Molecular characterization of EcCLP1, a new putative cathepsin L protease from Echinococcus canadensis.

Echinococcus granulosus sensu lato is a platyhelminth parasite and the etiological cause of cystic echinococcosis (CE), a zoonotic and neglected disease that infects animals and humans worldwide. As a part of the biological arsenal of the parasite, cathepsin L proteases are a group of proteins that are believed to be essential for parasite penetration, immune evasion, and establishment in the tissues of the host. In this work, we have cloned and sequenced a new putative cathepsin L protease from Echinococcus canadensis (EcCLP1). The bioinformatic analysis suggests that EcCLP1 could be synthesized as a zymogen and activated after proteolytic cleavage. The multiple sequence alignment with other cathepsin proteases reveals important functional conserved features like a conserved active site, an N-linked glycosylation residue, a catalytic triad, an oxyanion hole, and three putative disulfide bonds. The phylogenetic analysis suggests that EcCLP1 could indeed be a cathepsin L cysteine protease from clade 1 as it grouped with cathepsins from other species in this clade. Modeling studies suggest that EcCLP1 has two domains forming a cleft where the active site is located and an occluding role for the propeptide. The transcriptomic analysis reveals different levels of cathepsin transcript expression along the different stages of the parasite life cycle. The whole-mount immunohistochemistry shows an interesting superficial punctate pattern of staining which suggests a secretory pattern of expression. The putative cathepsin L protease characterized here may represent an interesting tool for diagnostic purposes, vaccine design, or a new pharmacological target for antiparasitic intervention.

Title: Caractérisation moléculaire d'EcCLP1, une nouvelle protéase putative de type cathepsine L d'Echinococcus canadensis. Abstract: Echinococcus granulosus sensu lato est un Plathelminthe parasite et la cause étiologique de l'échinococcose kystique (EK), une maladie zoonotique et négligée qui infecte les animaux et les humains dans le monde entier. En tant que partie de l'arsenal biologique du parasite, les protéases de type cathepsine L sont un groupe de protéines considérées comme essentielles à la pénétration du parasite, l'évasion immunitaire et son établissement dans les tissus de l'hôte. Dans ce travail, nous avons cloné et séquencé une nouvelle protéase putative de type cathepsine L d'Echinococcus canadensis (EcCLP1). L'analyse bioinformatique suggère qu'EcCLP1 pourrait être synthétisée sous forme de zymogène et activée après clivage protéolytique. L'alignement de séquences multiples avec d'autres protéases de type cathepsine révèle d'importantes caractéristiques fonctionnelles conservées telles qu'un site actif conservé, un résidu de glycosylation lié à N, une triade catalytique, un trou oxyanion et trois liaisons disulfure putatives. L'analyse phylogénétique suggère qu'EcCLP1 pourrait en effet être une protéase de type cathepsine L du clade 1 car elle se regroupe avec les cathepsines d'autres espèces de ce clade. Les études de modélisation suggèrent qu'EcCLP1 possède deux domaines formant une fente où se trouve le site actif et un rôle d'occlusion pour le propeptide. L'analyse transcriptomique révèle différents niveaux d'expression du transcrit de la cathepsine au cours des différentes étapes du cycle de vie du parasite. L'immunohistochimie de montages entiers montre un intéressant motif de coloration ponctuée superficielle qui suggère un modèle d'expression sécrétoire. La protéase putative de type cathepsine L caractérisée ici peut représenter un outil intéressant à des fins de diagnostic, de conception de vaccins ou une nouvelle cible pharmacologique pour une intervention antiparasitaire.

Analysis of vaccination strategy against cystic echinococcosis developed in the Province of Río Negro, Argentina: 12 years of work.

Cystic echinococcosis (CE) is a zoonosis caused by species of the complex Echinococcus granulosus, sensu lato in their larval stage. It is an endemic disease in the province of Río Negro, where small farmers generally have both sheep and goats. Lamb vaccination with EG95 was incorporated in 2009 with very good results: in fact, it contributed to a significant drop in prevalence of infection in both sheep and goats, when determined by necropsy and serology in 2018. In the design of the activity, it was decided not to vaccinate goats in order to minimize the operational requirements of vaccination and comments from producers about the rarity of observing hydatid cysts in goat viscera were considered. OBJECTIVE: To identify causes which can still generate infection in dogs, and to detect species/genotypes in circulation in the province of Río Negro. MATERIALS AND METHODS: In indigenous reserves comprised within the area of lamb vaccination with 3 doses of EG95, (dose 1 in December, dose 2 in January and dose 3 in December of the year following, at the time of application of dose 1 to the new lambs). Prevalence in adult goats and sheep was determined by necropsy and serology (ELISA). Infective species/genotypes present in the work area and in the rest of the province of Río Negro were identified by Cox1 mitochondrial gene sequencing. Epidemiological analysis was completed with surveys among farmers about slaughter habits for human consumption. RESULTS: Through serology and necropsy, infection rates in vaccinated and nonvaccinated sheep were significantly different (21% versus 66%). Non-vaccinated sheep and non-vaccinated goats were also significantly different in that there was less infection in goats compared to sheep (7% versus 66% for necropsy, 30% versus 61% for serology); After many years of sheep vaccination the infection positives were low, and differences between vaccinated sheep and non-vaccinated goats turned out non-significant (21% versus 7%). With reference to epidemiology and control along the period 2018-2022, PZQ dosing of dogs 4 times a year was maintained, and 2 extra deworming tasks were introduced together with dose 1 and 2 of EG95, performed by the veterinary vaccination team, ensuring the ingestion of PZQ by dogs. Assessment of animal slaughter for consumption in 41 producers showed that 21 of them slaughter a monthly average of 18 goats (an average of 0.43 goat per month per farm) and 36 in all slaughter 35 old sheep in a year (average of 0.85 sheep per month per farm). With respect to identification of species/genotypes as from 2010, genotypes G1 have been found in 11 sheep (out of which 6 belong to vaccination zone) and genotypes G7, in one pig. A goat cyst within vaccination zone turned out unfertile and it was not possible to sequence it. CONCLUSION: Design and implementation of a vaccine programme combined with the use of PZQ resulted as cost-effective, since it was possible to maintain the vaccine over time, with clear impact on prevalence decrease in sheep and goats.

Cystic echinococcosis in South America: systematic review of species and genotypes of Echinococcus granulosus sensu lato in humans and natural domestic hosts.

OBJECTIVE: To systematically review publications on Echinococcus granulosus sensu lato species/genotypes reported in domestic intermediate and definitive hosts in South America and in human cases worldwide, taking into account those articles where DNA sequencing was performed; and to analyse the density of each type of livestock that can act as intermediate host, and features of medical importance such as cyst organ location. METHODS: Literature search in numerous databases. We included only articles where samples were genotyped by sequencing since to date it is the most accurate method to unambiguously identify all E. granulosus s. l. genotypes. Also, we report new E. granulosus s. l. samples from Argentina and Uruguay analysed by sequencing of cox1 gene. RESULTS: In South America, five countries have cystic echinococcosis cases for which sequencing data are available: Argentina, Brazil, Chile, Peru and Uruguay, adding up 1534 cases. E. granulosus s. s. (G1) accounts for most of the global burden of human and livestock cases. Also, E. canadensis (G6) plays a significant role in human cystic echinococcosis. Likewise, worldwide analysis of human cases showed that 72.9% are caused by E. granulosus s. s. (G1) and 12.2% and 9.6% by E. canadensis G6 and G7, respectively. CONCLUSIONS: E. granulosus s. s. (G1) accounts for most of the global burden followed by E. canadensis (G6 and G7) in South America and worldwide. This information should be taken into account to suit local cystic echinococcosis control and prevention programmes according to each molecular epidemiological situation.

Human hydatid disease in Peru is basically restricted to Echinococcus granulosus genotype G1.

A molecular PCR study using DNA from 21 hydatid cysts was performed to determine which strain type is responsible for human infection in Peru. The mitochondrial cytochrome c oxidase subunit 1 (CO1) gene was amplified in 20 out of 21 samples, revealing that all but 1 sample (19/20, 95%) belonged to the common sheep strain (G1). The remaining samples belonged to the camel strain (G6). The G1 genotype was most frequently found in human cases of cystic hydatid disease (CHD) in Peru. Local control measures should focus primarily on decreasing dog and sheep infection rather than intermediate reservoirs.

Sequencing, bioinformatic characterization and expression pattern of a putative amino acid transporter from the parasitic cestode Echinococcus granulosus.

We have sequenced and partially characterized an Echinococcus granulosus cDNA, termed egat1, from a protoscolex signal sequence trap (SST) cDNA library. The isolated 1627 bp long cDNA contains an ORF of 489 amino acids and shows an amino acid identity of 30% with neutral and excitatory amino acid transporters members of the Dicarboxylate/Amino Acid Na+ and/or H+ Cation Symporter family (DAACS) (TC 2.A.23). Additional bioinformatics analysis of EgAT1, confirmed the results obtained by similarity searches and showed the presence of 9 to 10 transmembrane domains, consensus sequences for N-glycosylation between the third and fourth transmembrane domain, a highly similar hydropathy profile with ASCT1 (a known member of DAACS family), high score with SDF (Sodium Dicarboxilate Family) and similar motifs with EDTRANSPORT, a fingerprint of excitatory amino acid transporters. The localization of the putative amino acid transporter was analyzed by in situ hybridization and immunofluorescence in protoscoleces and associated germinal layer. The in situ hybridization labelling indicates the distribution of egat1 mRNA throughout the tegument. EgAT1 protein, which showed in Western blots a molecular mass of approximately 60 kD, is localized in the subtegumental region of the metacestode, particularly around suckers and rostellum of protoscoleces and layers from brood capsules. The sequence and expression analyses of EgAT1 pave the way for functional analysis of amino acids transporters of E. granulosus and its evaluation as new drug targets against cystic echinococcosis.

Echinococcus granulosus antigen B gene family: further studies of strain polymorphism at the genomic and transcriptional levels.

Echinococcus granulosus AgB gene family is constituted by five gene loci. In a previous study, we analyzed the strain variation of EgAgB2/B4 sequences. Here, we have analyzed, by SSCP and sequencing, 250 genomic clones of EgAgB1/B3/B5 gene cluster from five E. granulosus strains. Several new EgAgB genomic variants were found. EgAgB1 and EgAgB3 genomic sequences grouped E. granulosus strains by phylogenetic tools in two clusters: one formed by G1/G2 and the other by G5, G6/G7 strains, in accordance with other molecular markers. EgAgB5 genomic and cDNA sequences were only found in G1/G2 cluster. Reverse transcription-PCR analysis using subunit specific primers revealed that all the EgAgB genes were transcribed in G1 and G7 strains with the exception of EgAgB5 transcripts that were not detected in G7 strain. Interestingly, AgB2 transcripts that were probably processed by an aberrant splicing mechanism leading to a non-functional B2 protein were found in G7 strain.

Patent and pre-patent detection of Echinococcus granulosus genotypes in the definitive host.

The detection of Echinococcus granulosus in dogs is important for epidemiological surveillance and evaluation of cystic hydatic disease control programs. We report the efficacy of two PCR-based methods to detect patent and pre-patent infection in dogs experimentally infected with E. granulosus. The detection is based on amplification of a fragment of a mitochondrial gene (Mit-PCR) and a DNA repetitive element (Rep-PCR) of E. granulosus. We tested the ability of both methods to detect several genotypes of the parasite. Both PCR methods could detect E. granulosus in pre-patent and patent periods, even when microscopical observation of eggs resulted negative in fecal samples. The Mit-PCR produced the same amplification pattern for all the parasite genotypes tested while the amplification patterns with the Rep-PCR differed among groups of strains. Fecal samples collected from dogs of an endemic area were diagnosed with more sensitivity than arecoline hydrobromide purgation. These molecular methods could be applied in the confirmation of coproantigen-positive fecal samples and to verify the success of control programs.

Identification and intra-specific variability analysis of secreted and membrane-bound proteins from Echinococcus granulosus.

Echinococcus granulosus, the etiological agent of cystic hydatid disease, exists as a series of strains or genotypes, differing in biological features. Many of the secreted and membrane-bound proteins (S/M) from helminth parasites are involved in the host-parasite interplay and constitute potential targets for diagnosis, anti-parasitic drugs and vaccines. A number of E. granulosus S/M proteins were identified using the signal sequence trap technique. Six out of seven cDNA fragments of these newly identified proteins showed nucleotide and amino acid sequence variation. Inter-strain variation was reported for other characterized S/M proteins as the vaccine target EG95 and the major hydatid cyst fluid antigen, Antigen B (AgB). AgB is highly polymorphic, 101 different sequences related to AgB were reported so far and were grouped in 5 genes (EgB1-EgB5) and one pseudogene (EgB2p) exclusive of G5, G6/G7 genotypes. The significance of AgB polymorphism and possible consequences in diagnostic performance are discussed. The diagnostic value of the new protein variants detected in E. granulosus strains could be determined through standardized inter-laboratory studies as the recently done by the South American Network for Hydatid Serology.

Cystic echinococcosis in Argentina: evolution of metacestode and clinical expression in various Echinococcus granulosus strains.

Echinococcus granulosus hydatid cysts were examined in 41 patients from Neuquén and Tucumán provinces in Argentina. Sequencing of the mitochondrial cytochrome c oxidase subunit 1 (CO1) revealed in 19 patients common sheep strain (G1), in 6 patients Tasmania sheep strain (G2), in 1 patient cattle strain (G5), and in 15 patients camel strain (G6). In Argentina the only known is the domestic cycle that affects dogs and herbivorous, including ovine, swine, cattle and goats. These strains produced a total of 58.6% of primary liver infections, 29.2% primary in lung, 2.4% primary in spleen and 9.8% were multiorgan abdominal infections. The metacestode was classified using the evolutive stages proposed by WHO-IWGE (from CE1 to CE5). We estimated that CE1 cyst has a duration of about 22 years, CE2 of 14 years, CE3 of 10 years, CE4 of 19 years and CE5 was not determined. The active types CE1 and CE2 reached 75% of all cases from all strains. In 36 patients with cysts from G1, G5 and G6 strain, there were only two asymptomatic cases. The strains of the E. granulosus complex do not present important clinical differences; only G6 seems to have higher growth rate.

Evaluation of Toxoplasma gondii recombinant proteins for the diagnosis of recently acquired toxoplasmosis by an immunoglobulin G analysis.

The value of T. gondii recombinant antigens rRop2, rGra4, rGra7 and rSAG1m (mature version) or rSAG1ct (C-terminal version) in differentiating recently acquired from chronic infections was determined by IgG-ELISA. The general highest sensitivity was observed with rRop2 whereas rSAG1m was not recognized by any of the serum samples, suggesting an incorrect folding. rGra4 and rGra7 showed significant higher sensitivity and absorbance values with serum samples from recently infected individuals compared to those with chronic infection. In contrast, rRop2 and rSAG1ct did not show differences in the reactivity pattern between both groups of serum samples.

Cepas de Echinococcus granulosus y su importancia para el control de la hidatidosis en Argentina / Strain of Echinococcus granulosus and their importance for the control of the hydatidosis in Argentina

El agente causante de la hidatidosis (echinococcosis quística) en el hombre, es el cestode Echinococcus granulosus. Hasta el momento están descriptas nueve variantes genéticas: cepa oveja (genotipo G1), oveja de Tasmania (genotipo G2), cepa búfalo (genotipo G3), cepa caballo (genotipo G4), cepa vaca (genotipo G5), cepa camello (genotipo G6), cepa cerdo (genotipo G7), cepa cérvido (genotipo G8) y el genotipo G9. Estas variantes genéticas deben ser consideradas al desarrollar tareas de investigación y diagnóstico en E. granulosus y al implementar programas de control de la enfermedad. Para algunas de ellas se ha podido determinar diferencias en la infectividad para el hombre, porcentaje de quistes fértiles en diferentes hospedadores intermediarios y período prepatente en perros, entre otras. La identificación de cepas se basa en las diferencias de las características morfológicas, bioquímicas, epidemiológicas y actualmente en la caracterización del genoma usando técnicas moleculares. En la Argentina la hidatidosis es endémica, principalmente en áreas rurales, lo que permite el estudio de los patrones de transmisión, estructura genética y cepas de E. granulosus infectivas para el hombre. Hasta el momento estudios en el resto del mundo indican que la más importante fuente de infección para el hombre es la cepa oveja. Sin embargo, recientemente en Argentina se ha demostrado molecularmente que además de la cepa oveja, la cepa oveja de Tasmania y camello también son infectivas para el hombre en altas proporciones. En este trabajo multidisciplinario, llevado a cabo con personal de las áreas endémicas, el propósito fue conocer la situación epidemiológica de la hidatidosis en la Argentina, por medio de la caracterización molecular de aislamientos de E. granulosus presentes en el país. Analizar su variabilidad genética mediante regiones de ADN evolutivamente independiente, incluyendo moléculas involucradas en el diagnóstico y en la modulación de la respuesta inmune en el hombre, tratando de concentrar el análisis en aquellos aislamientos en los que hay pocos estudios realizados, como la cepa oveja de Tasmania, y la cepa camello y correlacionar dichas variantes con las caracterísitcas clínico-patológicas de la enfermedad. Los resultados obtenidos confirman la presencia de por lo menos cinco variantes genéticas: cepa oveja, oveja de Tasmania, vaca, camello y cerdo en la Argentina... (TRUNCADO)

Cepas de Echinococcus granulosus y su importancia para el control de la hidatidosis en Argentina / Strain of Echinococcus granulosus and their importance for the control of the hydatidosis in Argentina

El agente causante de la hidatidosis (echinococcosis quística) en el hombre, es el cestode Echinococcus granulosus. Hasta el momento están descriptas nueve variantes genéticas: cepa oveja (genotipo G1), oveja de Tasmania (genotipo G2), cepa búfalo (genotipo G3), cepa caballo (genotipo G4), cepa vaca (genotipo G5), cepa camello (genotipo G6), cepa cerdo (genotipo G7), cepa cérvido (genotipo G8) y el genotipo G9. Estas variantes genéticas deben ser consideradas al desarrollar tareas de investigación y diagnóstico en E. granulosus y al implementar programas de control de la enfermedad. Para algunas de ellas se ha podido determinar diferencias en la infectividad para el hombre, porcentaje de quistes fértiles en diferentes hospedadores intermediarios y período prepatente en perros, entre otras. La identificación de cepas se basa en las diferencias de las características morfológicas, bioquímicas, epidemiológicas y actualmente en la caracterización del genoma usando técnicas moleculares. En la Argentina la hidatidosis es endémica, principalmente en áreas rurales, lo que permite el estudio de los patrones de transmisión, estructura genética y cepas de E. granulosus infectivas para el hombre. Hasta el momento estudios en el resto del mundo indican que la más importante fuente de infección para el hombre es la cepa oveja. Sin embargo, recientemente en Argentina se ha demostrado molecularmente que además de la cepa oveja, la cepa oveja de Tasmania y camello también son infectivas para el hombre en altas proporciones. En este trabajo multidisciplinario, llevado a cabo con personal de las áreas endémicas, el propósito fue conocer la situación epidemiológica de la hidatidosis en la Argentina, por medio de la caracterización molecular de aislamientos de E. granulosus presentes en el país. Analizar su variabilidad genética mediante regiones de ADN evolutivamente independiente, incluyendo moléculas involucradas en el diagnóstico y en la modulación de la respuesta inmune en el hombre, tratando de concentrar el análisis en aquellos aislamientos en los que hay pocos estudios realizados, como la cepa oveja de Tasmania, y la cepa camello y correlacionar dichas variantes con las caracterísitcas clínico-patológicas de la enfermedad. Los resultados obtenidos confirman la presencia de por lo menos cinco variantes genéticas: cepa oveja, oveja de Tasmania, vaca, camello y cerdo en la Argentina... (TRUNCADO) (AU)

Several strains of Echinococcus granulosus infect livestock and humans in Argentina.

Mitochondrial cytochrome oxidase subunit 1 (CO1) sequencing, Southern blot of a repetitive DNA element and single strand conformation polymorphism of the 5' non-transcribed region of the cytosolic malate dehydrogenase (MDH) gene were used to determine the extent and distribution of Echinococcus granulosus genetic variation in Argentina. Five distinct strains of E. granulosus were shown to exist in the country. The common sheep, Tasmanian sheep, cattle and camel strains were identified in humans. Unlike the situation found in other countries, where the common sheep strain is the major source of human contamination, the Tasmanian sheep and camel strains produced a significant number of human infections in some regions of Argentina. This is the first report of cattle strain in humans in South America. Goats could be the natural intermediate host of the camel strain, which was not identified in humans from other regions so far. More than one genotype was identified in the same geographic area. These findings may have important consequences for human health and the control of hydatid disease. Within-strain differences were also observed, showing the potential of variation of E. granulosus.