Efficacy of thymol against Echinococcus granulosus protoscoleces

The aim of the present work was to determine the in vitro protoscolicidal effect of thymol against Echinococcus granulosus. Protoscoleces of E. granulosus were incubated with thymol at concentrations of 10, 5 and 1 μg/ml. The first signs of thymol-induced damage were observed between 1 and 4 days post-incubation. The maximum protoscolicidal effect was found with thymol at 10 μg/ml, viability reduced to 53.5 ± 11.9% after 12 days of incubation. At day 42, viability was 11.5 ± 15.3% and, reached 0% after 80 days. Thymol at concentrations of 5 and 1 μg/ml provoked a later protoscolicidal effect. Results of viability tests were consistent with the tissue damage observed at the ultrastructural level. The primary site of damage was the tegument of the parasite. The morphological changes included contraction of the soma region, formation of blebs on the tegument, rostellar disorganization, loss of hooks and destruction of microtriches. The data reported in this article demonstrate a clear in vitro effect of thymol against E. granulosus protoscoleces.     

Occurrence of the G3 Indian buffalo strain of Echinococcus granulosus in cattle

During a survey carried out to define the occurrence of Echinococcus granulosus in cattle bred in the province of Rieti (Central Italy), molecular diagnostics (PCR amplification and sequencing of a partial region of the mitochondrial CO1 gene) showed that 6/10 positive bovines harboured hydatid cysts (No.=16) genetically identical (95.8-100%) to the Indian buffalo genotype G3. As far the location of the 16 cysts, 11 of them were found in the lungs of three animals, whereas 5 cysts were in the liver of three parasitized hosts. The occurrence of genotype G3 in 60% of parasitized bovines living in an area never studied before provides more definite evidence about the existence of the strain in this region, and proves that cattle have to be considered a non-accidental host.     

Strain characterization of Echinococcus granulosus protoscoleces of cattle origin using the in vitro vesicular development

The aim of this work was to characterize the strain of protoscoleces of E. granulosus of cattle origin using the in vitro vesicular development. The in vitro development of these samples was compared to samples of sheep origin determined previously by genetic analyses as common sheep strain (G1). There were similarities between sheep and cattle samples not only in the time of microcysts formation, but also in the development process. Vesiculated protoscoleces and protoscoleces with posterior bladders appeared during the first week of incubation. After 14 days of culture, a laminated layer appeared like a fine membrane in one of the extremes of the protoscoleces. In the sheep samples, microcysts were observed between 19 and 20 days. In the cattle samples, microcysts appeared between 20 and 23 days. The coincidence between the development times and physiological characteristics found in the present study may indicate that the parasites from cattle and sheep were of the same strain.     

Praziquantel adversely affects protoscoleces of Echinococcus granulosus in vitro

Praziquantel was shown to have adverse effects on protoscoleces of Echinococcus granulosus in vitro. Exposure to one dose of praziquantel at a concentration of 10 micrograms/ml caused protoscoleces to die within 12 to 15 days. Protoscolecidal effects were more marked when cultures were exposed to the drug continuously by the addition of multiple doses. On the basis of these observations, there is a need to reappraise the metacestocidal potential of praziquantel, particularly with regard to the development of new drug regimes employing multiple dose or sustained release schedules. Possible reasons for the reduced susceptibility of protoscoleces and juvenile worms to praziquantel observed in this study are discussed.     

Ultrastructural study of in vitro larval development of Echinococcus granulosus protoscoleces

Through ultrastructural study of the morphological forms developed in vitro during protoscolex culture, we describe larval E. granulosus histogenesis. The transformation of the spined microtriches in the protoscolex into truncated microtriches that develop within the hydatid cyst is discussed. The paper also describes the mitochondria location change that occurs during the evolution; the mitochondria pass from the most internal area of the distal cytoplasm along the cytoplasmic extensions into the cytoplasm of tegumental cells. The ultrastructures of both the vesiculated protoscolex and the posterior bladder demonstrate that each state corresponds to the initial step on one of the two paths of in vitro vesicular development.     

Echinococcus granulosus: cellular territories and morphological regions in mature protoscoleces

Basic aspects of the generation, structure and function of Echinococcus granulosus protoscoleces are unknown. We review the work done on the structure and ultrastructure of the E. granulosus protoscolex and provide new data together with a comprehensive view of this form of the parasite. The surface, as observed by scanning electron microscopy, tightly correlates with five cellular territories characterized in the interior using light and transmission electron microscopy as well as a histochemical technique. Three of these territories are surrounded by a basal lamina that is also present in the internal side of the tegument, suggesting a complex internal organization. These cellular territories correlate with the expression of specific genes and the regionalization of DNA synthesis in protoscoleces. Additionally, a proposal to explain movements of the body of this form of the parasite in relation to the neck or to the germinal layer of the hydatid cyst is provided.     

Echinococcus granulosus: first report of microcysts formation from protoscoleces of cattle origin using the in vitro vesicular culture technique

The aim of this work was the achievement of microcysts formation from protoscoleces of E. granulosus of cattle origin using the in vitro vesicular culture technique. Vesiculated protoscoleces and protoscoleces with posterior bladders appeared during the first week of incubation. After 14 days of culture, a laminated layer appeared like a fine membrane in one of the extremes of the protoscoleces. On day 20, some microcysts with a complete laminated layer were observed. By day 48, microcysts completely developed could be observed. This is the first study where microcysts formation was obtained using protoscoleces of E. granulosus of cattle origin.     

Morphological characteristics of Echinococcus granulosus derived from buffalo in Iran

Cystic echinococcosis is a significant parasitic disease in Iran, where a variety of animals act as intermediate hosts. In this study, 25 isolates of Echinococcus granulosus obtained from water buffalo from various parts of Iran were characterized on the basis of the morphology of the metacestode and the adult worm. The characteristics of protoscoleces from the different studied areas were nearly similar. They showed 2 rows of alternating large and small hooks and their shapes were smooth in outline. In contrast to the protoscoleces, the adult rostellar hooks showed a rough outline. The results showed that the total length, the blade lengths of the large and small hooks and the number of hooks are almost similar to those isolated from sheep but significantly different from those isolated from camels. The growth rates of adult E. granulosus (total worm length, segmentation and maturation) of buffalo origin, at 35 and 41 days post-infection of dogs, were nearly comparable to the common sheep strain. The form of the strobila and the morphology of the reproductive system were also similar to those of sheep origin. This suggests that the common sheep strain (G1) of E. granulosus may also use buffaloes as its intermediate host.     

An experimental in vitro model for evaluating drugs against protoscoleces of Echinococcus granulosus

Pharmacological studies carried out on protoscoleces in vitro to standardize conditions that would permit a preliminary estimate of the efficacy of drugs with potential activity against Echinococcus granulosus are reported. Media such as PBS and Hanks solution, maintenance temperature, different pH values and concentrations of various solvents have been tested to check the effects on protoscolex survival in tubes in vitro. Mebendazole has been used as the pharmacological standard reference. Changes in the viability of protoscoleces have been used to demonstrate pharmacological activity. Best conditions were obtained employing Hanks solution and propylene glycol at low concentrations. Mebendazole was not completely effective at the concentrations achievable in human therapy. Linear, reproducible results demonstrated that Hanks solution provides an ideal medium for pharmacological studies. Among tested solvents, propylene glycol and dimethyl sulphoxide showed no lethal activity at low concentrations. At concentrations similar to those normally obtained in human sera, mebendazole, as in vivo, demonstrated only partial lethality for protoscoleces. The present study represents a new experimental approach to chemotherapy of hydatid disease.     

Studies on the mechanism of lysis of Echinococcus granulosus protoscoleces incubated in normal serum.

Brood capsules were obtained from freshly collected cysts of equine and ovine strains of Echinococcus granulosus. Protoscoleces were freed from brood capsules either by mechanical disruption or pepsin-HCI digestion. Preparations of protoscoleces studied included: mechanically released protoscoleces without further treatment, or incubated either in HCI pH 2.0 or in evaginating solution (containing Na taurocholate) for 24 h; pepsin-HCI released protoscoleces without further treatment or incubated in evaginating solution for 24 h or 7 days. Half of each preparation of ovine protoscoleces was fixed in absolute methanol. All fresh preparations of protoscoleces lysed rapidly when incubated in normal human serum. Studies with a fluorescein isothiocyanate (FITC) labelled sheep anti-human C3 antiserum revealed the presence of C3 on the surface of lysing protoscoleces. Antibody could not be detected on the surface of any of the preparations of fresh or methanol-fixed protoscoleces using direct or indirect fluorescent antibody tests suggesting that the classical pathway of complement activation was not involved in the lytic process. Strong evidence for lysis by the alternate pathway of complement activation was the lysis of protoscoleces which had been treated with pepsin-HCI and lysis of protoscoleces in guinea-pig serum deficient in C4 component of complement.     

In vitro assessment of praziquantel and a novel nanomaterial against protoscoleces of Echinococcus granulosus

The present study describes the activity of a nanomaterial on protoscoleces of Echinococcus granulosus, which exhibited morphological changes and apoptosis. Apoptotic changes were deduced on the basis of effector caspase activation and nucleosomal laddering. Invaginated protoscoleces maintained in vitro became evaginated and had hooks, presumptive suckers and stalks. Degenerative changes of protoscoleces were evidenced after treatment with praziquantel and nano-combination. Protoscoleces treated with praziquantel had distinct attestation of necrosis and nano-combination-treated protoscoleces had signatures of apoptosis.     

Observations on Echinococcus granulosus of cattle origin in Switzerland

Switzerland is one of the few countries where high fertility rates have been reported in cattle hydatid cysts and where the cattle/dog cycle is the most important for the maintenance of Echinococcus granulosus. The developmental and morphological characteristics of E. granulosus of Swiss cattle origin were studied and compared with that of E. granulosus of domestic animal origin from Great Britain and Australia, countries where bovine hydatid cysts are usually sterile and cattle play little role in the life-cycle of the parasite. Adult E. granulosus of Swiss cattle origin differed markedly in its developmental characteristics compared to other isolates, particularly in its rate of maturation in dogs, producing eggs as early as 35 days post-infection. The morphology of E. granulosus of Swiss cattle origin was characteristic and it could be easily distinguished from other isolates of the parasite. Further, E. granulosus of Swiss cattle origin was found to closely resemble that occurring in cattle in South Africa where high fertility rates have also been reported in bovine hydatid cysts. It is concluded that a strain of E. granulosus exists which is adapted to cattle and that further studies are required to determine whether this strain warrants formal taxonomic status as the species E. ortleppi which was originally described for the parasite of South African cattle origin.     

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.     

Determination of the minimum time of praziquantel therapy required for the in vitro treatment of protoscoleces of Echinococcus granulosus

Ovine and equine protoscoleces of Echinococcus granulosus were cultured for 26 days with our without praziquantel and viability assessed, by eosin exclusion, for cultures in various drug concentrations (50, 250 and 500 micrograms/l) and periods of exposure (1, 3 or 7 days (d] before removing/'rescuing' to drug-free medium. Drug efficacy was proportional to drug concentration and to length of exposure. At higher drug concentrations shorter exposures were required to produce the effect of continuous drug treatment, 1d therapy at 500 micrograms/l killing 96% ovine protoscoleces by day 14 whereas 7d therapy at 50 micrograms/l was required to produce a similar effect. Equine protoscoleces appeared marginally less susceptible than those of ovine origin. The relevance of the results in the need for peri-operative prophylaxis against spilled protoscoleces in man is discussed.     

Comparative cytotoxicity of secondary hydatid cysts, protoscoleces, and in vitro developed microcysts of Echinococcus granulosus.

Infection with the metacestode of Echinococcus granulosus is characterized by a concomitant immunity. Survival of established and developing hydatid cysts in the intermediate host implies a mechanism to modulate its immunological reactions. In order to investigate this mechanism, secondary hydatid cysts were isolated from intraperitoneally infected laboratory white mice (strain NMRI) 12 months p.i. A number of hydatid cysts were freed from the surrounding host adventitial tissue. Monolayer cultures of non-stimulated peritoneal macrophages of NMRI mice were prepared and incubated in the presence of the hydatid cysts. By means of a trypan blue exclusion test and by measuring the incorporation of tritium labelled uridine, it was found that the presence of hydatid cysts reduced the viability of the macrophages in vitro. Toxic substances are probably secreted since the medium of cultured hydatid cysts also displayed cytotoxic activity. Hydatid cysts with adventitia, as well as culture medium of those cysts, were less toxic. When toxins, partially purified from hydatid cyst fluid, were previously incubated on a collagen coated surface, a reduced level of toxicity was found, suggesting that collagen of the host adventitia may play a role in controlling the liberation of toxins by the hydatid cyst. Virtually no toxicity was exerted by protoscoleces or by the medium of cultured protoscoleces, in contrast to in vitro vesiculated protoscoleces (so called microcysts). The results reveal a novel feature of hydatid cysts that may play a role in the survival of the parasite in the immunized host.     

Preliminary study of the presence of antibodies against excretory-secretory antigens from protoscoleces of Echinococcus granulosus in dogs with intestinal echinococcosis

The aim of the present study was to analyze the antibody response against excretory-secretory antigens (ES-Ag) from Echinococcus granulosus protoscoleces, using sera from dogs infected with E. granulosus and other helminths. ES-Ag were obtained from the first 50 h maintenance of protoscoleces in vitro. Immunochemical characterization was performed by immunoblotting with sera from dogs naturally infected with E. granulosus (n = 12), sera from dogs infected with helminths other than E. granulosus (n = 30), and helminth-free dog sera (n = 20). These findings were compared to those obtained from a somatic extract of protoscoleces (S-Ag). ES-Ag only showed four cross-reacting proteins of 65, 61, 54, and 45-46 kDa. Antigens with apparent masses of 89 and 50 kDa in ES-Ag and of 130 and 67 kDa in S-Ag were identified by sera of dogs infected with E. granulosus only, whereas a protein of 41-43 kDa was recognised by the majority of the sera from dogs with non-echinococcal infection. Employing ELISA to study the same sera, S-Ag revealed higher immunoreactivity than ES-Ag, but also showed higher cross-reactivity levels when sera from dogs with non-echinococcal infection were assayed in immunoblotting.     

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.