Toxocara canis: Molecular basis of immune recognition and evasion

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Veterinary Parasitology 193 (2013) 365 374Contents lists available at SciVerse ScienceDirectVeterinary Parasitologyjo u rn al hom epa ge : www.elsev ier .com/ locate /vetparToxoca e reRick M. MInstitute of Imm ories, Wa r t i c l e i n f oKeywords:AntibodiesDiagnosisLarva migransMucinsSurface coata b s t r a c tToxocara canis has extraordinary abilities to survive for many years in the tissues of diversevertebrate species, as well as to develop to maturity in the intestinal tract of its deni-1. IntroduToxocaraworm of dopotential fotoxocariasis Tel.: +44 1E-mail add0304-4017/$ http://dx.doi.otive canid host. Human disease is caused by larval stages invading musculature, brain andthe eye, and immune mechanisms appear to be ineffective at eliminating the infection.Survival of T. canis larvae can be attributed to two molecular strategies evolved by the par-asite. Firstly, it releases quantities of excretorysecretory products which include lectins,mucins and enzymes that interact with and modulate host immunity. For example, onelectin (CTL-1) is very similar to mammalian lectins, required for tissue inammation, sug-gesting that T. canis may interfere with leucocyte extravasation into infected sites. Thesecond strategy is the elaboration of a specialised mucin-rich surface coat; this is looselyattached to the parasite epicuticle in a fashion that permits rapid escape when host antibod-ies and cells adhere, resulting in an inammatory reaction around a newly vacated focus.The mucins have been characterised as bearing multiple glycan side-chains, consisting ofa blood-group-like trisaccharide with one or two O-methylation modications. Both thelectins and these trisaccharides are targeted by host antibodies, with anti-lectin antibodiesshowing particular diagnostic promise. Antibodies to the mono-methylated trisaccharideappear to be T. canis-specic, as this epitope is not found in the closely related Toxocara cati,but all other antigenic determinants are very similar between the two species. This distinc-tion may be important in designing new and more accurate diagnostic tests. Further toolsto control toxocariasis could also arise from understanding the molecular cues and stepsinvolved in larval development. In vitro-cultivated larvae express high levels of four mRNAsthat are translationally silenced, as the proteins they encode are not detectable in culturedlarvae. However, these appear to be produced once the parasite has entered the mam-malian host, as they are recognised by specic antibodies in infected patients. Elucidatingthe function of these genes, or analysing if micro-RNA translational silencing suppressesproduction of the proteins, may point towards new drug targets for tissue-phase parasitesin humans. 2013 Elsevier B.V. All rights reserved.ction canis is the most prevalent intestinal round-gs, foxes and other canid species, with zoonoticr human beings. In many temperate countries, is the most common helminth infection and31 650 5511; fax: +44 131 650 5450.ress: a signicant morbidity (Despommier, 2003; Hotezand Wilkins, 2009; Rubinsky-Elefant et al., 2010; Smithet al., 2009).The nematode has many salient biological features,which contribute to its continuing presence in animaland human populations. Firstly, it is able to invade anextraordinarily wide range of hosts, from invertebrates andpoultry (Galvin, 1964) through to mice and man (Strubeet al., 2013); for all but the denitive (canid) species,these represent intermediate hosts which can, through see front matter 2013 Elsevier B.V. All rights reserved.rg/10.1016/j.vetpar.2012.12.032ra canis: Molecular basis of immunaizels unology and Infection Research, University of Edinburgh, Ashworth Laboratcognition and evasionest Mains Road, Edinburgh EH9 3JT, United Kingdom366 R.M. Maizels / Veterinary Parasitology 193 (2013) 365 374predation, allow the parasite to reach its nal denitivehost species. Secondly, the larval stage is able to enter along-term developmental arrest, which allows it to sus-pend life cycle progression whilst in a paratenic host, and toresume macanid specital arrest opregnancy colostrum tet al., 2011)T. canis that are nits infectiviimpossible.longevity: iple, larvae were able tWithin widely incluvous systemvisceral larvand Rocha, the brain acern in the(OT) a recorotoxocariaprevalence and ndinget al., 1985infected wicacy becauthe incompWisniewsk2. ImmunPathogesystem reatures associlipopolysachydrates. Ssentinel cepresence ofspecic resCurrently, fterns (PAparasite, buby the strothis infectioresponse isciated withPrete et al.,Such a a specic scytokines Iquently, IL-class-switceosinophilstion (Beavehuman beiulated by tparasite (Del Prete et al., 1991). However, no individual Tcell specic antigen or epitope has yet been dened fromT. canis.Recently, the distinction between innate and adap-arms o identily proequiri). Indee popnse t IL-5, earasiteected nd late4+ T cg thattive Tyeneratitive eostic lot reoyed. respeed, an reactin whiction inere arti-TESd, usinositiv3% in c of th et al.n trop; Magreportsz et a) and 9o 27% ) and esia (sis reme que relevations gcant isotypse, tistosomugh re larva is whetion anssibly as infed to tionallyappinganismturation to the adult stage only once reaching aes. Thirdly, even in female canids, developmen-ccurs so that larvae can dwell in tissues untiloccurs, and then migrate transplacentally or viao infect the foetal or newborn pup ( highly prevalent in all canid populationsot treated regularly with anthelmintics, andty to wild species renders elimination almost Furthermore, the arrested state has remarkablen experimentally infected monkeys for exam-remained viable in the tissues for 9 years ando infect mice on transfer (Beaver, 1962).the paratenic host, T. canis larvae can migrateding the liver, musculature and the central ner-, causing the well-characterised syndrome ofa migrans (VLM) (Beaver et al., 1952; Carvalho2011; Schantz, 1989). The propensity to invadend the eye has given rise to particular con- human population, with ocular toxocariasisgnised syndrome (Good et al., 2004), and neu-sis (NT) inferred from cognitive decits, higheramong epilepsy cases (Quattrocchi et al., 2012), larvae in post-mortem brain samples (Hill). Current options for treatment of humansth tissue-dwelling larvae are of uncertain ef-se of the covert nature of the infection andlete resolution of symptoms (Othman, 2012;a-Ligier et al., 2012).e recognitionns are recognised initially by the innate immunecting to intrinsically foreign molecular signa-ated with xenogeneic species, such as bacterialcharide, unmethylated DNA and fungal carbo-uch signals are essential for innate immunells, such as dendritic cells, to react to the infectious organisms and to initiate pathogen-ponses from the adaptive immune system.ew such pathogen-associated molecular pat-MPs) have been dened for any helmintht their existence in T. canis can be surmisedng adaptive immune response that occurs inn. The major feature of this adaptive immune production of T. canis-specic antibodies, asso- CD4+ T-helper type 2 cell (Th2) activity (Del 1991).Th2 response is characterised by release ofubset of mediators, in particular the type 2L-4, -5, -10 and -13, during infection. Subse-4 promotes B cell differentiation and antibodyhing, while IL-5 drives the differentiation of, a marked feature of human Toxocara spp. infec-r et al., 1952). T cell responses to T. canis inngs are clearly of the Th2 type, and are stim-he excretorysecretory (ES) antigens of thetive withrapidout r2012innatrespoducethe pin inf10) ain CDcatinadapGdendiagnern bemplwithbe usbodyand iinfecThof anworlserop7 to 213.9%(Won50% i1987larly (Jaro2007ing t2008IndoncariaThmostinfecsigniIgG4intenschisalthoceralissueinfecor posuchRelatfunctby trmechf immunity has become much more blurredcation of innate lymphoid cells, which canduce Type-2 cytokines (Neill et al., 2010) with-ng activation by dendritic cells (Smith et al.,ed, it has been known for some time that someulations of cells can participate in the Th2o T. canis, for example non-B-non-T cells pro-ven in T cell-decient nude mice infected with (Takamoto et al., 1995). Moreover, eosinophiliamice shows a biphasic response with early (day (day 21) peaks; while the later peak is absentell-decient mice, the earlier one is intact, indi- it is generated by the innate rather than thepe-2 response (Takamoto et al., 1998).ion of specic antibodies provides the mostvidence for infection and is the basis for alltests for toxocariasis; with ELISA and West-activity to T. canis ES (TES) antigens generallyHowever, the diagnostic eld is still evolvingct to the optimal target antigens that shouldd more critically to the interpretation of anti-vity to an infection which is frequently coverth symptoms do not necessarily correlate withtensity or antibody titre (Smith et al., 2009).e now extensive data on the seroprevalence antibodies in human populations around theg the established ELISA. In older studies,ity in Europe ranged from 3 to 7% in adults andhildren (Gillespie et al., 1993), while in the USAose over 6 years of age were antibody-positive, 2008). Remarkably, seroprevalences exceededical settings such as the Caribbean (Bundy et al.,naval et al., 1994). More recent studies simi- seroprevalences of 14.5% in Polish teenagersl., 2010), 13% in Turkish children (Dogan et al.,% in Iranian under-10s (Fallah et al., 2007), ris-in Brazilian Amazonia (Rubinsky-Elefant et al.,4582% in different rural districts of Sulawesi,Hayashi et al., 2005). Hence widespread toxo-ains of great concern in most parts of the world.stion of which serum antibody isotypes arent still remains to be explored. Most humanenerate antibodies of the IgG1 subclass, withlevels of both IgM and IgE (Smith, 1993). Thee, which can be predominant in other, moresue helminth infections such as lariasis andiasis (Maizels et al., 1995) is less prominent,portedly more evident in active cases of vis-migrans (Obwaller et al., 1998). An unresolvedther the expression of IgE correlates with actived/or invasion of certain tissue sites in the host,mediates only bystander allergic-type reactionsection-related rashes (Magnaval et al., 2006).his question is whether particular isotypes are important in tissue immunity, for example larvae or activating Fc-dependent protections.R.M. Maizels / Veterinary Parasitology 193 (2013) 365 374 367Recent analyses of differential expression proles ofanti-Toxocara spp. antibody isotypes, studying childrenreceiving thiabendazole therapy, found that specic IgEand eosinophilia declined within the rst year, with IgAand IgG4 fainformationnew immuthe success3. ImmuniAlthoughave centrimmune resworms is aof researchcine againstimmunity tsite.Intestinastood; takimodels of postrongylupredicted tthe parasiteparticipatinhas been renumerous (Junginger is involved strategies timmunity, of helminthImmunistood, althohosts, but to generaterodents hanoted that adult wormsurviving chtibility to lawith UV-irret al., 1991dence of prolarvae to pmore likely2011). A motion and adresultant imtion.4. ToxocarMany, bcara spp. in(reviewed with T. canmore pronoindividualssymptoms rhinitis (Yariktas et al., 2007). Mice infected with T. canisdeveloped compromised lung function for up to 60 days,associated with bronchioalveolar eosinophilia and serumIgE production (Pinelli et al., 2005). Moreover, using a pro- in mie modeumin Pinelli a brose huer, 20ter to a whicies aral imever, it denitsts suost maasionng-terpressnse, wediatoing ortinal trody, r inamit is imanisme celle tissuels anprodurate su dama a pr otherly resissing reosinrelate, 1999-5T mbelow), 2008)ge in linishe(Takameddine inabro, in ated. ophilsn 24 hh et alling at later times (Elefant et al., 2006). Such may prove useful in addressing the need fornological markers that could be used to chart of therapy in drug-treated patients.ty in the denitive hosth almost all immunological studies on T. canised a round the tissue-migrating larvae, theponse of the nal canid host to intestinal adultlso of central importance. A long-term aim in this area is to develop an effective vac- canine toxocariasis, which ideally would buildo both tissue- and intestinal stages of the para-l immunity to ascarid worms is poorly under-ng a comparative approach, based on animaltaxonomically distant nematodes (e.g. Nip-s brasiliensis, Trichuris muris), it would behat Th2-dependent mechanisms act to expels, but the cell types and molecular mediatorsg in expulsion are as yet unknown. Notably, itported that Foxp3+ regulatory T cells are morein dogs with intestinal nematode infectionset al., 2012), suggesting that mucosal tolerancein persistence of infection. Thus, interventionalo neutralise Tregs could promote protectiveas has been reported in some animal models infection (Taylor et al., 2007).ty to the larval stage is similarly poorly under-ugh important not only to the denitive canidalso in the human setting. To date, attempts protective anti-larval immunity in laboratoryve had mixed results. While an early reportimmunisation with somatic extracts of eggs ors resulted in approximately half the number ofallenge larvae (Izzat and Olson, 1970), suscep-rval infection was unaltered in mice immunisedadiated eggs or larval ES antigens (Abo-Shehada). Likewise, a more recent study found no evi-tection by administering a second challenge ofreviously infected mice, other than they were to sequester in the brain (Kolbekov et al.,re systematic approach, comparing immunisa-juvant regimes and following more closely themune response, would help clarify this ques-a spp. and allergyut not all, studies have linked human Toxo-fection with exacerbation of allergic diseaseby Pinelli and Aranzamendi (2012)). Childrenis seroreactivity have been reported to showunced IgE and eosinophilia than seronegative as well as greater propensity for airway allergicsuch as asthma (Buijs et al., 1997) and allergictocolto thovalbogy (Ondispo(Coopcounesis,allerggeneHowto itstal hothe h5. EvLoan imrespolar minvadintesthe blittlefore, mechOnin thMaizious geneothertainlymanylargeexprehypean unet IL(see et al.chanis dimtrols 6. ShThin vitincubeosinwithi(Fattace which induces an airway allergic responsel antigen ovalbumin, T. canis infection prior tochallenge was found to exacerbate lung pathol- et al., 2008).ader scale, infection with T. canis might pre-man beings towards development of asthma08). Such pro-allergic effects seemingly run recent interpretation of the hygiene hypoth-h suggests that parasitic helminths dampennd other immunopathologies through theirmune suppressive properties (Maizels, 2005). is important to recognise that T. canis is adaptedive host rather than to intermediate or acciden-ch as the human, and in the maladapted setting,y react more vigorously and pathologically. of the immune responsem survival of parasitic helminths in the host isive feat in the face of the well-armed immunehich mobilises multiple cell types and molecu-rs, including high-afnity antibodies, to attackganisms. T. canis larvae invade from the hostact and disseminate throughout the tissues ofemaining in an extracellular state, generatingmatory reaction at the site of infection. There-plicit that T. canis is able to disable host effectors in an extremely effective fashion. type, associated with immunity to helminthses, is the eosinophil (Klion and Nutman, 2004;d Balic, 2004), which is able to exgorge nox-cts such as major basic protein, as well asperoxides (through eosinophil peroxidase) andging free radicals. Although eosinophilia is cer-ominent feature of toxocariasis, as indeed in helminth infections, it appears that T. canis isstant to attack by this cell type. In mice over-an IL-5 transgene (IL-5T) and with resultantophilia, T. canis larvae are unharmed, althoughd helminth N. brasiliensis is eliminated (Dent). Notably, when N. brasiliensis is introducedice in the presence of T. canis ES antigens, their survival is greatly enhanced (Giacomin. Conversely, in IL-5-decient mice there is noarval survival, although pathology in the lungd compared to wild-type (IL-5-sufcient) con-oto et al., 1997).g of the surface coatility of eosinophils to kill T. canis was observedexperiments in which cells and larvae are co-While in the presence of specic antibodies, rapidly adhered to the parasite surface, but the larvae had escaped from surrounding cellsl., 1986), leaving the eosinophils still attached368 R.M. Maizels / Veterinary Parasitology 193 (2013) 365 374to material which had clearly become detached from thesurface (Badley et al., 1987a).These observations of shedding cells bound to the para-site surface have an interesting link to electron microscopicobservationdistal to anlarvae are can be visuathe same dieach cuticlefuzzy coat cferritin, indNotably, thvae with anphysical baby T. canis lWhile temployed pmonoclonaMAbs desiglive larvae, strated by imcoat (Page ies were usdistinct secgland opensecretory cet al., 1992aThe Tcn-the surfaceride glycanare expresspally MUC-et al., 2000bound by tGerm Agglutherefore srich assembfurther covmolecular cture.7. TES antiantigensWhile thlular attackgeneral fearesponse toinammato(Kuroda et dose of JapaPavri, 1987the suppresin murine tunderpin soIt is likesite infectioproducts, sously, TES c(Giacomin eboth eosinophils (Sugane and Oshima, 1984) and alterna-tively activated macrophages (Allen and MacDonald, 1998).However, TES is a complex mixture of differing molecularcomponents, which require further characterisation if thec immdersto Savigrkableof ES a TESoses. Wibed bith maDa (Mverlap.bsequPAGE t., 1987iqueso acidh degr of TEal sectis releaver suencingng (Bobinanels, 19f TES ) whileised.TES-26S-26 dylethed Tdant mtode 2ly in Ce the other s mar contalogouone, Snthus Kber of but alas no kTES-32S-32 , 2000ences val mbers oigned s of a labile surface coat from the larval parasite,d separated from the nematode cuticle. Whentreated with a lipophilic stain, a fuzzy coatlised, 1020 nm thick and lying approximatelystance from the cuticle surface, while following annulation and fold (Page et al., 1992b). Thean be stained with polycationic ligands such asicating that it bears an overall negative charge.e coat is lost within 2 h of co-incubating lar-tibodies reactive to the surface, elucidating thesis of the original report of antibody sheddingarvae in vitro (Smith et al., 1981).he rst observations of antibody sheddingolyclonal sera, later studies conrmed this withl antibody (MAb) reagents. In particular, twonated Tcn-2 and Tcn-8, which bind strongly toand which are rapidly shed, were also demon-muno-electron microscopy to bind the surfaceet al., 1992b). Interestingly, the same antibod-ed to stain sections of larvae, and identied tworetory bodies within the larva, the oesophagealing into the buccal cavity, and the mid-bodyolumn which is a ducted secretory pore (Page).2 and Tcn-8 monoclonal antibodies which bind coat, recognise related but distinct trisaccha- side-chains (Schabussova et al., 2007), whiched on the family of mucin polypeptides, princi-1, -2 and -3 collectively termed TES-120 (Loukasb). The coat and the secretory column are alsohe N-acetylglucosamine-specic lectin, Wheattinin (Page et al., 1992c). While the fuzzy coateems likely to represent a mucin- and glycan-ly, it is not yet determined if the mucins arealently modied in any way, or linked to otheromponents, in order to form a stable coat struc-gens and immune modulation by TESe ability of larvae to escape antibody and cel- is unusual among helminth parasites, a moreture is modulation of the systemic immune infection. In T. canis infected mice, the pro-ry responses of macrophages are dampenedal., 2001), while susceptibility to a sub-lethalnese encephalitis virus is increased (Gupta and). Recent work has reported an expansion ofsive T cell subset of Foxp3+ regulatory T cellsoxocariasis (Othman et al., 2011), which mayme of these in vivo that both local and systemic effects of para-n on host immunity are mediated by secreteduch as the TES antigens. As mentioned previ-an inhibit the protective effects of eosinophilst al., 2008) and is able to induce recruitment ofspecibe underemation suchpurpdescrsis, w400 ksive oclearSuSDS-et altechnaminA hig40%logicTES iOsequbindirecomMaizsets o-120acter7.1. TEphatirenamabunnemaviousWhilfromdiffersion,homoanemhelianumspp., yet h7.2. TEet al.sequof larmemreassunomodulatory effects of the parasite are tood.ny (1975) rst reported that T. canis larvae show longevity in vitro, allowing long-term collec-ntigens over many months. As detailed below, proved to be extremely useful for diagnosticithin TES, the molecular components were rsty their molecular weight on SDS-PAGE analy-jor bands evident at 26, 32, 45, 55, 70, 120 andaizels et al., 1984); even at this stage the exten- between surface proteins and those in TES wasent studies of TES used 1- and 2-dimensionalo more precisely classify components (Badleyb; Meghji and Maizels, 1986), together with such as biosynthetic labelling with radioactives (Page and Maizels, 1992; Sugane et al., 1985).ee of glycosylation was observed, amounting toS by weight (Meghji and Maizels, 1986). Histo-ons of liver from infected mice also showed thatsed from larvae in vivo (Parsons et al., 1986).bsequent years, a combination of peptide (Loukas et al., 1999b), monoclonal antibodywman et al., 1993; Maizels et al., 1987) andt DNA techniques (Gems et al., 1995; Gems and96; Maizels et al., 2000) has characterised threeproteins and glycoproteins (TES-26, -32/70 and the other components remain to be fully char-: Tc-PEB-1is a homologue of mammalian phos-anolamine (PE)-binding protein and wasc-PEB-1. It was identied initially from anRNA bearing at its 5 end the conserved a2-nt spliced leader (SL1) sequence found pre-aenorhabditis elegans (Krause and Hirsh, 1987).T. canis PEB protein is similar to homologuesspecies in functionally binding PE, its structurekedly in including a 72-aa N-terminal exten-ining a tandemly repeated six-cysteine motifs to the potassium channel toxin of the seatocihactis helianthus (Gems et al., 1995). The S.-toxin (ShKT) domain gures prominently in aother secreted proteins not only from Toxocaraso from other nematode parasites, although asnown functional role. and -70: C-type lectins (CTLs)(Loukas et al., 1999b) and TES-70 (Loukasa) were isolated by matching tryptic peptidefrom gel-puried proteins to an EST databaseRNA sequences (Tetteh et al., 1999). Both aref the C-type lectin family; hence TES-32 wasat Tc-CTL-1. This is homologous to mammalianR.M. Maizels / Veterinary Parasitology 193 (2013) 365 374 369mannose-binding protein, and modelling studies whichindicated that Tc-CTL-1 does indeed bind mannose residueswere experimentally conrmed (Loukas et al., 1999b).Within the EST database were two related sequences withminor aminCTL-2 and -by an indep(Yamasaki eTc-CTL-1and monocexpressed iWhether this secreted ronment, reto be well-cTc-CTL-1 Mcati (Kenneet al., 1991)tion, there antibodies procyonis (Breaction wiand mice inantibodies tTES-70 Tc-CTL-4, win vitro. Althtied, this targeting hoantibodies other Toxoclectins migh1991).7.3. TES-12As withcorrespondpresence ofserved sequwithout thement of spmost highlydesignated larvae expresive Serineof O-linkeddescribed pmucins are3 (Loukas eMUC-4 andture and thresidues (Lotandemly rN- and C-te8. GlycansInitial ctent of galac40% by totMaizels, 1986). However, there is only a limited degree ofN-glycoslyation, of CTL-1 and -4, as revealed by molecu-lar weight shifts following N-glycanase digestion of totalTES products. In contrast, O-linked sugars represent therity ofucin fass sp to be-Me)GAc, d bearsbly, thntical cturalcitiess of the, 2001odies armed tono-mpresenture (SbsequAc, oe or th loss e-Gal, 2012es in TinantAc-Glcmunoe origd, 197gh fotivity ester extraS has iagnoensitivstic terns th-reacter of lood gcular-wherefoodies t26 an, 1991,combtion ootein as et antidition also rin beto acid variants which have been designated Tc-3. A near-identical sequence was later describedendent group as a proteoglycan core proteint al., 1998). is also one of the major larval surface proteins,lonal antibody localisation has shown that it isn the cuticle of the parasite (Page et al., 1992a).e cuticular form bears any modication, and/ordirectly from the cuticle into the larval envi-mains to be determined. Tc-CTL-1 also appearsonserved within the Toxocara genus; the anti-ab Tcn-3 reacts with proteins in both Toxocarady et al., 1987) and Toxocara vitulorum (Page, albeit of differing molecular weights. In addi-is signicant cross-reactivity of Tc-CTL-1 withgenerated to the raccoon ascarid Baylisascarisoyce et al., 1988). However, there is no cross-th ES from Toxascaris leonina (Page et al., 1991)fected with Ascaris suum do not develop serumo a further C-type lectin, now referred to ashich has been shown to bind to canine cellsough the mammalian ligand has yet to be iden-is the rst instance of a TES product directlyst receptors (Loukas et al., 2000a). Monoclonalto Tc-CTL-4 also react with products in ES ofara species, indicating that secretion of C-typet be a shared hallmark of this genus (Page et al.,0: mucins TES-26, the original isolation of cDNA clonesing to TES-120 antigens was facilitated by the the SL1 trans-spliced leader sequence. The con-ence of the SL1 permitted full-length cloning, requirement for a cDNA library or develop-ecic probes (Gems and Maizels, 1996). The expressed gene encoded a mucin sequence,MUC-1. Further studies established that T. canisss a set of mucin-like glycoproteins with exten--Threonine rich domains which act as sites glycosylation, forming a surface fuzzy coatreviously (Page et al., 1992b). The principal MUC-1 (Gems and Maizels, 1996), -2 and -t al., 2000b) with less abundant components -5 (Doedens et al., 2001), differing in struc-e relative proportions of Serine or Threonineukas et al., 2000b). Most strikingly, all containepeated ShKT domains at either or both of therminal regions, anking the central mucin core. of T. canishemical analysis of TES revealed a high con-tose and N-acetylgalactosamine, amounting toal weight of the secreted products (Meghji andmajothe mMthem2(4-O3GalNsugarNotais idea struspecithesiet al.antibconthe mtope strucSu3GalNfucosstratethe Met al.specia domGlcN9. ImThTizarthrousensiand Wwormof TEcial dthe sagnoconcecrossnumbThe bmoleand tantib(TES-et al.Redetec32 pr(LoukserumIn adwereprote the released carbohydrates, mostly linked toamilies (MUC-1 to -5).ectrometry of the O-linked sugars determined two related trisaccharides, 2-O-Me-Fuc1-al1-3-GalNAc, and 2-O-Me-Fuc1-2Gal1-iffering only in whether the central galactose an O-methyl side chain (Khoo et al., 1991).e non-O-methylated form of this trisaccharideto the human blood group H antigen, providing explanation for the expression of blood group by T. canis (Smith et al., 1983). Chemical syn- Toxocara spp.-specic glycan structures (Amer) provided material for ELISA with monoclonalnd human sera (Schabussova et al., 2007). Thishat the species-specic Mab, Tcn-2, recognisesethylated trisaccharide, while the shared epi-t in other Toxocara species is the dimethylatedchabussova et al., 2007).ently, synthesis of the glycan Fuc1-2Gal1-mitting methylation of either the terminale intermediate galactose, was used to demon-of antibody binding, formally conrming that residue is essential for this reaction (Koizumi). In addition, analysis of the N-linked glycanES antigens has been undertaken, identifying and relatively simple biantennary Man2Man-NAc side chain (Khoo et al., 1993).-diagnosisinal reports from De Savigny (de Savigny and7; de Savigny et al., 1979) provided a break-r immuno-diagnosis of toxocariasis, as theand specicity of TES in assays such as ELISAn Blot proved to be far superior to use of wholects (Van Knapen et al., 1983). Indeed, the usebeen successfully translated into a commer-stic kit (Jacquier et al., 1991). Nevertheless,ity and specicity of TES-based immunodi-sts vary around the 90% level, and there areat the presence of blood group-like or otherive carbohydrates could be responsible for afalse-positive results (Smith et al., 1984, 2009).roup-like antigens are more abundant on highereight TES components (TES-120 and TES-400),re a Western blotting procedure to assay humano the lower molecular weight TES componentsd TES-32) has also been developed (Magnaval 2002).inant proteins are now being developed forf specic antibodies for toxocariasis. The TES-(which corresponds to the C-type lectin CTL-1al., 1999b)) was shown to be detected bybodies from all 9 toxocariasis patients tested., sera from animals carrying T. cati infectioneactive, reecting the cross-reactivity of thisween Toxocara species (Kennedy et al., 1987).370 R.M. Maizels / Veterinary Parasitology 193 (2013) 365 374In a standard ELISA assay, some 43% of sera from otherhelminth infections cross-reacted with whole TES, but thiswas reduced to 2% when tested against recombinant CTL-1 adsorbed at an optimal antigen concentration (Yamasakiet al., 2000)(12%) of chCTL-1 (TESshould be CTL-1 was required 8 Mfolded versimmunoreawell as lineMost diabodies. In assay, it wa(as many pspecicity wtotal IgG (Nto developthree recom1 or TES-3TES120). W100% specidetect all sashowed crofrom other As well structures immuno-ditoxocariasisture 2-O-Mno antibodmonomeththe case wrestricted tUsing poELISA, circuserum of inf1984), and (Luo et al., found in boples, with tha species-sption (Gilles10. GenomanalysesDespite and zoonottion availabCurrently, tHowever, thpublished sequence oreported (JeSimilarlyexplored (GEST projectgenes and dmost of the major secreted antigens and a number of novelgene families, many of which are listed in Table 1 (Tettehet al., 1999). A further 1975 gene clusters were assembledfrom 5000 Sanger sequencing reads and can be searchedp://neencings and most opositeng onlgene stious toire c pro the lay molel (22it trandationA binstreamAs ha et al notab well-cros etAs ha that arval site disestinglspondr geneed witch traelegangene wgenic ved (C of can exprees strouturepandivasionge boes to dy beiner res the ssary inrch ne T. canvene tth theld spoby T. unomoave th. In a subsequent evaluation, sera from 26/215ildren from Pernambuco, Brazil, recognised Tc--32) (De Andrade Lima Colho et al., 2005). Itnoted that in both studies, recombinant Tc-produced as inclusion bodies in bacteria and urea solubilisation, indicating that a correctlyion of the same antigen may show increasedctivity as this would detect conformational asar epitopes.gnostic serological tests measure total IgG anti-a comparative study to test an IgG4-specics noted that sensitivity was markedly reducedatients do not mount an IgG4 response) butas greatly enhanced compared to measuringoordin et al., 2005). These authors went on an IgG4-specic diagnostic assay employingbinant antigens (Tc-PEB-1 or TES-26, Tc-CTL-2 also named TES-30USM, and Tc-MUC-1 orhile none of the antigens individually showedcity, a combination of the three was able tomples serologically reactive to native TES, andss-reactivity with only 24% of serum samplesinfections (Mohamad et al., 2009).as recombinant proteins, the specic glycanassociated with T. canis also hold promise foragnosis. It was found that sera from human patients reacted to the di-O-methylated struc-e-Fuc1-2(4-O-Me)Gal1-3-GalNAc, althoughies were detected to the species-specicylated form Schabussova et al. (2007). As is oftenith glycan epitopes, human antibodies wereo the IgM and IgG2 isotypes.lyclonal antibodies against TES in a sandwichlating parasite antigens can be detected in theected dogs, particularly pups (Matsumura et al.,in serum of children with suspected infection1999). Similarly, circulating antigen could beth human and experimental mouse serum sam-e monoclonal antibody Tcn-2 which recognisesecic repetitive epitope that facilitates detec-pie et al., 1993; Robertson et al., 1988).ics, transcriptomics and other molecularthe importance of T. canis in both veterinaryic contexts, there is relatively little informa-le at the genomic and transcriptomic levels.he parasite genome has yet to be sequenced.e 14,162-bp mitochondrial sequence has been(Jex et al., 2008), and the 273-Mb genomef the related parasite A. suum was recentlyx et al., 2011)., the T. canis transcriptome is relatively under-asser, 2013). Over 10 years ago a small-scale on larval cDNAs was able to identify 128 distinctespite its limited scope, this study characterisedat httsequlectinever,or deholditein ambireperspeciInlatorsmalinhibdegramiRNdownmiRN(Chenmostvides(AmbmiRNnotedthe ldespIntercorreeithepressof eain C. ANT transachiementgenestudi11. FExand emanastudialreafurthTES anecereseadoesinteror boshousion immcan More recently, a female adult cDNA effort yielded 79 genes, including 2 C-type an As16 homologue (Zhou et al., 2011). How-f the identied genes have yet to be annotatedd in public databases, with NCBI for exampley 28 distinct non-mitochondrial/ribosomal pro-equences at the time of writing. Much moredatasets are required to establish the proteinof T. canis and to identify patterns of stage-tein expression in the parasite life few years, a new class of biological regu-cules has been dened with the discovery of-nucleotide) RNA species which bind to andslation of target mRNAs and mediate their within the cell. Most documented cases ofding are to the 3 untranslated region (3 UTR) of the translational stop codon. Abundantve been described in several nematode species., 2011; Poole et al., 2010; Winter et al., 2012),ly the free-living worm C. elegans, which pro-haracterised conserved sequences such as let-7 al., 2003; Hammell et al., 2009). Although nove yet been described in T. canis, it had been4 abundant novel transcript (ANT) genes fromtage shared highly similar 3 UTR sequencessimilar coding sequences (Tetteh et al., 1999).y, in the larval parasite, no evidence of proteinsing to the ANT genes was found, indicating that transcription or mRNA translation was sup-hin the arrested stage. Within the 3 UTR tractsnscript were motifs similar to those targeteds by let-7, and when the 3 UTR of a T. canisas inserted downstream of a GFP construct inC. elegans, suppression of gene expression wasallister et al., 2008). Thus, although the involve-onical miRNA mechanisms in control of larvalssion has yet to be directly demonstrated, thesengly suggest that this is the case. prospects and prioritiesng our understanding of immune recognition by T. canis is a priority if we are to appropriatelyth human and veterinary disease. Moleculardate have identied key products which areg used in new diagnostic assays, and doubtlessnement will lead to the replacement of nativeprincipal diagnostic reagent. Beyond this verynovation, two general and interrelated areas ofed to be addressed: by what molecular meansis suppress host immunity, and how can weo provide immunological protection in either denitive and paratenic host? These questionstlight the highly evolved state of immune eva-canis, with every probability of dening newdulatory molecules. In this way, future worke way for rational immunological tools, mostR.M. Maizels / Veterinary Parasitology 193 (2013) 365 374 371Table 1Dened proteins of Toxocara canis.Abbreviation Name TES componentif applicableAccessionnumber(s)Length (inclsignalsequence ifpresent)Notes ReferenceTc-ANT-3 Abundant novel transcript-3 EU792508;ACF19852271 aa Callister et al.(2008)Tc-ANT-5 Abundant novel transcript-5 EU792509;ACF19853489 aa Callister et al.(2008)Tc-ANT-30 Abundant novel transcript-30 EU792510;ACF19854843 aa Callister et al.(2008)Tc-ANT-34 Abundant novel transcript-34 EU792511;ACF19855608 aa Callister et al.(2008)Tc-AQP-1 Aquaporin AAC32826 310 aa Loukas et al.(1999a)Tc-ARK-1 Arginine kinase ABK76312;AFJ95132UnpublishedTc-CPL-1 Cathepsin L-like cysteineproteaseAAC48340 360 aa Loukas et al. (1998)Tc-CPZ-1 Cathepsin Z-like cysteineproteaseAF143817 307 aa Falcone et al.(2000)Tc-CTL-1 C-type lectin-1 TES-32 AF041023 219 aa Binds mannose Loukas et al.(1999b)Proteoglycan core protein AB009305 219 aa Differs by 3 aa fromaboveYamasaki et al.(1998)Tc-CTL-2 C-type lectin-2 219 aa 17% aa divergence fromCTL-1Tetteh et al. (1999)Tc-CTL-3 C-type lectin-3 220 aa 13% aa divergence fromCTL-1Tetteh et al. (1999)Tc-CTL-4 C-type lectin-4 TES-70 AF126830 288 aa Binds canine cell surface Loukas et al.(2000a)Tc-GLB-1 Pseudocoelomic globin AAL56430;AAL58703;AAL58704171 aa Minor sequence variants UnpublishedTc-GLB-2 Intracellular globin AAL56428;AAL56429153 aa 50% identity to GLB-1 UnpublishedTc-LDH-1 Lactate dehydrogenase AAB07368 92 aa Partial sequence UnpubishedTc-MUC-1 Mucin-1 TES-120 AAB05820;U39815176 aa 15.7 kDa Serine-richpeptide backbone with 2ShKT domains, 39.7 kDatotal mass; 120 kDaapparent mobility onSDS-PAGEGems and Maizels(1996)Tc-MUC-2 Mucin-2 TES-120 AF167707 182 aa 16.2 kDa peptide with 2ShKT domains; 47.8 kDatotal.Loukas et al.(2000b)Tc-MUC-3 Mucin-3 TES-120 AF167708 269 aa 26.0 kDa threonine-richpeptide with 4 ShKTdomains, 45.0 kDa total.Loukas et al.(2000b)Tc-MUC-4 Mucin-4 TES-120 AF167709 191 aa 26.0 kDa threonine-richpeptide with 4 ShKTdomains, 45.0 kDa total.Tetteh et al. (1999)Tc-MUC-5 Mucin-5 AF167710 316 aa 26.0 kDa threonine-richpeptide with 4 ShKTdomains, 45.0 kDa total.Doedens et al.(2001)Tc-MHC-1 Myosin heavy chain CAC28360 1814 aa Obwaller et al.(2001)Tc-MLC-1 Myosin light chain U25057 148 aa UnpublishedTc-NPA-1 Nematode polyprotein BAA14015;AAD01628;AAB26196140 aa Fragments of longerpolyproteinChristie et al.(1993) andunpublishedTc-PEB-1 Phosphatidylethanolamine-bindingproteinTES-26 P54190,U29761262 aa PE-binding domain fusedwith 2 ShKT domainsGems et al. (1995)Tc-PRO-1 Prohibitin AAB53231 274 aa Loukas and Maizels(1998)Tc-SLO-1 Calcium activated channel ACJ64718 1123 aa UnpublishedTc-SOD-1 CuZn superoxide dismutase AAB00227 190 aa UnpublishedNote: Gene abbreviations follow the convention of two-letter species abbreviation, a three-letter code for gene name (capitalised when referring to a proteinproduct) and a number to distinguish related members of the same gene family.372 R.M. Maizels / Veterinary Parasitology 193 (2013) 365 374importantly vaccines for use in canid species, to mimimise,if not altogether eradicate, this important infection.Conict of interestNone.AcknowledThroughthe work oson, David GDebbie Calllate Huw SmFunding:Wellcome TReferencesAbo-Shehada,nity to ToxAllen, J.E., Maceration m20, 2412Ambros, V., LeNAs and o807818.Amer, H., HomethylateToxocara lBadley, J.E., Gmediated the role 133143.Badley, J.E., GrAnalysis ocochemica593600.Beaver, P.C., 19ical eosinoBeaver, P.C., SChronic eoBowman, D.D.stage larvaAscaridoidBoyce, W.M., sis of larvToxocara cimmunoasBuijs, J., BorsbJansen, G.tations anelementarBundy, D.A.P., tionships prevalencParasitol. 3Callister, D.M.dant novesequencesof gene exCarvalho, E.A.Achildren. JChen, M.X., AiY.C., Tian, cantonensiand femalChristie, J.F., Dnematodeterminal sClin. Exp. 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Parasitol. 129,Toxocara canis: Molecular basis of immune recognition and evasion1 Introduction2 Immune recognition3 Immunity in the definitive host4 Toxocara spp. and allergy5 Evasion of the immune response6 Shedding of the surface coat7 TES antigens and immune modulation by TES antigens7.1 TES-26: Tc-PEB-17.2 TES-32 and -70: C-type lectins (CTLs)7.3 TES-120: mucins8 Glycans of T. canis9 Immuno-diagnosis10 Genomics, transcriptomics and other molecular analyses11 Future prospects and prioritiesConflict of interestAcknowledgementsReferences


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