Scolicidal effects of Olea europaea and Satureja khuzestanica extracts on protoscolices of hydatid cysts

Treatment of hydatid disease is mainly surgical, with medical treatment being reserved as a coadjuvant treatment. Use of effective scolicidal agents during surgery of cystic echinococcosis is essential to reduce the recurrence rate. The goal of this study was to evaluate the in vitro scolicidal effects of hydroalcoholic extracts of Satureja khuzestanica leaves and aqueous extracts of Olea europaea leaves on hydatid cyst protoscolices. Echinococcus granulosus protoscolices were collected from the liver of sheep infected with the hydatid cyst. Various concentrations of plant extracts were used in different exposure times for viability assay of protoscolices. Among the olive leaf extracts tested, 0.1% and 0.01% concentrations had strong scolicidal effects in 120 min. S. khuzestanica 0.1% had very strong scolicidal effects in 30, 60, and 120 min of exposure times and the mortality rate decreased with the lower concentration. The finding have shown that the scolicidal activity of S. khuzestanica against cystic echinococosis protoscolices were more effective, while the O. europaea extract showed less effects.     

In Vitro Effects of SB202190 on Echinococcus granulosus

Spillage of cyst contents during surgical operation is the major cause of recurrence after hydatid cyst surgery. Instillation of a scolicidal agent into a hepatic hydatid cyst is the most commonly employed measure to prevent this complication. SB202190 is a pyridinyl imidazole derivative and is known to be a specific inhibitor of p38 MAPK. In the present study, the scolicidal effect of SB202190 was investigated. Freshly isolated Echinococcus granulosus protoscolices were subjected to SB202190 treatment (10, 20, 40, and 80 µM), and the effects on parasite viability were monitored by trypan blue staining. Corresponding effects were visualized by scanning and transmission electron microscopy. Dose-dependent protoscolex death within a few days of SB202190 treatment was observed. Although the in vitro scolicidal effect of SB202190 was satisfactory, the in vivo efficacy of this drug and also possible side effects remain to be further investigated.     

Echinococcus granulosus: pre-culture of protoscoleces in vitro significantly increases development and viability of secondary hydatid cysts in mice

We describe a method for obtaining improved secondary infections of Echinococcus granulosus that involves culturing protoscolex larvae in vitro prior to inoculation into mice. This approach provides a far superior method for obtaining secondary echinococcosis infections in mice compared with the traditional method of direct inoculation of protoscoleces (PSC) where the majority of parasites are killed by the host. We obtained a high rate of recovery both in terms of secondary cyst numbers and their viability. After 50 weeks post-infection (p.i.), brood capsules were formed and the first PSC developed in each of the capsules. After 56 weeks p.i., the fastest developing brood capsule contained four PSC. The approach will prove valuable for investigating parasite development and the host-parasite interaction in secondary echinococcosis.     

Precocious development of hydatid cysts in a macropodid host

This study describes the pathological changes associated with an experimental infection of captive wallabies with Echinococcus granulosus. Adult and juvenile tammar wallabies (Macropus eugenii) were infected orally with 0, 1,000, 2,500 or 8,000 E. granulosus eggs. Lung cyst progression was monitored by chest radiography every 4 months until 16 months p.i. Animals were necropsied from 9 to 16 months after infection. Cysts were detected radiographically from 4 months onwards. The number of cysts per animal varied from one to 10 and the majority (36/40) of cysts established in the lungs. Infection rate was low (35.5%), but cyst development was more rapid and onset of fertility much earlier than has been recorded in sheep. Cyst growth resulted in loss of functional lung capacity, up to an estimated 28% within 14 months of infection. Degenerative changes in cysts were less common in tammars than has been reported in sheep, with gross degeneration of cysts identified in only two animals. Complications associated with lung cyst development included fatal anaphylaxis, pneumothorax and atelectasis. Seven of the 11 infected tammars died or were euthanased as a result of infection during the experiment. From the parasite's perspective, infection of this host allows a shortened life cycle and correspondingly greater biotic potential. We believe this is the first published study that demonstrates the susceptibility of tammar wallabies to hydatid disease and confirms their suitability as a laboratory model for studying the disease in macropodids.     

Two cases of primary splenic hydatid cyst in Greece

Cystic disease of the spleen is an uncommon entity in general population. Most cases result from parasitic infection by Echinococcus granulosus, a form called splenic hydatid disease (SHD), with a reported frequency of 0.5-6.0% within abdominal hydatidosis. On the contrary, an isolated splenic involvement of hydatid disease is very uncommon even in endemic regions. Two cases of primary SHD managed with open and laparoscopic radical surgery in our department are reported herein. Primary SHD is a rare entity with non-specific symptoms underlying clinical suspicion by the physician for prompt diagnosis. Surgical treatment is the mainstay therapy, while laparoscopic approach when feasible is safe, offering the advantages of laparoscopic surgery.     

Construction of In Vivo Fluorescent Imaging of Echinococcus granulosus in a Mouse Model

Human hydatid disease (cystic echinococcosis, CE) is a chronic parasitic infection caused by the larval stage of the cestode Echinococcus granulosus. As the disease mainly affects the liver, approximately 70% of all identified CE cases are detected in this organ. Optical molecular imaging (OMI), a noninvasive imaging technique, has never been used in vivo with the specific molecular markers of CE. Thus, we aimed to construct an in vivo fluorescent imaging mouse model of CE to locate and quantify the presence of the parasites within the liver noninvasively. Drug-treated protoscolices were monitored after marking by JC-1 dye in in vitro and in vivo studies. This work describes for the first time the successful construction of an in vivo model of E. granulosus in a small living experimental animal to achieve dynamic monitoring and observation of multiple time points of the infection course. Using this model, we quantified and analyzed labeled protoscolices based on the intensities of their red and green fluorescence. Interestingly, the ratio of red to green fluorescence intensity not only revealed the location of protoscolices but also determined the viability of the parasites in vivo and in vivo tests. The noninvasive imaging model proposed in this work will be further studied for long-term detection and observation and may potentially be widely utilized in susceptibility testing and therapeutic effect evaluation.     

In vitro protoscolicidal effects of hypertonic glucose on protoscolices of hydatid cyst

To evaluate the protoscolicidal effects of various concentrations of hypertonic glucose, live protoscolices of sheep were exposed to 10%, 15%, 25% and 50% glucose solutions. Cetrimide (0.5%), silver nitrate (0.5%) and hypertonic saline (20%) were used as positive controls, while physiological saline was used as a negative control. After 1, 2 and 5 min, the protoscolicidal effects were determined by 1% eosin. A 25% glucose solution had no significant protoscolicidal effect. However, a 50% glucose solution revealed higher protoscolicidal effect than 0.5% silver nitrate but weaker effect than 0.5% cetrimide; the effect was comparable with that of 20% hypertonic saline. The results showed that hypertonic glucose solution is highly effective in killing protoscolices of Echinococcus granulosus in vitro.     

An Imported Case of Echinococcosis of the Liver in a Korean Who Traveled to Western and Central Europe

Echinococcus granulosus, an intestinal tapeworm of dogs and other canids, infects humans in its larval stage and causes human echinococcosis or hydatid disease. In the Republic of Korea, 31 parasite-proven human echinococcosis cases have been reported, most of which were imported from the Middle East. We recently examined a 61-year-old Korean man who had a large cystic mass in his liver. ELISA was negative for tissue parasitic infections, including echinococcosis, cysticercosis, paragonimiasis, and sparganosis. The patient underwent surgery to remove the cyst, and the resected cyst was processed histopathologically for microscopic examinations. In sectioned cyst tissue, necrotizing protoscolices with disintegrated hooklets of E. granulosus were found. In some areas, only freed, fragmented hooklets were detected. The patient had traveled to western and central Europe in 1996, and had no other history of overseas travel. We report our patient as a hepatic echinococcosis case which was probably imported from Europe.     

Scolicidal Effects of Black Cumin Seed (Nigella sativa) Essential Oil on Hydatid Cysts

Surgery remains the preferred treatment for hydatid cyst (cystic echinococcosis, CE). Various scolicidal agents have been used for inactivation of protoscolices during surgery, but most of them are associated with adverse side effects. The present study aimed to evaluate the in vitro scolicidal effect of Nigella sativa (Ranunculaceae) essential oil and also its active principle, thymoquinone, against protoscolices of hydatid cysts. Protoscolices were aseptically aspirated from sheep livers having hydatid cysts. Various concentrations of the essential oil (0.01-10 mg/ml) and thymoquinone (0.125-1.0 mg/ml) were used for 5 to 60 min. Viability of protoscolices was confirmed by 0.1% eosin staining. Furthermore, the components of the N. sativa essential oil were identified by gas chromatography/mass spectroscopy (GC/MS). Our study revealed that the essential oil of N. sativa at the concentration of 10 mg/ml and its main component, thymoquinone, at the concentration of 1 mg/ml had potent scolicidal activities against protoscolices of Echinococcus granulosus after 10 min exposure. Moreover, thymoquinone (42.4%), p-cymene (14.1%), carvacrol (10.3%), and longifolene (6.1%) were found to be the major components of N. sativa essential oil by GC/MS analysis. The results of this study indicated the potential of N. sativa as a natural source for production of a new scolicidal agent for use in hydatid cyst surgery. However, further studies will be needed to confirm these results by checking the essential oil and its active component in in vivo models.     

Genetic Variability of Antigen B among Echinococcus granulosus Egyptian Isolates

Genetic polymorphisms of encoding antigen B2 gene (AgB2) in Echinococcus granulosus were studied using PCR-RFLP and DNA sequencing among 20 Egyptian isolates. Five isolates from different host origins (humans, camels, pigs, and sheep) were collected and used. All examined isolates of each host group gave very similar patterns of PCR-RFLP after restriction enzyme digestion with AluI, with the gene size of approximately 140 bp and 240 bp for sheep and human isolates, and approximately 150 bp and 250 bp for pig and camel isolates. No digestion pattern was obtained after incubation of all studied isolates with EcoRI. These results reveal high intra-group homogeneity. DNA sequence analysis highlighted that human infecting strain showed 100% identity with respect to sheep infecting isolate, 96% and 99% with pig and camel infecting isolates, respectively.     

Serodiagnosis of Echinococcosis by ELISA Using Cystic Fluid from Uzbekistan Sheep

According to increase of travel, the cases of imported echinococcosis have been increasing in Korea. The present study was undertaken to develop a serodiagnostic system for echinococcosis in Korea. For diagnosis of echinococcosis, the fluid of Echinococcus granulosus hydatid cysts was collected from naturally infected sheep in Uzbekistan. Also serum samples of infected patients who were surgically confirmed were collected in a hospital in Tashkent, Uzbekistan. According to the absorbance of 59 echinococcosis positive and 39 negative control serum samples, the cut-off value was determined as 0.27. The sensitivity and specificity of ELISA with hydatid fluid antigen were 91.5% and 96%, respectively. The antigen cross-reacted with the serum of some cysticercosis or clonorchiasis patients. However, immunoblot analysis on the cystic fluid recognized antigenic proteins of 7-, 16-, and 24-kDa bands in their dominant protein quantity and strong blotting reactivity. In conclusion, the present ELISA system using hydatid cyst fluid antigen from Uzbekistan sheep is sensitive and specific for diagnosis of echinococcosis cases.     

Echinococcus granulosus: lethal effect of low voltage direct electric current on hydatid cyst protoscoleces

We have studied a small scale method for killing hydatid cyst protoscoleces using low voltage direct electric current. After collecting hydatid cysts from infected organs of slaughtered animals, protoscoleces were cultured in four different media: hydatid cyst fluid, RPMI, normal saline, and Tris buffer, respectively. Protoscoleces from each of the above media were then transferred to an electrolysis device through which different electric current densities were applied. For measuring the survival rate of protoscoleces, flame cell movement and eosin staining was used. The results show that the survival rate of protoscoleces in hydatid fluid was dependent on the electric current density and the time of the applied current. Current densities of 62.5 mA/cm2 (11 V), 53.71 mA/cm2 (10 V), and 18.18 mA/cm2 (5 V) after 1, 2, and 3 min, respectively, killed all the parasites in the hydatid fluid. However, a current density of 7 mA/cm2 (9 V) in RPMI medium after 3 min was most effective.     

Prevalence of Cystic Echinococcosis in Slaughtered Sheep as an Indicator to Assess Control Progress in Emin County,Xinjiang, China

Hydatid disease imposing serious threat on human health and great loss in live¬stock pastoralism remains a major public health problem in western China. To assess and monitor the effect of control program on transmission dynamics, we used the prevalence of cystic echinococcosis in slaughtered sheep at slaughterhouse as an indicator during the period of 2007 to 2013 in Emin County, Xinjiang Uygur Autonomous Region, China. The results showed a significant decline trend of prevalence in all age groups during the 7 years when the control program was implemented; particularly, the rate was reduced by 72% after first 3 years. Among the sheep slaughtered, the age distribution evidenced that the prevalence increased significantly as the sheep grew older. The baseline data indicated that the rate was 4.5% at the age <1, 6.7% at age 2~, and reached to the highest 17.9% at age ≥4 years. Earlier response to the intervention pressure was seen in the sheep at the younger age. Significant decline started from 2008 at the age <1, from 2009 at age of 1~, 2010 at 2~ to 3~, and the latest, in 2012 at age ≥4. This study demonstrated that the prevalence of cystic echinococcosis in slaughtered sheep may be used as an indicator to assess and monitor the transmission status during and after control program providing information for betterment of performance to sustain control strength.     

Environmental Contamination by Echinococcus granulosus sensu lato Eggs in Relation to Slaughterhouses in Urban and Rural Areas in Tunisia

Hydatidosis has become a real concern for health care institutions and animal rearers in Tunisia. The Tunisian endemicity is aggravated by the growing number of dogs and the difficulty of getting rid of contaminated viscera because of the lack of equipment in most slaughterhouses. Therefore, microscopic and molecular tools were applied to evaluate the role of slaughterhouses in canine infection and Echinococcus granulosus sensu lato (s. l.) egg dissemination. Exposure risk to E. granulosus s. l. eggs in urban and rural areas was explored in order to implant preventive and adapted control strategies. Microscopic examinations detected taeniid eggs in 152 amongst 553 fecal samples. The copro-PCR demonstrated that 138 of 152 taeniid samples analyzed were positive for E. granulosus s. l. DNA. PCR-RFLP demonstrated that all isolated samples belonged to E. granulosus sensu stricto (s. s.). An important environmental contamination index (25.0%) by E. granulosus s. l. eggs was demonstrated. The average contamination index from the regions around slaughterhouses (23.3%; 95% CI: 17.7-28.9%) was in the same range as detected in areas located far from slaughterhouses (26.0%, 95% CI: 21.3-30.8%). Echinococcosis endemic areas were extended in both rural (29.9%, 95% CI: 24.8-34.9%) and urban locations (18.1%, 95% CI: 13.0-22.9%). The pathogen dissemination is related neither to the presence/absence of slaughterhouses nor to the location in urban or rural areas, but is probably influenced by human activities (home slaughtering) and behavior towards the infected viscera.     

Infection Status of Hydatid Cysts in Humans and Sheep in Uzbekistan

Uzbekistan is endemic of cystic echinococcosis (CE). In order to estimate endemicity of CE, we collected data from emergency surgery due to CE in 2002-2010 and also investigated the prevalence of hydatid cysts in the liver and lungs of sheep at an abattoir in Uzbekistan from July 2009 to June 2010. In 14 emergency hospitals, 8,014 patients received surgical removal or drainage of CE during 2002-2010, and 2,966 patients were found in 2010. A total of 22,959 sheep were grossly examined of their liver and lungs, and 479 (2.1%) and 340 (1.5%) of them were positive for the cyst in the liver and lungs, respectively. Echinococcus granulosus is actively transmitted both to humans and sheep, and CE is a zoonotic disease of public health priority in Uzbekistan.     

In Vitro Scolicidal Effects of Salvadora persica Root Extract against Protoscolices of Echinococcus granulosus

It has been known that Arak, Salvadora persica, has a number of medicinal properties. We tried to investigate in vitro scolicidal effect of root extracts of this plant against protoscolices from hydatid cysts of Echinococcus granulosus. Protoscolices were aseptically collected from sheep livers containing hydatid cysts. S. persica root extract was used in 10, 30, and 50 mg/ml concentration for 10, 20, and 30 min. The viability of protoscolices was ascertained by 0.1% eosin staining. Scolicidal activity of S. persica extract at a concentration of 10 mg/ml was 36.3%, 50.3%, and 70.8% after 10, 20, and 30 min of exposure, respectively. The scolicidal effect of this extract at a concentration of 30 mg/ml was 52.9%, 86.7%, and 100% after 10, 20, and 30 min of exposure, respectively. S. persica extract at a concentration of 50 mg/ml, meanwhile, killed 81.4%, 100%, and 100% of protoscolices after 10, 20, and 30 min, respectively. Also, the cytotoxic potential of S. persica was assessed on human liver cells (HepG2) using trypan blue exclusion test. No cytotoxic effect was observed on HepG2 cell line. The present study confirmed for the first time that the ethanolic extract of S. persica has high scolicidal power in vitro. However, in vivo effect of this material remains to be studied for treatment of echinococcosis in humans and herbivorous animals.     

Prevalence of Serum IgG Antibodies to Cystic Echinococcus Antigen among Patients in an Uzbekistan Emergency Hospital

Cystic echinococcosis (CE) is one of the most widespread zoonotic helminthiases, which can last an asymptomatic infection for several years. The purpose of this study was to demonstrate serum antibody prevalence of CE among asymptomatic people in Uzbekistan using ELISA. A total of 2,547 serum samples were collected, 66 from confirmed CE patients and 2,481 of patients with other diseases than CE at a hospital in Tashkent, Uzbekistan. The serum samples were screened for CE specific IgG antibodies by ELISA using cystic fluid antigen obtained from sheep. The serum antibody positive rate was 89.4% (59/66) in CE and 3.6% (89/2,481) in other disease patients. The present ELISA recognized 89.4% sensitivity and 96.4% specificity. The ELISA absorbance of positive samples was distributed 0.271-0.971 for CE and 0.273-0.887 for other disease patients. The other disease patients with high absorbance over 0.3 were 50 (2.0%) who were presumed to be active CE patients. The patients in their 40s showed the highest positive rate of 5.2% (P=0.181), and women were 4.4% while men were 3.1% positive (P=0.136). The data confirmed that there are many asymptomatic patients of CE in Tashkent. It is indicated that CE is an endemic disease of public health importance in Uzbekistan.     

Differential Expression of Hox and Notch Genes in Larval and Adult Stages of Echinococcus granulosus

This investigation aimed to evaluate the differential expression of HoxB7 and notch genes in different developmental stages of Echinococcus granulosus sensu stricto. The expression of HoxB7 gene was observed at all developmental stages. Nevertheless, significant fold differences in the expression level was documented in the juvenile worm with 3 or more proglottids, the germinal layer from infected sheep, and the adult worm from an experimentally infected dog. The notch gene was expressed at all developmental stages of E. granulosus; however, the fold difference was significantly increased at the microcysts in monophasic culture medium and the germinal layer of infected sheep in comparison with other stages. The findings demonstrated that the 2 aforementioned genes evaluated in the present study were differentially expressed at different developmental stages of the parasite and may contribute to some important biological processes of E. granulosus.     

Higher Seroprevalence of Hepatitis B Virus Antigen in Patients with Cystic Hydatid Disease than in Patients Referred to Internal Medicine Clinics in Turkey

Turkey remains an intermediate area for prevalence of hepatitis B virus (HBV) surface antigenemia. The sheep-raising areas of Turkey also pose a high risk for cystic hydatid disease (CHD). Both HBV infection and CHD are major public health issues particularly in eastern parts of Turkey; however, there is no data regarding HBV infection in patients who have had CHD. The aims of this study were to evaluate the association between HBV infection and CHD and suggest ways to reduce HBV infection which is still widespread in Turkey. A retrospective study was conducted with 94 adult patients with active CHD referred to the hepatology department, Yuzuncuyil University School of Medicine from December 2010 to December 2012. All subjects came from rural areas of the region and underwent ultrasonography of abdomen which detected CHD of the liver. All the patients were serologically positive for Echinococcus granulosus. The control group consisted of 500 patients (300 men and 200 women) referred to the internal medicine clinics for other reasons. The patients with CHD and in the control group were tested for the existence of HBs antigen according to the standard procedures. The seroprevalence of HBs antigen was significantly higher in patients with active CHD than those in the control group (12.7% vs 5.2%; P=0.0017). Our data indicate that there is significant association between HBV infection and CHD. All patients with CHD should be screened for HBV infection.     

An Imported Case of Cystic Echinococcosis in the Liver

A 25-year-old Uzbek male presented with right upper abdominal pain for 20 days. On radiologic studies, a huge cystic mass was noticed in the right liver which was suspected as parasitic. The patient received right hepatic segmentectomy (segment 7), and the surgically resected mass was confirmed as cystic echinococcosis (CE), measuring 10.5 cm in its diameter. The inner surface of the cyst was bile-stained. The patient was discharged on the 8th hospital day, and was rechecked 6 months after the surgical intervention without any evidence of recurrence. The present report describes findings of an imported case of CE which represented ultrasound images of the ''ball of wool''.