Cryopreservation of Echinococcus multilocularis metacestodes and subsequent proliferation in rodents (Meriones)

Four isolates of larval Echinococcus multilocularis originating from Switzerland (CH/1, CH/6 and CH/22) and Alaska (A/1) were used to prepare crude homogenate or small tissue fragments (STF) in Eagle's Minimal Essential Medium with Earle's salts (EMEM/A), or 0.2 g tissue blocks (TB) which were suspended in the same medium. After addition of dimethylsulfoxide or glycerol in final concentrations of 5% and 10% (v/v), respectively, aliquots of 1.0 ml, containing either 0.1 ml crude homogenate or STF, or one block of 0.2 g, were kept in cryotubes for 30 min at +2-4 degrees C (precooling phase), cooled subsequently to lower temperatures following a two-step or three-step schedule and finally plunged into liquid nitrogen (-196 degrees C). After storage for one week the samples were rapidly thawed at +37 degrees C for approximately 3 min, washed in fresh EMEM/A (37 degrees C) and transferred into the peritoneal cavity of Meriones for viability testing. As judged by histological examinations and metacestode weights of each 24 Meriones infected with cryopreserved homogenate, STF or TB, respectively, 46%, 87% or 100% contained viable, proliferating parasites. The best proliferation rate occurred when 10% glycerol was used as cryoprotectant and after precooling a three-step freezing schedule was employed (30 min at -28 degrees C, 30 min at -80 degrees C, transfer to liquid nitrogen). Cooling rates were determined as 0.7, 1.0 and 1.7 degrees C min-1 for the precooling phase, step 1 and step 2, respectively, and estimated as 65 degrees C min-1 for step 3. These results demonstrate that metacestodes of E. multilocularis can be successfully maintained by cryopreservation without losing their proliferative capacity in the intermediate host.     

Recent advances in the in vitro cultivation and genetic manipulation of Echinococcus multilocularis metacestodes and germinal cells

In order to elucidate host-parasite interactions and infection strategies of helminths at the molecular level, the availability of suitable in vitro cultivation systems for this group of parasites is of vital importance. One of the few helminth systems for which in vitro cultivation has been relatively successfully carried out in the past is the larval stage of the fox-tapeworm Echinococcus multilocularis, the causative agent of alveolar echinococcosis. Respective ‘first generation’ cultivation systems relied on the co-incubation of larval tissue, isolated from laboratory rodents, with host feeder cells. Although these techniques have been very successful in producing metacestode material for drug screening assays or the establishment of cDNA libraries, the continuous presence of host cells prevented detailed studies on the influence of defined host factors on larval growth. To facilitate such investigations, we have recently introduced the first truly axenic system for long-term in vitro maintenance of metacestode vesicles and used it to establish a technique for parasite cell cultivation. The resulting culture system, which allows the complete in vitro regeneration of metacestode vesicles from germinal cells, is a highly useful tool to study the cellular and molecular basis of a variety of developmental processes that occur during the infection of the mammalian host. Furthermore, it provides a solid basis for establishing transgenic techniques in cestodes for the first time. We consider it an appropriate time point to discuss the characteristics of these ‘second generation’ cultivation systems in comparison with former techniques, to present our first successful attempts to introduce foreign DNA into Echinococcus cells, and to share our ideas on how a fully transgenic Echinococcus strain can be generated in the near future.     

Effect of parenterally injected benzimidazole compounds on Echinococcus multilocularis and Taenia crassiceps metacestodes in laboratory animals.

In mice infected with metacestodes of Taenia crassiceps, the following compounds were at least partially effective when injected intraperitoneally at the dosage indicated: cambendazole (500 mg/kg), mebendazole (6.25 mg/kg), oxibendazole (500 mg/kg), 5-benzamido-2(4-thiazolyl)benzimidazole (500 mg/kg), 2-carboethoxyamino benzimidazole (125 mg/kg), and 2-carbomethoxyamino benzimidazole (500 mg/kg). The following were inactive at the dosage indicated: parbendazole (500 mg/kg), thiabendazole (1,000 mg/kg), and fenbendazole (1,000 mg/kg). Mebendazole, which showed some activity at 6.25 mg/kg, was highly active as a single intraperitoneal dose at 25 mg/kg. When injected subcutaneously, mebendazole was much less active than when given intraperitoneally. In mice infected with metacestodes of Echinococcus multilocularis, intraperitoneal injection of mebendazole at 75 to 150 mg/kg, daily for 3 days, was highly effective (95 to 100% reduction in cyst mass). In contrast, oral administration at 1,000 mg/kg, daily for 3 days, was only partially effective. The drug was also effective when given intraperitoneally to infected cotton rats. A water-soluble benzimidazole, carboxymethyleneamino cambendazole, was approximately 50% effective in mice when injected daily for 3 days at a dosage of 75 or 150 mg/kg. The results suggest that, in metacestode infections of medical importance, it may be possible to kill the parasite by delivering a drug to its immediate vicinity, and so to reduce the required dosage with respect to the host.     

Early-phase migration dynamics of Echinococcus multilocularis in two mouse strains showing different infection susceptibilities

The early-phase migration dynamics of Echinococcus multilocularis in the intermediate hosts remain largely unknown. We compared the parasite burden in the intestine, liver and faeces of DBA/2 and C57BL/6 mouse strains using parasite-specific quantitative PCR. Our results indicated that the parasites invaded mainly from the middle segments of the small intestine and completed migration to the liver within 24 h p.i. C57BL/6 mice had lower parasite DNA burdens in the intestine and liver but higher in the faeces than DBA/2 mice, suggesting that parasite invasion of the intestine may be a critical stage regulating susceptibility to E. multilocularis infection in mice.     

Glycosphingolipids with Gal beta 1----6Gal sequences in metacestodes of the parasite Echinococcus multilocularis.

Neutral glycosphingolipids of the metacestodes of Echinococcus multilocularis, an animal and human parasite, were resolved by high performance thin layer chromatography into 12 fractions. Nine of these fractions were permethylated, analyzed by electron impact-mass spectrometry, and submitted to methylation analysis by gas chromatography-mass spectrometry. Native fractions were analyzed by liquid secondary ion-mass spectrometry and degraded sequentially by exoglycosidases. In addition to a previously described galactosylceramide, a di-, a tri-, and a tetragalactosyl-ceramide having Gal beta 1-6Gal internal linkages were characterized. This type of carbohydrate chain has been described in glycolipids of a marine mollusk, Turbo cornutus (Matsubara, T., and Hayashi, A. (1981) J. Biochem. (Tokyo), 89, 645-650). In addition two novel fucolipids were found with the following structures: Fuc alpha 1-3Gal beta 1-6Gal-Cer and Gal beta 1-6(Fuc alpha 1-3)Gal beta 1-6Gal-Cer. Ceramides contained sphinganine and either nonhydroxy fatty acids with 16, 18, 26, and 28 carbon atoms, or hydroxy fatty acids, with 16 and 18 carbon atoms. Di-, tri-, and tetragalactosylceramides containing the Gal beta 1-6Gal disaccharide were found to be immunogenic in humans.     

Life cycles of Echinococcus multilocularis in relation to human infection

The cycle of Echinococcus multilocularus in natural and synanthropic hosts was investigated during 10 yr in an endemic focus of alveolar hydatid disease in the Massif Central of France. The natural cycle, involving red foxes, Vulpes vulpes, and voles, Arvicola terrestris, existed immediately surrounding a village in which human cases of alveolar hydatid disease occurred. Both foxes and free-ranging dogs could serve as the source of infection for the human population.     

Echinococcus multilocularis: susceptibility and responses to infection in inbred mice

Kroeze W. K. and Tanner C. E. 1987. Echinococcus multilocularis: susceptibility and responses to infection in inbred mice. International Journal for Parasitology17: 873–883. Of six strains of mice examined, C57L/J mice were the most susceptible to intraperitoneal infections with Echinococcus multilocularis. Five of the six strains developed splenomegaly during the infection. Changes in leukocyte levels in infected mice were most pronounced in the C57L/J and BALB/cJ strains. Two of the six strains of mice, C57BL/6J and C57BL/6J (bg^J), showed low specific IgG responses to E. multilocularis when measured using an ELISA. Many of the responses observed were directly correlated with the parasite burden in infected animals. It is concluded that susceptibility or resistance to E. multilocularis in mice is probably controlled by non-H-2 gene(s); additionally, hematological and immunological responses to infection, although correlated to parasite burden, varied among strains of mice, suggesting some degree of host genetic control of these responses.     

Effect of mouse strain variations and cortisone treatment on the establishment and growth of primary Echinococcus multilocularis hydatid cysts

The main purpose of this study was to determine the effects of cortisone on the number and size of primary Echinococcus multilocularis cysts developing in a moderately resistant strain of mice, i.e., C3H/HeJ. Computerized image analysis was used to measure the surface area occupied by hydatid cysts 10 wk after inoculation of the mice with E. multilocularis eggs. Our second objective was to compare the infectivity of primary E. multilocularis hydatid cysts in C57BL/6J-Ay/a (lethal yellow) mice with that in C57BL/6J-a/a (non-agouti black) mice. The data obtained show no difference between the C3H/HeJ and C57BL/6J-a/a strains of mice; yet, the image analysis method was able to detect a slight increase in the total cyst size within the Ay/a mutant of the C57BL/6J strain. Treatment of C3H/HeJ mice with cortisone drastically increased both the number of cysts and the average size of each cyst when the treatment occurred early in the infection.     

Observations on Echinococcus multilocularis in the definitive host

Six dogs were found to be susceptible to experimental infections with a European isolate of Echinococcus multilocularis from southern Germany. Two cats were only poorly susceptible. Adult worms were not evenly distributed throughout the small intestine and the majority of parasites were found in the posterior region. The mode of attachment of E. multilocularis in the dog was similar to that for E. granulosus with the adult worm extending its rostellum deep within a crypt of Lieberkühn. In cats only few worms were found to have penetrated deeply between the villi. E. multilocularis was found to possess a modified group of rostellar tegumental cells, morphologically and functionally identical to those described in E. granulosus and previously referred to as the "rostellar gland". By studying development in vivo and in vitro, the time required for the production of shelled eggs was demonstrated to be only 28 days. Concurrent experimental infections in dogs with E. multilocularis and E. granulosus revealed that both species will develop together in the same host. Their development was not retarded in any way by the presence of the other and both species were able to coexist in the same area of the intestine.     

High molecular mass glycans are major structural elements associated with the laminated layer of in vitro cultivated Echinococcus multilocularis metacestodes

The laminated layer of the larval stage (metacestode) of the cestode parasite Echinococcus multilocularis is composed largely of carbohydrates, which form a tight microfibrillar meshwork around the entire metacestode. Since this laminated layer is the only parasite structure which is in constant contact with host immune and non-immune cells, and appears largely resistant to physiological and immunological reactions of the host, it most likely carries out important functions with regard to host-parasite interactions. In infected hosts, the metacestode is usually concentrically covered by host connective tissue cells and large amounts of collagen, causing a dense scar-like fibrosis, and it is likely that host-derived components are incorporated into the laminated layer at the host-parasite interface. Therefore, in order to obtain information on the molecular composition of this structure, we used parasite larvae which were generated through in vitro cultivation and thus were largely devoid of interfering host components. Lectin fluorescence on section-labelling of metacestodes embedded in LR-White suggested that the laminated layer is largely composed of N-acetyl-beta-D-galactosaminyl, and alpha- and beta-D-galactosyl residues, as well as of the core structure of O-linked carbohydrate chains, N-acetylgalactosamine-beta-1.3-galactose, while N-linked glycopeptides and alpha-D-mannosyl residues and/or glucosyl residues were found mainly within the germinal layer, and within the cellular mass and the surface of developing protoscoleces. Lectin-gold EM confirmed these findings. The laminated layer was isolated from in vitro cultivated metacestodes by urea extraction, and the ultrastructure of the purified laminated layer was assessed comparatively with respect to the laminated layer of intact parasites. The glycan composition was determined using SDS-PAGE and lectin blotting. This work has laid the basis for a more detailed dissection of the molecular composition of the laminated layer.     

Observations on the development of Echinococcus multilocularis in cats

The development of a European isolate of Echinococcus multilocularis was compared in cats and dogs at the end of the prepatent period. Echinococcus multilocularis established in all dogs and cats, but worm recovery was significantly greater from dogs than from cats. Overall, worms in cats were not as advanced as those in dogs in terms of development and maturation, but there was no evidence of retarded development or stunted forms. These results confirm that dogs are highly susceptible to E. multilocularis, whereas cats have lower and more variable recovery rates. However, because cats produce thick-shelled eggs of E. multilocularis after experimental and natural infections, they have to be regarded as potential sources of infection both for intermediate and accidental hosts, including humans. However, their general role in the epidemiology of the infection has yet to be determined.     

Echinococcus multilocularis: identification of proteins inducing antibody formation in metacestode infection

An approach to the identification of parasite proteins which are immunogenic in natural infections is described, using the infection with the larval stage of Echinococcus multilocularis as a parasite model. Metacestode proteins were separated by SDS-polyacrylamide gel electrophoresis, and transferred electrophoretically to nitrocellulose sheets (Western blotting). Subsequently, immune recognition of the proteins was performed with various host sera and antigen-antibody complexes were detected enzymatically. Using homologous antisera, different patterns of immunogenic bands were revealed by sera of different host species. Cross-reactions with sera from individuals infected with unrelated helminths were analysed. Four proteins of E. multilocularis which failed to show any cross-reaction were identified.     

Diurnal variations in the activities of the glycogen metabolizing enzymes in mouse liver

The glycogen level in mouse liver was maximal during the night and decreased to the lowest level during the light period. The peak activity of phosphorylase alpha was observed during the light hours and thus paralleled the decline of hepatic glycogen concentrations. The period of rapid glycogen synthesis (1800-2200 hr) was immediately preceded by maximum glycogen synthase alpha activity. Significant diurnal rhythms for phosphorylase kinase and phosphorylase phosphatase activities were also observed and appear to play a role in regulating the diurnal rhythm of phosphorylase alpha activity.     

Effects of oral contraceptive pills on drug metabolizing enzymes and acid soluble sulfhydryl level in mouse liver.

The present paper reports the modulatory influence of two widely used combined oral contraceptive pills "OVRAL" and "NORACYCLINE" on hepatic phase I and II drug metabolizing enzymes and acid soluble sulfhydryl group of the mouse. Three different doses of the pills were used in this study i.e. D1 (1/2000th of a pill), D2 (1/200th of a pill) and D3 (1/20th of a pill). The sulfhydryl group increased significantly with the D1 and D2 dose of Ovral and the D2 dose of Noracycline. Dose D2 of both pills decreased cyt.P450 and cyt.b5 contents. D3 of Noracycline, however increased both the cytochrome levels. Dose D3 of Ovral and all three doses of Noracycline reduced the glutathione S-transferase activity.     

Ecology and epidemiology of Echinococcus multilocularis in Europe

Human alveolar echinococcosis (AE), caused by the larval stage of Echinococcus multilocularis has a high mortality rate in untreated patients. The life-cycle of E. multilocularis in Europe predominantly involves foxes as definitive hosts. However, experimental studies demonstrated a comparable biotic potential of E. multilocularis in dogs and raccoon dogs but an insignificant potential in cats. AE occurs in central and eastern Europe at low incidence rates. Recent studies in foxes have shown that E. multilocularis has a wider geographic range (including Italy) than previously thought. In recent years, increases in fox populations have been observed in many European countries, especially in urban areas. As a result, the E. multilocularis cycle is now established in the urban environment. This presents an increased risk of infection for a large human population. Based on these facts and new epidemiological data, possible intervention strategies are presented.     

Effect of Echinococcus multilocularis on the origin of acetyl-coA entering the tricarboxylic acid cycle in host liver

Carbon-13 nuclear magnetic resonance (NMR) spectroscopy was employed to investigate alterations in hepatic carbohydrate metabolism in Meriones unguiculatus infected with Echinococcus multilocularis. Following portal vein injections of an equimolar mixture of [1,2-13C2]acetate and [3-13C]lactate, perchloric acid extracts of the livers were prepared and NMR spectra obtained. Isotopomer analysis using glutamate resonances in these spectra showed that the relative contributions of endogenous and exogenous substrates to the acetyl-CoA entering the tricarboxylic acid cycle differed significantly between infected and control groups. The mole fraction of acetyl-CoA that was derived from endogenous, unlabelled sources (F(U)) was 0.50 +/- 0.10 in controls compared to 0.34 +/- 0.04 in infected animals. However, the fraction of acetyl-CoA derived from [3-13C]lactate (FLL) was larger in livers of infected animals than those from controls with values of 0.27 +/- 0.04 and 0.18 +/- 0.04, respectively. Similarly, the fraction of acetyl-CoA derived from [1,2-13C2]acetate (FLA) was larger in livers of infected animals compared to those in controls; the fractions were 0.38 +/- 0.01 and 0.32 +/- 0.07, respectively. The ratio of FLA:FLL was significantly smaller in the infected group with a value of 1.42 +/- 0.18 compared to 1.74 +/- 0.09 for the controls. These results indicate that alveolar hydatid disease has a pronounced effect on the partitioning of substrates within the pathways of carbohydrate metabolism in the host liver.     

Investigation on the occurrence of Echinococcus multilocularis in Central Italy

Background  

Feed contaminated with Salmonella spp. constitutes a risk of Salmonella infections in animals, and subsequently in the consumers of animal products. Salmonella are occasionally isolated from the feed factory environment and some clones of Salmonella persist in the factory environment for several years. One hypothesis is that biofilm formation facilitates persistence by protecting bacteria against environmental stress, e.g. disinfection. The aim of this study was to investigate the biofilm forming potential of Salmonella strains from feed- and fishmeal factories. The study included 111 Salmonella strains isolated from Norwegian feed and fish meal factories in the period 1991–2006 of serovar Agona, serovar Montevideo, serovar Senftenberg and serovar Typhimurium.     

Effect of chlorinated naphthalenes and terphenyls on the activities of drug metabolizing enzymes in rat liver

γ-Aminobutyric acid-α-ketoglutarate transaminase from pig brain is irreversibly inactivated by 4-amino-5-halopentanoic acids. Protection from inactivation by the natural substrates, the pH dependence of inactivation and the incorporation of 1.7 moles of radioactive inhibitor per mole of enzyme from (S)-[U-¹⁴C]-4-amino-5-chloropentanoic acid suggest a covalent adduct at the active site of the enzyme. A mechanism-based inactivation is proposed.