Very low frequency waves as selective probe for Cysticercus tenuicollis, Hydatid cyst and Coenurus cerebralis bio-analysis using single cell-signal recording.

Comparative electric behavior of Cysticercus tenuicollis, Hydatid cyst and Coenurus cerebralis at the Very Low Frequency (VLF) region has been studied in detail. This investigation could be significant, because of the economic and public health importance of these parasitic infections in domestic animals. In this report, a single cell signal recording technique has been adopted for comparison using a stainless steel (type: 316, diameter: ~ 300 µm, height: 2.00 cm) two identical electrode system, implanted on the surface of the tested cysts with inter electrode distance of 0.50 cm at a ~ 6.0 giga ohm (GΩ) sealed condition (based on the situation of the implanted electrode system). This process was achieved based on applying electrical interaction between the cysts and the VLF electrical signal. Relative to the measured time domain signal (Current-time diagram), the frequency domain (Current-frequency diagram) was estimated via applying a "Discrete Fast Fourier Transform" (DFFT) algorithm at a fixed time interval (5.0 min). Factors, having important influence on the sensitivity of the detection system including the type (waveform) of different alternating-current (AC) triggering stimulus signals (such as direct current, square wave, triangular, sin (t), etc.), the amplitude, as well as the frequency were optimized automatically through a written "Visual Basic 6" program by one-factor-at-a-time method. Direct applying this AC triggering VLF voltage to the cysts resulted in tracing an AC electrical current vs. time that considered as the time domain wave. However, this electrical current was decayed rapidly versus time during maximum 30.0 s time scale. Applying the DFFT algorithm to the measured time domain, resulted in accessing to the frequency domain at the selected frequency range between 2 and 5 kHz that was considered as the selected frequency for the selective differentiation of C. tenuicollis, Hydatid cyst and C. cerebralis. The related probable mechanism of this process may be attributed to the correlation between the triggering potential and the cyst's electrical surface charge (Zeta potential) as the current source under similar conditions. The results of this study may help to introduce a new detection system for in vivo recognition of the cysts in future.

Diode and Active Negative Resistance Behaviors of Helminth Eggs as a Novel Identification/Differentiation Probe.

Helminths have always been studied as one of the critically annoying pathogens of parasite classes due to their adverse effects on the ecosystem of human life. They have the potency to negatively affect their hosts as points of disease, infection, cancer, and death, but in this study, we found interesting electronic properties in Fasciola hepatica, Parascaris equorum (with and without larvae), Dicrocoelium dendriticum, Taenia multiceps, and Moniezia expansa eggs. This claim is attributed to some surprising characteristics such as significant diode behavior [forward bias, 5.36-11.17 (±0.01) V, versus the ground, GND] and backward bias (-45.0 to -125.0 (±7.0) V, versus the GND) and highly active negative resistance (-2.59 to -7.11) × 1015 (±1.5) Ω in the AC mode. These traits were measured by the "blind patch-clamp, single-unit recording" methodology using a three-microelectrode system, implanted onto each tested egg under giga ohm sealed conditions (6.28 ± 0.02 GΩ cm-1 and n = 4). All the characteristic parameters were simultaneously attributed to the helminth egg structure by acceptable reproducibility (percentage of relative standard deviation: > 5%) and high enough rectitude with enough differentiation in their magnitudes, relatively. The reliability of these results was further confirmed using multiple calibrated techniques such as alternative/direct current voltammetry. Also, the significant role of water molecules as the key medium in creating these properties is evaluated qualitatively. In addition, the study aims at introducing these interesting parameters as a new approach to the fabrication of bio-based electronic elements, which are considered as a novel class of helminth egg-detection and -identification probes.

Supercapacitance/Resistance Behaviors of Helminth Eggs as Reliable Recognition and Direct Differentiation Probe.

Parasitic helminths are usually known as undesired pathogens, causing various diseases in both human and animal species. In this study, we explore supercapacitance/resistance behaviors as a novel probe for rapid identification and direct differentiation of Fasciola hepatica, Parascaris equorum (with and without larvae), Dicrocoelium dendriticum, Taenia multiceps, and Moniezia expansa eggs. This claim is attributed to some characteristics, such as grave supercapacitance/area, high-energy storage/area, large power/egg, huge permittivity, and great electrical break-down potential, respectively (Fasciola hepatica: 2,158, 0.485, 2.7 × 10-3, 267, 52.6, Parascaris equorum without larvae: 2,825, 0.574, 3.0 × 10-3, 351, 68.4, Parascaris equorum with larvae: 4,519, 0.716, 2.4 × 10-3, 1.96, 97.6, Dicrocoelium dendriticum: 1,581, 0.219, 2.8 × 10-3, 1.96, 48.8, Moniezia expansa: 714, 0.149, 2.2 × 10-3, 0.88, 35.2, Taenia multiceps: 3,738, 0.619, 4.7 × 10-3, 4.63, 84.4), and durable capacitance up to at least 15,000 sequential cycles at different scan rates (between 2.0 × 10-4 and 120.0 V s-1) as well as highly differentiated resistance between 400 and 600 Ω. These traits are measured by the "Blind Patch-Clamp" method, at the giga ohm sealed condition (6.18 ± 0.12 GΩ cm-1, n = 5). Significant detection ranges are detected for each capacitance and resistance with gradient limits as large as at least 880 to 1,000 mF and 400 to 600 Ω depending on the type of helminth egg. The effect of water in the structure of helminth eggs has also been investigated with acceptable reproducibility (RSD 7%-10%, n = 5). These intrinsic characteristics would provide novel facilitators for direct helminth egg identification in comparison with several methods, such as ELISA, PCR, and microscopic methods.

Effects of Zataria multiflora essential oil on the germinative cells of Echinococcus granulosus.

BACKGROUND: Novel and more efficient compounds are urgently required for medical treatment of cystic echinococcosis (CE). Germinative cell culture of Echinococcus granulosus could be used for anti-echinococcosis agent tests and other biological studies on CE. This study was performed to establish an in vitro cell culture model for E. granulosus germinative cells and to evaluate the lethal effect of Zataria multiflora essential oil (ZMEO) on the cultured cells. METHODS: The inner surface of germinal layers of CE cysts was scraped, and the obtained materials were trypsinized to obtain a suspension of single germinative cells. Medium 199 was used as the basic culture medium and was supplemented with fetal bovine serum, 2-mercaptoethanol, L-cysteine, L-glutamine, glucose, sodium pyruvate, hydatid fluid, amphotericin B and antibiotics. The cells were cultured at a concentration of 104 cells/ml of culture medium and incubated at 37 °C. The culture medium was replaced every 7 days. Chemical composition of ZMEO was identified by GC-MS analysis. ZMEO was tested at concentrations of 0.5-8 mg/ml. Viability of the cells was assessed by trypan blue exclusion assay. RESULTS: A significant increase in the cell number was evident at 20, 30 and 45 days after cultivation. At 45 days of cultivation, the number of cells was approximately five-fold higher than on the first day. In GC-MC analysis, carvacrol, p-cymene, g-terpinene and thymol were found to be the main compounds of ZMEO. The lethal effect of ZMEO on the germinative cells at concentrations of 6, 7 and 8 mg/ml was 100% after 60, 25 and 7 min of exposure, respectively. CONCLUSIONS: At 45 days of cultivation, the cell concentration was suitable for the desired in vitro experiments. A high lethal effect of ZMEO on the germinative cells of E. granulosus may be considered an opportunity for the introduction of a novel, more effective and safer therapeutic agent for treatment of CE using an herbal product.

Evaluation of the efficacy of Zataria multiflora essential oil versus albendazole in patients infected with liver cystic echinococcosis: A nonrandomized clinical trial.

BACKGROUND: Cystic echinococcosis (CE) is a life-threatening disease in many countries. Albendazole, as the drug of choice for medical treatment of CE, is accompanied by adverse effects and may be ineffective in 20%-40% of cases; hence, new and more effective compounds are urgently needed to optimize the management of the disease. This study was performed to evaluate the efficacy of Zataria multiflora essential oil (ZMEO) versus albendazole against human liver CE. MATERIALS AND METHODS: In this nonrandomized and single-blinded clinical trial, thirty patients who were infected with liver CE were divided into two groups (15 in each) and treated with albendazole (800 mg daily) and ZMEO (60 mg daily), respectively. Albendazole and ZMEO were administered orally for 180 consecutive days. The volume of hydatid cysts was measured by ultrasonography before and 2, 4 and 6 months after the start of treatment. Simultaneously, biochemical analysis was performed on the blood samples of patients to assess the possible side effects of the two treatment regimens. RESULTS: Two, 4 and 6 months after the start of treatment, ZMEO indicated a significantly higher ability in reduction of the volume of the hydatid cysts, compared to albendazole (P < 0.05). The mean values of aspartate aminotransferase, alanine transaminase and alkaline phosphatase were also significantly lower in the patients treated with ZMEO in comparison to those treated with albendazole (P < 0.05). No clinical adverse effects were observed in the patients treated by ZMEO. CONCLUSION: From the point of view of efficacy and safety, ZMEO indicated a significant superiority to albendazole. Hence, ZMEO may be considered as an alternative for albendazole in the medical treatment of liver CE.

Evaluation of the efficacy of albendazole sulfoxide (ABZ-SO)-loaded chitosan-PLGA nanoparticles in the treatment of cystic echinococcosis in laboratory mice.

Albendazole is known as the drug of choice for medical treatment of cystic echinococcosis (CE). Albendazole sulfoxide (ABZ-SO), as the main active metabolite of albendazole, has low efficacy in the disease due to low water solubility and poor absorptivity. PLGA nanoparticles (NPs) enhance the dissolution of poorly soluble drugs, and chitosan (CS) coating enhances oral drug delivery of NPs. In this study, the efficacy of ABZ-SO-loaded CS-PGLA NPs in the treatment of CE was evaluated in laboratory mice. ABZ-SO-loaded CS-PGLA NPs were prepared by nanoprecipitation and characterized by dynamic light scattering method and scanning electron microscopy. Thirty mice were intraperitoneally infected by 1000 protoscoleces of Echinococcus granulosus. Ten months later, the mice were allocated into 3 groups: groups 1 and 2 were treated with ABZ-SO and ABZ-SO-loaded CS-PGLA NPs, respectively, and the mice in group 3 remained untreated as the control group. The drugs were administered by gavage for 45 days at a daily dose of 10 mg/kg. Finally, all mice were opened and the cysts were collected, counted, weighed, and measured separately. The therapeutic effect of ABZ-SO in the number, weight, and volume of the cysts were not statistically significant compared with those in ABZ-SO-loaded CS-PGLA NPs and the control group. However, the therapeutic effect of ABZ-SO-loaded CS-PGLA NPs in the weight and volume of cysts were statistically significant when compared with that in the control group (p ˂ 0.05). In conclusions, this study revealed that ABZ-SO-loaded CS-PGLA NPs could enhance the therapeutic efficacy of ABZ-SO in the treatment of CE in laboratory mice.

Molecular Characterization of Fasciola spp. from a Donkey (Equus asinus) Using Partial Sequencing of cox1 and nad1.

BACKGROUND: Fasciola hepatica as an important parasite affects health of humans and animals in some tropical and subtropical areas of the world, including Iran. Little is known about the molecular diversity of Fasciola in Equidae. Therefore, this study aimed to characterize the genetic polymorphisms among parasites. METHODS: Eight adult Fasciola spp. isolates were collected from a working donkey after necropsy in Shiraz, southwestern Iran, in 2018. Primarily, various parameters were measured morphologically. Subsequently, DNA was extracted from each fluke and molecular markers of cytochrome C oxidase (cox1) and NADH dehydrogenase 1(nad1) from individual Fasciola isolates were amplified using PCR assay and sequence data were employed for molecular and phylogenetic analysis. Genetic diversity between isolates was evaluated by comparing the sequences of these two mitochondrial regions. RESULTS: Based on the morphological and analyzed mitochondrial sequences, all of eight donkey isolates (100%) were identified as F. hepatica. Moreover, nine and five nucleotide polymorphisms were identified in the cox1and nad1 region sequences, respectively. CONCLUSION: Accordingly, phylogenetic data revealed five and four haplotypes among donkey isolates based on the cox1and nad1 markers. Similarly, some of these haplotypes have been previously reported from different host species in Iran as well as all around the world.

The Lethal Effect of a Nano Emulsion of Satureja hortensis Essential Oil on Protoscoleces and Germinal Layer of Hydatid Cysts.

BACKGROUND: New scolicidal agents and novel therapeutic drugs are essential for better management of the zoonotic infection, hydatid disease. This study evaluated the effect of a nanoemulsion (NE) of Satureja hortensis essential oil (SHEO) on protoscoleces and germinal layer of hydatid cysts. METHODS: This study was conducted from July to October 2016 in Shiraz University, Shiraz, Iran. Gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) were performed to identify the main components of SHEO. To determine the scolicidal power of the NE of SHEO, live protoscoleces of hydatid cysts were exposed to two concentrations (1 and, 2 mg/mL) of the NE and incubated at 37 °C for 10 and 20 min. To evaluate the anti-hydatid effect of the NE of SHEO, the collected hydatid cysts from the abdominal cavities of the experimentally infected mice were immersed in the NE (0.5 mg/ml) and incubated at 22 °C for 24 h. RESULTS: Carvacrol and γ-terpinene were the major components of the SHEO. NE of SHEO at the concentrations of 1 and 2 mg/mL showed 100% scolicidal power after 20 and 10 min respectively. Exposure of the hydatid cysts to the NE of SHEO resulted in crumpling of their germinal layer and detachment of this layer from the laminated layer. CONCLUSION: NE of SHEO showed a strong scolicidal activity as well as a profound lethal effect on the germinal layer of hydatid cysts. Accordingly, this product may be used as a natural scolicidal agent in hydatid cyst surgery. Furthermore, it may be used as a therapeutic tool for treatment of hydatid disease.

Histopathological and Molecular Evaluation of the Experimentally Infected Goats by the Larval Forms of Taenia multiceps.

BACKGROUND: Introduction of Taenia multiceps and T. gaigeri as two separate species have been recognized mainly on morphological grounds. This experimental study was conducted to test whether cerebral and non-cerebral forms of Coenurus cerebralis belong to one origin or they are originated from two different tape worms. METHODS: Two groups of dogs were infected with the cerebral and muscular sources of the coenuri cysts. About two months later the eggs were collected from the fecal samples to be used to experimentally infect other healthy goats. Histopathological and molecular evaluation was conducted in two groups of goats that were challenged with T. multiceps eggs obtained from the infected dogs by brain and muscular sources of coenuri cysts in School of Veterinary Medicine of Shiraz University, Shiraz, Iran in 2015. All aberrant sites of predilection of the metacestode in goats were muscles, heart, diaphragm and lungs. The brain and spinal cord were carefully dissected and examined but the cysts were not found in these locations. In addition, the molecular genetic markers of mitochondrial DNA (CO1 and ND1) were applied to resolve the questionable relationship between T. multiceps and T. gaigeri. RESULTS: The larval stages of T. multiceps in brain and in other aberrant sites, which showed similar morphological criteria, were monophyletic species. CONCLUSION: Therefore, T. gaigeri must be considered taxonomically invalid.

Enhancement of the Therapeutic Effect of Albendazole on Cystic Echinococcosis using a Herbal Product.

PURPOSE: Albendazole is the drug of choice for chemotherapy of cystic echinococcosis (CE) but 40% of patients do not respond to it satisfactorily. With regard to the previously reported scolicidal activity of Zataria multiflora, this study was done to evaluate the efficacy of the co-administration of albendazole and Z. multiflora aromatic water (AW) on hydatid cysts in a murine model. MATERIALS AND METHODS: Laboratory mice were infected with 1,500 viable protoscoleces. After development of hydatid cysts, the infected mice were allocated into four treatment and one control group. The mice of groups 1, 2, 3, and 4 were treated orally with albendazole (100 mg/kg), Z. multiform AW (100 ml/l), albendazole (100 mg/kg) - Z. multiform AW and albendazole ((50 mg/kg) - Z. multiform AW respectively. Two months later, all the mice were euthanized and necropsied and the number, size and weight of hydatid cysts were compared between different groups. RESULTS: The size of the largest cysts, as well as the total weight of cysts, were significantly lower (p < .05) in the mice treated with albendazole (50 mg/kg) - Z. multiflora AW in comparison to those treated with albendazole (100 mg/kg) alone. CONCLUSIONS: The results of this study revealed the superiority of co-administration of albendazole and Z. multiflora AW to albendazole alone in the treatment of CE in laboratory mice. Since Z. multiflora AW is a safe and useful drink, it may be used successfully to enhance the therapeutic effect of albendazole in the patients infected with hydatid disease.

Epidemiology of taeniosis, cysticercosis and trichinellosis in Iran: A systematic review.

BACKGROUND: The aim of this review was to establish the current epidemiology of taeniosis, cysticercosis and trichinellosis among humans and animals in Iran by carrying out a comprehensive assessment of published articles reporting on these foodborne zoonotic diseases across the country. METHODS: The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline was used in the search for relevant published articles reporting on cysticercosis, taeniosis and trichinellosis in Iran using a number of appropriate key words. The search was conducted through PubMed, Web of Science, Google Scholar, SpringerLink, SCOPUS, WHOLIS, FAO and CDC. Published scientific articles including journals, books and book chapters reporting on cysticercosis, taeniosis and trichinellosis in Iran for the period between 1967 and 2018 were selected. RESULTS: A total of 37 articles met the search criteria and were incorporated in this review. Of these, 10 (27%) reported on human taeniosis, 15 (40.5%) on cysticercosis (10 on Taenia saginata and five on Taenia spp. cysticercosis) and 12 (32.5%) on trichinellosis. T. saginata was implicated in all human taeniosis cases. All Taenia spp. cysticercosis cases were reported among domesticated pigs and wild animals. A case of neurocysticercosis was reported in a male patient at Shohada Hospital in Tehran. Eleven (91.7%) of the 12 studies reported on trichinellosis among wild animals, while one (8.3%) study detected anti-Trichinella IgG in 8 (2.2%) of the 364 at-risk human beings tested. Nevertheless, most of these studies were carried out in northern Iran. CONCLUSION: This review found T. saginata to be the most prevalent and of greater economic and public health significance in Iran. However, T. solium and Trichinella spp. were of little significance to human health. More studies should focus on other regions besides northern Iran.

Influence of hydatid disease on the pregnancy outcomes: An experimental study.

AIM: Hydatid disease during pregnancy may lead to various complications in both mother and child. Accordingly, enough knowledge is required to manage these complications. This study was done to evaluate the interaction between hydatid disease and pregnancy in laboratory mice. METHODS: Twenty female BALB/c mice (Mus musculus) were divided into test and control groups, 10 animal in each. The mice of the test group were infected intraperitoneally by injection of 1000 protoescolices. Six months after infection, the mice of test and control groups were mated with male mice for a week. After parturition, the size and weight of babies were measured and compared between two groups. RESULTS: While all the mice of control group delivered healthy babies, 3 out of 10 mice of test group showed no pregnancy. The infected mice delivered lower number of babies than the mice of control group. The mean size of fetuses or babies obtained from the infected mice were statistically lower than those obtained from the mice of control group (P = 0.000). The weight of babies delivered by the infected mice not only at the time of birth (P = 0.005) but also 10 days after birth (P = 0.021), were significantly lower than those delivered by the mice of control group. The level of progesterone and estradiol in the mice of test group were significantly lower (P = 0.009) and higher (P = 0.043), respectively, in comparison to the mice of control group. CONCLUSION: Hydatid disease during pregnancy may considerably affect the pregnancy outcomes.

A new method for laboratory rearing of Galba truncatula, the intermediate host of Fasciola hepatica.

In this study a relatively large and open top aquarium was designed, constructed and introduced as a suitable habitat for nutrition, growth and development as well as for egg laying and breeding of Galba truncatula under laboratory conditions. The soil and water used in the aquarium were collected from the locality in which the snails were collected. The aquarium was placed in a laboratory with temperature of 18-32 ºC and relative humidity of 22-37% respectively, according to the season. The artificial light was controlled by a light timer, giving 12 h of light and 12 h of darkness. The snails were fed with dried lettuce leaves, Cyperus alternifolius (aquatic plant), Spirulina (algae), Orthotrichum rupestre (moss) and cuttlebone (a supplementary source of calcium). Approximately five weeks after the start of study, there was evidence of reproduction and success in rearing of G. truncatula by the appearance of eggs and small snails (0.1-0.5 mm) in the aquarium. In conclusion, large scale laboratory rearing of G. truncatula is a feasible task. The method may be improved by balancing the temperature and relative humidity as well as by optimizing the soil type, the water quality and the type of food.

In vitro and in vivo antihydatid activity of a nano emulsion of Zataria multiflora essential oil.

In in vitro process of this study, protoscoleces of the hydatid cysts were exposed to two concentrations of nano emulsion (NE) of Zataria multiflora essential oil (ZMEO) (1 and 2mg/mL) for 10 and 20min. Viability of protoscoleces was confirmed using 0.1% eosin staining. For in vivo studies, sixteen laboratory mice were infected intraperitoneally by 1500 live protoscoleces. Five months after infection, the infected mice were divided into treatment and control groups. The mice of treatment group received the NE of ZMEO (20mg/kg) orally via their drinking water while the mice of control group received no treatment. Two months after the start of treatment, all of the mice were necropsied and the hydatid cysts were collected. Subsequently, the numbers, sizes and weights of the collected cysts were compared between the mice of two groups. The results of in vitro scolicidal assays showed that the scolicidal power of NE of a ZMEO at concentration of 1mg/mL was 88.01%, and 100% after 10 and 20min respectively. NE of ZMEO showed 100% scolicidal power at a concentration of 2mg/mL after 10min (comparing to 4.46% for the control group). The results of in vivo studies revealed that the size of the largest cysts as well as the total number of the cysts were significantly lower in the mice treated with NE of ZMEO (P<0.05). In conclusion, NE of ZMEO may be considered as a natural scolicidal agent and a potential therapeutic tool for treatment of hydatid disease.

In vitro ovicidal activity of Peganum harmala seeds extract on the eggs of Fasciola hepatica.

Peganum harmala seeds extract has been previously reported to have antimicrobial and other medicinal properties. The aim of this study was to evaluate the ovicidal activity of the methanolic extract of P. harmala seeds against the eggs of F. hepatica. The phenolic compounds of the methanolic extract of P. harmala seeds were identified by HPLC analysis. Catechin, rutin, p-Coumaric acid, chloregenic acid and hesperetin were found to be the major phenolic compounds. F. hepatica eggs were collected from the gall bladder of naturally infected sheep. The eggs were exposed to two concentrations of P. harmala seeds extract (1 and 3 mg/mL) for 24 and 48 h. To investigate the effect of the P. harmala seeds extract on the miracidial formation, the treated eggs were incubated at 28 °C for 14 days. The results indicated that F. hepatica eggs were susceptible to the methanolic extract of P. harmala seeds. Following 24 h exposure of the eggs to P. harmala seeds extract with concentrations of 1 and 3 mg/mL, the miracidial formation reduced to 5 and 2.2 % respectively (compared with 60 % for the control group). Following 48 h of exposure of the eggs to P. harmala seeds extract with 1 mg/mL concentration, the miracidial formation reduced to 0.5 %. In this exposure time, no miracidial formation was observed in the eggs exposed to P. harmala seeds extract with concentration of 3 mg/mL. Therefore, the results of this study indicated that P. harmala seeds extract has high ovicidal activity against the eggs of F. hepatica. Accordingly, this extract may have the potential flukicidal activity against the immature and mature F. hepatica.

Ovicidal effect of the methanolic extract of ginger (Zingiber officinale) on Fasciola hepatica eggs: an in vitro study.

Fasciolosis is of considerable economic and public health importance worldwide. Little information is available on the ovicidal effects of anthelminthic drugs. The use of ovicidal anthelmintics can be effective in disease control. In this study, the effectiveness of the methanolic extract of ginger (Zingiber officinale) on the eggs of Fasciola hepatica is investigated. Fasciola hepatica eggs were obtained from the gall bladders of naturally infected sheep and kept at 4 °C until use. The eggs were exposed to varying concentrations of ginger extract (1, 5, 10, 25 and 50 mg/mL) for 24, 48 and 72 h. To investigate the effect of the ginger extracts on the miracidial formation, the treated eggs were incubated at 28 °C for 14 days. The results indicated that F. hepatica eggs are susceptible to the methanolic extract of Z. officinale. The ovicidal effect of ginger extract at a concentration of 1 mg/mL with 24, 48 and 72 h treatment time was 46.08, 51.53 and 69.09 % respectively (compared with 22.70 % for control group). The ovicidal effect of ginger extract at a concentration of 5 mg/mL after 24 h was 98.84 %. One hundred percent ovicidal efficacy was obtained through application of ginger extract at concentrations of 5 and 10 mg/mL with a 48 and 24 h treatment time respectively. The in vitro ovicidal effect of the methanolic extract of Z. officinale was satisfactory in this study, however, in vivo efficacy of this extract, remains for further investigation. To the best of our knowledge, this is the first report on the ovicidal effect of Z. officinale against F. hepatica eggs.

Controversial aspects of the life cycle of Fasciola hepatica.

Fasciola hepatica is a well-known helminth parasite, with significant economic and public health importance all over the world. It has been known since more than 630 years ago and a considerable research work has been carried out on the life cycle of this important parasite. In the hepatic phase of the life cycle of F. hepatica, it is assumed that the young flukes, after about 6-7 weeks of migration in the liver parenchyma, enter into the bile ducts of the definitive hosts and become sexually mature. Even though the secretion of cysteine peptidases including cathepsin L and B proteases by F. hepatica may justify this opinion, because of several scientific reasons and based on the experimental studies conducted in different animals (reviewed in this article), the entry of parasites into the bile ducts, after their migration in the liver parenchyma seems to be doubtful. However, considering all the facts relating to the hepatic and biliary phases of the life cycle of F. hepatica, two alternative ideas are suggested: 1) some of the migrating juvenile flukes may enter into the bile ducts immediately after reaching the liver parenchyma while they are still very small, or 2) when newly excysted juvenile flukes are penetrating into the intestinal wall to reach the liver through the abdominal cavity, a number of these flukes may enter into the choleduct and reach the hepatic bile ducts, where they mature. According to the previously performed natural and experimental studies in different animals and human beings, the supporting and opposing evidences for the current opinion as well as the evidences that might justify the two new ideas are reviewed and discussed briefly. In conclusion, our present knowledge about the time and quality of the entry of F. hepaticas into the bile ducts, seems to be insufficient, therefore, there are still some dark corners and unknown aspects in this field that should be clarified.

Comparison of cerebral and non-cerebral coenurosis by genetic markers of glycolytic enzyme (enolase) and mitochondrial sequences in sheep and goats.

Coenurosis is a debilitating disease caused by the larval stage of Taenia multiceps. The larval stage of T. multiceps appears in two cerebral and non-cerebral forms known as Coenurus cerebralis and Coenurus gaigeri,respectively. This leads to the hypothesis that there are genetic intraspecific variabilities within this species. Molecular analysis in the present study showed that C. cerebralis and C. gaigeri were 100% genetically identical based on exonic region of enolase (ENO) gene and both mitochondrial (cox1 and nad1) markers. In addition, an intronic mutation at ENO gene (position: 1171) was detected in the Iranian C. cerebralis. The phylogenetic analyses based on the ENO, cox1, nad1 sequence data also suggest that T. gaigeri may not be distinct from T. multiceps and there is only one single valid species within T. multiceps.

Application of polymerase chain reaction on cerebrospinal fluid for diagnosis of cerebral coenurosis in small ruminants.

Sheep and goats serve as intermediate hosts for the canine tapeworm Taenia multiceps. The cysts produced by the intermediate stage of parasite are usually found in the cerebral hemispheres of small ruminants, and the resulting disease is commonly known as coenurosis. Coenurosis is clinically manifested in the form of various nervous symptoms, depending on the exact location of the cyst. The variety of neurological symptoms contributes to the complexity of clinical diagnosis and reinforces the need for a more specific and acceptable diagnostic approach. We demonstrated here, for the first time, that the T. multiceps DNA is present in the cerebrospinal fluid (CSF) of the infected sheep and goats. In addition, the molecular genetic marker of the mitochondrial DNA was applied phylogenetically to show that our isolates together with other T. multiceps strains comprised a monophyletic group that is a sister to Taenia krabbei. Pairwise comparison between the cox1 sequences of our study and other T. multiceps genotypes existing in the GenBank showed similarity ranging from 98 to 100%. Accordingly, the polymerase chain reaction (PCR) can be used for amplification of DNA of the parasite originated from the CSF and provides a valuable method for accurate identification of coenurosis cases.

Experimental cerebral and non-cerebral coenurosis in goats: A comparative study on the morphological and molecular characteristics of the parasite.

This experimental study was conducted to test whether cerebral and non-cerebral forms of Coenurus cerebralis belong to one origin or they are originated from two different tape worms. In the first step of the study, two groups of dogs were orally infected with the protoscoleces of cerebral and non-cerebral cysts and four months after infection, the adult worms were collected and morphologically characterized. Then the obtained eggs from two groups of adult worms were orally inoculated to two groups of goats to trace the predilection sites and also to compare the morphological and molecular characteristics of the larval stage of the parasites. The results showed that, both cerebral and non-cerebral coenuri, produced morphologically similar worms in the experimentally infected dogs. We observed only non-cerebral coenurus cysts in two groups of experimentally infected goats and the morphological characteristics of the cysts of two different groups were also similar. The molecular findings also showed that the cysts of two groups were 100% identical to each other based on the CO1 and nad1 sequences. In conclusion, our morphological and molecular findings reinforce the evidence that Taenia multiceps is the single origin of both cerebral and non-cerebral form of C. cerebralis but in goats, the cysts show more tendency to non-cerebral organs.