Zoonootiliste paelusside liikidekompleksi Echinococcus granulosus sensu lato taksonoomia ja geneetiline mitmekesisus

Väitekirja elektrooniline versioon ei sisalda publikatsioonePõistang-paelussi on liikide kompleks, mille vastsevorm põhjustab inimestel ja kariloomadel haigust nimega tsüstiline ehhinokokkoos. Selle tagajärjel hakkab vaheperemehes (tavaliselt rohusööja või inimene) maksas või kopsus arenema vedelikuga täidetud tsüst. Sümptomid on varieeruvad ning sõltuvad sellest, mis organis tsüst arenema hakkab, kus ta paikneb antud organis ning kui suureks ta kasvab. Teadaolevalt on inimeselt leitud kõige suurema tsüsti kaaluks olnud ca 3 kg. Inimese endani jõuab see parasiit reeglina läbi koera, kes on parasiidile lõpp-peremeheks. Haiguse ravimata jätmisel võib see lõppeda nii inimese kui looma jaoks surmaga. Kuna antud parasiidi geneetiline mitmekesisus on väga lai siis on põistang-paelussi puhul valitsenud segadus liikide arvus. Mitmest liigist see koosneb? Erinevaid variante ehk genotüüpe on tuvastatud 9 (nummerdatud G1-G8, G10). Seejuures on nendel erinevatel geneetilistel variantidel erinevused elutsüklites (nt osad suudavad inimest nakatada, teised mitte), levikupiirkondades (ühed põhjapoolkeral, teised rohkem lõunas) ja ka haiguse sümptomite tõsiduses (G1 variandiga on haigus nt kiirema kulgemisega ja tõsisemate tagajärgedega). Nende mainitud erinevuste põhjal on leitud, et tegelikkuses koosneb põistang-paeluss hoopis mitmest liigist. Seejuures ei ole aga jõutud ühtsele arusaamisele, et kas G6-G8 ja G10 moodustavad ühe, kaks või kolm liiki. Siiani pole ka ära tõestatud, et G1-G3 peaksid üks liik olema. Kuna tegemist on tõsiste tagajärgedega haigusega, siis on oluline, et nii arstidel kui veterinaaridel oleks ühine arusaam sellest, millisest haigustekitajast juttu on. Leviku piiramiseks on oluline info ka potentsiaalsete levikumustrite tuvastamine. Käesolevas doktoritöö tulemused näitasid, et siiani segadust põhjustanud G6-G8 ja G10 moodustavad kaks liiki – koduloomadel levinud G6/G7 on üks, ning metsloomade G8/G10 moodustavad teise liigi. Seejuures tõestasime esmakordselt ka G1/G3 liigilist kuuluvust. Samuti analüüsisime inimestelt kõige sagedamini leitud variantide levikumustreid ning leidsime, et loomakaubandusel on antud parasiidi levikule olnud suur mõju.Echinococcus granulosus is a tapeworm species complex which causes cystic echinococcosis in humans and livestock animals. The larval form of this parasite is in the form of a fluid filled cyst, which typically develops in the liver or in the lungs of the intermediate host (herbivores, humans). Symptoms depend on the size and location of the cyst. So far the biggest cyst found in one patient has been about the size of 3 kg. Humans frequently get the infection through contact with dogs, who are the final hosts for this parasite. Left untreated, the disease can result in the death of the infected intermediate host. The genetic diversity of this parasite is considerably high and there has been no unanimous decision of how many species there actually are in this complex. The different variants or in other words – genotypes – that have been confirmed are numbered G1-G8 and G10. Over the years the evidence has been accumulating that there are a number of major differences between these genotypes and it has been suggested to regard a few genotypes as one species. However, no decision has been reached for G6-G10 – are they one, two or three species? No evidence has been presented to regard G1-G3 as one species either. However, for accurate communication between doctors and veterinarians, it is vital to reach a unanimous decision about the number of species and their names. Moreover, for potential control efforts to stop the spread of this disease, it is important to also collect data on the distribution patterns of this parasite. In the current thesis we show that the domestic genotypes (G6/G7) form one species, and the genotypes frequently associated with wildlife (G8/G10) form another. We also provided conclusive evidence to regard G1/G3 as one one species. What is more, we also looked at the distribution patterns and population structure of the genotypes most frequently associated with human infections, and found that animal transportation has had a major influence on the current population structure for this parasite species complex

Cystic echinococcosis in Iceland: a brief history and genetic analysis of a 46-year-old Echinococcus isolate collected prior to the eradication of this zoonotic disease

Cystic echinococcosis (CE) is considered the most severe parasitic disease that ever affected the human population in Iceland. Before the start of eradication campaign in the 1860s, Iceland was a country with very high prevalence of human CE, with approximately every fifth person infected. Eradication of CE from Iceland by 1979 was a huge success story and served as a leading example for other countries on how to combat such a severe One Health problem. However, there is no genetic information on Echinococcus parasites before eradication. Here, we reveal the genetic identity for one of the last Echinococcus isolates in Iceland, obtained from a sheep 46 years ago (1977). We sequenced a large portion of the mitochondrial genome (8141 bp) and identified the isolate as Echinococcus granulosus sensu stricto genotype G1. As G1 is known to be highly infective genotype to humans, it may partly explain why such a large proportion of human population in Iceland was infected at a time . The study demonstrates that decades-old samples hold significant potential to uncover genetic identities of parasites in the past

Monoclonal antibody-based localization of major diagnostic antigens in metacestode tissue, excretory/secretory products, and extracellular vesicles of Echinococcus species

Alveolar (AE) and cystic echinococcosis (CE) are severe parasitic zoonoses caused by the larval stages of Echinococcus multilocularis and E. granulosus sensu lato, respectively. A panel of 7 monoclonal antibodies (mAbs) was selected against major diagnostic epitopes of both species. The binding capacity of the mAbs to Echinococcus spp. excretory/secretory products (ESP) was analyzed by sandwich-ELISA, where mAb Em2G11 and mAb EmG3 detected in vitro extravesicular ESP of both E. multilocularis and E. granulosus s.s. These findings were subsequently confirmed by the detection of circulating ESP in a subset of serum samples from infected hosts including humans. Extracellular vesicles (EVs) were purified, and the binding to mAbs was analyzed by sandwich-ELISA. Transmission electron microscopy (TEM) was used to confirm the binding of mAb EmG3 to EVs from intravesicular fluid of Echinococcus spp. vesicles. The specificity of the mAbs in ELISA corresponded to the immunohistochemical staining (IHC-S) patterns performed on human AE and CE liver sections. Antigenic small particles designated as ‘‘spems’’ for E. multilocularis and ‘‘spegs’’ for E. granulosus s.l. were stained by the mAb EmG3IgM, mAb EmG3IgG1, mAb AgB, and mAb 2B2, while mAb Em2G11 reacted with spems and mAb Eg2 with spegs only. The laminated layer (LL) of both species was strongly visualized by using mAb EmG3IgM, mAb EmG3IgG1, mAb AgB, and mAb 2B2. The LL was specifically stained by mAb Em2G11 in E. multilocularis and by mAb Eg2 in E. granulosus s.l. In the germinal layer (GL), including the protoscoleces, a wide staining pattern with all structures of both species was observed with mAb EmG3IgG1, mAb EmG3IgM, mAb AgB, mAb 2B2, and mAb Em18. In the GL and protoscoleces, the mAb Eg2 displayed a strong E. granulosus s.l. specific binding, while mAb Em2G11 exhibited a weak granular E. multilocularis specific reaction. The most notable staining pattern in IHC-S was found with mAb Em18, which solely bound to the GL and protoscoleces of Echinococcus species and potentially to primary cells. To conclude, mAbs represent valuable tools for the visualization of major antigens in the most important Echinococcus species, as well as providing insights into parasite-host interactions and pathogenesis

First isolation of Echinococcus granulosus sensu lato genotype 7 in the archipelago of Cape Verde

There are no scientific data available on the occurrence of the Echinococcus granulosus sensu lato (s.l.) cluster in definitive hosts (domestic dogs), intermediate hosts (domestic livestock) nor humans in Cape Verde. In this pilot study, environmental dog fecal samples (n = 369) were collected around food markets, official slaughterhouses, as well as home and small business slaughter spots in 8 of the 9 inhabited islands from the Cape Verde archipelago, between June 2021 and March 2022. Additionally, during the same period, 40 cysts and tissue lesions were opportunistically collected from 5 islands, from locally slaughtered cattle (n = 7), goats (n = 2), sheep (n = 1) and pigs (n = 26). Genetic characterization by a multiplex polymerase chain reaction assay targeting the 12S rRNA gene confirmed the presence of E. granulosus s.l. in fecal and tissue material. In total, 17 cyst samples from Santiago (n = 9), Sal (n = 7) and São Vicente (n = 1) and 8 G6/G7-positive dog fecal samples from Santiago (n = 4) and Sal (n = 4) were identified as E. granulosus s.l. G7 by sequence analysis (nad2, nad5 and nad1 genes). This study discloses the transmission of E. granulosus s.l. G7, in pig, cattle and dog in Cape Verde

Monoclonal antibody-based localization of major diagnostic antigens in metacestode tissue, excretory/secretory products, and extracellular vesicles of Echinococcus species

Alveolar (AE) and cystic echinococcosis (CE) are severe parasitic zoonoses caused by the larval stages of Echinococcus multilocularis and E. granulosus sensu lato, respectively. A panel of 7 monoclonal antibodies (mAbs) was selected against major diagnostic epitopes of both species. The binding capacity of the mAbs to Echinococcus spp. excretory/secretory products (ESP) was analyzed by sandwich-ELISA, where mAb Em2G11 and mAb EmG3 detected in vitro extravesicular ESP of both E. multilocularis and E. granulosus s.s. These findings were subsequently confirmed by the detection of circulating ESP in a subset of serum samples from infected hosts including humans. Extracellular vesicles (EVs) were purified, and the binding to mAbs was analyzed by sandwich-ELISA. Transmission electron microscopy (TEM) was used to confirm the binding of mAb EmG3 to EVs from intravesicular fluid of Echinococcus spp. vesicles. The specificity of the mAbs in ELISA corresponded to the immunohistochemical staining (IHC-S) patterns performed on human AE and CE liver sections. Antigenic small particles designated as ‘‘spems’’ for E. multilocularis and ‘‘spegs’’ for E. granulosus s.l. were stained by the mAb EmG3$_{IgM}$, mAb EmG3$_{IgG1}$, mAb AgB, and mAb 2B2, while mAb Em2G11 reacted with spems and mAb Eg2 with spegs only. The laminated layer (LL) of both species was strongly visualized by using mAb EmG3$_{IgM}$, mAb EmG3$_{IgG1}$, mAb AgB, and mAb 2B2. The LL was specifically stained by mAb Em2G11 in E. multilocularis and by mAb Eg2 in E. granulosus s.l. In the germinal layer (GL), including the protoscoleces, a wide staining pattern with all structures of both species was observed with mAb EmG3$_{IgG1}$, mAb EmG3$_{IgM}$, mAb AgB, mAb 2B2, and mAb Em18. In the GL and protoscoleces, the mAb Eg2 displayed a strong E. granulosus s.l. specific binding, while mAb Em2G11 exhibited a weak granular E. multilocularis specific reaction. The most notable staining pattern in IHC-S was found with mAb Em18, which solely bound to the GL and protoscoleces of Echinococcus species and potentially to primary cells. To conclude, mAbs represent valuable tools for the visualization of major antigens in the most important Echinococcus species, as well as providing insights into parasite-host interactions and pathogenesis

First report of highly pathogenic Echinococcus granulosus genotype G1 in dogs in a European urban environment

BACKGROUND: Echinococcus granulosus and E. multilocularis are tapeworm parasites of major medical and veterinary importance, causing cystic and alveolar echinococcosis, respectively. Both diseases are listed among the most severe parasitic diseases in humans, representing 2 of the 17 neglected diseases prioritised by the World Health Organisation. However, little is known about the role of urban animals in transmission of both parasite species. FINDINGS: A sensitive non-invasive genetic method was used to monitor E. granulosus and E. multilocularis infection among dog faecal samples collected from an urban area in Estonia in 2012–13. Out of 181 dog faecal samples analysed, 2.2% tested positive for E. granulosus, determined by sequencing as genotype G1. None of the samples tested positive for E. multilocularis. CONCLUSIONS: We report contamination of an urban environment with highly pathogenic E. granulosus G1 disseminated by dogs, and a potential risk to human health

First isolation of Echinococcus granulosus sensu lato genotype 7 in the archipelago of Cape Verde

There are no scientific data available on the occurrence of the Echinococcus granulosus sensu lato (s.l.) cluster in definitive hosts (domestic dogs), intermediate hosts (domestic livestock) nor humans in Cape Verde. In this pilot study, environmental dog fecal samples (n = 369) were collected around food markets, official slaughterhouses, as well as home and small business slaughter spots in 8 of the 9 inhabited islands from the Cape Verde archipelago, between June 2021 and March 2022. Additionally, during the same period, 40 cysts and tissue lesions were opportunistically collected from 5 islands, from locally slaughtered cattle (n = 7), goats (n = 2), sheep (n = 1) and pigs (n = 26). Genetic characterization by a multiplex polymerase chain reaction assay targeting the 12S rRNA gene confirmed the presence of E. granulosus s.l. in fecal and tissue material. In total, 17 cyst samples from Santiago (n = 9), Sal (n = 7) and São Vicente (n = 1) and 8 G6/G7-positive dog fecal samples from Santiago (n = 4) and Sal (n = 4) were identified as E. granulosus s.l. G7 by sequence analysis (nad2, nad5 and nad1 genes). This study discloses the transmission of E. granulosus s.l. G7, in pig, cattle and dog in Cape Verde

Molecular characterization of Echinococcus granulosus sensu lato genotypes in dromedary camels from extreme Sahara of Algeria based on analysis of nad2 and nad5 genetic markers

Cystic echinococcosis is parasitic disease caused by the metacestodes belonging to the Echinococcus granulosus sensu lato (s.l.) species complex. Cystic echinococcosis is of considerable economic and public health importance. It is endemic in both livestock and humans in North African countries, including Algeria. The present study aimed to characterize E. granulosus s.l. genotypes in dromedary camels (Camelus dromedarius) from the extreme Sahara of Algeria, using recently developed mitochondrial genetic markers (NADH dehydrogenase subunit 2 and NADH dehydrogenase subunit 5) for reliable identification of different genotypes. A total of 75 Echinococcus cysts were collected from 49 dromedary camels, including 65 and 10 cysts from 45 and four camels originating from two slaughterhouses of Tindouf and Illizi provinces, respectively. E. granulosus sensu stricto (s.s.) G1 and G3 were identified in camels from both areas based on nad5 (649 bp) gene sequences, whereas E. granulosus s.l. G6 was identified in camels from Tindouf region based on concatenated nad5 and nad2 gene sequences (total 1336 bp). Identified samples clustered into 11 different haplotypes (ALG1-ALG11), including four haplotypes (ALG8-ALG11) for E. granulosus s.s. G1, one haplotype (ALG7) for E. granulosus s.s. G3, and six haplotypes (ALG1-ALG6) for E. granulosus s.l. G6. The present study provides valuable molecular data, including genotyping and haplotypic variability, on E. granulosus s.l. in dromedary camels from two regions in the extreme Sahara of Algeria. Future characterization of the G1, G3, and G6 samples based on sequencing of complete mitochondrial genomes would be of considerable significance for a more comprehensive understanding of molecular epidemiology of CE in Algeria

First detection of zoonotic tapeworm Echinococcus granulosus sensu lato genotype G7 in continental Italy.

The larval stage of the species complex Echinococcus granulosus sensu lato (s.l.) is the cause of a widespread zoonotic disease known as cystic echinococcosis (CE). The disease is highly prevalent in southern Italy and represents a serious public health issue. The main aim of this study was to characterize E. granulosus s.l. genotypes from wild boar on a continental area of Italy (Campania region), using recently developed mtDNA markers of nad2 and nad5 for reliable identification of different genotypes. Here, nad5 (680 bp) allowed for a clear identification of G1 and G3, whereas a combination of nad2 (714 bp) and nad5 (1394 bp in total) did the same for genotype G7 and its haplogroups G7a and G7b. The results of this study revealed for the first time the presence of genotype G7 in continental Italy. While haplogroup G7b was previously shown to be restricted to the islands of Corsica and Sardinia, here we demonstrate that haplogroup G7b is also present on the mainland of Italy. This work has implications in designing future strategies to reduce CE in Italy