Echinococcus Multilocularis Eggs Research Articles

肝単包虫症を親子に認めた症例

Echinococcosis is a disease in which a cystic lesion develops mainly in the liver. It is caused by exposure to eggs of Echinococcus multilocularis and E. granulosus. Fourteen years ago, we reported a patient with unilocular hydatidosis of the liver in our hospital. This time, we report a unilocular liver cyst in his mother. Based on the familial history and magnetic resonance imaging (MRI) findings, we suspected echinococcosis, and cystectomy was conducted. In Japan, E. granulosus in very rare; therefore, our experience is limited. It is very important to cooperate with specialized laboratories who have encountered parasites from foreign countries and to examine other members of an affected patient’s family.

Time course of the antibody response in humans compared with rats experimentally infected with hepatic alveolar echinococcosis

Human alveolar echinococcosis (AE) is caused by the accidental ingestion of Echinococcus multilocularis eggs. Early detection is essential as surgical resection is the only treatment for a complete cure. However, details are unclear about changes in the antibody response during the initial stages of infection, yet such information is useful for early serodiagnosis. Therefore, a long-term investigation was performed into the time course of the antibody response before 'positive' detection. Patient sera were used for enzyme-linked immunosorbent assay (ELISA) and Western blotting (WB) analysis using crude antigens extracted from E. multilocularis protoscoleces. Rats were experimentally infected with AE and similarly analysed by ELISA and WB. Among the markers for diagnoses, the 18kDa band (main marker) appeared last in the preoperative stages and disappeared first after operation in a WB test. Although the 18kDa antigen can be useful as a marker for AE diagnosis, it cannot contribute to the detection of some patients before the 18kDa band appearance. To avoid misdiagnosis, different diagnostic antigens such as the 26-28 and 7-8kDa bands should also be considered. These bands tend to appear earlier than the 18kDa band and thus offer the potential for early detection of AE. We first observed changes in the antibody response in a relatively early stage after infection in human AE cases. Notably, changes in the antibody response of two intermediate species were similar. These findings provide valuable information for the early detection of human AE cases in the future.

Case studies emphasising the difficulties in the diagnosis and management of alveolar echinococcosis in rural China

BackgroundHuman alveolar echinococcosis (AE) is caused by the accidental ingestion of the eggs of the fox tapeworm Echinococcus multilocularis. AE occurs frequently in rural western China due to the poor levels of hygiene, the close contact of people with dogs, and the lack of appropriate facilities for the correct and rapid diagnosis of the disease.FindingsWe describe a case of a patient with hepatic AE, and AE metastases of the brain. She was mistakenly diagnosed with suspected undifferentiated metastatic cancer of the liver and brain, and with a pulmonary bacterial infection, but was subsequently correctly diagnosed during a follow-up field survey for echinococcosis. The diagnosis of brain AE was confirmed by pathological examination of tissue biopsies removed during neurosurgery. We also briefly describe other symptomatic and asymptomatic AE cases, identified by chance, likely due to the inadequate facilities available in rural communities in China for AE diagnosis and management, since the rapid and accurate diagnosis of metastatic AE requires a high level of expertise in the appropriate diagnostic procedures.ConclusionsThis report highlights the necessity for an upgrade in the diagnosis, treatment, prevention and control of AE in rural China.

Primary alveolar echinococcosis: Course of larval development and antibody responses in intermediate host rodents with different genetic backgrounds after oral infection with eggs of Echinococcus multilocularis

We investigated parasite establishment, subsequent larval development and antibody responses in gerbils, cotton rats and 4 inbred mouse strains until 16 weeks post inoculation (p.i.) with 200 eggs of Echinococcus multilocularis. The rate of parasite establishment in the liver determined at 4 weeks p.i. was highest in DBA/2, followed by AKR/N, C57BL/10 and C57BL/6 mice, whereas gerbils harboured few parasite foci. The accurate number of liver lesions in cotton rats could not be determined due to rapid growth and advanced multivesiculation of the parasite observed at 2 weeks p.i. The course of larval development was most advanced in DBA/2 mice with mature protoscolex formation at 16 weeks p.i., followed by AKR/N harbouring metacestodes with sparsely distributed immature protoscoleces. On the other hand, C57BL/6 and C57BL/10 mice had infertile metacestodes without any protoscolex formation. The parasite growth in mice was totally slower than those in gerbils and cotton rats. Specific IgG and IgM responses against 3 types of native crude antigens of larval E. multilocularis were evaluated using somatic extracts of and vesicle fluid of metacestode, and somatic extracts from purified protoscoleces. The 4 mouse strains demonstrated basically similar kinetics with apparent IgG and IgM increases at 9 weeks p.i. and thereafter, except C57BL/10, exhibited higher levels of IgM against crude antigens at some time point of infection. On the other hand, a follow-up determination of specific IgG and IgM levels against recombinant antigens from larval E. multilocularis revealed that each mouse strain showed different antibody-level kinetics. The findings in the present study demonstrate that the course of host–parasite interactions in primary alveolar echinococcosis, caused by larval E. multilocularis, clearly varies among intermediate host rodents with different genetic backgrounds.

Hepatic Gene Expression Profile in Mice Perorally Infected with Echinococcus multilocularis Eggs

BackgroundAlveolar echinococcosis (AE) is a severe chronic hepatic parasitic disease currently emerging in central and eastern Europe. Untreated AE presents a high mortality (>90%) due to a severe hepatic destruction as a result of parasitic metacestode proliferation which behaves like a malignant tumor. Despite this severe course and outcome of disease, the genetic program that regulates the host response leading to organ damage as a consequence of hepatic alveolar echinococcosis is largely unknown.Methodology/Principal FindingsWe used a mouse model of AE to assess gene expression profiles in the liver after establishment of a chronic disease status as a result of a primary peroral infection with eggs of the fox tapeworm Echinococcus multilocularis. Among 38 genes differentially regulated (false discovery rate adjusted p≤0.05), 35 genes were assigned to the functional gene ontology group <immune response>, while 3 associated with the functional group <intermediary metabolism>. Upregulated genes associated with <immune response> could be clustered into functional subgroups including <macrophages>, <APCs>, <lymphocytes, chemokines and regulation>, <B-cells> and <eosinophils>. Two downregulated genes related to <lymphocytes, chemokines and regulation> and <intermediary metabolism>, respectively. The <immune response> genes either associated with an <immunosupression> or an <immunostimulation> pathway. From the overexpressed genes, 18 genes were subsequently processed with a Custom Array microfluidic card system in order to assess respective expression status at the mRNA level relative to 5 reference genes (Gapdh, Est1, Rlp3, Mdh-1, Rpl37) selected upon a constitutive and stable expression level. The results generated by the two independent tools used for the assessment of gene expression, i.e., microarray and microfluidic card system, exhibited a high level of congruency (Spearman correlation rho = 0.81, p = 7.87e-5) and thus validated the applied methods.Conclusions/SignificanceBased on this set of biomarkers, new diagnostic targets have been made available to predict disease status and progression. These biomarkers may also offer new targets for immuno-therapeutic intervention.

The first instance of a cat excreting Echinococcus multilocularis eggs in Japan

A cat excreting Echinococcus multilocularis eggs was recently identified in Hokkaido, representing the first such observation in Japan. The cat was raised free-range and frequently ate rodents. Fecal egg examination revealed eggs of taeniids (EPG: 440) and Spirometra spp. (EPG: > 1000). PCR targeting part of the mitochondrial cytochrome c oxidase subunit I gene of E. multilocularis was positive with DNA from 3 single isolated taeniid eggs, and sequence analysis of one amplicon confirmed E. multilocularis. The results indicated that the eggs of E. multilocularis distributed in Hokkaido can be excreted in cat feces, and suggested the necessity of further studies to clarify whether the eggs excreted in cat feces are infective and thus whether cats can serve as infectious source to humans in Japan.

Imported disease of dogs and cats exotic to Ireland: Echinococcus multilocularis

Changes in legislation that facilitate the movement of animals within the European Union may increase the risk that some microbial and parasitic organisms, currently exotic to Ireland, will be introduced by travelled pet animals. It is possible that the fox tapeworm, Echinococcus multilocularis, might be introduced in that manner from any of the several member states in which it is endemic. Red foxes are the principal definitive hosts of E. multilocularis but dogs and cats can also be infected. Infection in the definitive host is of little clinical significance, but aberrant infection of humans results in alveolar echinococcosis, a debilitating disease that has a high mortality rate. Humans acquire the organism by ingestion of Echinococcus multilocularis eggs excreted by definitive hosts; the larval metacestodes develop primarily in the liver, in the initial asymptomatic phase as small, well-encapsulated cysts. Over time, perhaps five to 15 years, progressive local infiltration and secondary cyst development at distant sites occur with resultant clinical signs. Patients with infiltrative liver disease present with cholestatic jaundice, epigastric pain, fatigue, weight loss and hepatomegaly. If left untreated, the disease can be fatal.This paper recounts the life cycle of the parasite, and discusses the control measures on which its exclusion from Ireland depend. Strict adherence to the routine worming of travelled dogs with praziquantel, at appropriate doses, 24 to 48 hours prior to entry into the country will minimise the likelihood of introduction of this zoonosis.

Experimental alveolar echinococcosis in pigs, lesion development and serological follow up

Liver lesions were found in 6/6 pigs 7 months after oral inoculation with 5000 or 35,000 Echinococcus multilocularis eggs. However, lesion morphology differed considerably among the animals. The largest lesions (3–8 mm in diameter) were found in a single pig and smaller lesions (1.5–3 mm) in three pigs. These lesions were clearly circumscribed and had pronounced central necroses and dystrophic calcifications. In contrast, most of the smallest (usually <1.5 mm in diameter) found in two other pigs, had small compact fibrotic areas and blurred borders with obvious fibrous infiltrations into the interlobular tissues. E. multilocularis specific DNA was detected by PCR in all lesion types, but metacestode viability, as assessed by in vivo intraperitoneal inoculations in jirds, could not be demonstrated. Within 1 month post inoculation, all pigs developed specific IgG antibody responses against a battery of different antigens (metacestode, cyst fluid, and protoscoleces-derived native E. multilocularis and E. granulosus antigens, affinity purified Em2G11 antigen, antigen B, recombinant Em II/3–10 antigen). Two different reaction patterns were recorded. In the two pigs with the small lesions, pronounced reactions against all crude antigens with peaks 3–5 months p.i. and clearly elevated levels until the end of the experiment were noted. In all other pigs, antibody reactions remained low in all cases. In conclusion, we demonstrated two types of E. multilocularis metacestode development in pigs with distinct immunological response patterns.

Modified Sugar Centrifugal Flotation Technique for Recovering Echinococcus multilocularis Eggs From Soil

Among soil-transmitted parasitic diseases, alveolar hydatidosis due to the ingestion of Echinococcus multilocularis eggs is becoming a serious problem in Hokkaido, the northern most island of Japan. Dissemination of the infection far from the endemic areas can occur if motor vehicles transmit soil contaminated with eggs. No appropriate and validated method for recovering the taeniid eggs from soil is available. A modified sugar centrifugal flotation technique, using a sucrose solution of specific gravity 1.27 and 0.05% Tween-80, was evaluated as a method to successfully recover eggs from soil. Contamination levels as low as 10 eggs per gram could be detected. This method may be useful to determine the prevalence of E. multilocularis, its transmission, and the potential for by monitoring soil contamination with eggs.

Lethal effects of freezing Echinococcus multilocularis eggs at ultralow temperatures.

Methods for killing Echinococcus multilocularis eggs within stool or intestinal samples, without damaging the diagnostic value of the sample, would significantly reduce the risk of animal health providers acquiring alveolar hydatid disease. The first objective of this study was to determine whether E. multilocularis eggs located in fox intestines can survive storage at -70 C for at least 4 days. Results showed that none of 72,000 E. multilocularis eggs remained infectious to defined strains of mice under these conditions, yet, similar eggs recovered from nonfrozen carcasses stored at 4 C for the same time period were viable. The structural identities of adult worms and eggs were not significantly altered by the freezing and thawing processes. These results indicate that ultracold temperatures can be used to kill or inactivate E. multilocularis eggs, making them safe to handle when diagnosing this parasite in definitive hosts. The second objective of this study was to determine whether E. multilocularis eggs could survive freezing to -70 C if commonly used cryopreservation protocols were used. The use of the cryoprotectant solution, 5% dimethyl sulfoxide-35% saline-60% lamb serum, with a -1 C/min freezing rate was unable to prevent the eggs from being killed by freezing to -70 C. Rapid cooling by plunge freezing into liquid nitrogen was also lethal to E. multilocularis eggs. Only a few of the many potential cryopreservation protocols were tested in this study, so it is not yet possible to completely rule out the possibility of preserving these eggs at ultralow temperatures, but it does indicate that temperatures below -70 C are lethal to eggs even under favorable storage conditions.

18 ハエ類がヒトの多包虫感染に関与する可能性の検討 : 野外ハエ類の多包条虫虫卵摂取について

18 ハエ類がヒトの多包虫感染に関与する可能性の検討 : 野外ハエ類の多包条虫虫卵摂取について

Immunisation with Salmonella typhimurium-delivered glyceraldehyde-3-phosphate dehydrogenase protects mice against challenge infection with Echinococcus multilocularis eggs

Recombinant glyceraldehyde-3-phosphate dehydrogenase of the cestode parasite Echinococcus multilocularis was expressed in Escherichia coli and in Salmonella typhimurium. The potential of different forms of the recombinant antigen to protect BALB/c mice against oral challenge infections with E. multilocularis eggs was evaluated. Oral or intraperitoneal immunisation with live attenuated S. typhimurium as a carrier for recombinant glyceraldehyde-3-phosphate dehydrogenase of the E. multilocularis resulted in significant protection, reducing the number of developing metacestodes up to 79.8%. The sera of protected animals did not contain detectable amounts of antibody against glyceraldehyde-3-phosphate dehydrogenase of E. multilocularis. By contrast, although anti-glyceraldehyde-3-phosphate dehydrogenase of E. multilocularis antibodies were detectable in the sera, immunisation with E. coli-expressed recombinant glutathione-S-transferase-fusion protein or with glyceraldehyde-3-phosphate dehydrogenase of E. multilocularis fused to a 6HIS-tag failed to protect the animals against oral challenge infections. These data emphasise that antigen delivery systems play a critical role in vaccination and the induction of protective immunity against helminth parasites.

Experimental investigations on the B and T cell immune response in primary alveolar echinococcosis.

Susceptibility/resistance of the intermediate host to alveolar echinococcosis (AE) seems to be based on hitherto unknown immunological mechanisms, possibly involving the activation of different CD4+ T cell immune responses (Th1/Th2). Mice of two strains previously characterized as 'susceptible' (C57BL/6 J) and 'resistant' (C57BL/10 J) to secondary AE were orally infected with eggs of Echinococcus multilocularis and the course of infection was analysed by macroscopical, pathohistological and immunohistochemical examinations of the lymphocytes and cytokines participating in the periparasitic granulomas and by serological examinations of cytokines and E. multilocularis-specific antibodies. Although differences in the extent of parasitic growth were seen between the two groups, the composition of the granulomas was quite similar with CD4+ cells being the dominant lymphocyte subpopulation, succeeded by B cells and CD8+ cells. Interferon (IFN)-gamma-, interleukin (IL)-2- and IL-4-expressing cells could not be detected in the lesions of the early phase of the infection, possibly indicating the host's immunosuppression, but were present at the end. IL-10 was the most prominent cytokine throughout the course of the disease. Serological analyses of the cytokine concentrations revealed small amounts at the beginning and high levels at the end of the infection. The pattern of cytokine response was similar for IL-4 in both strains, but different for IL-2 and IL-10 in the late phase, when the C57BL/10 J strain developed higher levels than the C57BL/6 J strain. Correspondingly only small amounts of immunoglobulin (Ig)M, IgG1, IgG2a and IgG3 could be detected at the beginning of disease, followed by higher levels at the end. The courses of antibody titres were similar in both groups except IgG3, which was more pronounced in the C57BL/10 J strain. Parasite-specific IgG2b could neither be detected in the C57BL/6 J nor in the C57BL/10 J strain by the test system used. The results of the study suggest both subsets of CD4+ T cells (Th1 and Th2) being involved in murine primary alveolar echinococcosis. A strict differentiation of mice in susceptible and resistant animals based on the activation of different CD4+ T cell immune responses (Th1 'resistant' and Th2 'susceptible') should be avoided.

Intestinal and systemic humoral immunological events in the susceptible Balb/C mouse strain after oral administration of Echinococcus multilocularis eggs.

The aim of the study was to investigate the systemic and, for the first time, the intestinal humoral events in the susceptible Balb/C mouse strain after oral administration of Echinococcus multilocularis eggs. Thirty-one mice were divided into three groups; W-2, W-8 and control group. Each mouse of the W-2 and W-8 groups was orally infected with 1,500 E. multilocularis eggs, two weeks and eight weeks before sacrifice respectively. Control group mice received phosphate buffer saline. Measurement of anti-E. multilocularis and non-specific IgG, IgA and IgM, and of a transudation marker, albumin, were performed in serum and intestinal washings by a time-resolved immunofluorometric assay. These results were complemented by microscopic examination of the intestinal mucosa. This infection model is well-suited to the study of mucosal immunity during alveolar echinococcosis. It showed a major specific intestinal response in the early stage of the disease whereas the systemic response predominated later in the disease. Histopathological studies and calculation of the relative coefficient of excretion of Ig also confirmed that the presence of the parasite, even during a short period, was responsible for a local immunological and inflammatory response and for a change in mucosal permeability. Mucosal immunity could thus play a role in tolerance induction against E. multilocularis that could be a prerequisite for the subsequent development of the larvae in the liver, and for the occurrence of the parasitic disease, alveolar echinococcosis.

Détection des oeufs d'Echinococcus multilocularis Lueuckart, 1863 dans les fèces de renard (Vulpes vulpes Linnaeus, 1758) par amplification en chaîne par polymérase

The polymerase chain reaction (PCR) was applied to the identification of eggs of Echinococcus multilocularis in faeces from foxes. The test was positive in three of six faeces samples from foxes which were harbouring adult worms, and in one of four samples from foxes in which no adult E. multilocularis was found in the intestines. These initial results show that it is possible to use PCR to identify E. multilocularis eggs in faeces. PCR can be used to complement examination of intestinal contents, showing that the distribution of eggs in faeces is uneven. The sensitivity of the test was estimated to be 50 eggs in 5 g of faeces. Further work is needed to confirm these initial results before the test can be used more widely.

Primary pulmonary involvement in rodents infected with Echinococcus multilocularis.

Pulmonary cysts developed in four gerbils and one cotton rat infected orally with Alaskan strain Echinococcus multilocularis eggs. In three cases, liver and lung cysts were similar. Two rodents, however, showed typical fertile pulmonary cysts while the liver foci were small and sterile or necrotic. For these reasons, the lung infections were considered to be primary in origin.

Biologic Identification of the Cestode Echinococcus multilocularis Isolated from Foxes in North Dakota

The eggs from Echinococcus multilocularis recovered from red foxes in North Dakota were fed to cotton rats (Sigmodon hispidus) and infection in this host was obtained. This is biological verification of the occurrence of this species in the continental United States. Adult cestodes isolated from red foxes (Vulpes vulpes) in North Dakota were identified morphologically by Leiby and Olsen (1964) as Echinococcus multilocularis, Leuckart, 1863. This was the first time this species had been found in the continental United States and marked an extension of its geographic range. Biologic and morphologic studies of the cystic stages which developed in an experimentally infected rodent host have confirmed the identification of the organisms as E. multilocularis, indistinguishable from the species isolated in Alaska (Rausch, 1956). Gravid worms collected in North Dakota were suspended in water and given by stomach cannula to groups of cotton rats (Sigmodon hispidus). Individual rats received 10 to 15 worms each. The first three rats were inoculated 2 days after receipt of the worm material; the second group of three was fed 7 days later, and the final group was fed after the adult worms and some of the expelled eggs had been stored in water in the cold for 3 weeks. All but one of the cotton rats became infected. Figure 1 is a picture of an animal autopsied 8 weeks after experimental infection with small cystic clumps of E. multilocularis which are typical for infections in cotton rats (Sadun et al., 1957). In this animal there were abundant scolices in the cysts, many of which were mature although the infection was only 8 weeks old. A second animal autopsied at 6 weeks also contained some mature cysts. The scolices in cysts from two 4-month-old infections were examined with low-power magnification and could not be differentiated from scolices collected from rats infected for the Received for publication 29 March 1965. * U. S. Department of Health, Education, and Welfare, Public Health Service, Communicable Disease Center, Atlanta, Georgia. t Minot State College, Minot, North Dakota. same length of time with the Alaskan isolates which are being maintained at the Communicable Disease Center. The number of scolices per cyst of the North Dakota isolate was more numerous than that of the Alaskan strain. Two animals died with heavy experimental infections. The number of rostellar hooks on the scolices of E. multilocularis recovered from two animals with primary infections autopsied 8 months after feeding varied from 24 to 33 with a mean of 26.9 hooklets. This is comparable to the observations made by Cameron (1960). When the scolices were washed and subinoculated by intraperitoneal injection into four young cotton rats and three young gerbils FIGURE 1. Experimental infection of a cotton rat (Sigmodon hispidus) with eggs of Echinococcus multilocularis collected from a red fox (Vulpes vulpes) in North Dakota, USA.