Immunological follow-up of hydatid cyst cases

Hydatid disease is caused by Echinococcus granulosus. In this study, we aimed to investigate the benefit of monitoring cases with hydatid cyst by means of immune components in patients in a long-term follow-up after surgery. Eighty-four preoperative and postoperative serum samples from 14 cases undergoing surgery for hydatid disease were evaluated in terms of immune parameters, such as total and specific IgE, IgG, IgM, IgA and complement. Total and specific IgE were determined by ELISA. Specific IgG levels were measured by indirect hemagglutination.Total IgG, IgM, IgA and complement (C3 and C4) were detected by nephelometry. Imaging studies were also carried out during the follow-up. In none of the patients hydatid cysts were detected during the follow-up. Total IgE levels in the sera of the patients decreased to normal six months after surgery. Although specific IgE against echinococcal antigens decreased one year after operation, levels were still significantly high. There were no changes in the levels of anti-Echinococcus IgG and total IgG in follow-up period. Additionally, other parameters, such as IgA, IgM, C3 and C4, were not affected.     

Further observations on the specificity of antigen 5 of Echinococcus granulosus.

The presence of IgE antibodies to antigen 5 of Echinococcus granulosus was detected by means of radioimmunoelectrophoresis in the sera of two of six patients infected with E. multilocularis. Sera from three of these patients gave a precipitin band in gel diffusion tests identical to that produced by a monospecific rabbit anti-E. granulosus antigen 5 serum, when tested against whole hydatid fluid. Sera from 19 individuals infected with Fasciola hepatica, 20 with Schistosoma mansoni, and 5 with with Taenia saginata showed no detectable antibodies against antigen 5 of E. granulosus, The monospecific rabbit anti-E. granulosus antigen 5 serum did not react in immunodiffusion with homologous antigen when absorbed with either 4 mg/ml of whole hydatid fluid or with 200 mg/ml of a soluble E. multilocularis extract. Absorption of the monospecific antiserum with crude antigens of either F. hepatica, Onchocerca volvulus, S. mansoni, or T. saginata did not abolish the reaction with antigen 5. It appears, therefore, that antigen 5 can no longer be considered specific for E. granulosus, but is also present in E. multilocularis. In the light of this observation, some reevaluation of immunodiagnostic tests in hydatid disease will be necessary.     

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.     

Immunology and molecular biology of Echinococcus infections

Echinococcus spp. are the etiological agents of hydatid disease in man and other intermediate hosts. Many questions regarding the factors which determine susceptibility/resistance to hydatid disease, and the factors which influence the viability and fertility of hydatid cysts, remain to be answered. Recent research into the effects of hydatid infection on the immune system of the host has provided some insights into the host-parasite relationships. Immunochemical and recombinant DNA techniques are being applied to improve diagnosis of hydatidosis in man and E. granulosus infection in dogs, and also in the development of vaccines against infection with taeniid cestode larvae. The successes which have been achieved in these areas are likely to provide valuable tools for the control of cystic hydatidosis in man. These recent studies in the areas of immunobiology, serological diagnosis and vaccination are reviewed.     

Molecular characterization of Echinococcus granulosus in Tunisia and Mauritania by mitochondrial rrnS gene sequencing

Cystic hydatid disease is a zoonotic parasitic disease caused by the cestode Echinococcus granulosus and represents a major public health problem in many countries around the world, including North Africa. E. granulosus exists as a series of genetic variants or strains which differ in a wide variety of criteria that impact on the epidemiology, pathology and control of cystic hydatid disease. Nucleotide sequencing of the mitochondrial rrnS gene was here used to characterize 38 E. granulosus isolates collected from different regions and hosts in Tunisia and Mauritania. The results obtained reveal a significant genetic differentiation between E. granulosus hydatid cysts identified as belonging to the G1 genotype and to the G6/G7 cluster using the rrnS gene as marker, and indicate the circulation of the common sheep strain (G1) in all host species from Tunisia and the camel/pig strain cluster (G6/G7) in camel from Mauritania. Other investigations, using this method, are necessary for further genetic analysis of a wider range of isolates from different host species in order to more fully understand the genetic structure of E. granulosus populations and their transmission dynamics in this and neighbouring African countries.     

Identification of Echinococcus granulosus microRNAs and their expression in different life cycle stages and parasite genotypes

The aetiological agent of cystic hydatid disease, the platyhelminth parasite Echinococcus granulosus, undergoes a series of metamorphic events during its complex life cycle. One of its developmental stages, the protoscolex, shows a remarkable degree of heterogeneous morphogenesis, being able to develop either into the vesicular or strobilar direction. Another level of complexity is added by the existence of genotypes or strains that differ in the range of intermediate hosts where they can develop and form fertile cysts. These features make E. granulosus an interesting model for developmental studies. Hence, we focused on the study of the regulation of gene expression by microRNAs (miRNAs), one of the key mechanisms that control development in metazoans and plants and which has not been analysed in E. granulosus yet. In this study, we cloned 38 distinct miRNAs, including four candidate new miRNAs that seem to be specific to Echinococcus spp. Thirty-four cloned sequences were orthologous to miRNAs already described in other organisms and were grouped in 16 metazoan miRNA families, some of them known for their role in the development of other organisms. The expression of some of the cloned miRNAs differs according to the parasite life cycle stage analysed, showing differential developmental expression. We did not detect differences in the expression of the analysed miRNAs between protoscoleces of two parasite genotypes. This work sets the scene for the study of gene regulation mediated by miRNAs in E. granulosus and provides a new approach to study the molecules involved in its developmental plasticity and intermediate host specificity. Understanding the developmental processes of E. granulosus may help to find new strategies for the control of cystic hydatid disease, caused by the metacestode stage of the parasite.     

Echinococcus granulosus in sheep: transfer from ewe to lamb of 'Arc 5' antibodies and oncosphere-killing activity, but not protection.

Fourteen ewes were orally dosed with 2000 E. granulosus eggs at 2 weeks of age, were mated at 19 months, and produced lambs when the cysts were 2 years old. One week after parturition, all 14 ewes had 'Arc 5' antibodies in their serum, as did 11/14 of their lambs. Fourteen uninfected ewes were immunized three times before parturition with E. granulosus eggs injected intramuscularly. Cysts grew at the first, or first and second site, but not the third, indicating that the ewes were immune prior to parturition. Most sera from these ewes and their lambs, and from 14 control ewes and their lambs, produced precipitin arcs with hydatid cyst fluid, but no 'Arc 5'. All lambs were challenged with 500 eggs 2 weeks after birth. At necropsy, cyst numbers within groups ranged from 3 to > 200, but there was no significant difference between the three groups of lambs. The immunized ewes did not pass a protection to their lambs that was effective when the lambs were challenged. 'Arc 5' antibodies were induced by prolonged infection with cysts, and were not seen in the sera of ewes immunized with eggs, although the eggs developed into cysts at the injection sites. 'Arc 5' antibodies did not protect lambs against infection, and were not correlated with protection in ewes. Subcutaneous injection of oncospheres into four ewes from each group at the time of lamb challenge showed that the immunized ewes were immune to this method of challenge, but the infected and control ewes were not.(ABSTRACT TRUNCATED AT 250 WORDS)     

Echinococcus granulosus down regulates the hepatic expression of inflammatory cytokines IL-6 and TNF alpha in BALB/c mice

Hydatid disease is caused by the metacestode of Echinococcus granulosus. Different experimental models have been used to understand hydatid disease. In current studies BALB/c mice were used to evaluate the hepatic response of IL-6 and TNF alpha triggered by Echinococcus granulosus. BALB/c mice were intraperitoneally infected with protoscoleces from E. granulosus; hydatid cysts appeared on the liver eight weeks after inoculation. The RNA extracted from hepatic sections was used for RT-PCR amplification with primers for IL-6, TNF alpha, IL-10, TGF beta and G3PDH. In situ cytokine expression was assessed by FISH. Complete parasite cysts on the liver surface were observed 16 weeks after infection; controls were negative. The expression of IL-6 and TNF alpha was normal at baseline and declined progressively eight weeks after infection; in some animals such expression was abrogated 16 weeks after infection. On the other hand IL-10 and TGF beta were increased progressively. Controls expressed the cytokines normally. Present results suggest that E. granulosus induces a local immunosupression probably mediated by IL-10 and TGF beta; therefore it seems possible that such a mechanism would assist the parasite in escaping the harmful host cell-mediated response.     

Medical treatment of pulmonary hydatid disease: for which child?

There have been many encouraging studies on medical treatment of pulmonary hydatid disease due to Echinococcus granulosus infection. Our aims were to demonstrate the safety and efficacy of medical treatment in pulmonary hydatid disease and to describe a pediatric population who would benefit from medical treatment, especially in respect to the diameter of the hydatid cyst. All patients were treated with mebendazole or albendazole. Treatment outcome was defined as cure, improvement or failure. Among 82 patients, 34.1% were cured, 34.1% improved and 31.8% failed. When 102 cysts were individually evaluated, 36.31% were cured, 32.4% improved and 31.3% failed. The cure and the failure rates were statistically insignificant in cysts treated with mebendazole and albendazole; however statistically significantly more cysts were improved with albendazole. The results were statistically insignificant between continuous and cyclic albendazole treatment. The mean size of successfully treated cysts was 5.3+/-3.4 cm, but "failed" for cyst with a mean size of 7.3+/-4.3 cm. There was a positive, weak and statistically significant correlation between the cyst size and treatment results. The major complication was infection. We suggest that selected pediatric patients with uncomplicated pulmonary hydatid cysts sized less than 5 cm should have a trial of medical treatment with a very close follow up.     

Control of hydatid disease in Wales.

OBJECTIVES--To evaluate the success of the south Powys hydatid control programme by analysis of trends in cystic disease in humans and sheep and dog infestation. DESIGN--A review of hospital admissions for human hydatid disease in 1984-90, abattoir prevalence surveys of hydatid cysts in adult sheep, arecoline acetarsol and coproantigen surveys of prevalence of Echinococcus infestation in dogs. SETTING--All hospitals in England and Wales, three abattoirs, and dog populations in mid ands south east Wales. SUBJECTS--Residents of England and Wales admitted to hospital between 1984 and 1990 with a new diagnosis of human hydatid disease (International Classification of Diseases (ICD), ninth revision, code 122) acquired in the United Kingdom. RESULTS--The average annual incidence of human hydatid disease in Powys, mid-Wales, fell from 3.9x10(-5) in 1974-83 to 2.3x10(-5) in 1984-90. Age specific incidence rates in Wales declined over this period only in children, and no cases occurred in children (<15 years) in Powys. Two Welsh children who lived in Gwent and mid-Glamorgan were infected. Prevalence of hydatid cysts in old sheep from south Wales declined during the control period, but in 1993 prevalence of cysts was 13%. Prevalence of E granulosus infestation was zero in the control area in 1993, but it was 2.4% in Powys dogs outside the control area in 1989 and 9.2% in dogs in Gwent in 1991. CONCLUSIONS--Human hydatid disease has been successfully controlled in south Powys but cystic echinococcosis is still endemic in sheep in mid-Wales, and there is a focus of infection in humans, sheep, and dogs in the bordering areas of Gwent and mid-Glamorgan. There is considerable potential for an upsurge in human cases if control measures are relaxed.     

Bovine (Bos taurus) humoral immune response against Echinococcus granulosus and hydatid cyst infertility

Echinococcus granulosus, the agent of hydatid disease, presents an indirect life cycle, with canines (mainly dogs) as definitive hosts, and herbivores and human as intermediary ones. In intermediary hosts fertile and infertile cysts develop, but only the first ones develop protoscoleces, the parasite form infective to definitive hosts. We report the presence of bovine IgGs in the germinal layer from infertile cysts (GLIC), in an order of magnitude greater than in the germinal layer from fertile cysts (GLFC). When extracted with salt solutions, bovine IgGs from GLIC are associated with low or with high affinity (most likely corresponding to non specific and antigen specific antibodies, respectively). Specific IgGs penetrate both the cells of the germinal layer and HeLa cultured cells and recognize parasitic proteins. These results, taken together with previous ones from our laboratory, showing induction of apoptosis in the germinal layer of infertile hydatid cysts, provide the first coherent explanation of the infertility process. They also offer the possibility of identifying the parasite antigens recognized, as possible targets for immune modulation.     

Echinococcus granulosus protoscolex formation in natural infections

Echinococcus granulosus is a parasitic platyhelminth that is responsible for cystic hydatid disease. From the inner, germinal layer of hydatid cysts protoscoleces are generated, which are are the infective forms to the dog. Systematic studies on the cell biology of E. granulosus protoscolex formation in natural infections are scarce and incomplete. In the present report we describe seven steps in the development of protoscoleces. Cellular buds formed by a clustering of cells emerge from the germinal layer of hydatid cysts. The buds elongate and the cells at their bases seem to diminish in number. Very early on a furrow appears in the elongated buds, delimiting anterior (scolex) and caudal (body) regions. Hooks are the first fully-differentiated structures formed at the apical region of the nascent scolex. In a more advanced stage, the scolex shows circular projections and depressions that develop into suckers. A cone can later be seen at the center of the hooks, the body is expanded and a structured neck is evident between the scolex and the body. During protoscolex development this parasitic form remains attached to the germinative layer through a stalk. When fully differentiated, the stalk is cut off and the infective protoscolex is now free in the hydatid fluid.     

Echinococcus granulosus genotypes circulating in alpacas (Lama pacos) and pigs (Sus scrofa) from an endemic region in Peru

The identification of the genotypes of Echinococcus granulosus present in livestock and wild animals within regions endemic for cystic echinococcosis (CE) is epidemiologically important. Individual strains display different biological characteristics that contribute to outbreaks of CE and that must be taken into account in the design of intervention programs. In this study, samples of hydatid cysts due to E. granulosus were collected from alpacas (4) in Puno and pigs (8) in Ayacucho in Peru, an endemic region for CE. Polymerase chain reaction amplification and DNA sequencing of specific regions of the mitochondrial cytochrome C oxidase subunit 1 and NADH dehydrogenase subunit 1 genes confirmed the presence of a strain common to sheep, the G1 genotype, in alpacas. Two different strains of E. granulosus were identified in pigs: the G1 and the G7 genotypes. This is the first report of the G1 genotype of E. granulosus in alpacas in endemic regions of CE in Peru.     

Human and canine echinococcosis infection in informal, unlicensed abattoirs in Lima, Peru

Echinococcus granulosus infections are a major public health problem in livestock-raising regions around the world. The life cycle of this tapeworm is sustained between dogs (definitive host, canine echinococcosis), and herbivores (intermediary host, cystic hydatid disease). Humans may also develop cystic hydatid disease. Echinococcosis is endemic in rural areas of Peru; nevertheless, its presence or the extension of the problem in urban areas is basically unknown. Migration into Lima, an 8-million habitant's metropolis, creates peripheral areas where animals brought from endemic areas are slaughtered without veterinary supervision. We identified eight informal, unlicensed abattoirs in a peripheral district of Lima and performed a cross-sectional study in to assess the prevalence of canine echinococcosis, evaluated by coproELISA followed by PCR evaluation and arecoline purge. Eight of 22 dogs (36%) were positive to coproELISA, and four (18%) were confirmed to be infected with E. granulosus tapeworms either by PCR or direct observation (purge). Later evaluation of the human population living in these abattoirs using abdominal ultrasound, chest X-rays and serology, found 3 out of 32 (9.3%) subjects with echinococcal cysts in the liver (two viable, one calcified), one of whom had also lung involvement and a strongly positive antibody response. Autochthonous transmission of E. granulosus is present in Lima. Informal, unlicensed abattoirs may be sources of infection to neighbouring people in this urban environment.     

Epidemiology of hydatid disease in Sardinia: a study of fertility of cysts in sheep

Hydatidosis, caused by Echinococcus granulosus, is a cyclozoonotic disease of economic significance in Sardinia. The life-cycle involves stray and sheep dogs as definitive hosts and sheep, pigs, goats and cattle as intermediate hosts. The most important intermediate host is sheep, due to home slaughtering with ready access of the viscera to dogs. This survey was undertaken in 1987 to ascertain the epidemiological significance of sheep in maintaining the life-cycle. A total of 700 (91.3%) of 767 sheep harboured hydatid cysts. The frequency distribution of the number of hydatid cysts was over-dispersed. Of 497 infected sheep, 7.6% had fertile cysts, 75.7% sterile cysts and 16.7% fertile + sterile 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.     

The action of albendazole on hydatid cysts in sheep experimentally infected with eggs of Echinococcus granulosus

According to the Brazilian Agricultural Ministry, the prevalence of cystic echinococcosis in the state of Rio Grande do Sul, Brazil has decreased between 1990 and 1998, possibly due to the common use of albendazole to control parasites in sheep. Therefore, albendazole (ABZ) action was tested on hydatid cysts of sheep experimentally infected with eggs of Echinococcus granulosus in Uruguaiana County (RS) with the aim of evaluating the observed changes in the parasite's prevalence. The sheep were divided into three groups of 15 animals each and treated as follows. Group 01 was a control (without treatment), groups 02 and 03 received 7.5 mg/kg and 15 mg/kg, respectively, of ABZ orally for 12 months, at intervals between 36 and 45 days. At the end of the studied period the animals were necropsied and the viability of the hydatid cysts was evaluated. Lung and hepatic cysts of sheep treated with ABZ were reduced in size. Degeneration and evident calcification were seen in 87.3% (group 02) and 75.7% (group 03), which were significantly different (P < 0.01) from the 17.3% demonstrated in the control group. It could be concluded that ABZ used early and over long periods, even in small doses, is capable of promoting lesions that devitalize the cysts. This leads to a decrease in fertility of E. granulosus in sheep and could be the reason why the prevalence of cystic echinococcosis has decreased in recent years.     

Molecular identification of unilocular hydatid cysts from domestic ungulates in Ethiopia: implications for human infections

To identify the etiologic agents of cystic echinococcosis in Ethiopia, unilocular hydatid cysts were collected from 11 sheep, 16 cattle and 16 camels slaughtered in abattoirs of Aweday, Jijiga, Haramaya and Addis Ababa during June 2010 to February 2011. A PCR-based DNA sequencing of the mitochondrial cytochrome oxidase c subunit 1 gene (cox1) was conducted for 40 cysts. The majority of cysts (87.5%) were identified as Echinococcus granulosus sensu stricto and the rest as Echinococcus canadensis. The fertile cysts of E. granulosus s.s. were found only from sheep, although it occurred in all the host species. The predominance of E. granulosus s.s. has important implications for public health since this species is the most typical causative agent of human cystic echinococcosis worldwide. The major cox1 haplotype of E. granulosus s.s. detected in Ethiopia was the same as that has been reported to be most common in Peru and China. However, a few cox1 haplotypes unique to Ethiopia were found in both of the two Echinococcus species. The present regional data would serve as baseline information in determining the local transmission patterns and in designing appropriate control strategies.     

Application of antigens from Taenia hydatigena cyst fluid for the immunodiagnosis of human hydatidosis

Fluid was collected from cysts of Taenia hydatigena in 60 adult sheep and fluid from each animal pooled separately. By double diffusion antigen 5 was demonstrated in all pools but one. The criteria are described for selection and standardization of these preparations for use as antigens for the immunodiagnosis of human hydatid disease. Sera from 50 persons harbouring hydatid cysts and from 50 patients with other disease conditions were examined by the arc-5 double-diffusion test, using two antigens prepared from Echinococcus granulosus and T. hydatigena cyst fluids, respectively. The results showed that a higher diagnostic sensitivity was obtained with the hydatid antigen. The significance of the findings is discussed in terms of their application to human immunodiagnosis in areas where hydatidosis, but not cysticercosis, is rare in livestock.