Search

Hidatidoza je svjetska zoonoza uzrokovana larvalnim stadijem trakavice Echinococcus, koja je endemična u mnogim dijelovima svijeta. Ehinokokoza je relevantan zdravstveni problem u nerazvijenim područjima gdje ne postoji veterinarska kontrola. Patogenost ehinokokne ciste ovisi o mehaničkom učinku same ciste. Najčešća lokacija hidatidnih cističnih lezija je u jetri s nižom prijavljenom incidencijom u bilo kojem drugom organu ili tkivu. Dijagnoza ehinokokoze ovisi o kliničkim, radiološkim i serološkim nalazima. Uključuje kombinaciju medicinske anamneze, fizikalnog pregleda, laboratorijskih testova i slikovnih pretraga poput ultrazvuka, CT – a ili MRI – a. Osnova je liječenja kirurški zahvat koji se kombinira s antihelmintikom albendazolom. Preventivni programi usmjereni su na dehelmintizaciju pasa, koji su konačni domaćini. Hydatid disease is a global zoonosis caused by the larval stage of the Echinococcus tapeworm, which is endemic in many parts of the world. Echinococcosis is a relevant health problem in underdeveloped areas where there is no veterinary control. The pathogenicity of the hydatid cyst depends on the mechanical effect of the cyst itself. The most common location of hydatid cystic lesions is the liver, with lower reported incidences in any other organ or tissue. The diagnosis of echinococcosis relies on clinical, radiological, and serological findings. It combines a medical history check, physical examination, lab testing, and imaging tests such an MRI, CT scan, or ultrasound. The cornerstone of treatment is surgical intervention combined with the anthelmintic drug albendazole. Preventive programs are focused on deworming dogs, which are the definitive hosts

Susceptibility to Echinococcus multilocularis infection considerably varies among intermediate (mostly rodents) and dead-end host species (e.g. humans and pig), in particular regarding intestinal oncosphere invasion and subsequent hepatic metacestode development. Wistar rats are highly resistant to infection and subsequent diseases upon oral inoculation with E. multilocularis eggs, however, after immunosuppressive treatment with dexamethasone, rats become susceptible. To address the role of the cellular innate immunity, Wistar rats were individually or combined depleted of natural killer (NK) cells, macrophages (MΦ) and granulocytes (polymorphonuclear cells, PMN) prior to E. multilocularis egg inoculation. Although NK cell and MΦ depletion did not alter the resistance status of rats, the majority of PMN-depleted animals developed liver metacestodes within 10 weeks, indicating that PMN are key players in preventing oncosphere migration and/or development in Wistar rats. In vitro studies indicated that resistance is not caused by neutrophil reactive oxygen species or NETosis. Also, light microscopical examinations of the small intestine showed that oral inoculation of E. multilocularis eggs does not elicit a mucosal neutrophil response, suggesting that the interaction of oncospheres and neutrophils may occur after the former have entered the peripheral blood. We suggest to consider granulocytes as mediators of resistance in more resistant species, such as humans.

Wild mustelids and canids are definitive hosts of Taenia and Versteria spp. while rodents act as natural intermediate hosts. Rarely, larval stages of these parasites can cause serious zoonoses. In Europe, four cases of Taenia martis cysticercosis have been diagnosed in immunocompetent women, and two cases in zoo primates since 2013. In North America, a zoonotic genotype related but distinct from Versteria mustelae has been identified in 2014, which had caused a fatal infection in an orangutan and liver- and disseminated cysticercoses in two severely immune deficient human patients in 2018, respectively. Additionally, we could attribute a historic human case from the USA to this Versteria sp. by reanalysing a published nucleotide sequence. In the last decades, sporadic zoonotic infections by cysticerci of the canid tapeworm Taenia crassiceps have been described (4 in North America, 8 in Europe). Besides, 3 ocular cases from North America and one neural infection from Europe, all in immunocompetent patients, 6 cutaneous infections were described in severely immunocompromised European patients. Correspondingly, besides oral infections with taeniid eggs, accidental subcutaneous oncosphere establishment after egg-contamination of open wounds was suggested, especially in cases with a history of cutaneous injuries at the infection site. Taenia multiceps is mainly transmitted in a domestic cycle. Only five human coenurosis cases are published since 2000. In contrast, T. serialis coenurosis (1 human case since 2000) is primarily transmitted by wild canids. The etiological diagnosis of exotic cysticercoses is challenging. Usually, clinical material does not allow for a morphological identification, and serological tests are not available. These limitations have partly been overcome by molecular tools. Without claiming any dramatic emergence of cysticercoses and coenuroses transmitted by wild carnivores, further sporadic cases of such ‘exotic’ infections have to be expected., Graphical abstract Taenia crassiceps cysticerci from the abdomen of an infected rodent.Image 1, Highlights • Wild canids and mustelids transmit rare but potentially fatal cysticercoses and coenuroses. • Martens and weasels can rarely transmit dangerous parasitic infections. • Tapeworm eggs may contaminate wounds and develop locally. • In North America, the mustelid tapeworm Versteria causes severe human infections. • Molecular analyses from minute clinical material allows for a specific diagnosis.

Cerebral coenurosis, caused by the larvae of Taenia multiceps (Coenurus cerebralis), is a fatal central nervous system disease in sheep and other herbivores and occasionally humans. Comparative transcriptomic profiles of the developmental stages of the parasite remain unknown. In this study, RNA sequencing was used to determine the transcriptome profiles of different stages of the life cycle of T. multiceps, including Oncosphere, Coenurus cerebralis (Pro with Cyst), and Adult (Adu), as well as scolex-neck proglottids (Snp), immature–mature proglottids (Imp), and gravid proglottids (Grp) of the adult stage. A total of 42.6 Gb (average 6.1 Gb) Illumina pair-end reads with a 125-bp read length were generated for seven samples. The total number of differentially expressed genes (DEGs) in the various life stages ranged from 2,577 to 3,879; however, for the tissues of the adult worm, the range was from 1,229 to 1,939. Kyoto Encyclopedia of Genes and Genomes analysis showed that the DEGs mainly participated in cellular and metabolic processes, binding and catalytic activity, genetic information processing, and environmental information processing. In addition, a large number of genes related to development and parasite–host interaction were identified. Quantitative reverse transcription-polymerase chain reaction confirmed that the levels of 28 selected DEGs were consistent with those determined using RNA sequencing. The present study provides insights into the mechanisms of the development and parasitic life of T. multiceps.

Taenia solium oncosphere protein TSOL18 is the host-protective antigen against porcine cysticercosis. Little attention has been given to use it as target molecule in immunodiagnostic tests. The objective of this paper is to describe the immunodiagnostic potential of monoclonal antibodies (MoAbs) raised against conformational epitopes of TSOL18. Three murine IgG1 MoAbs (25D12C1, 21C2D2, 10H1F2) against three different conformational epitopes of TSOL18 were produced and evaluated with an inhibition enzyme-linked immunosorbent assay (i-ELISA) for the detection of anti-TSOL18 and anti-oncosphere antibodies. Serum samples from pigs immunized with TSOL18 inhibited the binding of the three MoAbs to TSOL18 antigen in i-ELISA. The highest inhibition of anti-TSOL18 antibodies in immunized pigs was observed with MoAb 25D12C1. Ten field sera (12.19%) from 82 non-vaccinated and non-infected pigs showed anti-oncosphere antibodies inhibiting the binding of MoAb 25D12C1. Anti-oncosphere antibodies in pigs experimentally infected with T. solium eggs inhibited the binding of MoAb 25D12C1 from 2 to 8 week-post infection. It is concluded that MoAb 25D12C1 has excellent immunodiagnostic potentials to detect anti-oncosphere antibodies in the intermediate hosts at early exposure to T. solium eggs. Further investigations on potential use of MoAb 25D12C1 in a capture antigen ELISA for the detection of post-oncospheral antigens in infected pigs cannot be overemphasized., Graphical abstract Unlabelled Image, Highlights • We describe the potential usefulness of the monoclonal antibody 25D12C1 to TSOL18 protein. • Serum samples from pigs immunized with TSOL18 inhibited the binding of 25D12C1 in an inhibition enzyme-linked immunosorbent assay (i-ELISA) for the detection of anti-TSOL18 and anti-oncosphere antibodies. • Anti-oncosphere antibodies in pigs infected with T. solium eggs inhibited also the binding of 25D12C1. • It is concluded that MoAb i-ELISA using 25D12C1 has the potential to identify animals exposed to T. solium infection.

Cystic echinococcosis, caused by infection with the larval stage of the cestode Echinococcus granulosus, is a chronic zoonosis. The lifecycle of the E. granulosus parasite includes three consecutive stages that require specific gene regulation or protein expression to survive environmental shifts between definitive hosts and intermediate hosts. The aim of the present study is to screen and analyze the stage differential antigens to be considered for vaccine development against E. granulosus. By using the iTRAQ (isobaric tags for relative and absolute quantification) method, the differentially expressed proteins were selected from the three consecutive developmental stages of E. granulosus: oncosphere, adult tapeworms, and protoscolex. Through a bioinformatics analysis including Clusters of Orthologous Groups (COG), Gene Ontology (GO), and pathway metabolic annotation, we identified some proteins of interest from each stage. The results showed that a large number of differentially expressed proteins (375: oncosphere vs. adult, 346: oncosphere vs. protoscolex, and 391: adult vs. protoscolex) were identified from the three main lifecycle stages. Analysis of the differential protein pathways showed that these differential proteins are mainly enriched in metabolic pathways, Huntington's diseases, Alzheimer's diseases, and ribosome metabolic pathways. Interestingly, among these differential proteins, expression levels of paramyosin, HSP60, HSP70, HSP90, cathepsin L1, cathepsin D, casein kinase, and calmodulin were significantly higher in the oncosphere than in the adult or protoscolex (p 0.05). We hope our findings will help to identify potential targets for diagnosis or for therapeutic and prophylactic intervention.Analyse protéomique comparative basée sur iTRAQ de différents stades de développement d’Echinococcus granulosus.L’échinococcose kystique, causée par une infection au stade larvaire du cestode Echinococcus granulosus, est une zoonose chronique. Le cycle de vie du parasite E. granulosus comprend trois étapes consécutives qui nécessitent une régulation génétique ou une expression de protéines spécifiques pour survivre aux changements environnementaux entre les hôtes définitifs et les hôtes intermédiaires. Le but de la présente étude est de cribler et d’analyser les antigènes différentiels de stade à considérer pour le développement de vaccins contre E. granulosus. En utilisant la méthode iTRAQ (étiquettes isobares pour la quantification relative et absolue), les protéines différentiellement exprimées ont été sélectionnées parmi les trois stades de développement consécutifs d’E. granulosus : l’oncosphère, les ténias adultes et le protoscolex. Grâce à une analyse bioinformatique comprenant les grappes de groupes orthologues (COG), l’ontologie des gènes (GO) et l’annotation des voies métaboliques, nous avons identifié certaines protéines d’intérêt à chaque étape. Les résultats ont montré qu’un grand nombre de protéines exprimées différentiellement (375 : oncosphère vs adulte, 346 : oncosphère vs protoscolex et 391 : adulte vs protoscolex) sont identifiées pour les trois étapes principales du cycle de vie. L’analyse des voies différentielles des protéines a montré que ces protéines différentielles sont principalement enrichies dans les voies métaboliques, la maladie de Huntington, la maladie d’Alzheimer et les voies métaboliques des ribosomes. Fait intéressant, parmi ces protéines différentielles, les niveaux d’expression des protéines paramyosine, HSP60, HSP70, HSP90, cathepsine L1, cathepsine D, caséine kinase et calmoduline étaient significativement plus élevés dans l’oncosphère que chez l’adulte ou le protoscolex (p 0,05). Nous espérons que nos résultats pourront identifier des cibles potentielles pour un diagnostic ou une intervention thérapeutique ou prophylactique.

The fine structure of the infective hexacanths of Echinococcus multilocularis was examined with particular emphasis on the functional ultrastructure of penetration glands and nerve cells directly involved in the mechanism of initial host infection. The oncosphere contains two types of penetration glands, PG1 and PG2, that differ slightly in size and form a large U-shaped bi-nucleated syncytial structure. The arms of each gland at each end of the U, directed towards the hook region, exit into the tegument peripheral layer between the median and lateral hook pairs. The lobate nuclei of PG1 and PG2 contain prominent spherical nucleoli surrounded by several large electron-dense islands of heterochromatin. The syncytial cytoplasm of both types of glands is rich in free ribosomes, polysomes, several mitochondria, and heavy accumulations of discoid secretory granules of moderate to high electron density. The secretory granules, sg1 and sg2, differ in their ultrastructure and electron density; the sg2 are much smaller and more flattened in shape. A common characteristic for sg1 and sg2, evident under high magnification, is their high electron density and discoidal shape, with two bi-concave surfaces. Both sg1 and sg2 are frequently grouped in characteristic parallel stacks, the “rouleau”-shaped assemblages with sometimes six to ten granules. Two nerve cells of neurosecretory type are situated in the central part of the hexacanth, each one in a deep U-shaped invagination between the two penetration glands. The nuclei of nerve cells contain several large heterochromatin islands closely adjacent to their nuclear membranes. Their cytoplasm is characterized by having membrane-bound, dense-cored neurosecretory-like granules not only in nerve cell perikarya but also in the elongated nerve processes frequently adjacent to gland arms and to both somatic or body musculature, including the complex system of hook muscles. The results of the present study, when supported with literature data on oncospheres of other cestode species, allow for a better understanding of the important role and coordinated functions of three structural components, i.e., oncospheral hooks, penetration glands, and nerve cells, in the mechanism of intermediate host infection. Presence or absence of nerve cells in oncospheres of various cestodes is reviewed, and perspectives on the value and application of research on functional morphology of oncospheres are discussed.

The origin, differentiation and functional ultrastructure of oncospheral or egg envelopes in Echinococcus multilocularis Leuckart, 1863 were studied by transmission electron microscopy (TEM) and cytochemistry. The purpose of our study is to describe the formation of the four primary embryonic envelopes, namely vitelline capsule, outer envelope, inner envelope and oncospheral membrane, and their transformation into the oncospheral or egg envelopes surrounding the mature hexacanth. This transformation takes place in the preoncospheral phase of embryonic development. The vitelline capsule and oncospheral membrane are thin membranes, while the outer and inner envelopes are thick cytoplasmic layers formed by two specific types of blastomeres: the outer envelope by cytoplasmic fusion of two macromeres and the inner envelope by cytoplasmic fusion of three mesomeres. Both outer and inner envelopes are therefore cellular in origin and syncytial in nature. During the advanced phase of embryonic development, the outer and inner envelopes undergo great modifications. The outer envelope remains as a metabolically active layer involved in the storage of glycogen and lipids for the final stages of egg development and survival. The inner envelope is the most important protective layer because of its thick layer of embryophoric blocks that assures oncospheral protection and survival. This embryophore is the principal layer of mature eggs, affording physical and physiological protection for the differentiated embryo or oncosphere, since the outer envelope is stripped from the egg before it is liberated. The embryophore is very thick and impermeable, consisting of polygonal blocks of an inert keratin-like protein held together by a cementing substance. The embryophore therefore assures extreme resistance of eggs, enabling them to withstand a wide range of environmental temperatures and physicochemical conditions.

Transmission electron microscopy (TEM) was used to study the ultrastructure of the uterus and egg morphology in the enigmatic bothriocephalidean tapeworm Tetracampos ciliotheca. The uterine wall, underlain by well-developed muscle bundles, consists of a syncytial epithelium which is characterized by the abundance of free ribosomes, mitochondria and cisternae of granular endoplasmic reticulum (GER). On the apical surface of the uterine epithelium, there is an abundant network of cytoplasmic microlamellae projecting into the uterine lumen. The lumen is filled with freely lying eggs which are located close to the uterine wall but do not contact with the microlamellae of the uterine epithelium. The developed eggs possess an oncosphere surrounded by four envelopes: (1) a thin egg shell; (2) an outer envelope; (3) a syncytial, ciliated inner envelope; and (4) the oncospheral membrane. The mature hexacanth is armed with three pairs of oncospheral hooks, as well as somatic and hook muscles and five types of cells (1) binucleated subtegumental cell, (2) somatic cells, (3) penetration gland cells, (4) nerve cells and (5) germinative cells. Considering the relative scarcity of descriptive and comparative studies on the ultrastructure of the uterus and egg morphology in the order Bothriocephalidea, we concluded that Tet. ciliotheca displays a unique type of egg development. Based on these results, we discuss plausible ideas relating to the function of these structures for consideration in future studies.

Background Taenia multiceps is a harmful tapeworm and its larval form (coenurus cerebralis) is the causative agent of coenurosis, a disease affecting the health of herbivores, resulting in great economic loss to animal husbandry. Heat-shock proteins (HSPs), expressed in all prokaryotes and eukaryotes, act as molecular chaperones and can affect pathogenicity. Methods Herein, cDNAs of T. multiceps genes Tm-HSP60 and Tm-p36 were cloned and molecularly characterised by bioinformatics analyses. The immunogenicity and immunoreactivity of recombinant rTm-HSP60 and rTm-p36 proteins were investigated by immunoblotting and indirect ELISA was established to evaluate their serodiagnostic potential. Tissue localisation and transcriptional level at different life stages of T. multiceps were determined by immunohistochemical and quantitative real-time PCR analyses. Result The 533 residue rTm-HSP60 and the 314 residue rTm-p36 proteins share typical highly conserved features of HSPs. Tm-p36 shares structural characteristics with metazoan small HSPs, with two N-terminal α-crystallin domains. Compared with Tm-p36, Tm-HSP60 displayed stronger immunogenicity, and the indirect ELISA based on rTm-HSP60 exhibited a sensitivity of 83.3% and a specificity of 87.5%, while rTm-p36 was not suitable to develop indirect ELISA. Tm-HSP60 was widely distributed in all stages of T. multiceps, albeit at relatively low levels, while Tm-p36 was specifically distributed in the protoscolex and oncosphere. Conclusions The sequence, structural and functional analyses of these two HSPs indicates that they may play important roles in the life-cycle of T. multiceps as molecular chaperones. Tm-HSP60 displayed stronger immunogenicity compare to Tm-p36, and has the potential for antibody detection. Tm-p36 was strongly associated with the activation of oncospheres and has potential interest for vaccination. Electronic supplementary material The online version of this article (10.1186/s13071-019-3352-8) contains supplementary material, which is available to authorized users.

Taenia solium is known to cause human cysticercosis while T. saginata does not. Comparative in vitro and in vivo studies on the oncosphere and the postoncospheral (PO) forms of T. solium and T. saginata may help to elucidate why cysticercosis can occur from one and not the other. The aim of this study was to use in vitro culture assays and in vivo models to study the differences in the development of the T. solium and T. saginata oncosphere. Furthermore, this study aimed to evaluate the expression of cytokines and metalloproteinases (MMPs) in human peripheral blood mononuclear cells (PBMCs), which were stimulated by these oncospheres and PO antigens. T. solium and T. saginata activated oncospheres (AO) were cultured in INT-407 and HCT-8 intestinal cells for 180 days. The T. solium began to die while the T. saginata grew for 180 days and developed to cysticerci in INT-407 cells. Rats were inoculated intracranially with AO and PO forms of either T. saginata or T. solium. Rats infected with T. solium AO and PO forms developed neurocysticercosis (NCC), while those infected with the T. saginata did not. Human PMBCs were stimulated with antigens of AO and PO forms of both species, and the production of cytokines and metalloproteinases (MMPs) was measured. The T. solium AO antigen stimulated a higher production of IL-4, IL-5, IL-13, IFN-γ, and IL-2 cytokines compared to T. saginata AO. In the PO form, the T. saginata PO antigen increased the production of IL-4, IL-5, IL-13, IFN-γ, IL-1β, IL-6, IL-10, TNF-α and IL-12 cytokines compared to T. solium, suggesting that this global immune response stimulated by different forms could permit survival or destruction of the parasite depending of their life-cycle stage. Regarding MMPs, T. solium AO antigen stimulated a higher production of MMP-9 compared to T. saginata AO antigen, which may be responsible for altering the permeability of intestinal cells and facilitating breakdown of the blood-brain barrier during the process of invasion of host tissue., Author summary Taenia solium and Taenia saginata are two parasites that cause the tissue infection cysticercosis in their intermediate hosts, pigs and cows, respectively. One major difference between them is that T. solium can also cause neurocysticercosis in the human brain, while T. saginata cannot. Neurocysticercosis is thought to be the major cause of adult-onset seizures in developing countries. It is not well understood why only T. solium can survive in human tissue; however, the host inflammatory response likely plays an important role. The authors found that human immune cells stimulated with T. solium in the early stages of the parasite life cycle produced a more robust cytokine response than T. saginata. However, in the mature stage, which occurs once T. solium reaches the brain, T. solium antigens stimulated a lower inflammatory response compared to T. saginata, suggesting the parasite is able to manipulate the host immune response in some way to evade destruction. These findings may support the differences in growth observed by the authors when rat brains were inoculated with either parasite species. This study provides new insights into the different ways T. solium and T. saginata activate the immune response to survive and develop within the host.

Several studies have been performed to determine specific antigens for the diagnosis of tapeworms. One of these antigens is Tso31, which is used to differentiate Taenia solium and Taenia saginata in human feces. The aim of the present work was the molecular characterization of this protein in different tapeworm specimens collected in Peru: T. omisa (n = 6), T. hydatigena (n = 7), T. taeniaeformis (n = 4), T. pisiformes (n = 1), T. multiceps (n = 7), and T. solium (n = 10). Total DNA was extracted from each proglottid using a commercial DNA kit for tissue. A nested PCR was used to amplify a fragment of the previously described oncosphere-specific protein Tso31 gene. The nested PCR products were analyzed by 1.5% agarose gel electrophoresis and visualized after ethidium bromide staining. All nested PCR-positive products were sequenced and their sequences were compared. Of all the tapeworms analyzed, only T. solium and T. multiceps amplified the Tso31 gene. All sequences were identical for each species. Our T. solium Tso31 showed 100% similarity when compared with published GenBank sequences. The difference between T. solium and T. multiceps Tso31 samples was 8.1%. In conclusion, our results show that the tsol31 gene is not exclusive to T. solium.

A 22-year-old man, originally from India, presented to an emergency department in Canada with a new-onset seizure and collapse associated with a few seconds of right eye and tongue deviation. This event occurred following 6 weeks of difficulty concentrating and unilateral left-sided headache. On examination, no focal neurological deficits were found. A non-contrast CT head was requested which showed a left-sided occipital lesion. A follow-up brain MRI revealed the ‘hole-with-dot’ lesion pathognomonic for neurocysticercosis (NCC). NCC is caused by the tapeworm Taenia solium and is the most common parasitic infection of the central nervous system, endemic to many low-income countries worldwide. T. solium is transmitted among humans via the faecal–oral route, with pigs acting as an intermediate host. Humans are a definitive host for the intestinal tapeworm which produces eggs containing an infective embryo (oncosphere). This embryo can actively cross the …

EgKI-1, a member of the Kunitz type protease inhibitor family, is highly expressed by the oncosphere of the canine tapeworm Echinococcus granulosus, the stage that is infectious to humans and ungulates, giving rise to a hydatid cyst localized to the liver and other organs. Larval protoscoleces, which develop within the hydatid cyst, have been shown to possess anti-cancer properties, although the precise molecules involved have not been identified. We show that recombinant EgKI-1 inhibits the growth and migration of a range of human cancers including breast, melanoma and cervical cancer cell lines in a dose-dependent manner in vitro without affecting normal cell growth. Furthermore, EgKI-1 treatment arrested the cancer cell growth by disrupting the cell cycle and induced apoptosis of cancer cells in vitro. An in vivo model of triple negative breast cancer (MDA-MB-231) in BALB/c nude mice showed significant tumor growth reduction in EgKI-1-treated mice compared with controls. These findings indicate that EgKI-1 shows promise for future development as an anti-cancer therapeutic.

EgKI-1, a member of the Kunitz type protease inhibitor family, is highly expressed by the oncosphere of the canine tapeworm Echinococcus granulosus, the stage that is infectious to humans and ungulates, giving rise to a hydatid cyst localized to the liver and other organs. Larval protoscoleces, which develop within the hydatid cyst, have been shown to possess anti-cancer properties, although the precise molecules involved have not been identified. We show that recombinant EgKI-1 inhibits the growth and migration of a range of human cancers including breast, melanoma and cervical cancer cell lines in a dose-dependent manner in vitro without affecting normal cell growth. Furthermore, EgKI-1 treatment arrested the cancer cell growth by disrupting the cell cycle and induced apoptosis of cancer cells in vitro. An in vivo model of triple negative breast cancer (MDA-MB-231) in BALB/c nude mice showed significant tumor growth reduction in EgKI-1-treated mice compared with controls. These findings indicate that EgKI-1 shows promise for future development as an anti-cancer therapeutic.

Neurocysticercosis is a parasitic brain disease caused by the larval form (Cysticercus cellulosae) of the Taenia solium and is the leading cause of preventable epilepsy world-wide. However, the pathophysiology and relation to the wide range of clinical features remains poorly understood. Axonal swelling is emerging as an important early pathological finding in multiple neurodegenerative diseases and causes of brain injury, but has not been well-described in neurocysticercosis. Histological analysis was performed on human, rat and porcine NCC brain specimens to identify axonal pathology. Rat infection was successfully carried out via two routes of inoculation: direct intracranial injection and oral feeding. Extensive axonal swellings, in the form of spheroids, were observed in both humans and rats and to a lesser extent in pigs with NCC. Spheroids demonstrated increased immunoreactivity to amyloid precursor protein and neurofilament indicating probable impairment of axonal transport. These novel findings demonstrate that spheroids are present in NCC which is conserved across species. Not only is this an important contribution towards understanding that pathogenesis of NCC, but it also provides a model to analyze the association of spheroids with specific clinical features and to investigate the reversibility of spheroid formation with antihelmintic treatment.

Neurocysticercosis (NCC), the infection of the human central nervous system (CNS) with larval cysts of Taenia solium causes widespread neurological morbidity. Animal models are crucial for studying the pathophysiology and treatment of NCC. Some drawbacks of current NCC models include differences in the pathogenesis of the model and wild-type parasite, low rates of infection efficiency and lack of reproducibility. We describe a novel porcine model that recreates infection in the CNS with high efficiency. Activated oncospheres, either in a high (45,000–50,000) or low (10,000) dose were inoculated in the common carotid artery of 12 pigs by ultrasound-guided catheterization. Following oncosphere injection, either a high (30 mL) or low (1–3 mL) volume of saline flush was also administered. Cyst burden in the CNS was evaluated independently according to oncosphere dose and flush volume. Neurocysticercosis was achieved in 8/12 (66.7%) pigs. Cyst burden in the CNS of pigs was higher in the high versus the low oncosphere dose category (median: 4.5; interquartile ranges [IQR]: 1–8 and median: 1; IQR: 0–4, respectively) and in the high versus the low flush volume category (median 5.5; IQR: 1–8 and median: 1; IQR: 0–2, respectively), although not statistically different. All cysts in the CNS were viable, whereas both viable and degenerated cysts were found in the musculature. Carotid injection of activated oncospheres in pigs is effective in reproducing NCC. Oncosphere entry into the CNS by way of vasculature mimics wild-type infection, and provides a useful alternative for future investigations on the pathogenesis and antiparasitic treatment of NCC.

Objective To analyse the genetic variability of EG95 sequences and provide guidance for EG95 vaccine application against Echinococcus granulosus ( E. granulosus ). Methods We analysed EG95 polymorphism by collecting total 97 different E. granulosus isolates from 12 different host species that originated from 10 different countries. Multiple sequence alignments and the homology were performed by Lasergene 1 (DNASTAR Inc., Madison, WI), and the phylogenetic analysis was performed by using MEGA5.1 (CEMI, Tempe, AZ, USA). In addition, linear and conformational epitopes were analysed, including secondary structure, NXT/S glycosylation, fibronectin type III (FnIII) domain and glycosylphosphatidylinositol anchor signal (GPI-anchor). The secondary structure was predicted by PSIPRED method. Results Our results indicated that most isolates overall shared 72.6–100% identity in EG95 gene sequence with the published standard EG95 sequence, X90928. However, EG95 gene indeed has polymorphism in different isolates. Phylogenetic analysis showed that different isolates could be divided into three subgroups. Subgroup 1 contained 87 isolates while Subgroup 2 and Subgroup 3 consisted of 3 and 7 isolates, respectively. Four sequences cloned from oncosphere shared a high identity with the parental sequence of the current vaccine, X90928, and they belonged to Subgroup 1. However, in comparison to X90928, several amino acid mutations occurred in most isolates besides oncosphere, which potentially altered the immunodominant linear epitopes, glycosylation sites and secondary structures in EG95 genes. All these variations might change their previous antigenicity and thereby affecting the efficacy of current EG95 vaccine. Conclusions This study reveals the genetic variability of EG95 sequences in different E. granulosus isolates, and proposed that more vaccination trials would be needed to test the effectiveness of current EG95 vaccine against distinct isolates in different countries.

Taenia pisiformis larvae cause significant health problems to rabbits. At present, it is not known whether the recombinant antigen from the T. pisiformis oncosphere is able to confer protective immunity against T. pisiformis larval infection. The full-length cDNA was cloned into a pET32a (+) vector, and the recombinant protein was then expressed in BL21 (DE3) cells. Vaccination with the purified rTpUbc2 coupled with QuilA was carried out in New Zealand rabbits to evaluate the immunoprotective effect against T. pisiformis infection. The full-length open reading frame of the TpUbc2 gene was 444 bp, and encoded a 16.63-kDa protein. Finally, rTpUbc2 was used to evaluate the ability to induce immunoprotective responses in rabbits. A 79.3-90.8% reduction (P0.01) in the recovery of larvae was observed in the experimental group compared to the control group. Specific anti-rTpUbc2 antibodies from immunized rabbits had significantly higher levels of IgG (P0.01) compared to the control group; however, no significant difference in IgA levels was found between groups (P0.05). Our data support the use of rTpUbc2 as a potential candidate to develop a vaccine against T. pisiformis larvae.

Echinococcus granulosus is a parasite that can be seen throughout the world. So far, five species of genus Echinococcus have been identified as parasite in people: E.granulosus, E.multilocularis, E.vogeli, E.oligarthrus, E.shiquicus. Larval (metacestod) form of parasite settles in internal organs of hoofed animals (cattle, goats, pigs, horses, sheep, etc.) and human; the adult form is found in small intestine of final host, canine. Disease caused by parasite called as "Cystic echinococcosis" (CE) is an important health problem and causes economic losses in many countries including our country that livestock is common. Infective eggs cause infections in intermediate hosts by taking oral way and rarely inhalation. Received egg opens in the stomach and intestines of intermediate host and oncosphere is released. Oncosphere quickly reaches the lamina propria of the villus epithelium by its histolytic enzymes and hooks. It usually transported from here to the liver and lungs, less frequently, muscle, brain, spleen, kidney and to other organs through the veins. By molecular studies, five species have been validated taxonomically and 10 different variants or strains of E.granulosus have been identified. Host and developmental differences between strains may negatively affect control studies and fight against the parasite. This study aimed to determinate E.granulosus strains obtained from cyst material of different intermediate hosts from different regions of Turkey by molecular methods. In the study, 25 human, 8 cattle, 6 sheep and 2 goat cysts material has been collected. Total genomic DNA was isolated from protoscoleces in cyst fluid and analyzed by PCR with COX-1 (L) and COX-1 (S) genes specific primers. DNA sequence analysis for each PCR product has been made. DNA sequence analysis results evaluated phylogenetically by MEGA analyze and BLAST software. As a result of this study, all isolates were identified as E.granulosus sensu stricto (G1) by DNA sequence analysis. CE is a major public health problem for our country so we believe that obtained data from this study is an important source for parasite control, effective diagnosis, treatment techniques, eradication, vaccination and drug development. Similar studies will be beneficial to cover all other regions of Turkey and to develop effective and successful control programs.

Taenia solium causes cysticercosis in pigs and taeniasis and neurocysticercosis in humans. Oncosphere antigens have proven to be effective as vaccines to protect pigs against an experimental infection with T. solium. A pair-matched vaccination trial field, using a combination of two recombinant antigens, TSOL16 and TSOL18, was undertaken in rural villages of Peru to evaluate the efficacy of this vaccine under natural conditions. Pairs of pigs (n=137) comprising one vaccinated and one control animal, were allocated to local villagers. Animals received two vaccinations with 200μg of each of TSOL16 and TSOL18, plus 5mg Quil-A. Necropsies were performed 7 months after the animals were distributed to the farmers. Vaccination reduced 99.7% and 99.9% (p

Highlights ► Experimental Taenia solium challenge of piglets vaccinated with recombinant antigens. ► Protection of piglets immunized with the TSOL16 recombinant antigen. ► Assessment of antibody responses in pigs vaccinated with recombinant antigens., Recombinant antigens from the oncosphere stage of the parasite Taenia solium were expressed in Escherichia coli. The TSOL16, TSOL45-1A and TSOL45-1B recombinant antigens, each consisting of fibronectin type III (FnIII) domain S, were produced as fusion proteins with glutathione S-transferase (GST) and maltose binding protein (MBP). Groups of pigs were immunized twice with the GST fusions of the antigens and boosted a third time with the MBP fusions prior to receiving a challenge infection with T. solium eggs. The TSOL16 antigen was found to be capable of inducing high levels of immunity in pigs against a challenge infection with T. solium. Immunological investigations identified differences in immune responses in the pigs vaccinated with the various antigens. The results demonstrate that the TSOL16 antigen could be a valuable adjunct to current porcine vaccination approaches and may allow the further development of new vaccination strategies against T. solium cysticercosis.

Cysticercosis is a public health problem in several developing countries. The oncosphere protein TSOL18 is the most immunogenic and protective antigen ever reported against porcine cysticercosis, although no specific epitope has been identified to account for these properties. Recent evidence suggests that protection might be associated with conformational epitopes. Linear epitopes from TSOL18 were computationally predicted and evaluated for immunogenicity and protection against porcine cysticercosis. A synthetic peptide was designed based on predicted linear B cell and T cell epitopes that are exposed on the surface of the theoretically modeled structure of TSOL18. Three surface epitopes from TSOL18 were predicted as immunogenic. A peptide comprising a linear arrangement of these epitopes was chemically synthesized. The capacity of the synthetic peptide to protect pigs against an oral challenge with Taenia solium proglottids was tested in a vaccine trial. The synthetic peptide was able to produce IgG antibodies in pigs and was associated to a reduction of the number of cysts, although was not able to provide complete protection, defined as the complete absence of cysts in necropsy. This study demonstrated that B cell and T cell predicted epitopes from TSOL18 were not able to completely protect pigs against an oral challenge with Taenia solium proglottids. Therefore, other linear epitopes or eventually conformational epitopes may be responsible for the protection conferred by TSOL18.

Taenia ovisis a cestode parasite infecting primarily sheep as intermediate hosts and dogs as definitive hosts. The first highly effective, recombinant vaccine against a parasitic organism was developed againstT. ovisinfection in sheep. Three separate host-protective antigens (To16, To18, and To45W) have been cloned from the oncosphere of the parasite. We localize these antigens in the oncosphere by using quantitative immunogold labeling and transmission electron microscopy. The three antigens were uniquely associated with penetration gland cells. The cytoplasm and secretory granules of both penetration gland type 1 and type 2 cells exhibited statistically significant levels of staining for each of the three antigens. The intensity of labeling of the penetration gland type 1 cell was approximately three to five times greater (P< 0.01) compared to the level of staining intensity seen in the penetration gland type 2 cell. In activated oncospheres, secretory blebs were found to contain granules with a structure similar to those observed in the penetration gland cells. The granules within the secretory blebs were shown to stain specifically for the presence of each of the three host-protective antigens. The absence of surface location of theT. ovisantigens suggests that the parasite may not be susceptible to vaccine-induced antibody- and complement-mediated attack until some postoncospheral development has occurred after infection of the intermediate host.

TSOL18 is a recombinant protein that has been shown in repeated experimental trials to be capable of protecting pigs against challenge infection with the cestode parasite Taenia solium. Antibodies raised by the vaccine are capable of killing the parasite in an in vitro culture and it is believed that antibody and complement-mediated killing of invading parasites is the major protective immune mechanism induced by vaccination with TSOL18. Investigations were undertaken to characterize whether the principal antibody specificities raised by TSOL18 in pigs were against linear or conformational determinants. TSOL18 was expressed in two truncated forms representing either the amino terminal portion or the carboxy terminal portion, with the two truncations overlapping in sequence by 25 amino acids. The original protein (designated TSOL18N(-)) and the two truncations (TSOL18N(-)-1 and TSOL18N(-)-2) were used in inhibition ELISA. TSOL18N(-) was shown to be capable of completely inhibiting the binding of pig anti-TSOL18N(-) antibodies to TSOL18N(-) in ELISA. However, neither TSOL18N(-)-1 nor TSOL18N(-)-2, either alone or when combined together, was capable of inhibiting any detectable amount of reactivity of pig anti-TSOL18N(-) antibodies with TSOL18N(-). It is concluded that the dominant antibody specificities, and probably the host-protective specificities, of TSOL18 are conformational epitopes.

Echinococcus multilocularis causes an important zoonotic cestode disease. The metacestocle stage proliferates in the liver of intermediate hosts including human and rodents and forms multiple cysts. Recently, members of a transmembrane protein tetraspanin (TSP) family have been used as vaccines against schistosomosis, or as diagnostic antigens for cysticercosis. In this study, seven tetraspanins of E. multilocularis, designated as TSP1 to TSP7, were evaluated for their protective potential against primary alveolar echinococcosis. The large extracellular loop (LEL) region of these tetraspanins was cloned from a full-length enriched cDNA library of E. multilocularis metacestodes and expressed in Escherichia coli as a fusion protein with thioredoxin. Recombinant TSPs were applied as vaccines against an E. multilocularis primary experimental infection in BALB/c mice. Cyst lesions in the livers of vaccinated and non-vaccinated mice were counted. The cyst lesion reduction rates induced by the seven tetraspanins in vaccinated vis-à-vis non-vaccinated mice were: 87.9%, 65.8%, 85.1%, 66.9%, 73.7%, 72.9% and 37.6%. Vaccination conferred protective rates to mice ranging from 0% (TSP5, 6, 7) to maximally 33% (TSP1, 3). The results indicated that recombinant tetraspanins have varying protective effects against primary alveolar echinococcosis and could be used in vaccine development.

Echinococcus multilocularis causes an important zoonotic cestode disease. The metacestode stage proliferates in the liver of intermediate hosts including human and rodents and forms multiple cysts. Recently, members of a transmembrane protein tetraspanin (TSP) family have been used as vaccines against schistosomosis, or as diagnostic antigens for cysticercosis. In this study, seven tetraspanins of E. multilocularis, designated as TSP1 to TSP7, were evaluated for their protective potential against primary alveolar echinococcosis. The large extracellular loop (LEL) region of these tetraspanins was cloned from a full-length enriched cDNA library of E. multilocularis metacestodes and expressed in Escherichia coli as a fusion protein with thioredoxin. Recombinant TSPs were applied as vaccines against an E. multilocularis primary experimental infection in BALB/c mice. Cyst lesions in the livers of vaccinated and non-vaccinated mice were counted. The cyst lesion reduction rates induced by the seven tetraspanins in vaccinated vis-à-vis non-vaccinated mice were: 87.9%, 65.8%, 85.1%, 66.9%, 73.7%, 72.9% and 37.6%. Vaccination conferred protective rates to mice ranging from 0% (TSP5, 6, 7) to maximally 33% (TSP1, 3). The results indicated that recombinant tetraspanins have varying protective effects against primary alveolar echinococcosis and could be used in vaccine development.

Taenia multiceps is a cestode parasite, the larval stage of which encysts in the brain of sheep, goats and cattle causing an often fatal condition. The parasite also causes zoonotic infections in humans. Homologues of the recombinant oncosphere vaccine antigens from Taenia ovis and other Taenia species were identified in T. multiceps. Sequencing of the associated T. multiceps genes and cloning of the encoding mRNA has revealed conserved features in the genes and proteins. The T. multiceps oncosphere proteins, designated Tm16 and Tm18, contain a predicted secretory signal and fibronectin type III domain. The recombinant Tm16 and Tm18 proteins were successfully expressed in Escherichia coli as fusion proteins with GST. The antigens, formulated with Quil A adjuvant, were tested in a vaccine trial in sheep. The antigens stimulated immunity in sheep against challenge infection with T. multiceps eggs. Five of nine control sheep died due to a challenge infection with T. multiceps whereas none of 20 vaccinated animals died as a result of the parasite challenge (P=0.001). In addition, vaccination with the Tm16 protein, or Tm16 plus Tm18, induced significant protection against the number of parasites encysting in the brain as a result of the challenge infection (P=0.023, P=0.015, respectively). No clear relationship was apparent between the level of specific serum antibody in vaccinated animals and either the presence or absence of parasites or the number of parasites that occurred in some of the vaccinated animals. We believe this study is the first description of recombinant vaccine-related investigations for T. multiceps. The recombinant oncosphere antigens identified may allow development of effective vaccination strategies against T. multiceps infection in sheep. They raise the potential for the development of a combined vaccine with the Echinococcus granulosus EG95 antigen for prevention of T. multiceps as well as preventing the transmission of cystic hydatid disease.

Background The transitional period between the oncosphere and the cysticercus of Taenia solium is the postoncospheral (PO) form, which has not yet been completely characterized. The aim of this work was to standardize a method to obtain T. solium PO forms by in vitro cultivation. We studied the morphology of the PO form and compared the expression of antigenic proteins among the PO form, oncosphere, and cysticerci stages. Methodology/Principal Findings T. solium activated oncospheres were co-cultured with ten cell lines to obtain PO forms, which we studied at three stages of development–days 15, 30, and 60. A high percentage (32%) of PO forms was obtained using HCT-8 cells in comparison to the other cell lines. The morphology was observed by bright field, scanning, and transmission electron microscopy. Morphology of the PO form changed over time, with the six hooks commonly seen in the oncosphere stage disappearing in the PO forms, and vesicles and microtriches observed in the tegument. The PO forms grew as they aged, reaching a diameter of 2.5 mm at 60 days of culture. 15–30 day PO forms developed into mature cysticerci when inoculated into rats. Antigenic proteins expressed in the PO forms are also expressed by the oncosphere and cysticerci stages, with more cysticerci antigenic proteins expressed as the PO forms ages. Conclusions/Significance This is the first report of an in vitro production method of T. solium PO forms. The changes observed in protein expression may be useful in identifying new targets for vaccine development. In vitro culture of PO form will aid in understanding the host-parasite relationship, since the structural changes of the developing PO forms may reflect the parasite’s immunoprotective mechanisms. A wider application of this method could significantly reduce the use of animals, and thus the costs and time required for further experimental investigations., Author Summary Neurocysticercosis is caused by T. solium, which is a neglected disease. The postoncospheral (PO) form is an intermediate form between the oncosphere, which is the larva, and the fully developed cysticercus, which is a cyst with a scolex. The morphology, development, and protein and antigen expression of the PO form have not previously been characterized. Here, we report the novel in vitro cultivation of T. solium PO forms and characterize the morphology, development, and expression of antigenic proteins. This new method will allow for better study of this transitional form, which is very difficult to study in the intermediate host. With the increased availability of secreted proteins and antigens, in vitro cultivation will help improve diagnostic assays and provide new targets for vaccine development to block transmission.

Ultrastructural characteristics of the oncospheral hook morphogenesis in the taeniid cestode Echinococcus multilocularis Leuckart, 1863, a parasite of medical and veterinary importance, are described. Oncospheral hook primordia appear at the preoncospheral phase of the embryonic development. Within six specialised cells of the so-called oncoblasts, high concentration of mitochondria, numerous ribosomes and extended Golgi regions are involved in hook development. During hook growth, the blade and base gradually protrude outside the oncoblast plasma membrane. The nucleated oncoblast persists around the handles of fully formed hooks. Simultaneously with the hook primordium elongation and transformation into a blade, handle and base, the hook material differentiates into an electron-dense cortex and a less dense inner core. The exit of the blade of each mature hook, protruding from the oncosphere, is surrounded by a circular, septate desmosome and two rigid, dense rings on either side. The pattern of oncospheral hook morphogenesis in E. multilocularis is compared with that of other previously examined cyclophyllidean cestodes. Though oncoblasts have never been observed around the mature hooks, their remnants are often still visible in the fully developed infective oncospheres in particular in some taeniid species so far examined in this respect. The origin and formation of oncospheral hooks in E. multilocularis, evidently differs from that of the rostellar hooks. Thus, although the hooks may have slight similarity at the gross level, they are neither analogous nor homologous structures.

Humans are definitive hosts of three species of the Taenia genus, namely Taenia solium, Taenia saginata and Taenia asiatica. The relative novelty of the latter explains the lack of knowledge concerning certain relevant aspects related to this parasite, such as its definite geographical distribution and whether its eggs can infect humans or not. So far, only the eggs of T. solium are known to be infective for humans, producing cysticercosis. Although eggs contain the infective stage, the oncosphere, there is a lack of research on the ultrastructure of eggs of human taeniids. We show, for the first time, the ultrastructure of eggs of T. asiatica by means of SEM and TEM analyses. We detected all the envelopes, namely the egg shell, vitelline layer, outer embryophoric membrane, embryophore, granular layer, basal membrane, oncospheral membrane and oncospheral tegument. Hooks surrounded by myofibrils and glycogen-like particles, the two types of secretory granules of the penetration glands, as well as several nuclei and mitochondria were also revealed in the oncospheres. In addition to the already known structures in eggs from other Taenia species, the presence of two types of small vesicles is described herein, possibly corresponding to exosomes and ectosomes because of their shape and size, which could participate in the host/parasite intercellular communication.

Infections due to Taenia solium in humans (taeniasis/cysticercosis) remain a complex health problem, particularly in developing countries. We identified two oncosphere proteins that might protect the porcine intermediate host against cysticercosis and therefore help prevent disease in humans. One of these proteins was further identified by two-dimensional gel electrophoresis and micro-sequencing. The gene encoding this protective protein was also identified, cloned and characterized. The native 31.5 kDa protein Tso31 has four variants at the cDNA level. The longest sequence from which the others seem to derive, encodes a 253 amino acid peptide. The predicted protein has a molecular weight of 25.1 kDa, one putative N-glycosylation site, two fibronectin type III domains, and one C terminal transmembrane domain. The gene structure of the protein consists of four exons and three introns. The finding of one gene and four different cDNAs for Tso31 suggests the existence of a possible mechanism of differential splicing in this parasite. The Tso31 protein is exclusive to T. solium oncospheres with a putative protein structure of an extra-cellular receptor-like protein. The Tso31 protein was expressed as a recombinant protein fused to GST and tested in a vaccine to determine its effectiveness in protecting pigs against cysticercosis. Only two pigs out of eight vaccinated were protected and although the total median number of cyst decreased in vaccinated pigs compared to controls this decrease was not statistically significant (P = 0.09).

The specific mechanisms underlying Taenia solium oncosphere adherence and penetration in the host have not been studied previously. We developed an in vitro adhesion model assay to evaluate the mechanisms of T. solium oncosphere adherence to the host cells. The following substrates were used: porcine intestinal mucosal scrapings (PIMS), porcine small intestinal mucosal explants (PSIME), Chinese hamster ovary cells (CHO cells), epithelial cells from ileocecal colorectal adenocarcinoma (HCT-8 cells), and epithelial cells from colorectal adenocarcinoma (Caco-2 cells). CHO cells were used to compare oncosphere adherence to fixed and viable cells, to determine the optimum time of oncosphere incubation, to determine the role of sera and monolayer cell maturation, and to determine the effect of temperature on oncosphere adherence. Light microscopy, scanning microscopy, and transmission microscopy were used to observe morphological characteristics of adhered oncospheres. This study showed in vitro adherence of activated T. solium oncospheres to PIMS, PSIME, monolayer CHO cells, Caco-2 cells, and HCT-8 cells. The reproducibility of T. solium oncosphere adherence was most easily measured with CHO cells. Adherence was enhanced by serum-binding medium with >5% fetal bovine serum, which resulted in a significantly greater number of oncospheres adhering than the number adhering when serum at a concentration less than 2.5% was used ( P < 0.05). Oncosphere adherence decreased with incubation of cells at 4°C compared with the adherence at 37°C. Our studies also demonstrated that T. solium oncospheres attach to cells with elongated microvillus processes and that the oncospheres expel external secretory vesicles that have the same oncosphere processes.

We compared the peptidase activities of the excretory/secretory (E/S) antigens of oncospheres of Taenia solium and related, but nonpathogenic, Taenia saginata. Taenia solium and T. saginata oncospheres were cultured, and the spent media of 24-, 48-, 72-, and 96-hr fractions were analyzed. Activities for serine peptidases (chymotrypsin-, trypsin-, and elastase-like), cysteine peptidases (cathepsin B-, cathepsin L-, and calpaine-like), and aminopeptidase (B-like peptidases) were tested fluorometrically with peptides coupled to 7-amino-4-methylcoumarin. In both species, the E/S antigens showed cysteine, serine, and aminopeptidase activities. Although no particular peptidase had high activity in T. solium, and was absent in T. saginata, or vice versa, different patterns of activity were found. A chymotrypsin-like peptidase showed the highest activity in both parasites, and it had 10 times higher activity in T. solium than in T. saginata. Trypsin-like and cathepsin B-like activities were significantly higher in T. solium. Minimal levels of cathepsin B were present in both species, and higher levels of elastase-like and cathepsin L-like activity were observed in T. saginata. Taenia solium and T. saginata have different levels and temporal activities of proteolytic enzymes that could play a modulator role in the host specificity for larval invasion through penetration of the intestinal mucosa.

The tapeworm Echinococcus granulosus is responsible for cystic echinococcosis (CE), a cosmopolitan disease which imposes a significant burden on the health and economy of affected communities. Little is known about the molecular mechanisms whereby E. granulosus is able to survive in the hostile mammalian host environment, avoiding attack by host enzymes and evading immune responses, but protease inhibitors released by the parasite are likely implicated. We identified two nucleotide sequences corresponding to secreted single domain Kunitz type protease inhibitors (EgKIs) in the E. granulosus genome, and their cDNAs were cloned, bacterially expressed and purified. EgKI-1 is highly expressed in the oncosphere (egg) stage and is a potent chymotrypsin and neutrophil elastase inhibitor that binds calcium and reduced neutrophil infiltration in a local inflammation model. EgKI-2 is highly expressed in adult worms and is a potent inhibitor of trypsin. As powerful inhibitors of mammalian intestinal proteases, the EgKIs may play a pivotal protective role in preventing proteolytic enzyme attack thereby ensuring survival of E. granulosus within its mammalian hosts. EgKI-1 may also be involved in the oncosphere in host immune evasion by inhibiting neutrophil elastase and cathepsin G once this stage is exposed to the mammalian blood system. In light of their key roles in protecting E. granulosus from host enzymatic attack, the EgKI proteins represent potential intervention targets to control CE. This is important as new public health measures against CE are required, given the inefficiencies of available drugs and the current difficulties in its treatment and control. In addition, being a small sized highly potent serine protease inhibitor, and an inhibitor of neutrophil chemotaxis, EgKI-1 may have clinical potential as a novel anti-inflammatory therapeutic., Author Summary The hydatid tapeworm Echinococcus granulosus is able to survive in its mammalian hosts for many years without being digested by proteases. Two E. granulosus Kunitz proteins with potent protease inhibitory properties were identified and characterized. These Kunitz proteins may provide protection to the parasite from proteolytic digestion. These newly identified proteins are promising targets for developing new control interventions against echinococcosis, and one has potential as a novel anti-inflammatory therapeutic.

Dogs are pivotal in Echinococcus granulosus transmission to humans, and dog vaccination provides a very practical and cost-effective prevention strategy. We vaccinated dogs with soluble native proteins isolated from protoscoleces of E. granulosus and induced significant suppression of worm growth and egg production. Accordingly, we tested for vaccine efficacy using recombinant proteins derived from a developmentally regulated gene family (egM) specifically expressed in mature adult E. granulosus worms. Three egM genes--egM4, egM9, and egM123--were subcloned into an expression vector that expressed the molecules as soluble glutathione S-transferase (GST) fusion proteins in Escherichia coli. The 3 fusion proteins were purified for dog vaccination trials (3 doses of 80 microg/protein/dog) in which the dogs were challenged and then necropsied 45 days after infection. Compared with worms in the control dogs that received GST, the 3 recombinant proteins induced a very high level of protection (97%-100%) in terms of suppression of worm growth and, especially, of egg development and embryogenesis. We have thus shown that vaccination of the dog host against E. granulosus is feasible when recombinant proteins are used. Because the egg stage is crucial in the echinococcal life cycle, successful suppression of egg development by vaccination would halt transmission to intermediate hosts, thereby effecting long-term control.

Freshly released eggs from four species of the cestode Eubothrium (Eubothrium crassum, Eubothrium fragile, Eubothrium rugosum, and Eubothrium salvelini) were subjected to morphological and morphometric analysis. The eggs of the two freshwater species, E. rugosum and E. salvelini, were ovoid with a lobed embryophore whereas the eggs of the two marine species, E. crassum and E. fragile, were more circular with a smooth embryophore. However, the morphological differences between species were not readily evident to permit their clear distinction from one another. To discriminate species, a forward stepwise linear discriminant analysis, using six of the seven measured metric characters made on the eggs, was used, which gave 100% correct classification of two species, E. rugosum and E. salvelini, and a high proportion of correct classification for E. crassum (98%) and E. fragile (83%). Of the latter two species, one specimen of E. crassum and five specimens of E. fragile were misclassified between the respective groups. The principal characters used in the classification of the species were the width of the egg, the length of the mediolateral hooks, and the width of the oncosphere. To provide more information on the life cycle of each species, the eggs were used in a series of infection trials to identify appropriate intermediate hosts. Experimental infections with freshwater copepods were successful when exposed to the eggs of E. salvelini, partially successful when exposed to the eggs of marine E. crassum with 10% of the copepods becoming infected, but no infections were obtained when the eggs of E. fragile were used.

Infection by larval parasites can have severe consequences on intermediate hosts that affect transmission, fecundity and fitness of the host, and host population structure. This study examines the pathogenic effects of cysticercoid larvae of the hymenolepidid cestode Microsomacanthus hopkinsi (Schiller, 1951) on its amphipod intermediate host, Hyalella azteca Saussure, 1858. There was a significant, positive relationship between oncosphere consumption, cysticercoid burden, and age in short-term experiments in which groups of H. azteca were exposed individually to single egg packets of M. hopkinsi during instars 1, 2, 3, 4, 6, 8, and 9; however, there was no correlation between oncosphere consumption and the intensity of infection in the amphipod hosts within each instar. The mean number of moults over a 14 day experimental period was significantly less in infected amphipods than in their respective controls. In short-term experiments, the greatest mortality appeared to be limited to amphipods exposed during the earliest instars; little mortality was observed in amphipods exposed during instar 4 or later. Long-term experiments revealed a significant negative effect of infection on the overall life span of both male and female H. azteca exposed individually to a single egg packet during instar 4. Of 72 females infected during instar 4 and provided with mates during instar 6, only 1 and 4 produced broods in instars 8 and 9, respectively, compared with 58 and 57 of 72 control females. Broods produced by infected females were significantly smaller than those of control females. Infected individuals were less likely to mate successfully. The results are discussed in terms of their consequences for transmission, host fitness, and potential effects on host populations.

Two recombinant Taenia solium oncosphere antigens, designated TSOL18 and TSOL45-1A, were investigated as vaccines to prevent transmission of the zoonotic disease cysticercosis through pigs. Both antigens were effective in inducing very high levels of protection (up to 100%) in three independent vaccine trials in pigs against experimental challenge infection with T. solium eggs, which were undertaken in Mexico and Cameroon. This is the highest level of protection that has been achieved against T. solium infection in pigs by vaccination with a defined antigen. TSOL18 and TSOL45-1A provide the basis for development of a highly effective practical vaccine that could assist in the control and, potentially, the eradication of human neurocysticercosis.

We tested the possible diagnostic utility of five Taenia saginata oncosphere-derived synthetic peptides in T. solium neurocysticercosis (NC). The five peptides correspond to protein sequences with high antigenic indexes that were cloned from a T. saginata oncosphere cDNA library. The test samples consisted of cerebrospinal fluid (CSF) samples randomly collected from patients referred from Mexican and Brazilian neurological institutes. Indirect enzyme-linked immunosorbent assays (ELISA) were carried out with the peptides either unconjugated or coupled to carrier proteins, and were compared with results obtained using T. solium cyst fluid as a positive control. For active inflammatory NC, the higher sensibility (93%) and specificity (85%) was obtained with peptides HP6-2 and Ts45W-1, respectively, coupled to ovalbumin, in both Mexican and Brazilian patients. Examining the results of the individual peptide assays in combination, in some instances, improved the sensitivity to 100%.

This study investigated the protective capacity of the recombinant Taenia saginata Tso18 antigen administered as a DNA vaccine in the Taenia crassiceps murine model of cysticercosis. This Tso18 DNA sequence, isolated from a T. saginata oncosphere cDNA library, has homologies with Taenia solium and Echinococcus sp. It was cloned in the pcDNA3.1 plasmid and injected once intramuscularly into mice. Compared to saline-vaccinated control mice, immunization reduced the parasite burden by 57.3-81.4%, while lower levels of non-specific protection were induced in control mice injected with the plasmid pcDNA3.1 (18.8-33.1%) or a plasmid with irrelevant construct, pcDNA3.1/3D15 (33.4-38.8%). Importantly, significant levels of protection were observed between the pcDNA3.1/Tso18 plasmid and pcDNA3.1/3D15 plasmid immunized mice. Mice immunized with pTso18 synthesized low levels of, primarily IgG1 sub-class, antibodies. These antibodies were shown to recognize a 66 kDa antigen fraction of T. crassiceps and T. solium. Splenocytes enriched in both CD4+CD8- and CD4-CD8+ T cells from these vaccinated mice proliferated in vitro when exposed to antigens from both T. solium and T. crassiceps cestodes. Immunolocalization studies revealed the Tso18 antigen in oncospheres of T. saginata and T. solium, in the adult tapeworm and in the tegument of T. solium cysticerci. The protective capacity of this antigen and its extensive distribution in different stages, species and genera of cestodes points to the potential of Tso18 antigen for the possible design of a vaccine against cestodes.

The life-threatening diseases alveolar and cystic echinococcoses are caused by larvae of the tapeworms Echinococcus multilocularis and E. granulosus, respectively. In both cases, intermediate hosts, such as humans, are infected by oral uptake of oncosphere larvae, followed by asexual multiplication and almost unrestricted growth of the metacestode within host organs. Besides surgery, echinococcosis treatment relies on benzimidazole-based chemother- apy, directed against parasite beta-tubulin. However, since beta-tubulins are highly similar between cestodes and humans, benzimidazoles can only be applied at parasitostatic doses and are associated with adverse side effects. Mostly aiming at identifying alternative drug targets, the nuclear genome sequences of E. multilocularis and E. granulosus have recently been characterized, revealing a large number of druggable targets that are expressed by the metacestode. Furthermore, recent cell biological investigations have demonstrated that E. multilocularis employs pluripotent stem cells, called germinative cells, which are the only parasite cells capable of proliferation and which give rise to all differentiated cells. Hence, the germinative cells are the crucial cell type mediating prolif- eration of E. multilocularis, and most likely also E. granulosus, within host organs and should also be responsible for parasite recurrence upon discontinuation of chemotherapy. Interestingly, recent investigations have also indicated that germinative cells might be less sensitive to chemotherapy because they express a beta-tubulin isoform with limited affinity to benzimidazoles. In this article, we briefly review the recent findings concerning Echinococcus genomics and stem cell research and propose that future research into anti-echinococcosis drugs should also focus on the parasite's stem cell population.