Echinococcus granulosus Sensu Stricto and Echinococcus multilocularis in a Gray Wolf (Canis lupus) in Turkey: Further Evidence for Increased Risk of Alveolar Echinococcosis in Urban Areas.

OBJECTIVE: The aim of this study was to identify Echinococcus species by morphological and molecular means. METHODS: A dead gray wolf (Canis lupus) was found near Erzurum province and brought to the parasitology laboratory. Sedimentation and counting technique (SCT) and polymerase chain reaction (PCR) analysis were conducted. RESULTS: The SCT implications indicated that the wolf had a substantial worm burden (62,720 and 49,280 parasites) due to a co-infection of E. granulosus s.l. and E. multilocularis. Genus/species-specific PCR was used to analyze DNA extracted from adult worms and confirmed as E. granulosus s.s. and E. multilocularis, utilizing COI and 12S rRNA gene sequence analysis, respectively. CONCLUSION: This report presents the first co-detection of E. granulosus s.s. and E. multilocularis in a gray wolf found in an urban area in a highly endemic area for human echinococcosis in northeastern Turkey. The results emphasize that AE is not only a problem of rural areas, but also occurs in urban areas, which may pose a threat to public health. Therefore, surveillance in urban areas is crucial. The need to develop new control strategies for domestic and wildlife in the study area is also highlighted.

Cystic echinococcosis in slaughtered sheep in Erzurum province, Turkey.

Cystic echinococcosis (CE) is an important zoonotic infection caused by the larval stages of the genus Echinococcus. Turkey is a highly endemic region for CE and the disease is one of the major public health problems. The study was aimed to assess the situation of the CE in sheep in Turkey and also to provide data on circulating genotypes in the country. A total of 3319 sheep at slaughter were screened during the study. The prevalence of CE in the study area was 31.7% (1052/3319). The lungs were the most frequently CE infected organ (50%, 526/1052). Microscopic examination revealed that overall cyst fertility was 68.1%. Molecular analysis of partial fragments of 12S and COI gene regions were included for 351 selected cyst samples and all of them were identified as E. granulosus sensu stricto. Sequence analysis showed that the predominant genotype in the study areas was G1 (77.1%), and the rest were G3 (22.9%). The prevalence rate of CE in sheep in the study area is lower compared to previous years except for one province. Considering the high cyst fertility rate and the predominance of E. granulosus G1 which is particularly pathogenic to humans, calls for serious control measures like public awareness about the disease, sufficient dog deworming programs, continuity of monitoring the disease should be taken.

Long-term outcomes of endovenous laser ablation, n-butyl cyanoacrylate, and radiofrequency ablation for treatment of chronic venous insufficiency.

BACKGROUND: In the present retrospective, single-center study, we evaluated the long-term effectiveness and reliability of endovenous laser ablation (EVLA), endovenous n-butyl cyanoacrylate (NBCA) application, and radiofrequency ablation (RFA) in the management of chronic venous insufficiency (CVI). METHODS: The medical records of patients who had undergone EVLA, NBCA, or RFA for CVI from January 1, 2014 to January 1, 2017 were reviewed. The medical records included data on sex, age, body mass index, American Society of Anesthesiologists score, and symptoms at admission. The great saphenous vein diameter, CEAP (Clinical, Etiology, Anatomy, and Pathophysiology) classification, and venous clinical severity score were also recorded. All the patients were followed up with physical examinations and color Doppler ultrasound scan at the first week and 6 and 12 months after treatment. After 12 months, the follow-up examinations were performed annually. RESULTS: A total of 232 patients who had undergone lower limb CVI surgical treatment (EVLA, n = 77; NBCA, n = 73; RFA, n = 82). The mean follow-up time was 67.5 ± 4.7 months. The procedure duration was significantly shorter for the NBCA group (13.5 minutes) vs that for the EVLA (31.7 minutes) and RFA (27.9 minutes) groups (P = .001). The pain score was highest in the EVLA group (P = .001). The EVLA group had also experienced a significantly greater incidence of complications and a longer time to return to daily activities (P = .001). The post hoc analysis revealed comparable occlusion success among the three groups on the first postoperative day and at 6, 12, and 24 months postoperatively. However, significantly better occlusion rates were found for RFA vs EVLA at 3 and 5 years of follow-up (P = .024 and P = .011, respectively). The success of NBCA and RFA was similar at 3 and 5 years of follow-up (P = .123 and P = .330, respectively). CONCLUSIONS: The outcomes showed similar early postoperative occlusion success among all three CVI treatment techniques. However, RFA resulted in a significantly higher success rate compared with EVLA at 3 and 5 years of follow-up. Additionally, the NBCA and RFA procedures achieved comparable long-term success. However, EVLA was associated with significantly greater complication rates and pain scores and a longer time to return to daily activities. The NBCA procedure had a significantly shorter operation time compared with the other procedures.

The situation of echinococcosis in stray dogs in Turkey: the first finding of Echinococcus multilocularis and Echinococcus ortleppi.

Echinococcosis, caused by larval stage of the genus Echinococcus, is one of the most important zoonotic diseases worldwide. The purpose of this study was to determine the presence and prevalence of Echinococcus species in stray dogs of Erzurum, a highly endemic region for cystic echinococcosis (CE) and alveolar echinococcosis (AE) in Turkey. The study samples consisted of 446 stray dog faecal specimens collected from an animal shelter in Erzurum, Turkey, between October 2015 and February 2016. The faecal samples were collected from individual dogs for the isolation of taeniid eggs using the sequential sieving and flotation method (SSFM). Molecular analyses and sequencing revealed the prevalence of Echinococcus spp. as 14.13% (63/446) in faecal samples. The stray dogs harboured five different Echinococcus spp.: E. granulosus s.s. (G1/G3) (n = 41), E. equinus (G4) (n = 3), E. ortleppi (G5) (n = 1), E. canadensis (G6/G7) (n = 3) and E. multilocularis (n = 16). E. granulosus s.s. was the most abundant species. Surprisingly, the occurrence of E. multilocularis in dogs was revealed for the first time in Turkey. E. ortleppi was also reported for the first time in Turkey. These findings highlight a significant public health risk for human AE and CE, presenting useful baseline data on Echinococcus spp. infection in dogs for designing control strategies.

Echinococcus multilocularis in Red Foxes in Turkey: Increasing risk in urban.

This study was conducted to determine the occurrence of E. multilocularis in foxes and environmental fecal contamination by E. multilocularis in Erzurum, the most highly endemic region for AE in Turkey. The study materials consisted of 50 red fox carcasses collected from 20 counties of Erzurum, Turkey, between October 2015 and February 2016. After the application of the sedimentation and counting technique (SCT), E. multilocularis was identified through the identification of typical morphological structures. Fox fecal samples (n = 600) were also collected from these counties for the isolation of taeniid eggs using the sequential sieving and flotation method (SSFM). Then, the collected adult worms and taeniid eggs were subjected to molecular and sequence analyses. Mature E. multilocularis parasites were found in 42% (21/50) of the fox intestines, with a mean number of 7,806 (150-31,644). The severity of infection was higher in carcasses obtained from the central district (48.6%, 17/35) than in those obtained from the peripheral district (26.7%, 4/15). The prevalence of environmental fecal contamination with E. multilocularis was 10.5% (63/600) in fecal samples collected from all counties of Erzurum. This infection rate was higher in the central district (32.1%, 36/112) than in the peripheral district (5.5%, 27/488; P < 0.0001). In conclusion, contrary to expectation, the prevalence of E. multilocularis positivity was high in urban areas. This could have been due to alterations in the dietary habitats of definitive and intermediate hosts. Therefore, new control strategies are essential to eliminate human AE cases in the future as urbanization advances.

Molecular Characterization of Echinococcus multilocularis and Echinococcus granulosus from Cysts and Formalin-Fixed Paraffin-Embedded Tissue Samples of Human Isolates in Northeastern Turkey.

Erzurum province of Turkey is known to be highly endemic for alveolar echinococcosis (AE) and cystic echinococcosis (CE). In this study, we confirmed Echinococcus multilocularis cases, searched genetic variations of the isolates, and-for the first time-determined the genotypes of Echinococcus granulosus s.l. infecting humans in the province. A total of 5 alveolar and 106 hydatid cysts as well as 23 formalin-fixed paraffin-embedded (FFPE) samples that were diagnosed as AE were collected from hospitals between 2015 and 2017. Partial sequences of two mitochondrial genes were amplified to detect E. multilocularis and E. granulosus sensu lato with conventional polymerase chain reactions (PCRs) and genotypes confirmed by sequencing. PCR amplification of a partial 12S rRNA gene on an alveolar cyst and FFPE tissue samples yielded the expected bp in 5 cysts and 19 of 23 FFPE samples; all Erzurum E. multilocularis isolates were confirmed by sequencing. Phylogenetic analysis of the isolates indicated that some of them were identical to European isolates, whereas some of them were identical to Asian isolates. Off all hydatid cyst samples, 101 (95.2%) yielded the expected bp (94 with 12S rRNA-PCR and 7 with COI-PCR). Sequence analysis showed that 98 (97%) of them corresponded to the G1 genotype, whereas 3 (3%) corresponded to the G3 genotype. Results of the study emphasize that E. multilocularis isolates of Erzurum, based on short sequencing, are similar to both European and Asian isolates, and the G1 genotype of E. granulosus is the main causative agent of human CE in Erzurum.

Distinguishing Echinococcus granulosus sensu stricto genotypes G1 and G3 with confidence: A practical guide.

Cystic echinococcosis (CE), a zoonotic disease caused by tapeworms of the species complex Echinococcus granulosus sensu lato, represents a substantial global health and economic burden. Within this complex, E. granulosus sensu stricto (genotypes G1 and G3) is the most frequent causative agent of human CE. Currently, there is no fully reliable method for assigning samples to genotypes G1 and G3, as the commonly used mitochondrial cox1 and nad1 genes are not sufficiently consistent for the identification and differentiation of these genotypes. Thus, a new genetic assay is required for the accurate assignment of G1 and G3. Here we use a large dataset of near-complete mtDNA sequences (n = 303) to reveal the extent of genetic variation of G1 and G3 on a broad geographical scale and to identify reliable informative positions for G1 and G3. Based on extensive sampling and sequencing data, we developed a new method, that is simple and cost-effective, to designate samples to genotypes G1 and G3. We found that the nad5 is the best gene in mtDNA to differentiate between G1 and G3, and developed new primers for the analysis. Our results also highlight problems related to the commonly used cox1 and nad1. To guarantee consistent identification of G1 and G3, we suggest using the sequencing of the nad5 gene region (680 bp). This region contains six informative positions within a relatively short fragment of the mtDNA, allowing the differentiation of G1 and G3 with confidence. Our method offers clear advantages over the previous ones, providing a significantly more consistent means to distinguish G1 and G3 than the commonly used cox1 and nad1.

Global phylogeography and genetic diversity of the zoonotic tapeworm Echinococcus granulosus sensu stricto genotype G1.

Echinococcus granulosus sensu stricto (s.s.) is the major cause of human cystic echinococcosis worldwide and is listed among the most severe parasitic diseases of humans. To date, numerous studies have investigated the genetic diversity and population structure of E. granulosus s.s. in various geographic regions. However, there has been no global study. Recently, using mitochondrial DNA, it was shown that E. granulosus s.s. G1 and G3 are distinct genotypes, but a larger dataset is required to confirm the distinction of these genotypes. The objectives of this study were to: (i) investigate the distinction of genotypes G1 and G3 using a large global dataset; and (ii) analyse the genetic diversity and phylogeography of genotype G1 on a global scale using near-complete mitogenome sequences. For this study, 222 globally distributed E. granulosus s.s. samples were used, of which 212 belonged to genotype G1 and 10 to G3. Using a total sequence length of 11,682 bp, we inferred phylogenetic networks for three datasets: E. granulosus s.s. (n = 222), G1 (n = 212) and human G1 samples (n = 41). In addition, the Bayesian phylogenetic and phylogeographic analyses were performed. The latter yielded several strongly supported diffusion routes of genotype G1 originating from Turkey, Tunisia and Argentina. We conclude that: (i) using a considerably larger dataset than employed previously, E. granulosus s.s. G1 and G3 are indeed distinct mitochondrial genotypes; (ii) the genetic diversity of E. granulosus s.s. G1 is high globally, with lower values in South America; and (iii) the complex phylogeographic patterns emerging from the phylogenetic and geographic analyses suggest that the current distribution of genotype G1 has been shaped by intensive animal trade.

Genetic diversity and phylogeography of the elusive, but epidemiologically important Echinococcus granulosus sensu stricto genotype G3.

Cystic echinococcosis (CE) is a severe parasitic disease caused by the species complex Echinococcus granulosus sensu lato. Human infections are most commonly associated with E. granulosus sensu stricto (s.s.), comprising genotypes G1 and G3. The objective of the current study was to provide first insight into the genetic diversity and phylogeography of genotype G3. Despite the epidemiological importance of the genotype, it has remained poorly explored due to the ambiguity in the definition of the genotype. However, it was recently demonstrated that long sequences of mitochondrial DNA (mtDNA) provide a reliable method to discriminate G1 and G3 from each other. Therefore, we sequenced near-complete mtDNA of 39 G3 samples, covering most of the known distribution range and host spectra of the genotype. The phylogenetic network revealed high genetic variation within E. granulosus s.s. G3 and while G3 is significantly less prevalent worldwide than G1, the genetic diversity of both of the genotypes is equally high. We also present the results of the Bayesian phylogeographic analysis, which yielded several well-supported diffusion routes of genotype G3 originating from Turkey and Iran, suggesting the Middle East as the origin of the genotype.

Echinococcus multilocularis in a Eurasian lynx (Lynx lynx) in Turkey.

Echinococcus multilocularis is the causative agent of alveolar echinococcosis (AE), one of the most threatening zoonoses in Eurasia. Human AE is widespread in the Erzurum region of Turkey, but the situation of the disease in intermediate and definitive hosts is unknown. A Eurasian lynx (Lynx lynx) was killed in a traffic accident in the north of Erzurum, and was taken to our laboratory. Sedimentation and counting technique (SCT), DNA isolation and polymerase chain reaction (PCR) analysis were performed. The SCT results showed that the lynx was infected with E. multilocularis with a medium (745 worms) worm burden. The DNA of adult worms obtained from the lynx was analyzed with a species-specific PCR, and the worms were confirmed to be E. multilocularis by 12S rRNA gene sequence analysis. This is the first report of E. multilocularis from Eurasian lynx in Turkey.

First detection of Echinococcus multilocularis in rodent intermediate hosts in Turkey.

Echinococcus multilocularis is the causative agent of alveolar echinococcosis (AE), a potentially fatal zoonotic disease. Large parts of Turkey are considered as endemic for E. multilocularis. The aim of this study was to determine the occurrence of metacestode of E. multilocularis in wild rodents in Erzurum, an endemic region for human AE in Turkey. During the sampling period, a total of 498 rodents were trapped in twenty counties of Erzurum Province. Suspected lesions were observed on the livers of 48 rodents, and then partial fragment of mitochondrial 12S rRNA gene was PCR-amplified. Five liver samples exhibited E. multilocularis infection. The prevalence of E. multilocularis for Microtus spp. was 1·3%. All of the infected rodents had fertile metacestodes. Infected rodents were morphologically and molecularly analysed and were confirmed to be Microtus irani by the mitochondrial cytochrome b gene sequence analysis. This is the first report of the presence of E. multilocularis in rodent intermediate hosts in Turkey. Our findings of infected M. irani with protoscoleces show that this rodent can act as suitable intermediate host for E. multilocularis' life cycle in Turkey. However, there was a complete lack of data on the infection of carnivores from the country. An extensive survey is recommended to determine the prevalence of E. multilocularis in definitive hosts in this endemic region.

High-resolution phylogeography of zoonotic tapeworm Echinococcus granulosus sensu stricto genotype G1 with an emphasis on its distribution in Turkey, Italy and Spain.

Echinococcus granulosus is the causative agent of cystic echinococcosis. The disease is a significant global public health concern and human infections are most commonly associated with E. granulosus sensu stricto (s. s.) genotype G1. The objectives of this study were to: (i) analyse the genetic variation and phylogeography of E. granulosus s. s. G1 in part of its main distribution range in Europe using 8274 bp of mtDNA; (ii) compare the results with those derived from previously used shorter mtDNA sequences and highlight the major differences. We sequenced a total of 91 E. granulosus s. s. G1 isolates from six different intermediate host species, including humans. The isolates originated from seven countries representing primarily Turkey, Italy and Spain. Few samples were also from Albania, Greece, Romania and from a patient originating from Algeria, but diagnosed in Finland. The analysed 91 sequences were divided into 83 haplotypes, revealing complex phylogeography and high genetic variation of E. granulosus s. s. G1 in Europe, particularly in the high-diversity domestication centre of western Asia. Comparisons with shorter mtDNA datasets revealed that 8274 bp sequences provided significantly higher phylogenetic resolution and thus more power to reveal the genetic relations between different haplotypes.

First Molecular Characterization of Echinococcus multilocularis in Turkey.

This study aimed to find out the occurrence of Echinococcus multilocularis in foxes in Erzurum province, the highest endemic region for human alveolar echinococcosis in Turkey. The sedimentation and counting technique was used to reveal adult Echinococcus spp. in the intestines of foxes. One out of the 10 foxes was infected with E. multilocularis. The adult worms were analyzed morphologically and molecularly and were confirmed to be E. multilocularis by species-specific PCR. Pairwise comparisons between the 12S rRNA sequences of the E. multilocularis isolate from Erzurum and other E. multilocularis isolates showed 100% similarity of the Erzurum isolate with European isolates. With this study, the presence of E. multilocularis in a fox in Erzurum was confirmed by PCR, and molecular identification of E. multilocularis is reported for the first time in Turkey.

Detection of polymorphism in AgB1 gene from sheep, cattle and human isolates of echinococcus granulosus by SSCP.

Antigen B (AgB) is a major protein produced by the metacestode cyst of Echinococcus granulosus, the causative agent of cystic hydatid disease. E. granulosus AgB is a gene family of at least five gene loci (B1-B5), each one consisting of several minor variants. We used PCR-SSCP followed by DNA sequencing to evaluate sequence variation and polymorphism of AgB1 in 99 isolates which the 43 were from cattle, 25 of sheep and 31 of human. All samples were analyzed with 12S rRNA-PCR for the strain detection and all of were identified as G1-G3 cluster (E. granulosus sensu stricto). The 16 human, 35 cattle and 25 sheep isolates were yielded the 102 bp band by PCR and these samples were tested by SSCP. As results of the SSCP, different band profiles were detected one each of cattle and human isolates while the other 74 isolates showed same band patterns. The DNA sequence analysis was performed for these two isolates and the other selected 4 isolates and polymorphism was confirmed.

Molecular discrimination of sheep and cattle isolates of Echinococcus granulosus by SSCP and conventional PCR in Turkey.

Cystic echinococcosis (CE), caused by hydatid cysts, is a widespread and hazardous disease in humans and animals worldwide. The aim of the current study was to investigate the genetic characteristics of sheep and cattle isolates of Echinococcus granulosus obtained from eastern Turkey using Single Stranded Conformation Polymorphism (SSCP) analysis and conventional PCR method. A total of 54 isolates collected from Erzurum and Elazig provinces of east-Turkey were examined. The 31 of these were obtained from liver of sheep while 23 cattle isolates (12 of liver and 11 of lung) were tested. After the total genomic DNA isolation 12S rRNA gene of all isolates were examined by PCR for the aim of genetic characterization by conventional PCR and mitochondrial CO1 gene for SSCP analysis. The 12S rRNA-PCR yielded 254 bp of amplification product with all samples analyzed. Thus, these samples were identified as G1-G3 cluster (E. granulosus sensu stricto). At least two major single stranded bands were resolved for G1-G3 cluster and G5 in SSCP analysis. While the resolution of more than two additional single stranded bands in SSCP indicated the existence of G7 genotype. The SSCP analysis was identified the G5 and G7 while failed to G1 and G3. The present SSCP analysis classified all 54 cyst isolates from sheep and cattle as E. granulosus sensu stricto (G1-G3 cluster). However, some sequenced samples for G1 and G3 showed the same band patterns by SSCP.

Epidemiological survey and molecular characterization of Echinococcus granulosus in cattle in an endemic area of eastern Turkey.

Cystic echinococcosis (CE) is a zoonotic disease affecting mainly various species of livestock and humans. A survey of cystic echinococcosis in cattle was conducted from December 2008 to April 2009 in an endemic area of eastern Turkey. A total of 1758 cattle were examined and hydatid cysts were found in 33.9% of the cattle (595/1758). Most of the cattle (75.8%) had hydatid cysts only in the lungs, 10.9% only in the liver, 12.4% in the both liver and lungs, 0.7% in the spleen and 0.2% in the heart. 220 of these cysts were examined by PCR of 12S rRNA gene and sequencing of mt-CO1 gene. 147 of 220 cattle isolates showed the same band pattern with 12S rRNA analyses and were identified as G1-G3 complex (Echinococcus granulosus sensu stricto) and also 28 of these were confirmed by mitochondrial CO1 sequencing as G1 genotype. The other 73 samples that did not amplified with 12S rRNA gene specific primers were analyzed for mt-CO1 gene and only 7 samples yielded 446bp product and after the CO1 sequencing these were identified as G3 genotype. This study confirms the predominance of the sheep strain (G1 genotype) in Turkey and this is the most comprehensive genetic survey of cattle CE in Turkey.