Detection of Echinococcus multilocularis in the definitive host: coprodiagnosis by PCR as an alternative to necropsy

Recently, extensions of the range of Echinococcus multilocularis in Europe and North America and drastic increases in fox populations in Europe put an increasing proportion of the human population at risk of alveolar echinococcosis. To obtain data on the local infection pressure, studies of the prevalence of the parasite in the animals that transmit the parasite, foxes, dogs, and cats, are urgently required. Such investigations, however, have been hampered by the need for necropsy of the host animal to specifically diagnose infection with the parasite. In this study, a nested PCR and an improved method for DNA extraction were developed to allow the sensitive and specific diagnosis of E. multilocularis infections directly from diluted fecal samples from foxes. The target sequence for amplification is part of the E. multilocularis mitochondrial 12S rRNA gene. The specificity of the method was 100% when it was tested against 18 isolates (metacestodes and adult worms) of 11 cestode species, including E. granulosus. The sensitivity of the method was evaluated by adding egg suspensions and individual eggs to samples of diluted feces from uninfected foxes. The presence of one egg was sufficient to give a specific signal. To confirm the PCR results, an internal probe which hybridized only with E. multilocularis amplification products but not with the DNA of other cestodes was constructed. In order to investigate the applicability of this method for epidemiological studies, 250 wild foxes from a area in southern Germany where echinococcosis is highly endemic were examined by both necropsy and PCR of rectal contents. The sensitivity correlated with the parasites' number and stage of maturity. It ranged from 100% (>1,000 gravid worms) to 70% (<10 nongravid worms). On the basis of positive PCR results for 165 foxes, the sensitivity of the traditional and widely used necropsy method was found to be not higher than 76%. We therefore present this PCR system as an alternative method for the routine diagnosis of E. multilocularis in carnivores.     

Prevalence of Echinococcus multilocularis and other metacestodes and cestodes in the muskrat (Ondatra zibethicus LINK 1795) in Lower Saxony

Aim of the study was to examine the prevalence and regional distribution of Echinococcus multilocularis and other metacestodes and cestodes in muskrats in Lower Saxony, Germany. A total of 991 muskrats with similar numbers from all districts of the country were examined between January and December 1995. E. multilocularis metacestodes were found in 4.1% of the muskrats in the liver and other organs of the abdominal cavity. The majority of the E. multilocularis positive animals came from the south of Lower Saxony, where E. multilocularis is endemic, and single animals from the middle or the northern part of the country. All metacestodes found were fertile. Additionally, metacestodes from T. taeniaeformis were found in the livers of 42.3% of the muskrats, metacestodes from T. crassiceps were present in the abdominal cavity of 2.7%, T. polyacantha in 0.4% and T. martis in 3.4% of the animals. Adult cestodes of the family of the Anoplocephalidae were found in the small intestine of 1.5% of the muskrats. Due to autolysis a species differentiation of the latter was not possible.     

Infestation of water voles (Arvicola terrestris) with metacestodes of Echinococcus multilocularis in the canton Freiburg (Switzerland)

The results of an investigation on Echinococcus multilocularis-metacestodes in European water voles (Arvicola terrestris) are shown. The investigation materials were 28 water voles captured in Schermann-traps on a meadow in the canton of Fribourg and subsequently maintained in laboratory cages. The animals were patho-anatomically and histo-pathologically examined, a part of them additionally with an E. multilocularis-specific direct immunofluorescence-assay and/or a polymerase-chain-reaction. Radiographs were obtained from one animal, showing typical calcifications as they often occur with alveolar echinococcosis. Altogether 11 out of 28 (39%) animals proved to be infected with E. multilocularis. As far as we know this represents the highest percentage of infested intermediate hosts ascertained in Europe. However, fertile, i.e. able to infect, protoscolices were detected in only two cases. In spite of this small number of protoscolices it can be shown that A. terrestris is able to maintain the lifecycle of this cestode at least spatially and temporally limited. In conclusion the possible measures for reducing the infection risk of the people in the affected community are shortly discussed.     

T-cell activity associated with secondary infections and implanted cysts of Echinococcus granulosus in BALB/c mice

Selected cytokine profiles of lymphocytes were assessed in BALB/c mice infected with protoscoleces of Echinococcus granulosus. Late stages of the infection (three months+) were characterized by more dominant Th2 activity with elevated IL-4 and IL-10 and reduced IFN-gamma output by Con-A and antigen stimulated splenocytes. Circumparasitic leucocytes produced mainly IL-10 by five months post infection. A peak in IFN-gamma production in the first month of infection may suggest Th1 or Th0 activity at this time and this may be correlated with initial protoscolex death. In addition, cytokine profiles from mice implanted with intact hydatid cysts were also assessed. At two weeks post implantation all cysts were still viable and cytokine production was characterized mainly by elevated IL-10 production. However, at four months post implantation, some of the cysts from two mice had been killed whilst all cysts in the remaining mouse remained viable. In the mice where dead cysts were present, elevated levels of IFN-gamma were detected from splenocytes and circumparasitic cells. Elevated IL-4 was also evident with the splenocytes. In the mouse with viable cysts IFN-gamma production was reduced Results indicate that IFN-gamma(Th1) activity may be correlated with killing of both protoscoleces and established cysts of E. granulosus.     

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.     

A case of subcutaneous cysticercosis

We report the fourth case of subcutaneous cysticercosis infected in Japan since 1975. The patient noticed a subcutaneous nodule on his left shoulder without symptoms for three years. No remarkable changes were found in laboratory findings and physical examination after surgical excision of the subcutaneous nodule. The adult worm of Taenia solium could not be found in the intestine. Histological findings revealed a cystic structure with a fibrous capsule and a protoscolex with suckers, hooks, and calcareous corpuscula. It was identified as a Cysticercus cellulosae hominis based on morphological characteristics. The patient has been living in the Kanto area of Japan and has never been outside Japan since he was born.     

Testicular torsion after 12 hours. III

To identify the risk of pet ownership (i.e., cats and dogs) for alveolar echinococcosis caused by Echinococcus multilocularis, the habits and activities of 21 patients (histologic confirmation or positive serology with corresponding evidence on an ultrasonogram, radiograph, or computed tomography scan) in Austria during the period 1967-1997 were compared with the habits and activities of 84 controls matched by sex, age, and residence. Cat ownership (odds ratio (OR) = 6.47, 95% confidence interval (CI) 1.54-27.29) and hunting (OR = 7.83, 95% CI 1.16-52.77) were independent risk factors associated with alveolar hydatid disease. The study is not in agreement with the hypothesis that eating mushrooms or certain wild berries which grow near the ground are the main risk factors for acquiring this disease. No other behavior patterns or activities studied were identified as risk factors.     

Dideoxy fingerprinting: application to the genotyping of Echinococcus

Dideoxy fingerprinting is an efficient method for the detection of sequence variation in PCR-amplified DNA segments. It is a hybrid between single-strand conformation polymorphism and dideoxy sequencing, employing only one dideoxynucleotide in the sequencing reaction. Herein, we report the application of dideoxy fingerprinting to genetically type cestodes of the genus Echinococcus, utilising the mitochondrial cytochrome c oxidase subunit I as the gene sequence for analysis. All of the seven genotypes (G1, G4, G6, G8, O, V and M2) examined could be readily differentiated from one another by their characteristic and reproducible dideoxy fingerprinting profiles. Only subtle variation in profiles was detected among some of the eight isolates representing genotype G1, and no variation was detected between two samples of genotype G4 and of genotype M2. The capacity of dideoxy fingerprinting to detect all nucleotide variations over 150-250bp fragments indicates that it should be possible to distinguish among all of the genotypes of Echinococcus thus far described. Although employed herein to display sequence variation in the cytochrome c oxidase subunit I of Echinococcus, dideoxy fingerprinting could be used for the high-resolution analysis of nucleotide variations in other parasite genes, without the need for DNA sequencing. This has important implications for studying the genetic structure of parasite populations.     

Primary infection of dogs with Echinococcus granulosus: systemic and local (Peyer's patches) immune responses

Local and systemic lymphocyte proliferation and antibody production were tested in five dogs 35 days after primary experimental infection with Echinococcus granulosus. A significant cell proliferation was demonstrated by [3H] thymidine incorporation in mesenteric, popliteal and/or Peyer's patches (PPs) cells in response to E. granulosus protoscolex or adult worm antigen in three of five infected dogs, but not in five control animals. In contrast, blood mononuclear cells responded very weakly in only two of the infected dogs to parasite antigens. Elevated levels (compared with preinfection status) of protoscolex- and adult worm antigen-specific serum IgG were detected (ELISA) in four of the five dogs 35 days after infection. Furthermore, slightly elevated levels of parasite-specific IgE and IgA were observed in the sera of three and four in four infected dogs, respectively. Specific serum IgM was not significantly higher 35 days after infection than before infection. Local antibody production was studied in vitro using PPs, mesenteric and popliteal cells isolated from three infected and three uninfected dogs by ELISA using adult worm antigen. In two of three cultures of unstimulated PPs cells of infected dogs, parasite-specific IgG was detectable. Parasite-specific IgA and IgM were detected in one of the unstimulated PPs cell culture derived from an infected dog. Following in vitro stimulation with parasite antigen, PPs cells from two infected dogs showed increased parasite-specific IgG and PPs cells of all three infected dogs produced parasite-specific IgA. PPs cells from uninfected dogs did not produce significant quantities of parasite-specific antibodies and cells from mesenteric and popliteal lymph nodes of infected or uninfected dogs neither produced antibodies whilst in in vitro cultures.     

Radiological diagnosis of echinococcus disease in man (author's transl)

The position of radiological diagnostics in the context of the diagnosis of echinococcosis was discussed on the basis of 11 examined cases of Echinococcus cysticus (9) and Echinococcus alveolaris (2). General thoracic x-rays as well as radiological examination of the gastrointestinal track and the kidneys were used to establish the extent of echinococcosis and to determine the involvement of neighboring organs. Diagnosis of the liver is generally of major interest diagnostically. With a scintigram of the liver, parenchymatous lesions of 2-4 cm and larger can be diagnosed with an optimal examination technique. The scintigram used in connection with the arteriogram can clarify the extent of the parenchymatous lesions. The celiacogram is the most important of the morphological liver diagnostic methods. It can be augmented by the selective hepaticogramm. An exact localization and the relationship of the process to the vessels is possible only with the celiacogram. In addition, the exclusion of other liver processes for the differential diagnosis is possible with the angiogram. The direct splenoportography is for the most part, replaced today by the indirect splenoportogram, it is only used now for special problems.     

Ovine cysticercosis in the Albany region of Western Australia. 1. Survey of tapeworms of rural dogs

A survey of the cestode infections of 304 dogs from 134 properties in the Albany area of Western Australia was performed. Purgation was induced by oral administration of arecoline and the purge examined for cestodes. The cestodes found and their infection rates were: Dipylidium caninum 16.4%; Taenia spp 36.5%; T. ovis 8.9%; T. hydatigena 15.1%; T. pisiformis 15.1%; T. serialis 2.3%; Echinococcus granulosus 0.7%; Questionnaires completed on 133 of the 134 properties at the time the dogs were purged showed that:--73.6% of farmers were feeding raw sheep-meat and 24.8% were feeding raw offal to their dogs; dogs were treated on a regular basis with a cestocide on 23.3% of properties; dogs were allowed to roam on 21.8% of the properties and stray dogs were considered prevalent on 23.3% of properties. Suggestions for control of Cysticercus ovis are made based on an interpretation of the significance of these findings.     

The structure underlying physical performance measures for older adults in the community

Nucleotide sequence variations in a region of the mitochondrial cytochrome c oxidase subunit I (COI) gene (391 bp) were examined within seven species of the genus Taenia and two species of the genus Echinococcus, including ten isolates of T. taeniaeformis and six isolates of E. multilocularis. More than a 12% rate of nucleotide differences between taeniid species was found, allowing the species to be distinguished. In E. multilocularis, no sequence variation was observed among isolates, regardless of the host (gray red-backed vole, tundra vole, pig, Norway rat) or area (Japan, Alaska) from which each metacestode had been isolated. In contrast, six distinct sequences were detected among the ten T. taeniaeformis isolates examined. The level of nucleotide variation in the COI gene within T. taeniaeformis isolates except for one isolate from the gray red-backed vole (TtACR), which has been proposed as a distinct strain or a different species, was about 0.3%-4.1%, whereas the COI gene sequence for TtACR differed from those of the other isolates, with levels being 9.0%-9.5%. Phylogenetic trees were then inferred from these sequence data using two different algorithms.     

A new species of Acanthobothrium van Beneden, 1849 (Eucestoda:Tetraphyllidea: Onchobothriidae) in Dasyatis longus Garman (Chondrichthyes:Myliobatiformes:Dasyatididae) from Chamela Bay, Jalisco, Mexico

A new species of Acanthobothrium in Dasyatis longus from Chamela Bay, Jalisco, Mexico, is a member of a presumed clade of species diagnosed by being anapolytic or nearly so, having more than 100 testes per proglottis, with immature and mature proglottides wider than long to square, aspinose scolex, muscular bothridia fused to the scolex at their posterior ends, H- to V -shaped ovaries, relatively short symmetrical to asymmetrical ovarian arms that extend anteriorly to, or nearly to, the cirrus sac, and vitellaria arranged in fields rather than a single row of follicles. The new species most closely resembles Acanthobothrium terezae from the freshwater stingray Potamotrygon motoro in the following characters: bothridial hooks longer than 200 microns with inner hooks having bent asymmetrical prongs, an average of 130-140 testes per proglottis, and shallow genital atria located posterior to midline of proglottis. The new species differs from A. terezae by having outer hooks approximately the same size and shape as the inner hooks, inner hooks averaging 230 microns rather than 313 microns in total length, and cirrus sacs averaging 255 microns rather than 450 microns in length. The new species is unique among all described species of Acanthobothrium by having a cleft in the posterior margin of each apical bothridial pad. The apparent close relationship of the new species to one inhabiting a Neotropical freshwater stingray provides support for the hypothesized Pacific marine ancestry of Neotropical freshwater stingrays and raises the possibility that the Neotropical freshwater stingrays may not be monophyletic.     

Immunology and morphology studies on the proliferation of in vitro cultivated Echinococcus multilocularis metacestodes

The larval stage of Echinococcus multilocularis causes alveolar echinococcosis (AE) in various mammals, including humans. Traditionally metacestodes are maintained in the laboratory by serial transplantation passages into susceptible animals such as mice or gerbils. However, in animal models it has always been difficult to draw definite conclusions about the factors modulating metacestode differentiation, and investigations on gene expression and respective regulation have been hampered by the complexicity of the host-parasite interplay. This paper describes the maintenance and proliferation of E. multilocularis metacestodes as well as the formation of protoscolices in a chemically defined medium devoid of host influence. The interactive role of a heterologous human cell line (CACO2) in the in vitro development of metacestodes was also assessed. The morphology and ultrastructure of in vitro-generated metacestodes was studied using scanning (SEM) and transmission electron microscopy (TEM). Different cultivation procedures were analyzed in terms of expression of B- and T-cell epitopes and of the relevant laminated layer-antigen Em2; the exact localization of this antigen was further demonstrated by immunogold electron microscopy.     

Forests without primates: primate/plant codependency

Detailed studies of primates and fruiting trees have illustrated that these groups of organisms are involved in a very complex set of interactions, with primates relying on fruiting trees as important food resources and fruiting trees relying on frugivores for seed dispersal. Human activities that influence either primate seed dispersal or fruit production have the potential of having unanticipated effects on the other interactants. Here we evaluate what is known and what we still need to learn to evaluate the long-term consequences of disrupting the interactions between primates and tropical forest trees. We do this by first assessing the potential importance of primates at dispersing the seeds of tropical forest trees. Second, we consider possible consequences of hunting primates on recruitment in tropical tree communities. Third, we address the converse by considering the impacts of decreasing resource availability for primates through either logging or through the extraction of nontimber forest products. Finally, we provide a case study from Kibale National Park, Uganda, that contrasts seedling recruitment in 20 forest fragments in which primate seed dispersers have been dramatically reduced with seedling recruitment in areas that have an intact frugivore community. In comparison to the intact forest, the fragments had lower seedling density and fewer species of seedlings. Furthermore, a greater proportion of the seedlings were from small-seeded species that might not require primates for their dispersal, since they probably can be dispersed by small birds. All of these considerations suggest that disrupting the complex interactions between primates and fruiting trees can potentially have negative and possibly cascading effects on ecosystem processes.     

Premature response to luteinizing hormone of granulosa cells from anovulatory women with polycystic ovary syndrome: relevance to mechanism of anovulation

Polycystic ovary syndrome is the most common cause of anovulatory infertility. Anovulation in polycystic ovary syndrome is characterized by the failure of selection of a dominant follicle with arrest of follicle development at the 5-10 mm stage. In an attempt to elucidate the mechanism of anovulation associated with this disorder we have investigated at what follicle size human granulosa cells from normal and polycystic ovaries respond to LH. Granulosa cells were isolated from individual follicles from unstimulated human ovaries and cultured in vitro in serum-free medium 199 in the presence of LH or FSH. At the end of a 48-h incubation period, estradiol (E2) and progesterone (P) were determined in the granulosa cell-conditioned medium by RIA. In ovulatory subjects (with either normal ovaries or polycystic ovaries), granulosa cells responded to LH once follicles reached 9.5/10 mm. In contrast, granulosa cells from anovulatory women with polycystic ovaries responded to LH in smaller follicles of 4 mm. Granulosa cells from anovulatory women with polycystic ovaries were significantly more responsive to LH than granulosa cells from ovulatory women with normal ovaries or polycystic ovaries (E2, P < 0.0003; P, P < 0.03). The median (and range) fold increase in estradiol and progesterone production in response to LH in granulosa cell cultures from size-matched follicles 8 mm or smaller were E2, 1.0 (0.5-3.9) and P, 1.0 (0.3-2.5) in ovulatory women and E2, 1.4 (0.7-25.4) and P, 1.3 (0.3-7.0) in anovulatory women. Granulosa cells from anovulatory (but not ovulatory) women with polycystic ovaries prematurely respond to LH; this may be important in the mechanism of anovulation in this common endocrinopathy.     

Plasma estradiol concentrations and pharmacokinetics following transdermal application of Menorest 50 or Systen (Evorel) 50

In order to evaluate the applicability of anthelminthic treatment of wild foxes (Vulpes vulpes) to limit their infection with Echinococcus multilocularis, bait pellets, each containing 50 mg praziquantel, were repeatedly distributed in an area of 566 km2 where many foxes are infected, in southern Germany. After six baiting campaigns (15-20 baits/km2) over a period of 14 months, the prevalence of the cestode in foxes, initially 32%, had fallen to 4%. The effect was most pronounced in the central part of the treated area, where no positive fox was found in the 2 months before the end of the trial. The study was controlled for other factors that could influence the parasite's prevalence, such as the availability of intermediate hosts. While the potential of this baiting method to remove E. multilocularis from wild hosts has been demonstrated, the question of its long-term efficacy and other unresolved problems have to be addressed by consecutive studies before routine application can be recommended.     

Lack of H-2 gene influence on mouse susceptibility to secondary alveolar echinococcosis

To determine whether the development of hepatic Echinococcus multilocularis infection is influenced by major histocompatibility-linked genes, metacestode growth and host immune responses were compared in 4 C57BL/10 congenic murine strains of H-2b, H-2d, H-2k and H-2q haplotypes. Although the H-2q strain appeared slightly more resistant than the other strains, the 4 strains of mice developed comparable spleen cell proliferative response and Th1/Th2 cytokine production at 13 weeks p.i. A kinetic analysis, performed in 2 of these congenic strains, showed a similar pattern of parasite growth in these mice and failed to detect any significant difference in the production of parasite-specific IgM, IgG1 and IgG2, antibodies. Consequently, this study indicates that the control of secondary alveolar echinococcosis is not H-2 gene-linked.     

Wijerama House

We report the case of a non-Hodgkin's lymphoma in an isolated and not previously diagnosed "adult polycystic liver disease". In this case, a rheumatism-like presentation was associated with the discovery of multiple nodular and cystic lesions in the liver. Of these, the former were related to lymphoma involvement, the latter to pre-existent liver disease. The singularity of the case, not previously reported, and some difficulties met in diagnosing the two pathologies, justify the report.