 56-71

Schabussova I, Amer H, van Die I, Kosma P, Maizels RM. O-Methylated glycans from Toxocara are specific targets for antibody binding in human and animal infections. International Journal of Parasitology 2007, 37:97-109.

Scothorn, M. W., Koutz, F. R. and Groves, H. F. (1965). Prenatal Toxocara canis infection in pups. Journal of the American Veterinary Medical Association 146, 45-48

Smith, H. V. (1991). Immune evasion and immunopathology in Toxocara canis infection. Parasitic Nematodes -Antigens, Membranes and Genes. (ed. Kennedy, M. W.) pp 116-139. Taylor and Francis, London. 

Smith, H. V. (1993). Antibody reactivity in human toxocariasis. Toxocariasis: epidemiological, clinical and molecular perspectives. (ed. Lewis, J. a. M., R.M.) pp in press. Institute of Biology, London. 

Smith, H. V., Kusel, J. R. and Girdwood, R. W. A. (1983). The production of human A and B blood group like substances by in vitro maintained second stage Toxocara canis larvae : their presence on the outer larval surfaces and in their excretions/secretions. Clinical and Experimental Immunology 54, 625-633

Smith, H. V., Quinn, R., Kusel, J. R. and Girdwood, R. W. A. (1981). The effect of temperature and antimetabolites on antibody binding to the outer surface of second stage Toxocara canis larvae. Molecular and Biochemical Parasitology 4, 183-193

Smith, H., Holland, C., Taylor, M., Magnaval, J.-F., Schantz, P., Maizels, R.M. (2009) How common is human toxocariasis? Towards standardising our knowledge. Trends in Parasitology 25:182-188

Sprent, J. F. A. (1952). On the migratory behavior of the larvae of various Ascaris species in white mice. I. Distribution of larvae in tissues. Journal of Infectious Diseases 90, 165-176

Sprent, J. F. A. (1958). Observations on the development of Toxocara canis (Werner,1782) in the dog. Parasitology 48, 184-210

Sugane, K. and Oshima, T. (1983). Purification and characterization of excretory and secretory antigen of Toxocara canis larvae. Immunology 50, 113-120

Sugane, K. and Oshima, T. (1984). Induction of peripheral blood eosinophilia in mice by excretory and secretory antigen of Toxocara canis larvae. Journal of Helminthology 58, 143-147

Tetteh, K. K. A., Loukas, A., Tripp, C. and Maizels, R. M. (1999). Identification of abundantly-expressed novel and conserved genes from infective stage larvae of Toxocara canis by an expressed sequence tag strategy. Infection and Immunity 67, 4771-4779

Warren, E. G. (1969). Infections of Toxocara canis in dogs fed infected mouse tissues. Parasitology 59, 837-841