Echinococcus granulosus: in vitro maintenance of whole cysts and the assessment of the effects of albendazole sulphoxide and praziquantel on the germinal layer

SummaryPraziquantel (500 mg/kg) administered orally to BALB/c mice with secondary equine E. granulosus daily for 21, 30 or 30 + 30 days without the drug resulted in the majority of cysts, using bench criteria of turgidity and eosin exclusion, being assessed as ‘alive’. Ultrastructural examination of 54 of these ‘alive’ cysts did not support this conclusion. They all showed increased vesiculation of the germinal layer leading, in many, to the loss of its integrity. Increased mitochondrial numbers occurred frequently. The longer drug treatments appeared to have greater effects on the germinal layer of ‘alive’ cysts and there was no detectable re-establishment of structural organization within 30 days after drug withdrawal. Subjectively, there was no substantial difference between cysts from 4-month and 9-month infections or between affected peritoneal and hepatic cysts. Tissue from collapsed cysts was necrotic. Peak serum levels of praziquantel (6430–6136 μg/l) occurred 5–10 min after drug administration (500 mg/kg) and dropped rapidly to less than 10 μg/l at 3 h. In an in vitro study at praziquantel concentrations of 1000 and 5000 μg/l over a 10-day period, most cysts were judged ‘alive’ by bench criteria but showed ultrastructurally a time- and concentration-dependent loss of integrity identical to that seen in vivo. Turgidity and eosin exclusion therefore underestimate the effect of praziquantel and the results indicate that in vitro experiments can fulfil a legitimate preliminary role in a hydatid chemotherapy programme.

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Evidence of in vitro germinal layer development in Echinococcus granulosus cysts

Zeitschrift für Parasitenkunde Parasitology Research ◽

10.1007/bf00531634 ◽

1988 ◽

Vol 74 (6) ◽

pp. 558-562 ◽

Cited By ~ 12

Author(s):

F. Rodriguez-Caabeiro ◽

N. Casado

Keyword(s):

Echinococcus Granulosus ◽

Germinal Layer ◽

Layer Development

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Echinococcus granulosus (Cestoda): uptake of L-amino acids by secondary hydatid cysts

Parasitology ◽

10.1017/s0031182000081725 ◽

1988 ◽

Vol 96 (1) ◽

pp. 145-156 ◽

Cited By ~ 10

Author(s):

S. A. Jeffs ◽

C. Arme

Keyword(s):

Amino Acids ◽

Experimental Method ◽

Echinococcus Granulosus ◽

Cyst Wall ◽

Hydatid Cysts ◽

Germinal Layer ◽

Uptake Studies ◽

Laminated Layer

SUMMARYThe uptake of cycloleucine, L-proline, L-alanine and L-threonine by secondary hydatid cysts of Echinococcus granulosus (U.K. horse strain 3–8 mm in diameter, derived from Balb/c mice infected 300–400 days previously) occurs by passive diffusion into the cyst wall (laminated layer plus germinal layer) and by mediated mechanisms into the fluid-filled interior. The maximal concentrations of these compounds are achieved after incubation for 2 h in vitro and approach those in vivo. Kt and Vmax values describing the uptake of these compounds are given. The flux rates for these compounds are extremely slow compared to those obtained with the protoscolex. A rationale for standardizing the experimental method for uptake studies with hydatid cysts is described.

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Comparative analysis of miRNA expressions in different developmental stages of Echinococcus granulosus in mono-phasic and di-phasic culture systems

Infectious Disorders - Drug Targets ◽

10.2174/1871526520999201103192518 ◽

2020 ◽

Vol 20 ◽

Author(s):

Seifollah Mortezaei ◽

Ali Afgar ◽

Balal Sadeghi ◽

Mohammad Ali Mohammadi ◽

Seyed Mohammad Mousavi ◽

...

Keyword(s):

Echinococcus Granulosus ◽

Drug Targets ◽

Developmental Stages ◽

Ex Vivo ◽

Basic Research ◽

Experimental Investigations ◽

Intermediate Hosts ◽

Germinal Layer ◽

Probable Role

Background:: The dog tapeworm, Echinococcus granulosus, is a zoonotic parasite affecting human and livestock across the globe. Basic research on the molecular biology and genetics of E. granulosus improves our understanding of the biology and potential drug targets in various developmental stages of E. granulosus in both definitive and intermediate hosts. There has been increasing interest in identification of microRNAs in parasitic organisms. The purpose of the current study was to compare the activity of a selected profile of miRNAs in different developmental stages of E. granulosus. Methods:: Different developmental stages of the parasite were obtained from ex vivo as well as in vitro cultured E. granu-losus. MicroRNAs were extracted from the ex vivo germinal layer and invaginated protoscoleces as well as the in vitro gen-erated microcysts, evaginated protoscoleces and strobilated worms. Expression of the selected miRNAs was evaluated by RT-qPCR for each stage. Results:: Four out of five miRNAs were present and active in different developmental stages of E. granulosus. A significant over-expression of miR-61 was observed in germinal layer and during the protoscolex transformation into the microcysts, however miR-10 was more expressed in the mature strobilated forms than the other stages. Let-7 and miR-3489 showed a high expression in germinal layer. Conclusion:: Differential expression of four miRNAs among different in vitro and ex vivo developmental stages of E. granu-losus was documented in the present study. Further experimental investigations are required to elucidate the probable role of the miRNAs in bi-directional differentiation of protoscoleces either into the strobilated worm or to a secondary hydatid cyst and the potential of these miRNAs as drug targets.

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In Vitro and In Vivo Efficacy of DNA Damage Repair Inhibitor Veliparib in Combination with Artesunate against Echinococcus granulosus

Disease Markers ◽

10.1155/2020/8259820 ◽

2020 ◽

Vol 2020 ◽

pp. 1-11

Author(s):

Y. F. Li ◽

L. M. Wen ◽

J. Zhao ◽

G. D. Lv ◽

S. Lu ◽

...

Keyword(s):

Dna Damage ◽

Echinococcus Granulosus ◽

Structural Integrity ◽

Dna Damage Repair ◽

Combination Group ◽

Germinal Layer ◽

Drug Induced ◽

Damage Repair

Cystic echinococcosis (CE), caused by the cestode Echinococcus granulosus, is a worldwide chronic zoonosis. Albendazole (ABZ) and mebendazole are effective against CE, but a high dosage in a long-term period is usually required. In this study, we evaluate the effects of DNA damage repair inhibitor (i.e., Veliparib) in combination with artesunate (AS) on hydatid cysts. For the in vitro assay, protoscoleces of E. granulosus (E.g PSCs) were incubated with low AS (AS-L, 65 μM), moderate AS (AS-M, 130 μM), and high AS (AS-H, 325 μM), AS-L/M/H+Veliparib (10 μM), and ABZ (25 μM), respectively. The AS-H+Veliparib group showed the maximal protoscolicidal effects. Ultrastructural change revealed that germinal layer (GL) cells were reduced, and lipid droplets appeared. AS could induce DNA injuries in PSCs. The 8-OHdG was expressed in the PSCs and GL of the cysts in mice, especially in the presence of Veliparib. The most severe DNA damages were observed in the AS-H+Veliparib group. Meanwhile, the expression of ribosomal protein S9 (RPS9) gene in the AS-H+Veliparib group was significantly lower than that in the AS-H group. The in vivo chemotherapeutic effects of AS-L (50 mg/kg), AS-H (200 mg/kg), and AS-H+Veliparib (25 mg/kg) were assessed in experimentally infected mice. Upon 6 weeks of oral administration, ultrasonography was used to monitor the volume change of vesicles. Maximum potentiation was seen on day 15 with values (versus AS) of 34 (P<0.05) for AS-H + Veliparib. It led to the reduction of cyst weight (55.40%) compared with the model group (P<0.01), which was better than AS alone (52.84%) and ABZ-treated mice (55.35%). Analysis of cysts collected from AS-H+Veliparib-treated mice by transmission electron microscopy revealed a drug-induced structural destruction. The structural integrity of the germinal layer was lost, and the majority of the microtriches disappeared. In conclusion, our study demonstrates that AS or AS in combination with Veliparib is effective for treating CE, especially the combination group. On this basis, AS represented promising drug candidates in anti-CE chemotherapy.

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In vitro immunoregulatory activity and anti-inflammatory effect of Echinococcus granulosus laminated layer

Acta Tropica ◽

10.1016/j.actatropica.2021.105886 ◽

2021 ◽

Vol 218 ◽

pp. 105886

Author(s):

Sara Benazzouz ◽

Manel Amri ◽

Junhua Wang ◽

Samia Bouaziz ◽

Fahima Ameur ◽

...

Keyword(s):

Echinococcus Granulosus ◽

Anti Inflammatory ◽

Laminated Layer ◽

Inflammatory Effect

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Comparative proteomic profiling of newly acquired, virulent and attenuated Neoparamoeba perurans proteins associated with amoebic gill disease

Scientific Reports ◽

10.1038/s41598-021-85988-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Kerrie Ní Dhufaigh ◽

Eugene Dillon ◽

Natasha Botwright ◽

Anita Talbot ◽

Ian O’Connor ◽

...

Keyword(s):

Bacterial Community Composition ◽

Illumina Miseq ◽

Proteomic Profiling ◽

Farmed Fish ◽

Liquid Chromatography Tandem Mass ◽

Proteome Database ◽

Gill Disease ◽

The Impact ◽

In Vitro Maintenance

AbstractThe causative agent of amoebic gill disease, Neoparamoeba perurans is reported to lose virulence during prolonged in vitro maintenance. In this study, the impact of prolonged culture on N. perurans virulence and its proteome was investigated. Two isolates, attenuated and virulent, had their virulence assessed in an experimental trial using Atlantic salmon smolts and their bacterial community composition was evaluated by 16S rRNA Illumina MiSeq sequencing. Soluble proteins were isolated from three isolates: a newly acquired, virulent and attenuated N. perurans culture. Proteins were analysed using two-dimensional electrophoresis coupled with liquid chromatography tandem mass spectrometry (LC–MS/MS). The challenge trial using naïve smolts confirmed a loss in virulence in the attenuated N. perurans culture. A greater diversity of bacterial communities was found in the microbiome of the virulent isolate in contrast to a reduction in microbial community richness in the attenuated microbiome. A collated proteome database of N. perurans, Amoebozoa and four bacterial genera resulted in 24 proteins differentially expressed between the three cultures. The present LC–MS/MS results indicate protein synthesis, oxidative stress and immunomodulation are upregulated in a newly acquired N. perurans culture and future studies may exploit these protein identifications for therapeutic purposes in infected farmed fish.

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In vitro scolicidal effects of Sideritis perfoliata extract against Echinococcus granulosus

International Journal of Clinical Practice ◽

10.1111/ijcp.14498 ◽

2021 ◽

Author(s):

Tuncay Çelik ◽

Muhittin Önderci ◽

Mustafa Pehlivan ◽

Önder Yumrutaş ◽

Fatih Üçkardeş

Keyword(s):

Echinococcus Granulosus

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Characterization of excretory–secretory products from protoscoleces of Echinococcus granulosus and evaluation of their potential for immunodiagnosis of human cystic echinococcosis

Parasitology ◽

10.1017/s0031182004005670 ◽

2004 ◽

Vol 129 (3) ◽

pp. 371-378 ◽

Cited By ~ 20

Author(s):

D. CARMENA ◽

J. MARTÍNEZ ◽

A. BENITO ◽

J. A. GUISANTES

Keyword(s):

Echinococcus Granulosus ◽

Cystic Echinococcosis ◽

Secretory Protein ◽

Major Protein ◽

Healthy Donors ◽

Secretory Products ◽

Protein Components ◽

Human Cystic Echinococcosis

This study describes, for the first time, the characterization of excretory–secretory antigens (ES-Ag) from Echinococcus granulosus protoscoleces, evaluating their usefulness in the immunodiagnosis of human cystic echinococcosis. ES-Ag were obtained from the first 50 h maintenance of protoscoleces in vitro. This preparation contained over 20 major protein components which could be distinguished by 1-dimensional SDS–PAGE with apparent masses between 9 and 300 kDa. The culture of of protoscoleces from liver produced a greater variety of excretory–secretory protein components than those from lung. Determination of enzymatic activities of secreted proteins revealed the presence of phosphatases, lipases and glucosidases, but no proteases. These findings were compared to those obtained from somatic extracts of protoscoleces and hydatid cyst fluid products. Immunochemical characterization was performed by immunoblotting with sera from individuals infected by cystic echinococcosis (n=15), non-hydatidic parasitoses (n=19), various liver diseases (n=24), lung neoplasia (n=16), and healthy donors (n=18). Antigens with apparent masses of 89, 74, 47/50, 32, and 20 kDa showed specificity for immunodiagnosis of human hydatidosis. The 89 and 74 kDa components corresponded to antigens not yet described in E. granulosus, whereas proteins of 41–43 kDa and 91–95 kDa were recognized by the majority of the non-hydatid sera studied.

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Anti-echinococcal effect of verapamil involving the regulation of the calcium/calmodulin-dependent protein kinase II response in vitro and in a murine infection model

Parasites & Vectors ◽

10.1186/s13071-021-04618-4 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Hai-Jun Gao ◽

Xu-Dong Sun ◽

Yan-Ping Luo ◽

Hua-Sheng Pang ◽

Xing-Ming Ma ◽

...

Keyword(s):

Protein Kinase ◽

Mrna Expression ◽

Echinococcus Granulosus ◽

Calcium Content ◽

Mrna Levels ◽

Dependent Protein Kinase ◽

Calcium Calmodulin Dependent ◽

Dependent Protein

Abstract Background Echinococcosis, which is caused by the larvae of cestodes of the genus Echinococcus, is a parasitic zoonosis that poses a serious threat to the health of humans and animals globally. Albendazole is the drug of choice for the treatment of echinococcosis, but it is difficult to meet clinical goals with this chemotherapy due to its low cure rate and associated side effects after its long-term use. Hence, novel anti-parasitic targets and effective treatment alternatives are urgently needed. A previous study showed that verapamil (Vepm) can suppress the growth of Echinococcus granulosus larvae; however, the mechanism of this effect remains unclear. The aim of the present study was to gain insight into the anti-echinococcal effect of Vepm on Echinococcus with a particular focus on the regulatory effect of Vepm on calcium/calmodulin-dependent protein kinase II (Ca2+/CaM-CaMKII) in infected mice. Methods The anti-echinococcal effects of Vepm on Echinococcus granulosus protoscoleces (PSC) in vitro and Echinococcus multilocularis metacestodes in infected mice were assessed. The morphological alterations in Echinococcus spp. induced by Vepm were observed by scanning electron microscopy (SEM), and the changes in calcium content in both the parasite and mouse serum and liver were measured by SEM-energy dispersive spectrometry, inductively coupled plasma mass spectrometry and alizarin red staining. Additionally, the changes in the protein and mRNA levels of CaM and CaMKII in infected mice, and in the mRNA levels of CaMKII in E. granulosus PSC, were evaluated after treatment with Vepm by immunohistochemistry and/or real-time quantitative polymerase chain reaction. Results In vitro, E. granulosus PSC could be killed by Vepm at a concentration of 0.5 μg/ml or higher within 8 days. Under these conditions, the ultrastructure of PSC was damaged, and this damage was accompanied by obvious calcium loss and downregulation of CaMKII mRNA expression. In vivo, the weight and the calcium content of E. multilocularis metacestodes from mice were reduced after treatment with 40 mg/kg Vepm, and an elevation of the calcium content in the sera and livers of infected mice was observed. In addition, downregulation of CaM and CaMKII protein and mRNA expression in the livers of mice infected with E. multilocularis metacestodes was found after treatment with Vepm. Conclusions Vepm exerted a parasiticidal effect against Echinococcus both in vitro and in vivo through downregulating the expression of Ca2+/CaM-CaMKII, which was over-activated by parasitic infection. The results suggest that Ca2+/CaM-CaMKII may be a novel drug target, and that Vepm is a potential anti-echinococcal drug for the future control of echinococcosis.

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