First report of Echinococcus granulosus genotype 1 in a wild boar (Sus scrofa) from China.

Echinococcosis is a worldwide disease endemic to the western region of China. In 2023, echinococcosis was detected in one of 27 wild boars (Sus scrofa) in Yili Prefecture, Xinjiang, northwestern China. Histopathological staining and full sequence mitochondrial (mt) analysis were used to determine the infection genotype. Echinococcus granulosus was detected in the wild boar liver, and the cystic lesion characteristics indicated the E. granulosus genotype (G1). This case is the first confirmation of wild boar serving as a transmitter for the G1 genotype of E. granulosus within China. These findings suggest that surveillance is needed to assess the risk of E. granulosus sensu lato transmission to humans and wild animals.

Three species of Echinococcus granulosus sensu lato infect camels on the Arabian Peninsula.

We report on the genetic identity of 36 Echinococcus cysts that were collected during a recent slaughterhouse survey of 810 locally bred camels (dromedaries) in the Eastern Province of the Kingdom of Saudi Arabia. Analysis of a partial nad1 gene sequence showed that the majority (n = 29) belonged to E. granulosus sensu stricto, four to E. canadensis G6/7, and three to E. ortleppi. Eight of the 29 E. granulosus s.s. cysts contained protoscoleces; all other cysts were calcified and non-viable. This is the first report of the presence E. ortleppi from the Arabian Peninsula, a parasite that is typically transmitted via cattle. The results indicate widespread infection of camels with CE in eastern Saudi Arabia and an active role of camels in the lifecycles of at least E. granulosus s.s.. Complete cox1 haplotype analysis of 21 E. granulosus s.s. isolates shows that the majority of variants circulating in eastern Saudi Arabia is distinct from but closely related to haplotypes from neighboring countries in the Middle East, which indicates the presence of this parasite in KSA for a longer period of time. All isolates of E. granulosus s.s. in this study belonged to the G1 cluster, although the G3 genotype has previously also been reported from the Middle East.

Survey of coyotes, red foxes and wolves from Wyoming, USA, for Echinococcus granulosus s. l.

The paraphyletic group Echinococcus granulosus sensu lato is comprised of parasitic tapeworms of wild and domestic canids such as wolves (Canis lupus) and coyotes (Canis latrans), which serve as definitive hosts, and ungulates, which are the intermediate hosts. Members of this tapeworm group are characterized by both cosmopolitan distribution and zoonotic disease potential. This survey (conducted from 2012 through 2017) was designed to provide insight into the prevalence and distribution of this parasite in wild canids in Wyoming. Echinococcus sp. infections were documented in 14 of 22 gray wolves (63.6%), 1 of 182 coyotes (0.55%) and 0 of 5 red foxes (Vulpes fulva). Echinococcus granulosus s. l. was confirmed in 4 of these 14 specimens obtained from wolves with two parasite specimens corresponding morphologically with E. canadensis (G8/G10). These results suggest that wolves serve as the major definitive host of E. granulosus s. l. in Wyoming, while coyotes do not play an equivalent role. Limited sample size precludes evaluation of the importance of the red fox as a favorable definitive host. Whereas this study documents the occurrence of E. granulosus s. l. in Wyoming, the zoonotic disease risk does not appear to be high. Education remains the key to disease prevention, coupled with good hygienic practices by humans and anthelmintic treatment of domestic dogs exhibiting elevated risk of exposure.

Genetic diversity and transmission patterns of Echinococcus granulosus sensu stricto among domestic ungulates of Sardinia, Italy.

Cystic echinococcosis (CE), a parasitic zoonosis of public health and economic concern, is highly endemic in Sardinia, Italy. The study involved examining the intraspecific variability and demographic structure of Echinococcus granulosus sensu stricto (s.s.) in common hosts of this parasite. Molecular surveillance included the fragment amplification of a partial mitochondrial gene, cox1 (750 bp), for a total of 69 isolates derived from sheep (n = 52), cattle (n = 11), pigs (n = 4), and goats (n = 2). It was ascertained that E. granulosus s.s. was the primary agent of infection among these ungulates and G1 genotype was highly prevalent (79.71%). Considerable intraspecific variation was found, revealing the existence of 22 haplotypes with relatively high haplotype (0.8555 ± 0.033) and low nucleotide diversities (0.00281 ± 0.00030). Population demographics indicated an expanding parasitic population signifying negative deviation from neutrality indices. Little genetic differentiation was found between the subpopulations of E. granulosus s.s. in the island. Moreover, the geographic dispersal of genotypes G1 and G3 also indicated similarity between Sardinian and mainland Echinococcus granulosus s.s. populations reaffirming the sympatric occurrence and efficient transmission of G1 and G3 genotypes. Molecular survey of CE has the potential to yield baseline information on the infective genotypes among the intermediate hosts and helps in devising suitable control strategies for curtailing the disease.

Multiple haplotypes of Echinococcus granulosus sensu stricto in single naturally infected intermediate hosts.

Cystic echinococcosis is a disease that affects both humans and animals, caused by cryptic species complex belonging to the platyhelminth Echinococcus granulosus sensu lato (s.l.). This disease is distributed worldwide, with E. granulosus sensu stricto (s.s.) being the most widespread of the species. High genetic variability has been demonstrated within E. granulosus s.s. studying single cyst per infected animal identifying a number of different haplotypes. However, few studies have addressed the genetic diversity of this parasite within a single intermediate host with multiple Echinococcus cysts. To date, it remains unknown if specific haplotypes of E. granulosus s.s. produce differences in biological features of the cyst. Here, we use the full length of the mitochondrial gene cox1 to determine E. granulosus s.s. haplotypes in samples from both cattle and sheep which harboured more than one cyst in different areas in Chile, where this parasite is endemic. We found 16 different haplotypes in 66 echinococcal cysts from 10 animals, and both cattle and sheep can harbour up to five different haplotypes of E. granulosus s.s. in the same animal. Regarding cyst fertility, five animals had both fertile and infertile Echinococcus cysts in both single and multiple haplotype infections. There was no association between haplotype and cyst fertility, size, or adventitial layer characteristics. Sampling and sequencing every Echinococcus cyst found in the intermediate host reveals a high molecular variability. We speculate that multiple haplotype infections could also suggest that intermediate hosts come from hyperendemic areas.

Molecular characterization of Echinococcus granulosus in livestock of Al-Madinah (Saudi Arabia).

Echinococcus granulosus is the causative agent of cystic echinococcosis, which has serious impacts on human and/or animal health, resulting in significant economic losses. Echinococcus granulosus comprises a number of intra-specific variants or strains at the genetic level. In Saudi Arabia, few studies were performed on genetic variations in Echinococcus species. Therefore, the present study aimed to investigate the phenotypic and genetic characterization of hydatid cysts harboured by sheep and camels in Al-Madinah Al-Munawarah. Samples of hydatid cysts were collected from local sheep (n = 25) and camels (n = 8). The morphological criteria of protoscoleces were investigated. To investigate the molecular characterization, random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR), single-stranded conformation polymorphism (SSCP) were carried out. DNA was extracted from individual fertile cysts and subjected to RAPD-PCR analysis (using five arbitrary primers) and PCR amplification of cytochrome c oxidase I (cox1) and 12S ribosomal ribonucleic acid (12S rRNA) genes. The PCR products were subjected to SSCP analysis for genetic discrimination in E. granulosus isolates. In addition, partially sequencing of the mitochondrial DNA cox1 genes was achieved for assessing the phylogenetic positions of collected isolates using some global published sequence data of cox1 genes. The rostellar hooks of camel and local sheep isolates show remarkable variability in their dimensions. Five distinct SSCP patterns were identified in the 12S rRNA gene, showing intraspecific variations in E. granulosus of camels and local sheep. Sequencing of (cox1) genes of both local sheep and camels exhibit high similarity with those of the same gene (E. granulosus sensu stricto) published in NCBI BLAST.

Validation of a one-step PCR assay for the molecular identification of Echinococcus granulosus sensu stricto G1-G3 genotype.

The Italian National Reference Center for Echinococcosis (CeNRE, Sassari, Italy) set up a diagnostic protocol of "one-step-PCR" useful for the detection of E. granulosus sensu stricto (E.g.s.s.) and the identification of its genotype (G1-G3). The purpose of this work was to perform the validation of the "PCR E.g.s.s." method. The procedures were performed employing the criteria of the World Organization for Animal Health as well as of the Italian Accreditation Body (ACCREDIA) based on the Regulation UNI CEI EN ISO/IEC 17025. Positive DNA samples belonging to E. granulosus, E. ortleppi, E. multilocularis, E. canadensis species were used for the experiments. Analytical specificity evidenced primer pairs Cal (Calreticulin l gene of 1001 bp) with an specificity higher respect to Ef1 (Elongation-Factor 1 Alpha gene of 706 bp) and NAD (Dehydrogenase-subunit 1 gene of 219 bp). The analytical sensitivity presented the capability to detect a very low amount of parasite DNA corresponding to a concentration of 12.5 pg/µl; accuracy and precision related to the operator performance, along with repeatability and reproducibility, evidenced high concordance among results and demonstrated an excellent κ values of Cohen. According to the good performance related to the evaluated parameters, the method "PCR E.g.s.s." was suitable for the validation procedure, and consequently, to be undergone to the accreditation process. In conclusion, the results demonstrated an elevated robustness and reliable features of the "PCR E.g.s.s." able to perform a rapid diagnosis of E. granulosus in only "one step", hence, it is likely to avoid the sequencing step.

Evidence for camels (Camelus bactrianus) as the main intermediate host of Echinococcus granulosus sensu lato G6/G7 in Mongolia.

Cystic echinococcosis (CE), the parasitic disease caused by the larval stage of Echinococcus granulosus sensu lato (s.l.), is a global public health problem. In Mongolia, despite wide distribution of human CE, not enough information is available on the prevalence and molecular characterization of CE in livestock and its zoonotic linkage with human cases. We investigated the distribution of human CE cases and livestock population using statistical models to get insight into the zoonotic linkage. The incidence of human CE cases increased by a factor of 1.71 for one interquartile range increment in the density of the camel population. No significant association was observed with other livestock species. The samples collected from 96 camels and 15 goats in an endemic region showed a CE prevalence of 19.7% and 6.7%, respectively. All livestock CE were E. granulosus s.l. G6/G7 species of the E. granulosus s.l. complex. The genetic diversity was investigated using the haplotype network based on full cox1 gene analysis of the samples collected from livestock CE and nucleotide sequences previously reported from human CE and wild canids infection in Mongolia. Four haplotypes were identified within the livestock samples, two of which had not been previously reported. A common haplotype was identified among humans, camels, goats, and a wolf, all of which were within the same geographical area. A mixed infection of E. granulosus s.l. G6/G7 with different haplotypes in the intermediate host was identified. To the best of our knowledge, this is the most comprehensive description of the current epidemiological situation of CE in Mongolia with substantial evidence that camels might be the main intermediate host of E. granulosus s.l. G6/G7 in Mongolia. Moreover, our result presents the first report in the country to provide insight into the prevalence of E. granulosus s.l. G6/G7 in livestock.

Identification and molecular characterization of Echinococcus canadensis G6/7 in dogs from Corsica, France.

Recent surveys at slaughterhouses confirmed the presence of three different species of Echinococcus granulosus sensu lato in France: E. granulosus sensu stricto, E. ortleppi, and E. canadensis G6/7. The latter species was only identified on the French Mediterranean island of Corsica, with a high prevalence in pigs and wild boar. In order to investigate the life cycle of E. canadensis in this region, dog feces were collected in 31 municipalities, mainly from individual kennels. The analysis of fecal samples from 259 dogs by multiplex real-time PCR shows no infection by E. granulosus sensu stricto, but three dogs were infected by E. canadensis G6/7. Genetic analyses of mitochondrial genes (cox1, nad1, nad3, atp6) revealed in two dogs a haplotype previously identified in pigs. The third dog was infected by a new haplotype differing only from the two others from dogs by two mutations in the nad3 gene. This latter haplotype is genetically closer to those identified in pigs rather than those from wild boars. Analysis of questionnaires completed by the owners revealed that the sampled dog population was almost exclusively composed of hunting dogs that had been infrequently dewormed. Most of the owners (78%) leave carcasses of hunter-harvested wild boar in close proximity to their dogs. Nevertheless, genetic results seem to indicate that the three dogs were infected due to their consumption of a pig's infected viscera following home slaughtering. This study confirms the role of dogs as definitive hosts of E. canadensis G6/7 in Corsica. Further molecular studies, notably in human cases, are needed to assess the zoonotic impact of E. canadensis G6/7 in this region.

Molecular identification of cystic echinococcosis in humans and pigs reveals the presence of both Echinococcus granulosus sensu stricto and Echinococcus canadensis G6/G7 in the hyperendemic focus of the Republic of Moldova.

Cystic echinococcosis is caused by the parasitic species of the complex Echinococcus granulosus sensu lato. This disease is hyperendemic in the Republic of Moldova. Recent molecular analyses have revealed the exclusive presence of E. granulosus sensu stricto in sheep and cattle. Previous reports of prevalence in pigs suggest the potential presence of Echinococcus canadensis G6/G7, as this species is also reported in neighboring countries. The presence of cystic echinococcosis in pigs was specifically monitored at the slaughterhouse. In the meantime, human cases were genotyped for the first time. E. canadensis G6/G7 was identified in all ten pigs infected by E. granulosus s.l. One human case of infection by E. canadensis G6/G7 was also identified, while E. granulosus sensu stricto was found to be the cause for the 13 others. The description of one human case of E. canadensis G6/G7 has confirmed its zoonotic impact in the country. Future studies will be needed to estimate the relative proportion and distribution of both parasitic species in Moldova.

Prevalence and Molecular Characterization of Echinococcus granulosus Sensu Stricto in Northern Xinjiang, China.

Echinococcus granulosus is an important zoonotic parasite globally causing cystic echinococcosis (CE) in humans and animals. In this study, prevalence of CE and variation of cox1 gene sequence were analyzed with isolates E. granulosus collected from different areas in northern Xinjiang, China. The survey showed that 3.5% of sheep and 4.1% of cattle were infected with CE. Fragment of cox1 was amplified from all the positive sheep and cattle samples by PCR. In addition, 26 positive samples across the 4 areas were included. The isolates were all E. granulosus sensu stricto (s.s.) containing 15 haplotypes (Hap1-15), and clustered into 2 genotypes, G1 (90.1%, 91/101) and G3 (9.9%, 10/101). Hap1 was the most common haplotype (48.5%, 49/101). Hap9 were found in humans samples, indicating that sheep and cattle reservoir human CE. It is indicate that E. granulosus may impact on control of CE in livestock and humans in the region.

Dispersion of Echinococcus granulosus eggs from infected dogs under natural conditions in Patagonia, Argentina.

Cystic echinococcosis caused by Echinococcus granulosus is a major zoonosis of public health significance in the Patagonian region of Argentina. This investigation sought to test the hypothesis that the persistence and dispersion of the parasite eggs can be explained by physical and meteorological parameters along with final host infection and behaviour. This observational study was carried out over a five-year period within an enclosure where two dogs harbouring a worm burden ranging from 100 to 1000 mature adult E. granulosus, as well as two uninfected dogs, had previously been kept for six months. Environmental canine faeces, topsoil, pond water, and sediment samples were examined to control for the presence of eggs and coproantigens of the parasite using microscope-based techniques and copro-ELISA plus copro-Western Blot tests. The parasite eggs were detected up to 41 months later in faeces from infected dogs, soil and sediment, and coproantigen tests remained positive for up to 70 months in faeces. Overall, parasite eggs were found within a maximum distance of 115 m from the contaminated dog faeces deposition site. Our findings indicate that under Patagonian environmental conditions, egg persistence and dispersion seem to be related to the worm burden and habits of the infected dog, to prevailing wind direction and to the existence of low bushes as well as natural bodies of water. The present study is the first to provide direct evidence of interaction between bioclimatic conditions and E. granulosus egg dispersion under Patagonian field conditions.

Genotyping and phylogenetic analysis of unusually located hydatid cysts isolated from humans in north-east Iran.

Despite several studies conducted to determine the genotypes of cystic echinococcosis (CE) agents in humans and other intermediate hosts, the relationship between parasite genotype and clinical presentation of hydatidosis is yet to be well defined. The aim of this study was to compare the genotypes/haplotypes of CE agents of human extra-hepatopulmonary hydatid cysts and common hydatid cysts of the liver. A comparative analysis was carried out between partial cox1 sequences of ten extra-hepatopulmonary hydatid cysts, two liver cysts and reliable sequences from the GenBank database. All the studied hydatid cysts had the Echinococcus granulosus sensu stricto (G1-G2-G3) genotypes. The liver CE cysts were caused by common G1 genotype, while six of the extra-hepatopulmonary cysts had genotypes different from common G1 cysts. The sequences of these six isolates were identical to the G2 and G3 genotypes of E. granulosus sensu stricto; the kidney and peritoneum cysts and most of the brain cysts were identified as G2 genotype, while G3 genotype was only reported in a cyst belonging to the pelvic region. Given the observed differences between the sequences of hydatid cysts, it seems that the replacement of hydatid cysts in organs other than the liver and lungs can be related to their genotypes and probably intra-genotypic characteristics. It was hypothesized that in each geographic area, less frequent genotypes were likely to be more consistent with placement in the host's unusual organs.

Hydatidosis in slaughtered sheep and goats in India: prevalence, genotypic characterization and pathological studies.

The present study determined the prevalence of hydatid cysts in different organs of slaughtered hilly 'Gaddi' breed small ruminants-sheep (n = 230) and goats (n = 197)-in Kangra Valley of the north-western Himalayas, India. Hydatid cysts were found in 12.2% (n = 28) of sheep and 10.7% (n = 21) of goats. Pulmonary echinococcosis was more prevalent in slaughtered sheep and goats (sheep 56.36%; goats 62.90%) than hepatic echinococcosis (sheep 43.64%; goats 37.10%). Fertility rates were higher in hepatic (81.25%) and pulmonary cysts of sheep (83.87%) compared to goats. Molecular identification and genotypic characterization of Echinococcus granulosus isolates were based on mitochondrial cytochrome oxidase 1 gene (mtCO1). The genotypic characterization identified the isolated strain to be closely related to the G7 genotype. Histopathological examination revealed a thick coat of granulation tissue, causing fibrosis and inflammatory reaction composed of fibroblasts and mononuclear cells around the cysts. In the liver, hepato-cellular degeneration was prominent at the periphery of the cysts. The present study highlights the molecular confirmation and phylogenetic analysis of E. granulosus isolates with the prevalence of hydatidosis in a naïve host species and in an unexplored region. The findings are of significant medical and veterinary importance regarding development of control measures to check dissemination of hydatidosis.

Molecular phylogeny based on six nuclear genes suggests that Echinococcus granulosus sensu lato genotypes G6/G7 and G8/G10 can be regarded as two distinct species.

Tapeworms of the species complex of Echinococcus granulosus sensu lato (s. l.) are the cause of a severe zoonotic disease - cystic echinococcosis, which is listed among the most severe parasitic diseases in humans and is prioritized by the World Health Organization. A stable taxonomy of E. granulosus s. l. is essential to the medical and veterinary communities for accurate and effective communication of the role of different species in this complex on human and animal health. E. granulosus s. l. displays high genetic diversity and has been divided into different species and genotypes. Despite several decades of research, the taxonomy of E. granulosus s. l. has remained controversial, especially the species status of genotypes G6-G10. Here the Bayesian phylogeny based on six nuclear loci (7387 bp in total) demonstrated, with very high support, the clustering of G6/G7 and G8/G10 into two separate clades. According to the evolutionary species concept, G6/G7 and G8/G10 can be regarded as two distinct species. Species differentiation can be attributed to the association with distinct host species, largely separate geographical distribution and low level of cross-fertilization. These factors have limited the gene flow between genotypic groups G6/G7 and G8/G10, resulting in the formation of distinct species. We discuss ecological and epidemiological differences that support the validity of these species.

Human cystic echinococcosis in Turkey: a preliminary study on DNA polymorphisms of hydatid cysts removed from confirmed patients.

Cystic echinococcosis caused by the larval stages of Echinococcus granulosus sensu lato s.l is endemic in Turkey with a high public health impact particularly in rural areas. The aim of this study was to investigate the genetic variation and population structure of E. granulosus s.s using metacestode isolates removed from surgically confirmed patients originating from several regions in Turkey and to investigate the occurrence of autochthonous transmission. Using DNA extracted from a total of 46 human-derived CE isolates, we successfully analysed an 827-bp fragment within the cox1 mitochondrial gene and confirmed the causative agent of human cystic echinococcosis in patients included in this study to be Echinococcus granulosus s.s (G1 and G3 genotypes). The haplotype parsimony network consisted of 28 haplotypes arranged within three main clusters and the neutrality indices were both negative and significant indicating negative selection or population expansion. The assessment carried out in this study using GenBank nucleotide sequence data from Turkey for sheep and cattle hosts demonstrated the importance of autochthonous transmission with sheep, cattle and humans harbouring the same haplotypes. Further studies are required to investigate the biological significance, if any, of E. granulosus s.s haplotypes and the genetic variability of CE from human patients using longer nucleotide sequences and a larger sample set.

Genetic characterization of Echinococcus granulosus strains isolated from humans based on nad1 and cox1 gene analysis in Isfahan, central Iran.

Cystic echinococcosis (CE) is a medically important parasite-caused human disease. Humans may acquire the infection accidentally by ingestion of E. granulosus eggs. The parasite has a broad range of hosts and genotypes, which may affect its aetiological and biological characteristics. The present study aimed to determine the genetic characteristics of human isolates of E. granulosus in Isfahan, Iran. In this cross-sectional study, 50 surgically removed hydatid cysts were collected from hospitalized patients in Al-Zahra Hospital, Isfahan, Iran, over a period of 2 years (2015-2017). DNA was extracted from cyst material, and polymerase chain reactions (PCR) were performed targeting cox1 and nad1 genes. Amplicons were sequenced directly and the resulting sequences were aligned and analysed. Phylogenetic and genetic diversity analyses were also performed. Among the isolates, 43 (86%), 3 (6%) and 4 (8%) out of 50 were E. granulosus (G1), E. granulosus (G3) and E. intermedius (G6), respectively. In total, nine and eight haplotypes were identified by nad1 and cox1 gene analysis, respectively. The haplotype diversity index was higher by cox1 gene analysis (0.547) in G1 strains compared with nad1 (0.433). The G1 genotype was the most predominant isolate from human cases of CE, and the presence of G6 is indicative of an important role of camels in the development of human CE in Isfahan. This is the first report of the G3 genotype causing human CE in Isfahan. Moreover, cox1 gene analysis enables a higher resolution of the genetic diversity of the E. granulosus population compared with nad1 gene analysis.

Use of FTA® card methodology for sampling and molecular characterization of Echinococcus granulosus sensu lato in Africa.

Cystic Echinococcosis is a parasitic disease caused by the cestode Echinococcus granulosus widely distributed in Africa. Monitoring of this parasite requires access to cyst samples on intermediate hosts observed at the slaughterhouse. In order to facilitate sampling in the field and analysis, the French National Reference Laboratory for Echinococcus spp. has developed a tissue derived from DNA sampling with FTA® card technology. The DNA samples were taken by applying the FTA® paper on the germinal layer after opening the cysts. The sampling technique was validated using frozen cysts (n = 76) stored in the laboratory and from field samples (n = 134) taken at the slaughterhouse by veterinarian technicians during meat inspection in Morocco, Mali and Mauritania. DNA was extracted after several weeks of storage at room temperature. PCR assays were performed using primers for generic cestode (cox1) and amplified fragments were sequenced. All samples taken in the lab and 80% of field samples were capable of molecular characterization. Cyst-derived DNA from FTA® samples can be useful for easy sampling, storage and rapid, safe and cheap shipment. The use of the FTA methodology will facilitate studies in the field to investigate the presence and genetic characterization of E. granulosus sensu lato in African countries.

Multilocus sequence analysis of Echinococcus granulosus strains isolated from humans and animals in Iran.

Echinococcus granulosus is now considered a complex consisting of at least four species and ten genotypes. Different molecular targets have been described for molecular characterization of E. granulosus; however, in almost all studies only one or two of the targets have been used, and only limited data is available on the utilization of multiple loci. Therefore, we investigated the genetic diversity among 64 strains isolated from 138 cyst specimens of human and animal isolates, using a set of nuclear and mitochondrial genes; i.e., cytochrome c oxidase subunit 1 (cox1), NADH dehydrogenase subunit 1 (nad1), ATPase subunit 6 (atp6), 12S rRNA (12S), and Actin II (act II). In comparison to the use of molecular reference targets (nad1 + cox1), using singular target (act II or 12S or atp6) yielded lower discriminatory power. Act II and 12S genes could accurately discriminate the G6 genotype, but they were not able to differentiate between G1 and G3 genotypes. As the G1 and G3 genotypes belong to the E. granulosus sensu stricto, low intra-species variation was observed for act II and 12S. The atp6 gene could identify the G3 genotype but could not differentiate G6 and G1 genotypes. Using concatenated sequence of five genes (cox1 + nad1 + atp6 + 12S + act II), genotypes were identified accurately, and markedly higher resolution was obtained in comparison with the use of reference markers (nad1 + cox1) only. Application of multilocus sequence analysis (MLSA) to large-scale studies could provide valuable epidemiological data to make efficient control and management measures for cystic echinococcosis.

Molecular characterization of Echinococcus granulosus isolates from Bulgarian human cystic echinococcosis patients.

Although cystic echinococcosis (CE) is highly endemic in Bulgaria, there is still scarce information about species and/or genotypes of the Echinococcus granulosus complex that infect humans. Our study tackled the genetic diversity of E. granulosus complex in a cohort of 30 Bulgarian CE patients. Ten animal E. granulosus isolates from neighboring Greece were additionally included. Specimens were comparatively analyzed for partial sequences of five mitochondrial (mt) (cox I, nad I, rrnS, rrnL, and atp6) and three nuclear (nc) genes (act II, hbx 2, and ef-1α) using a PCR-sequencing approach. All 30 Bulgarian isolates were identified as E. granulosus sensu stricto (s.s.) and were showing identical sequences for each of the three examined partial nc gene markers. Based upon concatenated sequences from partial mtDNA markers, we detected 10 haplotypes: 6 haplotypes (H1-H6) clustering with E. granulosus s.s. (G1) and 4 haplotypes (H9-H13) grouping with E. granulosus s.s. (G3), with H1 and H10 being the most frequent in Bulgarian patients. The haplotypes H1, H4, and H11 were also present in Greek hydatid cyst samples of animal origin. In conclusion, E. granulosus s.s. (G1 and G3 genotypes) is the only causative agent found so far to cause human CE in Bulgaria. However, further studies including larger sample sizes and other additional geographic regions in Bulgaria will have to be performed to confirm our results.