Apoptosis as a possible mechanism of infertility in Echinococcus granulosus hydatid cysts

Echinococcus granulosus is a parasitic cestode causing hydatidosis in intermediate hosts (human and herbivorous). Most symptoms of the disease occur by the pressure exerted on viscera by cysts that are formed upon ingestion of the parasite eggs excreted by definitive hosts (canines). Protoscoleces, the developmental form of the parasite infective to definitive hosts, are formed in the germinal nucleated layer of fertile hydatid cysts. For unknown reasons, some cysts are unable to produce protoscoleces (infertile hydatid cysts). In this study, analysis of DNA fragmentation using TUNEL and agarose gel electrophoresis showed higher levels of apoptosis in infertile cysts as compared to fertile cysts. Additionally, caspase 3 was detected both in fertile and infertile cysts; the activity of this enzyme was found to be higher in infertile cysts. We conclude that apoptosis may be involved in hydatid cyst infertility. This is the first report on the presence of programmed cell death in E. granulosus.     

Mechanism of cholesterol absorption by the hydatid cysts of Echinococcus granulosus (Cestoda)

• 1.1. The mechanism of cholesterol absorption by hydatid cysts of Echinococcus granulosus was studied in function of time of incubation, size of cysts, temperature of incubation, concentration of cholesterol substrate and presence of metabolic inhibitor.
• 2.2. The results indicated that the uptake of cholesterol continuously increased with time of incubation, that small cysts absorbed cholesterol better than large ones, that the optimum temperature for uptake was 25°C and that uptake increased with increase in concentration of substrate.
• 3.3. The addition of the metabolic inhibitor 2, 4-dinitrophenol did not affect the process of absorption thus eliminating the participation of the mechanisms of active transport or facilitated diffusion.
• 4.4. It was suggested that mechanisms of simple diffusion and possibly exchange diffusion are operating in cholesterol absorption by hydatid cysts.

     

DNA damage, RAD9 and fertility/infertility of Echinococcus granulosus hydatid cysts

Hydatidosis, caused by the larval stage of the platyhelminth parasite Echinococcus granulosus, affects human and animal health. Hydatid fertile cysts are formed in intermediate hosts (human and herbivores) producing protoscoleces, the infective form to canines, at their germinal layers. Infertile cysts are also formed, but they are unable to produce protoscoleces. The molecular mechanisms involved in hydatid cysts fertility/infertility are unknown. Nevertheless, previous work from our laboratory has suggested that apoptosis is involved in hydatid cyst infertility and death. On the other hand, fertile hydatid cysts can resist oxidative damage due to reactive oxygen and nitrogen species. On these foundations, we have postulated that when oxidative damage of DNA in the germinal layers exceeds the capability of DNA repair mechanisms, apoptosis is triggered and hydatid cysts infertility occurs. We describe a much higher percentage of nuclei with oxidative DNA damage in dead protoscoleces and in the germinal layer of infertile cysts than in fertile cysts, suggesting that DNA repair mechanisms are active in fertile cysts. rad9, a conserved gene, plays a key role in cell cycle checkpoint modulation and DNA repair. We found that RAD9 of E. granulosus (EgRAD9) is expressed at the mRNA and protein levels. As it was found in other eukaryotes, EgRAD9 is hyperphosphorylated in response to DNA damage. Our results suggest that molecules involved in DNA repair in the germinal layer of fertile hydatid cysts and in protoscoleces, such as EgRAD9, may allow preserving the fertility of hydatid cysts in the presence of ROS and RNS.     

Proteomic characterisation of Echinococcus granulosus hydatid cyst fluid from sheep, cattle and humans

The hydatid cyst fluid (HCF) of Echinococcus granulosus is a complex biological mixture containing a wide range of proteins of both parasite and host origin. Using a combination of in- and off-gel protein fractionation techniques and tandem mass spectrometry 130 HCF proteins were identified from fertile cysts of sheep and human origin and infertile cysts from cattle. Forty-eight proteins were of parasite origin including Antigen 5 and Antigen B--the most abundant parasite proteins, thioredoxin, low-density lipoprotein receptors, cyclophilin and ferritin. Across the three host species the identified HCF proteins were broadly similar although, based on spectral counts, three proteins, including an antigen B isoform, were more abundant in sheep HCF compared with the fluids of cattle and human origin. Eighty-two host proteins were identified in HCF from the three species. Host plasma proteins were the most abundant, although approximately thirty of the host proteins that were identified are not considered constituents of plasma. The identification of parasite heat shock proteins and annexin A13 exclusively in infertile cysts, along with an increased spectral count for cathepsin B, supports the hypothesis of increased cellular stress and apoptosis as the cause of their infertility.     

The development of brood capsules and protoscolices in secondary hydatid cysts of Echinococcus granulosus. A histological study.

Using the Mongolian jird (Meriones unguiculatus) as a laboratory host for the cystic stage of the British horse strain of Echinococcus granulosus, the mode of development of brood capsules and protoscolices was studied histologically. The successive stages in development in secondary hydatid cysts are described and shown to correspond closely with previous observations on primary hydatid cysts. The usefulness of this experimental model for future ultrastructural studies is emphasized.     

The immunohomeostatic role of hydatid cyst toxins (Echinococcus granulosus).

It is obvious that during the evolution of a host-parasite relationship, in which one animal is needing the other, while the latter is trying to expel or destroy the former, both partners tend to reach an equilibrium. In many cases they are still far off, but in hydatidosis (Echinococcus granulosus) it would appear that a lot has been achieved. As the near equilibrium is mainly related to the state of the immunological relationship, it is in fact a case of immunohomeostasis. The immunological reactions of the host involve both humoral and cellular phenomena. The nature of the humoral response varies greatly according to host species, parasite strain and several factors of the infection and analysis procedures. The cell-mediated defense mechanism involves various types of cells, but activated macrophages appear to be most important. With regards to the modes of immunoevasion, attention is paid to the barrier role of the laminated layer, the inactivation of complement and the interference with the activities of host immune cells. The role of toxic substances released by protoscoleces and cysts is especially emphasized. The toxins appear extremely vulnerable to host macrophages and play a very important role in the establishment of a hydatid cyst. However, they have only a local influence and do not jeopardize the development of concomitant immunity. Observations in experimental secondary echinococcosis led to the conclusion that the establishment of a hydatid cyst depends on the speed of the parasite's toxic effect versus the host immunological reaction. Survival of the parasite is based on the continued balancing effects of both.     

Echinococcus granulosus: DNA extraction from germinal layers allows strain determination in fertile and nonfertile hydatid cysts

A method for the isolation of Echinococcus granulosus DNA from germinal layers of hydatid cysts is described. The method includes a hexadecyltrimethylammonium bromide/chloroform extraction and an adsorption to diatomaceous earth suspension. DNA suitable for polymerase chain reaction was obtained and used for parasite strain determination by mitochondrial cytochrome c oxidase I gene sequencing. Fertile and nonfertile cyst isolates from sheep, cattle, pigs, and humans were characterized. Hitherto, no direct parasite strain characterization has been made on nonfertile hydatid cysts, whereas here we report that nonfertile hydatid cysts were produced by sheep strain (G1 genotype) in sheep, cattle, and humans and by pig strain (G7 genotype) in pigs.     

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.     

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.     

Echinococcus granulosus: development of resistance to albendazole in an animal model

Gerbils with well developed peritoneal cysts of Echinococcus granulosus were randomized to albendazole 50 mg/kg/day or untreated control. Treated animals had less disease at post mortem after 3 months of treatment. Cysts were then taken from both albendazole-treated and control animals and cultured in vitro either with or without albendazole sulphoxide (Alb Sx) 500 micrograms/L for 14 days. Viability of cysts was then established by implantation of whole cysts into gerbils. Whilst naive cysts were affected by Alb Sx (only 2 cysts developed/gerbil) cysts from animals treated with albendazole were not sensitive to further therapy (6.4 cysts/gerbil).     

青海天峻地区犬细粒棘球绦虫感染情况初步调查

采用氢溴酸槟榔碱下泻法对天峻地区的犬进行了细粒棘球绦虫感染情况调查。共检查94只犬,检出阳性4只,感染率为4.26%,平均感染强度24.25条。     

Lipids in the laminated layer of liver, lung and daughter hydatid cysts of equine Echinococcus granulosus (Cestoda)

Lipids extracted from the laminated layers of horse liver and lung hydatids, including a daughter liver cyst, were analysed using TLC. No differences in lipid composition was detected in 11 liver cysts, whether from the same or different livers, and di- and triacylglycerols, cholesterol, wax and steryl esters, oleic acid, sphingomyelin, phosphatidyl choline, phosphatidyl inositol and ceramide hexosides were detected. The daughter cyst differed from its "parent" cyst in lacking diacylglycerols and wax and steryl esters. The lung cyst differed from the liver cysts in that cholesterol, wax and steryl esters and diacylglycerols were not detected.     

Rapid recovery of Echinococcus granulosus following 'successful' albendazole therapy in a gerbil model

Peritoneal Echinococcus granulosus in gerbils was treated with albendazole. Both early and late infections were studied; response to albendazole therapy and the ability of the parasite to recover after treatment was found to depend on dose and length of therapy. Even after treatment at 50 mg/kg for 2 months late infections retained the ability to recover over 3 months.