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Recent advances in the immunologyand diagnosis of echinococcosis Molecular Parasitology Laboratory, Australian Centre for International and Tropical Health and Nutrition, The Queensland Institute of Medical Researchand The University of Queensland, Brisbane, Queensland, Australia Molecular Parasitology Laboratory, Australian Echinococcosis is a cosmopolitan zoonosis caused by adult or larval stages of Centre for International and Tropical Healthand Nutrition, The Queensland Institute of cestodes belonging to the genus Echinococcus (family Taeniidae). The two major species of medical and public health importance are Echinococcus granulosus and Echinococcus multilocularis, which cause cystic echinococcosis and alveolar echi- Australia. Tel.: 161 7 3362 0401; fax 161 7 nococcosis, respectively. Both cystic echinococcosis and alveolar echinococcosis are serious diseases, the latter especially so, with a high fatality rate and poor prognosisif managed inappropriately. This review highlights recent advances in immunity to infection and vaccination against both parasites in their intermediate and definitive hosts and procedures for diagnosis of cystic echinococcosis and alveolar First published online 21 February 2006.
echinococcosis, including the value of immunodiagnostic and DNA approaches.
There is discussion also of progress in genomics and related technologies that isproviding valuable insights on the functional biology of the Echinococcus organ- isms. These studies will underpin future research that will reveal a better under-standing of the Echinococcus-host interplay, and suggest new avenues for the identification of additional targets for diagnosis, vaccination and chemotherapy.
Echinococcus granulosus; Echinococcusmultilocularis; echinococcosis; immunity toinfection; vaccination; diagnosis;immunodiagnosis.
fibrous capsule. Brood capsules and protoscoleces (PSC) bud off from the germinal membrane. Definitive hosts are Echinococcosis is a cosmopolitan parasitic zoonosis caused carnivores such as dogs, wolves and foxes. Sexual maturity of by adult or larval stages of cestodes belonging to the genus adult E. granulosus occurs in the host small intestine within Echinococcus (family Taeniidae). Larval infection (hydatid 4–5 weeks of ingesting offal containing viable PSC. Gravid disease; hydatidosis) is characterized by long-term growth of proglottids or released eggs are shed in the faeces and, metacestode (hydatid) cysts in the intermediate host. The following their ingestion by a human or ungulate host, an two major species of medical and public health importance oncosphere larva is released that penetrates the intestinal are Echinococcus granulosus and Echinococcus multilocularis, epithelium into the lamina propria. This is then transported which cause cystic echinococcosis (CE) and alveolar echino- passively through blood or lymph to the target organs where coccosis (AE), respectively. E. granulosus has a cosmopolitan it develops into a hydatid cyst. Since the life cycle relies on distribution (McManus et al., 2003) and E. multilocularis, carnivores eating infected herbivores, humans are usually a which is distributed in the northern hemisphere, is recog- ‘dead-end’ for the parasite. Adult worm infections of E.
nized as an emerging zoonosis in some regions of Europe multilocularis occur mainly in red and arctic foxes, although dogs and cats can also act as definitive hosts. Small mam- Hydatid cysts of E. granulosus develop in internal organs mals (usually microtine and arvicolid rodents) act as inter- (mainly liver and lungs) of humans and intermediate hosts mediate hosts. The metacestode of E. multilocularis is a (herbivores such as sheep, horses, cattle, pigs, goats and tumor-like multivesicular, infiltrating structure consisting of camels) as unilocular fluid-filled bladders. These consist of a numerous small vesicles embedded in stroma of connective parasite-derived inner nucleated germinal layer and an outer tissue; the larval mass usually contains a semisolid matrix acellular laminated layer surrounded by a host-produced rather than fluid (Eckert & Plazes, 2004). CE and AE are  2006 Federation of European Microbiological Societies FEMS Immunol Med Microbiol 47 (2006) 24–41 Published by Blackwell Publishing Ltd. All rights reserved Advances in immunology and diagnosis of echinococcosis both serious diseases, the latter especially so, with a high experimental infections of mice and sheep. After the onco- fatality rate and poor prognosis if careful clinical manage- sphere locates a target organ, the small hydatid cyst that commences development is immediately confronted by the In contrast to E. multilocularis, which appears to exhibit host immune responses, which are mainly cell-mediated, very limited genetic variation, an important feature of the especially involving infiltration of macrophages and eosino- biology of E. granulosus is that it comprises a number of phil cells, and low-level polarized Th1 responses. Antibody intraspecific variants or strains that exhibit considerable responses are weak and are, normally, undetectable in the variation at the genetic level. 10 distinct genetic types early two to three weeks following infection.
(genotypes G1–10) have been identified and this categoriza- There are extensive data on immune responses against the tion follows very closely the pattern of strain variation established cyst both from studies on patients with echino- emerging based on biological characteristics. The extensive coccosis and from experimentally infected animals (Zhang variation in nominal E. granulosus may influence life cycle et al., 2003a). The established parasite produces significant patterns, host specificity, development rate, antigenicity, quantities of antigens that modulate the immune responses transmission dynamics, sensitivity to chemotherapeutic and these include polarized Th2 responses, balanced with agents and pathology with important implications for the Th1 responses. The coexistence of elevated Th1 cytokines, design and development of vaccines, diagnostic reagents and especially interferon (IFN)-g, and Th2 cytokines including drugs. A detailed account of genetic variation in Echinococ- IL-4, IL-5, IL-6 and IL-10, has been recorded in most cus and its implications can be found in (McManus, 2006), hydatid patients where cytokine levels have been measured.
so this important topic will not be considered further here.
In addition, IgG, especially IgG1 and IgG4, IgE and IgM are In our earlier review on the immunology and diagnosis of elevated as the cyst grows and becomes established. When a echinococcosis (Zhang et al., 2003a), we considered immu- cyst dies naturally, is killed by chemotherapy treatment or is nity to infection in the intermediate and definitive hosts, removed by surgery, Th2 responses drop rapidly, and Th1 innate resistance, evasion of the immune system, develop- responses become dominant. IgG levels can be maintained ment of vaccines for use in intermediate and definitive hosts in humans for several years after the cyst has been removed.
and, in particular, we emphasized procedures for diagnosis Once a patient suffers relapse, the Th2 responses regenerate of CE and AE, including the value of immunodiagnostic and molecular approaches. Here we provide an update of recent In the early stages of echinococcal development, cellular progress in research on E. granulosus and E. multilocularis, responses may play a crucial role in protection against especially highlighting advances in immunology, vaccine infection. A repeat challenge experiment showed that mice development, diagnosis and functional expression of key given a second oncospheral challenge 21 days after the primary infection with E. granulosus produced very highlevels of protection (Zhang et al., 2001) but with a very low Immunity to infection in the intermediate antibody response (Zhang et al., 2003d) at the time of the secondary challenge. Early experiments in vitro showed thatneutrophils, in association with antibody, can bring about Although the host–parasite interplay, in most cases of echi- the killing of E. granulosus oncospheres (Rogan et al., 1992), nococcosis appears to be harmonious and clinically asympto- suggesting a possible role for antibody-dependent cell- matic for a long period after infection, the host does produce mediated cytotoxicity (ADCC) reactions although antibody a significant immune response against the early stages of levels against this stage are low and/or cell-mediated im- infection, while the parasite adapts highly effective evasive munity may induce killing. This is an important area that needs to be further explored as it may provide an under-standing of the mechanisms of protection against the onco- sphere with benefits for future vaccine design.
Clinical symptoms of CE reflect the presence of one or more A remarkable feature of CE infection is the coexistence of unilocular fluid-filled cysts. About 70% of cysts are formed both Th1 and Th2 responses. It is likely due to the presence in the liver, followed by the lungs (20%), with the remainder on echinococcal antigens of distinct epitopes for each T-cell involving other organs such as kidney, spleen, brain, heart subset as it has been shown that a single recombinant and bone. Clinical manifestations are mild in the early stage, protein can stimulate both types of response. The C- while as the cyst gradually grows, the parasite may physically terminal region of a heat shock protein, Eg2HSP70, induced damage tissues and organs, which can become dysfunctional significantly greater amounts of tumor necrosis factor at the later stages of echinococcosis. As discussed previously (TNF)-a, IFN-g, and IL-10 in Eg2HSP70-stimulated per- (Zhang et al., 2003a), immune responses against early E.
ipheral blood mononuclear cells (PBMC) from CE patients granulosus infection have mainly been investigated using compared with unstimulated cultures in all patients (Ortona FEMS Immunol Med Microbiol 47 (2006) 24–41  2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved et al., 2003). Furthermore, the antigens EgA31 and EgTrp common for CE infection in sheep (Zhang & Zhao, 1992; stimulated a significant amount of IL-12, IFN-g, IL-10 and Cabrera et al., 2003), and it most likely also happens in IL-6 cytokines in cytokine-producing splenocytes of BALB/c human populations in hyperendemic areas as patients with vaccinated with the two molecules compared with controls calcified cysts are often reported (Macpherson et al., 2004; Moro et al., 2005). Cytokines are likely to play a key role in The polarized T-cell responses are modulated by echino- coccal antigens. Dematteis et al. (2003) analysed whether the cytokine responses in early and late experimental infection (urticaria, itching and anaphylactic shock) often complicate with E. granulosus depend on the dose of parasites to which the course of CE. Some CE patients have elevated IgE levels the host is exposed. To this purpose BALB/c mice were to certain antigens, such as EgEF-1b/d, AE21 and EgTeg, that inoculated intraperitoneally (i.p.) with either 500 or 2000 appear to be associated with these allergic reactions (Coleb- protoscoleces. Splenocytes of mice were obtained at days 3, rook et al., 2002; Ortona et al., 2003; Vuitton, 2004).
7, 14 and 21 and also on week 37 post-infection and werecultured in vitro with protoscolex antigens. Type-1 and type- 2 cytokines were analysed in supernatants by enzyme-linkedimmunosorbent assay (ELISA). The lower number of pro- Human AE is a chronic and often fatal disease characterized toscoleces induced an early type-0 cytokine response, by slowly developing cysts, mainly in the liver. Pathologi- whereas the inoculation of 2000 protoscoleces induced an cally, the parasite destroys the liver parenchyma, bile ducts early Th2 response. Parasite growth was lower in the group and blood vessels resulting in biliary obstruction and portal inoculated with the low infective dose, which stimulated hypertension. In most late-stage cases a necrotic cavity, type-0 cytokine responses that may be protective, while containing a viscous fluid, may form in the liver. Like CE more protoscoleces may inhibit the protective Th0 or Th1 infection, Th1 responses predominate in the early stages of responses, invoking Th2 responses, which may be beneficial AE infection, with the immune response switching to a Th2 polarized profile in later progression (Shi et al., 2003; Wei To further investigate the role of T lymphocytes in the immune response to E. granulosus, Rigano et al. (2004) Another pathological characteristic of AE infection is the generated T-cell lines from patients with active, transitional strong cellular immune response elicited by E. multi- and inactive hydatid cysts and stimulated these using sheep locularis. This results in a large granulomatous infiltrate hydatid fluid (SHF) and antigen B (AgB). The cell lines from surrounding the parasitic lesions (Vuitton et al., 1989; a patient with an inactive cyst had a Th1 profile, while the Ricard-Blum et al., 1996; Grenard et al., 2001), which is a T-cell lines derived from seven patients with active and process simular to granuloma formation and the resulting transitional hydatid cysts had mixed Th1/Th2 and Th0 fibrosis associated with the pathology of schistosomiasis clones. The results showed that Th1 lymphocytes contribute (Wynn et al., 2004). The cells involved in the formation of significantly to the inactive stage of hydatid disease, with Th2 the periparasitic granuloma are mainly macrophages, myo- lymphocytes being more important in the active and transi- fibroblasts and T lymphocytes. In patients with abortive or tional stages. This group (Rigano et al., 1995) had previously dead lesions, a large number of CD4(1) T lymphocytes are shown that, in human subjects undergoing pharmacological present, whereas patients with active metacestodes display a treatment with albendazole/mebendazole, a Th1 cytokine significant increase in activation of predominantly CD8(1) profile, rather than a Th2 profile, typically dominates, T cells (Manfras et al., 2002), indicating that CD4(1) T cells indicating that Th1 responses have a role in the process of play a role in the killing mechanism. This is supported by cyst degeneration. An increased Th1-type cytokine IFN-g mouse experiments undertaken by Dai et al. (2004), who response has been suggested as a marker for monitoring AE infected mice with E. multilocularis of different genetic patient treatment (Dvoroznakova et al., 2004), whereas backgrounds including micro MT, nude, T-cell receptor measurement of serum IL-4 may be a useful marker for the follow up of patients with CE (Rigano et al., 1999).
(MHC)-I( À / À ) and MHC-II( À / À ) mice and found that Ultrasound can be used to classify cysts into different at 2 months post-infection, the parasite mass was more than clinical types according to the progression of cyst status 10 times higher in nude, TCR-b( À / À ) and MHC-II (Wang et al., 2003). There are no data showing cytokine ( À / À ) mice than in infected C57BL/6 wild-type (WT) profiles associated with these cyst categories and this is mice; furthermore these T-cell-deficient mice started to die clearly an area for future study. One aspect that is likely to of the high parasite load at this time-point. In contrast, be important is the influence of CD41 T-helper lympho- MHC-I( À / À ) and micro MT mice exhibited parasite cytes on the control of such immunological mechanisms as growth rates similar to those found in WT controls. These they may impact on treatment (Vuitton, 2004). Self-cure is findings clearly point to the major role that CD4(1) ab(1)  2006 Federation of European Microbiological Societies FEMS Immunol Med Microbiol 47 (2006) 24–41 Published by Blackwell Publishing Ltd. All rights reserved Advances in immunology and diagnosis of echinococcosis T cells play in limiting E. multilocularis proliferation, while parallels the production of TNF-a (Shi et al., 2003) indicat- CD8(1) T and B cells appeared to play a minor role in the ing that NO levels are enhanced by this cytokine. Antigens in control of parasite growth. In the absence of T cells, the laminated-layer of the cyst decrease NO production in especially CD4(1) or ab(1) T cells, the cellular immune vitro, indicating that E. multilocularis produces molecules response to infection was decreased which resulted in the that can modulate the host immune responses (Andrade lack of hepatic granuloma formation around the parasite. In addition, in T-cell-deficient mice, the expression of IFN-g Antibody levels were shown to be low early on in murine and other inflammatory cytokines (except for interleukin-6) (BALB/c) E. multilocularis infection but the levels of IgG1 were increased in association with a high parasite load.
and IgG3 increased significantly 8 weeks after challenge, and Thus, the relative protection mediated by CD4(1) ab(1) remained elevated throughout the 25 week period of ob- T cells against E. multilocularis infection seems not to be servation (Shi et al., 2003). The production of IL-2R and IFN-g dependent, but rather relies on the effector functions TNF-a by spleen cells from infected mice stimulated with of CD4(1) ab(1) T cells (Dai et al., 2004).
EmAg also increased significantly 8 weeks after infection, The precise role that each of the cytokines play in fibrosis while IL-2R sharply decreased after 12 weeks of infection.
and development of AE lesions remains to be determined.
During the period of 2–12 weeks after infection there was an Nevertheless, pretreatment of mice with interleukin (IL)-12 increase in IL-1 secretion. The levels of IL-1 and TNF-a was extremely efficient in preventing the development of rapidly increased during the 16 weeks postinfection. A high lesions and led to abortive parasitic vesicles surrounded by level of IFN-g was detected during the period of observa- fully efficient periparasitic immune cell infiltration and tion, and showed a peak at 12 weeks. These data indicate, as fibrosis (Emery et al., 1998). Also, 75% of mice treated with for E. granulosus, that Th1 is the major response in the early IFN-a-2a had no hepatic lesions and half were fully pro- stage of infection, which is replaced by a Th2 response in the tected. IFN-a-2a treatment markedly decreased the abnor- mally elevated production of IL-10 in both spleen cell Extracts from metacestodes of E. multilocularis cause cultures and peritoneal macrophage cultures from infected basophil degranulation, as well as the secretion of histamine, mice and restored phagocytosis and oxidative metabolism of IL-4 and IL-13, in a dose-dependent manner. IgE stripping macrophages. The treatment also inhibited IL-6 and IL-13 and resensitization of basophils indicating that the mechan- antigen-induced secretions in spleen cell cultures (Godot ism of IL-4 induction requires the presence of IgE on the et al., 2003) and may be useful for treatment of AE patients; cells (Aumuller et al., 2004). Echinococcus multilocularis may how these two Th1 cytokines may impact on the progression thus induce a Th2 response in their hosts by the induction of of AE is unknown but they likely act via inhibition of Th2 responses. IL-13 has clearly been shown to be a major factorin granuloma formation and the resulting fibrosis in schis- tosomal infection in the mouse model (Pearce & Mac- The life-cycles of E. granulosus and E. multilocularis include Donald, 2002), but there are no similar studies aimed at two hosts: an intermediate and a definitive host. Effective CE determining the role this cytokine plays in the hepatic control programs show that the prevention of transmission to either host can reduce or even eliminate the infection in Echinococcus multilocularis vesicle antigens have been human and livestock populations. Therefore, if either or shown to induce pro-inflammatory, regulatory and chemo- both hosts can be vaccinated, the effect will be to improve kine release by PBMC from patients (Eger et al., 2003). The and more rapidly expedite control. The sylvatic nature of the pro-inflammatory cytokines IL-1b and IL-18 were reduced lifecycle of E. multilocularis makes a vaccination approach to in echinococcosis patients; regulatory IL-10 was similar, but parasite vesicle-induced IL-8 was dominant and clearlyelevated in patients. Such selective and opposite dynamics of inflammatory cytokines and chemokine release mayprevent overwhelming and pathogenic inflammation, and Substantial progress has been made towards developing a constitute an appropriate response for attraction of effector practical, recombinant vaccine (EG95) for use against cells into the periparasitic tissues with the capacity to limit E. granulosus in sheep (Gauci et al., 2005). The EG95 E. multilocularis metacestode proliferation and dissemina- vaccine, cloned from the oncosphere, produces high levels of protection in terms of the reduction in the numbers of The production of nitric oxide (NO) by intraperitoneal hydatid cysts in sheep, goats and cattle following experi- macrophages of mice during secondary infection with E.
mental challenge in a number of countries, and natural multilocularis mediates immunosuppression at the early and challenge of sheep in China (Lightowlers & Heath, 2004).
late stages of infection (Dai et al., 2003). NO production First described nearly a decade ago, the EG95 vaccine has, FEMS Immunol Med Microbiol 47 (2006) 24–41  2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved however, yet to be introduced as part of any government- in the past two decades. As the major definitive hosts for E.
supported control program in areas endemic for hydatid granulosus, dogs play a pivotal role in the transmission of disease. Economic and political factors will likely impede the hydatid disease. Interruption of the parasite life cycle in the large-scale use of the vaccine (Gauci et al., 2005). Further dog host can provides a very acceptable and cost-effective developments in recombinant protein production and in complementary method for control by vaccination as there delivery of the EG95 vaccine may make it more amenable to are far fewer dogs than sheep on farms so that fewer animals widespread use. In this context, a commercial process was need vaccination and less vaccine doses would be necessary.
recently described for producing the recombinant vaccine in The fact that old animals have a lower abundance and/or E. coli (Manderson et al., 2005). Furthermore, Marsland prevalence rates of E. granulosus compared with young dogs et al. (2003) described the construction of a recombinant orf (Lahmar et al., 2001; Torgerson et al., 2003; Budke et al., (Parapoxvirus) virus expressing EG95 levels comparable to 2005; Buishi et al., 2005; Moro et al., 2005) provides that achieved by a similar vaccinia virus recombinant. This epidemiological evidence that canines may become resistant recombinant virus will be a valuable tool with which to to reinfection in nature. Similar results have been described assess the potential of recombinant orf viruses to deliver with E. multilocularis in naturally infected foxes (Hofer vaccine antigens to sheep. If the virus can pass vertically et al., 2000; Yimam et al., 2002; Losson et al., 2003). A from one generation to another, this would be a major step mathematical model has indicated that there is significant forward in the use of EG95 for the control of hydatid disease herd immunity in dogs under a relatively high infection Recent research indicates that the EG95 encoding gene It was found that when dogs were infected with belongs to a gene family of at least seven related genes, that E. granulosus, local and systemic specific antibodies and at least some of the proteins encoded by the eg95 gene family cellular responses were raised (Carmena et al., 2004, 2005; are expressed in other stages (immature and mature adult Moreno et al., 2004). While there was no relationship worms, protoscoleces) as well as in the oncosphere (Zhang between serum IgA responses and parasite burden at the et al., 2003b; Chow et al., 2004; Gauci et al., 2005). As end of the infection, an inverse association of antiparasite described earlier, E. granulosus exhibits extensive strain IgE and parasite load appeared to exist. No differences were variation, and variability of the eg95 gene in different isolates observed in the numbers of intestinal mast cells and goblet of E. granulosus may directly impact the effectiveness of the cells among all infected dogs (Moreno et al., 2004). Dogs EG95-based vaccine. Analysis of the eg95 gene family from infected with E. multilocularis also produced specific anti- E. granulosus collected in Xinjiang, in northwest China, bodies and T-cell responses to parasite antigens, although where hydatid disease is hyperendemic, showed the eg95 gene some modification of lymphocyte responses was apparent family was shown to comprise two basic cDNA sequence (Kato et al., 2005a), a situation also prevailing in the types but very limited sequence variation was evident in the Mongolian gerbil, a prednisolone-untreated rodent defini- EG95 protein from oncospheres (Zhang et al., 2003b). This tive host model (Kato et al., 2005b).
high degree of sequence conservation predicts that the In order to identify genes expressed only in mature adult vaccine will continue to be effective in China and elsewhere, worms that may encode targets for inhibiting egg produc- although this needs to be fully substantiated, particularly tion, thus forming the basis for developing a transmission should failures of the EG95 vaccine occur in the future.
blocking vaccine applicable to dogs, differences in mRNA A protein containing characteristic fibronectin III do- expression between immature adult worms and mature mains, related to EM95 (a homologue of EG95), that can adult worms of E. granulosus were determined using poly- induce significant levels of protection against challenge merase chain reaction-based differential display (DDRT- infection with E. multilocularis eggs in mice, has been PCR) (Zhang et al., 2003c). As a result, examination of the identified in E. multilocularis (Merckelbach et al., 2003).
deduced amino acid sequence of three of the corresponding Another recombinant E. multilocularis molecule, the 14–3–3 complimentary DNAs (cDNAs) (egM4, egM9 and egM123) protein, elicited a high degree of protection (97%) against a indicated they were cysteine-rich and contained a 24 amino primary egg challenge infection but no protection against acid repeat sequence, repeated four to six times. The repeat secondary infection in vaccinated mice (Siles-Lucas et al., regions were predominantly a helical in nature with inter- 2003); the results suggest the protective mechanisms were spersed turns, forming alternating zones of positive and effective only against the oncosphere.
negative charge. The functional significance of each of thecDNAs identified was unclear as none had significantsequence similarity to genes of known function. Never- theless, polypeptides encoded by egM4 and egM123 were In comparison with intermediate hosts of Echinococcus, recognized by antibodies in a serum pool from dogs immunization of canines has received very little attention experimentally infected with E. granulosus, suggesting they  2006 Federation of European Microbiological Societies FEMS Immunol Med Microbiol 47 (2006) 24–41 Published by Blackwell Publishing Ltd. All rights reserved Advances in immunology and diagnosis of echinococcosis could prove of value in serodiagnosis of definitive hosts. In exhibiting the requisite specificity and sensitivity is ever addition, pilot vaccine/challenge experiments in dogs with proteins encoded by egM4, egM9 and egM123 showed The lipoproteins antigen B (AgB) and antigen 5 (Ag5), encouraging results in terms of reduced egg production the major components of hydatid cyst fluid (HCF), have (Zhang, 2003), suggesting they may prove of value as been the two molecules that have received wide attention in components of a vaccine effective in the definitive host.
regards to diagnosis. Along with HCF, they are the mostwidely used antigens in current assays for immunodiagnosisof CE. Both antigens have been well characterized by immunoblotting and/or by immunoprecipitation of radi- Early diagnosis of CE and AE can provide significant olabeled antigen and sodium dodecyl sulphate-polyacryl- improvements in the quality of the management and treat- amide gel electrophoresis (SDS-PAGE). Although AgB and ment of both diseases. In most cases, the early stages of Ag5 have proved to be diagnostically valuable, there are infection are asymptomatic, so methods that are relatively difficulties related to their lack of sensitivity and specificity easy to use and that are cheap are required for large-scale and problems with the standardization of their use. Cross- epidemiological surveillance of populations at high risk.
reactivity with antigens from other parasites, notably other Immunodiagnosis provides such an approach and can, taeniid cestodes, is a major problem.
additionally, confirm clinical findings. The definitive diag-nosis for most human cases of CE is by physical imaging methods, such as radiology, ultrasonography, computedaxial tomography (CT scanning) and magnetic resonance Antigen B is a polymeric lipoprotein with a molecular imaging although such procedures are often not readily weight of 120 kDa. It can be measured in patient blood as available in isolated communities. Immunodiagnosis can circulating antigen and it has been suggested that AgB has an also play an important complementary role. It is useful not important role in the biology of the parasite and its relation- only in primary diagnosis but also for follow-up of patients ship with the host. AgB is a highly immunogenic molecule, a after surgical or pharmacological treatment. Additional characteristic that underpins its value in serodiagnosis. It advantages of immunodiagnosis include screening of large appears ladder-like under reduced condition on SDS-PAGE populations in communities from endemic areas, rapid with three bands (subunits AgB1, AgB2 and AgB3) with testing of individuals in remote areas where imaging equip- molecular sizes of approximately 8 or 12, 16 and 24 kDa, ment may not be readily available, for follow-up monitoring suggestive that it comprises polymers of 8 kDa subunits. The of subjects in endemic areas, and for confirmation of CE or smallest subunit has proved the most useful target in AE cases when physical imaging does not provide a defini- diagnostic studies. A possible new AgB subunit (AgB4) was tive diagnosis (see Qaqish et al., 2003; Hernandez et al., recently identified (Arend et al., 2004) and recent work 2005). Immunodiagnosis can also play a major role in the shows that AgB is encoded by a multigene family (Haag detection of AE infection, which is very important for early et al., 2006). Furthermore, AgB presents homology to, and commencement or treatment, because of the high associated shares apparent structural similarities with, helix-rich hy- drophobic ligand binding proteins (HLBPs) from othercestodes, and has fatty acid binding properties (Chemaleet al., 2005).
Two residues in the AgB sequence are critical for diagnosis The detection of circulating Echinococcus granulosus anti- (Gonzalez-Sapienza & Cachau, 2003). The N-terminal ex- gens in sera is less sensitive than antibody detection, which tension of the major subunit of AgB concentrates the remains the method of choice. CE serology has a very long immunoreactive B-cell epitopes of the native molecule. The history and almost all serological tests that have been nature of this immunodominance was analyzed using four developed have been used in the diagnosis of human cases.
monoclonal antibodies (mAbs) defining overlapping epi- There are considerable differences between the various tests, topes in this region of the AgB molecule. The minimal both in specificity and sensitivity. As the sensitivity of a test epitope requirements of these mAbs were determined using increases, so generally does the demand for improved phage display peptide libraries. The consensus sequences antigens in order that sufficient specificity can be achieved isolated with the mAbs, and alanine replacement analysis to take advantage of the greater sensitivity. An optimum test with synthetic peptides mapped the relevant molecular should be specific with high sensitivity. Insensitive tests have contacts within a short stretch corresponding to residues been replaced by the ELISA and immunoblotting (IB) in 17–24 of the AgB major subunit. Substitution of two critical routine laboratory applications (see, e.g. Nasrieh & Abdel- residues within this stretch produced a dramatic loss of Hafez, 2004) although the choice of diagnostic antigens antigenicity, as determined using patient sera.
FEMS Immunol Med Microbiol 47 (2006) 24–41  2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved To date, several AgB cDNAs have been cloned, expressed the Ag5 gene by reverse transcription-PCR on the basis of as recombinant proteins and used for diagnosis; in addition, the amino acid sequences of tryptic fragments. The nucleo- a number of AgB peptides have been synthesized and used in tide sequence indicated that Ag5 is synthesized as a single ELISA for diagnostic purposes. Peptide antigens have been polypeptide chain that is afterwards processed into single considered as a way to enhance specificity and efforts have disulphide-bridged 22 and 38 kDa subunits. Whereas the been made to define discrete epitopes of AgB and other 22-kDa component contains a highly conserved glycosami- molecules that could be mimicked by synthetic peptides.
noglycan-binding motif that may help to confine Ag5 in the Two of the recombinant subunit components (rAgB8/1 and host tissue surrounding the parasite, the 38 kDa subunit is rAgB8/2) of AgB have been used widely in diagnosis.
closely related to serine proteases of the trypsin family Virginio et al. (2003) showed that, of six purified recombi- (Lorenzo et al., 2003a). However, neither proteolytic activity nant proteins tested in ELISA for specific IgG with a panel of nor binding to protease inhibitors could be detected using sera from patients with surgically confirmed or immunolo- native purified Ag5. Thus it may be possible that Ag5 gically diagnosed CE, AgB8/2 provided the highest diagno- possesses a highly specific physiological substrate or, more stic sensitivity (93.1%) and specificity (99.5%). Further- likely, that trypsin-like folding has been recruited to fulfil more, different IgG isotypes showed dominance in the novel functions. Subsequently, this group (Lorenzo et al., response for each of the recombinant antigens. There was a 2005) prepared two recombinant forms of the antigen, clear predominance of IgG4 response for all antigens tested, namely, rAg5 (corresponding to the unprocessed polypep- indicating that this would be the subclass of choice to be tide chain of the antigen) and rAg5–38s (corresponding to assessed for these, and possibly other, recombinant proteins.
its 38 kDa subunit). Their antigenicities were compared to The detection by immunoblotting of antibodies specific for that of the native antigen using a human serum collection.
the 8 kDa subunit of antigen B and in particular the IgG4 There was a major drop in the reactivity of the sera, subclass expression, has also been advocated by Siracusano particularly against rAg5–38s, which was confirmed by et al. (2004) as a promising serodiagnostic tool.
analysis of the cross-reactivity of two panels of monoclonal To compare variability between laboratories, six South antibodies specific for rAg5–38s and the native antigen.
American groups double-blind tested six antigens against Using the chemically deglycosylated native antigen, these the same serum collection (Lorenzo et al., 2005). High inter- authors demonstrated that the reduced antigenicity of the center reproducibility was attained and the results showed recombinants was due to the loss of the sugar determinants, that HCF, native AgB and its recombinant AgB8/1 subunit and not to their misfolding. Inhibition experiments using had almost the same efficiency at 81.4, 81.3 and 81.9%, phosphorylcholine confirmed that this moiety also contri- respectively, with the diagnostic efficiencies for an AgB- butes to the reactivity of the antigen, but to a much lesser derived synthetic peptide peptide, the recombinant AgB8/2 extent. The presence of immunodominant highly cross- subunit, and recombinant cytosolic malate dehydrogenase reactive glycan moieties in the Ag5 molecule may involve a from E. granulosus (EgMDH) being less efficient at 76.8, parasite evasion mechanism, as was earlier shown for AgB 69.1 and 66.8%, respectively. Different regional batch pre- parations of HCF yielded different diagnostic performances Studies by Mahmoud & Abou Gamra (2004) have shown but the group recommended that recombinant AgB should that a purified alkaline phosphatase (EgAP) extracted from be used as the standard antigen in laboratory analysis.
E. granulosus hydatid cyst membranes possesses exceptional Using a novel approach, Chen et al. (2003) generated a diagnostic characteristics with 100% specificity without any genetically engineered antibody against recombinant AgB decrease in sensitivity (100%) with significant potential for that may have potential implications in immunological use in routine diagnosis and follow-up of CE patients. This treatment and drug targeting delivery.
mirrors the diagnostic value previously shown for purifiedalkaline phosphatase (pAP) from E. multilocularis metaces-todes (see Zhang et al., 2003a).
There have been few studies on Ag5 in recent years. Ag5 is a very high molecular weight (c. 400 kDa) lipoprotein com- plex composed of 57 and 67 kDa components that underreducing conditions dissociate into 38 and 22–24 kDa sub- In a significant advance, Li et al. (2003) used a pool of serum units. Historically, one of the most used immunodiagnostic samples from mice infected with oncospheres (eggs) of E.
procedures for CE was the demonstration of serum anti- granulosus to screen a cDNA library constructed with RNA bodies precipitating antigen 5 (arc 5) by immunoelectro- extracted from protoscolex larvae from sheep hydatid cysts.
phoresis or similar techniques. Although they did not carry One immunoreactive clone, designated EpC1, was shown to out any serodiagnostic studies, Lorenzo et al. (2003a) cloned encode a protein of 76 residues. The cDNA fragment was  2006 Federation of European Microbiological Societies FEMS Immunol Med Microbiol 47 (2006) 24–41 Published by Blackwell Publishing Ltd. All rights reserved Advances in immunology and diagnosis of echinococcosis subcloned into an expression vector, pET-41b(1), and the other parasite infection sera using camel hydatid cyst fluid as resulting recombinant EpC1 glutathione S-transferase antigen (Al-Sherbiny et al., 2004). Since the dipstick assay is (GST) fusion protein (rEpC1–GST) was expressed in Escher- extremely easy to perform with a visually interpreted result ichia coli and was affinity purified against the GST tag.
within 15 min, in addition to being both sensitive and Immunoglobulin G was the dominant antibody isotype specific, the test could be an acceptable alternative for use generated against rEpC1–GST. A total of 896 human serum in clinical laboratories lacking specialized equipment and samples were used to evaluate the diagnostic sensitivity and the technological expertise needed for western blotting or specificity of the fusion protein by immunoglobulin G immunoblotting; 324 serum samples from patients withCE, 172 from patients with neurocysticercosis, 89 frompatients with alveolar echinococcosis, and 241 from patients Application of serodiagnosis for evaluation of with other infections or clinical presentations, as well as 70 from confirmed-negative control subjects, yielded an overall An important area for study is the evaluation of the sensitivity of 92.2% and an overall specificity of 95.6%. The diagnostic potential of serodiagnostic procedures in the combined levels of sensitivity and specificity achieved with field. Qaqish et al. (2003) used ELISA to determine the the rEpC1-GST fusion protein for diagnosis of CE were seroprevalence of CE in different communities in Jordan.
unprecedented, taking into account the large panel of serum The rural-agricultural subjects were significantly more likely to be seropositive (11.4%) than the semi-Bedouin (5.0%) or In addition to testing recombinant antigen B subunits, Virginio et al. (2003) assessed the diagnostic potential in In another study, Hernandez et al. (2005) screened ELISA of a cytosolic isoform of malate dehydrogenase villagers in an area with high prevalence of CE in rural (EgcMDH), an EF-hand calcium-binding protein (Eg- Tacuarembo, Uruguay. The correlation between serological CaBP2), and a full-length (EgAFFPf) and a truncated form data and the incidence of risk factors carried out on 480 (EgAFFPt, aa 261370) of an actin filament fragmenting individuals who were examined by means of abdominal protein. These recombinant antigens yielded sensitivities sonography (local prevalence = 0.8%). Serum samples (305) between 58.6 and 89.7%, and three of them were considered were analysed by ELISA to determine specific IgG against crude antigens from E. granulosus. A total of 27 individuals Purified recombinant thioredoxin peroxidase of E. gran- exhibiting no detectable changes in abdominal sonographic ulosus (TPxEg) was used to screen sera from heavily infected examination were found to be seropositive (‘ultrasound mice and patients with confirmed hydatid infection. Only a normal group’). Of these individuals, nine were seroreactive portion of the sera reacted positively with the EgTPx-GST against purified AgB. A significant degree of correlation was fusion protein in Western blots (69.3% specificity and 39% found between seroreactivity and the incidence of some risk sensitivity with human sera), suggesting that EgTPx may factors (CE antecedent in the family, P o 0.005 and use of form antibody-antigen complexes or that responses to the rural water, P o 0.0001) among this group. Follow-up of EgTPx antigen may be immunologically regulated (Li et al., individuals of the ‘ultrasound normal group’ was carried out after 2 years to evaluate the implications of this serological A dipstick assay has been developed that exhibited 100% reactivity. No predictive value for cyst development was sensitivity and 91.4% specificity with 26 CE sera and 35 assessed with complementary image study; in contrast Table 1. Performance comparison of recombinant EpC1-GST protein with native hydatid cyst fluid antigen B (HCF-AgB) for human cysticechinococcosis (CE) serodiagnosis by immunoblotting (data summarized from Li et al. 2003, 2004) ÃPartially tested.
CE, cystic echinococcosis; AE, alveolar echinococcosis; NCC, neurocysticercosis.
FEMS Immunol Med Microbiol 47 (2006) 24–41  2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved transient antibodies were observed with both crude and In order to address this issue, Lawn et al. (2004) investi- purified antigen as approximately 60% of individuals be- gated whether concentrations of CE-specific IgG subclasses came negative when re-sampled. This study showed that 1–4 in ELISA using crude horse hydatid cyst fluid as an serodiagnostic data can prove useful for evaluating the antigen correlated better with disease activity than total IgG.
They studied a cohort of patients with symptomatic CE Deutz et al. (2003) investigated the seroprevalence of a treated with anthelminthic drugs and surgery and who were range of zoonotic pathogens, including E. granulosus and E.
followed up clinically and radiologically for several years multilocularis, in hunters originating from the south-eastern (Lawn et al., 2004). Changes in concentrations of antibodies Austrian federal states of Styria and Burgenland, and were correlated with clinical and radiologic outcome. At compared the results with other predisposed occupational diagnosis, concentrations of CE-specific total IgG, IgG1 and groups. The high seroprevalences for CE and AE, in addition IgG2 antibodies were significantly elevated in a greater to several other pathogens, demonstrated that hunters are proportion of patients compared with IgG3 and IgG4 particularly exposed to zoonotic pathogens, which is per- antibodies. The fact that using cyst fluid antigen rather haps not surprising giving their occupation and lifestyle than purified antigen (AgB or Ag5) yielded better responses could be that crude antigens enhance detection ofsubclass antibodies with different antigen specificities withIgG2 antibodies being most sensitive. Importantly, during post-treatment follow up, the IgG2 antibody response Despite the development of sensitive and specific techni- provided the best correlate of disease activity, with serum ques, such as immunoblotting and ELISA, the immunodiag- concentrations of CE-specific antibodies showing that IgG2 nosis of CE in clinical practice and population studies correlated most strongly with clinical outcome (Lawn et al., remains a complex task and three major problems still remain. The first problem is that most available screeningtests give a high percentage of false negative results which Immunodiagnosis of CE in animal intermediate can be as much as 50% with sera taken from communities in population surveys (Moro et al., 2005). To explore possiblefactors associated with false negative antibody response in In comparison with investigations in humans, relatively immunodiagnosis of CE patients, Xu et al. (2002) deter- little research has been directed toward the development of mined IgG subclasses (IgG1, IgG2, IgG3, IgG4) and IgA, immunodiagnostic techniques for E. granulosus infection in IgM and IgE in the sera of individuals with a negative total domesticated animals such as sheep and cattle. Currently, IgG response; they concluded that the seronegative response diagnosis of CE in intermediate hosts is based mainly on of total IgG in CE patients might be due to low levels of necropsy procedures. Accurate serological diagnosis of CE specific IgG, variant Ig antibody expression and/or forma- infection in livestock is difficult due to serological cross- tion of circulating immune complexes, and that the com- reactions with several other species of taeniid cestodes bined detection of IgG11IgA1IgM could enhance the including Taenia hydatigena and Taenia ovis. Furthermore, sensitivity of serological tests in CE patients (Xu et al., 2002).
natural intermediate host animals produce very poor anti- The second issue relates to putative false positive reac- body responses to infection compared with the relatively tions, the causes for which may be very complex. Cross- high levels of specific antibody seen in human infection. In reactivity with antibodies from other infections may be one sheep, the principal intermediate host of E. granulosus in reason for this false-positivity, but it may also be due to the most endemic regions of the world, antibodies to various fact that CE antibodies can remain in serum for long periods antigens including antigen 5 are detectable in the sera of following surgical removal or effective drug treatment of some, but not all infected sheep (‘nonresponders’). As with cysts, or even if the infection self-cures as discussed earlier.
human CE, the detection of circulating antigen does not The third problem is how to distinguish active or appear to be useful for diagnostic purposes.
progressive cases of echinococcosis from cured individuals.
Ibrahem et al. (2002) used AgB, partially purified from It has been shown in a number of studies that IgG, especially hydatid cyst fluid from camels or sheep, and a recombinant IgG1 and IgG4, can remain circulating in the human blood form of AgB (r-AgB) in an ELISA, to screen panels of serum system for more than 5 years (Bulut et al., 2001; Li et al., samples from slaughtered camels and sheep naturally in- 2003; Nasrieh & Abdel-Hafez, 2004), with IgA and IgM also fected with CE (Ibrahem et al., 2002). Seroreactivity, how- detectable in serum 3 years after surgical cyst removal (Doiz ever, was variable. Native AgB gave the highest sensitivity et al., 2002). In addition, significantly high levels of specific (97%) in ELISA for camel CE. In contrast, r-AgB gave lower IgE serum antibodies determined by ELISA were still detect- sensitivity for camel (84%) and sheep (28%) CE. The r-AgB- able one year after surgery (Bulut et al., 2001).
ELISA was, however, highly specific, yielding 90 and 95%  2006 Federation of European Microbiological Societies FEMS Immunol Med Microbiol 47 (2006) 24–41 Published by Blackwell Publishing Ltd. All rights reserved Advances in immunology and diagnosis of echinococcosis specificity, respectively, for natural camel and sheep CE emphasized earlier, AE is a very serious disease with a high fatality rate, so early detection is paramount in order that Kittelberger et al. (2002) carried out a very extensive successful management and treatment can commence.
study aimed at developing an immunological method for Em2, a species-specific native antigen isolated from the the identification of sheep infected with E. granulosus which metacestode of E. multilocularis (Gottstein, 1992) has been would allow the monitoring of animals imported into used successfully over a long period for immunodiagnosis of countries free from hydatidosis, and as an aid to countries human AE; the sensitivities of Em2 with ELISA vary where control schemes for the disease are in operation.
depending upon the geographical origin of the patient, Three ELISAs were developed and validated, using as anti- ranging between 77 and 92%. In addition, serology for gen purified 8 kDa AgB hydatid cyst fluid protein (8kDaE- antibodies against the Em2 antigen have been shown to be LISA), recombinant EG95 oncosphere protein (OncELISA) a useful method for identifying animals including cynomol- or a crude protoscolex preparation (ProtELISA). Sera used gus mokeys (Macaca fascicularis) (Bacciarini et al., 2004; for the assay validations were obtained from 249 sheep that Rehmann et al., 2005) and lowland gorillas (Gorilla g.
were infected either naturally or experimentally with E.
gorilla) (Rehmann et al., 2003) that might be infected granulosus and from 1012 non-infected sheep. The highest with E. multilocularis and are therefore at risk of developing diagnostic sensitivity was obtained using the ProtELISA at 62.7 and 51.4%, depending on the cut-off. Assay sensitivities The Em2plus ELISA, a combination of Em2 with a were lower for the 8kDaELISA and the OncELISA. Diag- recombinant protein designated II/3–10 (also termed nostic specificities were high, ranging from 95.8 to 99.5%, EM10), increased the sensitivity to 97% (Gottstein et al., depending on the ELISA type and cut-off level chosen. A few 1993). The Em2plus assay exhibits cross-reaction with CE sera from 39 sheep infected with T. hydatigena and from 19 (in 25.8% of cases) which is higher than the individual Em2 sheep infected with T. ovis were recorded as positive.
(5.6%) and 11/3–10 (6.5%), but limited cross-reactivity Western immunoblot analysis revealed that the dominant with other diseases. The Em2plus-ELISA has been commer- antigenic components in the crude protoscolex antigen cialized for clinical diagnosis of AE and for population preparation were macromolecules of about 70–150 kDa, most likely representing polysaccharides. This study demon- EM10 (Em II/3–10) shares almost complete identity to strated that the ProtELISA was the most effective immuno- the E. granulosus protein sequences EG10 and EGII.3 (Sako logical method of those assessed for detection of infection et al., 2002). Although the two species have similar se- with E. granulosus in sheep. Because of its limited diagnostic quences, recombinant EM10 protein showed very high sensitivity of about 50–60%, the assay would be useful for specificity to distinguish AE infection from CE infection in the detection of the presence of infected sheep on a flock human patients. It is not clear why these homologous basis but not for reliable identification of individual animals molecules exhibited such a different pattern of sero-recogni- infected with E. granulosus (Kittelberger et al., 2002).
tion. Sako et al. (2002) suggested that EG10 may be In a later study, Simsek & Koroglu (2004) investigated the expressed at a very low level in larval E. granulosus, but it antigenic characteristics of hydatid cyst fluid in sheep by may equally be that the transcription of EG10 is low or SDS-PAGE to evaluate the sensitivity and specificity of HCF- silenced. An 18 kDa antigen (Em18) from protoscoleces of ELISA and immunoblotting for diagnosis of sheep hydati- AE was reported as being a highly species-specific (96.8%) dosis. One band with a molecular weight of 116 kDa showed and sensitive (97%) antigen with potential not only for 88% sensitivity and 84% specificity in the immunoblot differentiation of AE from either CE or other helminth assay. Sensitivity (60%) was less but specificity was higher infections, but also for differentiation of active from inactive (94%) with the HCF-ELISA (Simsek & Koroglu, 2004).
AE (Ito et al., 1995; Akira, 1997). Subsequently, EM18 wasshown to be a fragment of the C-terminal of EM10 and therecombinant protein was recognized by 87.1 and 90.3% of 31 serum samples from AE patients in ELISA and immuno- The diagnosis of AE is based on similar findings and criteria blotting, respectively (Sako et al., 2002). Recombinant as in CE. These include case history, clinical findings, Em18-ELISA andEm18-immunoblot assays have proved morphological lesions identified by imaging techniques, invaluable for differentiating AE from CE infection (Ito PCR or immunofluorescence/immunohistochemistry, and et al., 2002), the former also being useful for evaluating the immunodiagnosis. Like CE, serodiagnosis of alveolar echi- efficacy of treatment in patients with AE (Fujimoto et al., nococcosis provides a complementary role to other proce- 2005). Epitope mapping indicated that the part of the dures in early detection of the infection. The methods are ReEm18 antigen sequence necessary for AE diagnosis occurs similar to those used for CE but serological tests for anti- in the N-terminal half to two-thirds of the entire sequence body detection are generally more reliable. As we have FEMS Immunol Med Microbiol 47 (2006) 24–41  2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved Full-length cDNA and genomic DNA encoding an 8-kDa in faeces. Overall, the available ELISA-based methods for subunit of antigen B from E. multilocularis (designated detection of circulating antibodies in canines have poor EmAgB8/1) were isolated from an E. multilocularis metaces- sensitivity, specificity is unclear and there is no correlation tode cDNA library and a protoscolex genomic DNA library, with worm burden so their usefulness, other than in respectively (Mamuti et al., 2004). Reverse transcription- population-based studies of canine hosts, is questionable PCR analysis revealed that the clone encoding EmAgB8/1 is predominantly transcribed in larval E. multilocularis. The Much more success has been achieved using the other mature form was expressed in Escherichia coli, and its major approach to immunological diagnosis of Echinococcus antigenic reactivity was compared with that its counterpart infection in the definitive host by detection of adult worm 8 kDa subunit of E. granulosus AgB (EgAgB8/1), by Western products in faeces using sandwich ELISA methodology and blotting and ELISA with serum samples from patients this technique has been widely used with successful detec- confirmed to have CE and AE. The sensitivity of EmAgB8/1 tion of E. granulosus and E. multilocularis (Lopera et al., was comparable to that of EgAgB8/1 for the serodiagnosis of 2003; Sanchez Thevenet et al., 2003; Benito & Carmena, echinococcosis. It is noteworthy that there was no cross- 2005; Cavagion et al., 2005; Moro et al., 2005; Reiterova reaction with sera from patients with cysticercosis, which et al., 2005). Although these tests for diagnosis of canine often cross-react when native antigens are used for serodiag- echinococcosis provide high specificity and sensitivity, little is known of the characteristics of antigenic molecules Direct detection or amplification of E. multilocularis present in faeces from infected animals. While initial nucleic acids in clinical samples is also considered useful attempts to determine the molecular weights of E. granulo- for the primary diagnostic identification of parasite materi- sus coproantigens by SDS-PAGE and Western blotting with als in biological specimens resected or biopsied from coproantigen reactive capture antibodies were equivocal, patients, and also for the assessment of the viability of they suggested the presence of a significant carbohydrate parasite samples after chemotherapy or other treatment.
component. Elayoubi et al. used SDS-PAGE and western Infections of humans with E. multilocularis, have been blot to show that the coproantigens are highly glycosyl- described with increasing frequency in Poland since 1994.
ated and contain b-galactose and N-acetyl-b-glucosamine In the attempt to verify these reports, specimens were (Elayoubi et al., 2003). Subsequently, supernatants prepared obtained from a group of Polish patients (Myjak et al., from E. granulosus-infected dog faecal samples and fractio- 2003). Liver lesions in patients with AE, diagnosed on the nated by size-exclusion fast protein liquid chromatography basis of results of histological and serological tests, were (FPLC) showed that the antigens are large molecular weight shown, by the presence of specific microsatellite sequences molecules that may be derived from the carbohydrate-rich and mitochondrial 12S rDNA, to contain E. multilocularis surface glycocalyx of adult worms, and are shed, released or DNA. These data provided unequivocal proof that human secreted during the life-span of the tapeworm (Elayoubi & infections with E. multilocularis occur in Poland. PCR- amplification of specific E. multilocularis DNA has proved Copro-antigen detection allows in vitam diagnosis of useful also for helping to diagnose unambiguously AE Echinococcus infections but, as well, several PCR-based infection in monkeys (Bacciarini et al., 2004).
protocols have been developed that allow identification ofEchinococcus DNA from eggs or from adult parasites. Theseprovide a highly complementary approach for positive and Diagnosis of echinococcosis in definitive hosts highly specific diagnosis of canines infected with E. granulo- The detection of Echinococcus infections in canines is sus and E. multilocularis (Cabrera et al., 2002; Abbasi et al., important for epidemiological surveillance and evaluation 2003; Deplazes et al., 2003; Casulli et al., 2004, 2005; Stefanic of echinococcosis control programs. Diagnosing Echinococ- et al., 2004; Varcasia et al., 2004; Reiterova et al., 2005) and cus infections in dogs and other definitive hosts is proble- environmental detection of Echinococcus eggs in soil samples matical as the eggs of taeniid cestodes are extremely similar, and thus identification by microscopic examination of the The efficacy of two PCR-based methods to detect patent faeces is risky and non-specific. Two major diagnostic and prepatent infection in dogs experimentally infected with methods have been extensively used in dogs, purgation with E. granulosus was recently compared (Naidich et al., 2006).
arecoline compounds and necropsy of the small intestine.
The detection was based on amplification of a fragment of Necropsy is the method of choice for foxes and other final the mitochondrial cox1 gene (Mit-PCR) and a repetitive hosts. Two immunodiagnostic approaches have been devel- element (Rep-PCR) from the genome of E. granulosus. The oped for diagnosis of E. granulosus and E. multilocularis ability of both methods to detect several genotypes of the infection in definitive hosts – assays for specific serum parasite were assessed. Both PCR methods could detect E.
antibody and detection of parasite products (coproantigens) granulosus during both prepatent and patent periods, even  2006 Federation of European Microbiological Societies FEMS Immunol Med Microbiol 47 (2006) 24–41 Published by Blackwell Publishing Ltd. All rights reserved Advances in immunology and diagnosis of echinococcosis when microscopical observation of eggs in faecal samples be 10-fold induced upon co-incubation of metacestode was negative. The Mit-PCR produced the same amplifica- vesicles with host cells which indicated a possible involve- tion pattern for all the parasite genotypes tested while the ment of parasite-determined, epidermal growth factor-like amplification patterns with the Rep-PCR differed among signal transduction systems in metacestode proliferation groups of strains. In addition, faecal samples collected from and development. Shortly thereafter, an E. multilocularis dogs from an endemic area in Argentina were diagnosed candidate receptor for Egfd was identified by Spiliotis et al.
with more sensitivity using the PCR methods than by (2003). The identified molecule, EmER, displayed clear arecoline hydrobromide purgation. These copro-PCR tests homologies to EGF receptors from different phylogenetic can thus be applied in the confirmation of coproantigen- origins and was expressed in metacestode and protoscolex. It positive fecal samples and to verify the success of control has not yet been experimentally shown that Egfd and EmER form a corresponding ligand–receptor pair. However, thefact that emer is the only E. multilocularis gene that encodesan EGF receptor strongly suggests that this is the case.
In another approach, Rosenzvit et al. (2006) used the signal sequence trap technique to identify genes coding for A series of recent studies have investigated the functional secreted and membrane-bound proteins from E. granulosus.
expression of genes from E. granulosus and E. multilocularis.
A number of isolated clones showed significant similarity All parasitic helminths, including Echinococcus process a with amino acid transporters, Krebs cycle intermediates large subset of their mRNA molecules via spliced leader transporters, presenilins and vacuolar protein sorter pro- trans-spling (Fernandez et al., 2002). In an important teins. Other cDNAs encoded secreted proteins without contribution, Brehm et al. (2003) described a rapid and homologues. All the mRNAs were expressed in proto- efficient method, ‘spliced leader differential display’, to scoleces and adult worms, but some of them were not found analyse gene expression patterns of in vitro cultivated in oncospheres. These identified putative E. granulosus protoscoleces and metacestode vesicles from E. multilocu- secreted and membrane-bound proteins are likely to play laris. The approach was further used to identify genes which important roles in the metabolism, development and survi- are induced in the E. multilocularis metacestode stage under growth-promoting conditions in the presence of host hepa- A series of more conventional studies have been under- tocytes. One mRNA, encoding a putative member of the taken on the cloning, characterization and functional ex- epidermal growth factor family of mitogens, was shown to pression of a number of molecules from both E. granulosus Table 2. Examples of molecules isolated from Echinococcus and their function Binding and hydrolyzing guanosine triphosphate (GTP) NERMAD-specific interaction partner of the ERM Mitochondrial malate dehydrogenase (mMDH) Smad family, important role in transforming growth Reducing thioredoxin and glutathione in the Actin filament fragmenting and nucleating activities Heart group of fatty acid binding protein FEMS Immunol Med Microbiol 47 (2006) 24–41  2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved and E. multilocularis which include regulatory factors, Alvite G, Di Pietro SM, Santome JA, Ehrlich R & Esteves A (2001) signalling factors, receptors and enzymes (Table 2).
Binding properties of Echinococcus granulosus fatty acid Currently, genomic and expressed sequence tag (EST) binding protein. Biochim Biophys Acta 1533: 293–302.
information on Echinococcus spp. is very limited compared Andrade MA, Siles-Lucas M, Espinoza E, Perez Arellano JL, with studies on the filaria and schistosomes although Gottstein B & Muro A (2004) Echinococcus multilocularis plans are in place to produce 60 000 ESTs to characterize laminated-layer components and the E14t 14–3–3 genes from E. granulosus and E. multilocularis metacestodes recombinant protein decrease NO production by activated rat (http://www.sanger.ac.uk/Projects/Echinococcus/). This new macrophages in vitro. Nitric Oxide 10: 150–155.
information will add significantly to the available Echinococ- Arend AC, Zaha A, Ayala FJ & Haag KL (2004) The Echinococcus cus genomic and cDNA sequences in GenBank and the granulosus antigen B shows a high degree of genetic variability.
current collection of 43000 EST clusters for trans-spliced Aumuller E, Schramm G, Gronow A, Brehm K, Gibbs BF, and non-trans-spliced cDNAs of E. granulosus and E. multi- Doenhoff MJ & Haas H (2004) Echinococcus multilocularis locularis available on the LophDB search base (http://zeldia.
metacestode extract triggers human basophils to release cap.ed.ac.uk/Lopho/LophDB.php), established by John interleukin-4. Parasite Immunol 26: 387–395.
Bacciarini LN, Gottstein B, Pagan O, Rehmann P & Grone A The new developments in genomics, proteomics (Che- (2004) Hepatic alveolar echinococcosis in cynomolgus male et al., 2003) and microarray analysis that are underway monkeys (Macaca fascicularis). Vet Pathol 41: 229–234.
or planned for the Echinococcus organisms will underpin Benito A & Carmena D (2005) Double-antibody sandwich ELISA future advances on the functional biology of these impor- using biotinylated antibodies for the detection of Echinococcus tant, emerging (Eckert & Deplazes, 2004) pathogens, pro- granulosus coproantigens in dogs. Acta Trop 95: 9–15.
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