Vaccination trials in Australia and Argentina confirm the effectiveness of the EG95 hydatid vaccine in sheep.

Experimental vaccine trials against hydatid disease have been undertaken in sheep using the EG95 recombinant vaccine. Challenge infection was with viable Echinococcus granulosus eggs obtained from a New Zealand isolate (dog/sheep cycle), an Australian isolate (dingo/wallaby cycle) and an Argentine isolate (dog/sheep cycle). Vaccination with EG95 conferred a high degree of protection against challenge with all three parasite isolates (protection range 96-100%). Taken together, the trials demonstrated that 86% of vaccinated sheep were completely free of viable hydatid cysts when examined approximately 1 year after challenge infection. Vaccination reduced the number of viable cysts by 99.3% compared with unvaccinated controls. These results suggest that the EG95 vaccine could have wide applicability as a new tool for use in hydatid control campaigns.     

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.     

Echinococcus granulosus (Cestoda: Taeniidae) infections in moose (Alces alces) from southwestern Quebec

Investigation of the distribution of larval Echinococcus granulosus in a moose population from southwestern Quebec revealed a distinct and stable pattern of infection with a prevalence of 44% (n = 580). Positive correlations between moose age and the intensity, mean cyst weight and biomass of the hydatid cysts suggested a process of continued parasite acquisition and cyst growth. The distribution of cyst sizes within individual moose provided circumstantial evidence of interaction between cysts, perhaps mediated through the host's immunological response.     

Natural history of cystic echinococcosis in humans

This study provides a contribution to understanding of the natural history of the hydatid cyst during its evolution in the human liver examining the high morphostructural variability of larval forms of Echinococcus granulosus. A detailed study of a large sample of intact cysts removed from patients surgically treated by means of total pericystectomy, has enabled to outline the different developmental stages of the parasite over time, up to its death and complete degeneration.     

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.     

Treatment of hepatic hydatid disease with mebedazole: preliminary results in four cases.

Mebendazole was given to four patients with hepatic hydatid disease. In three patients hydatidosis had remained after surgery, and in the fourth it could not be treated surgically. Mebendazole was given orally in maximum doses of 400-600 mg three times a day during courses lasting 21 to 30 days. Ultrasonic echotomography showed a complete regression of the intrahepatic cysts after four to 13 months in all four cases. In three patients the course of treatment had to be repeated. Mebendazole also induced clinical improvement and a progressive lowering of the concentration of specific IgE of Echinococcus granulosus. During treatment circulating blood levels of specific immune complexes of antigen 5 were increased. These observations indicate that mebendazole has a lethal effect on E granulosus cysts in primary hydatid disease in man and that the efficacy of chemotherapy can be assessed with ultrasonography and by measuring changes in the concentration of specific IgE of E granulosus and circulating immune complexes.     

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

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

Cystic echinococcosis in humans: our clinic experience

Echinococcosis in humans is a zoonotic infection caused by larval stages of cestode species of the Echinococcus genus. In cystic echinococcosis (CE), caused by Echinococcus granulosus, the liver is the first and the more frequent involved organ, followed by the lung. Heart, spleen, kidney and brain are usually less involved. The finding of a cyst in course of echinococcosis is usually fortuitous, during ultrasound examination, X-ray or CT. The Authors report 4 cases of human CE admitted to the Department of Infectious Diseases University of Naples "Federico II". Each case is peculiar both for the organ involved by the cysts and for the symptomatolgy. The abdominal pain, in case 1 caused by gallstones, allowed, by the ultrasound examination, to find several hydatid cysts in the liver, never symptomatic until then. The woman, in case 2, was operated for cysts in the lung, without receiving pharmacological prophylaxis. The same occurred in case 4, in which the lack of prophylaxis caused very serious relapses. In case 3, the young woman underwent an ultrasound examination because of an abdominal pain. A unique large cyst extended only in the spleen. The specific serology for immunoglobulin anti-E. granulosus resulted positive 1:61 (n.v. < 50). The Albendazole therapy caused the disappareance of pain, quickly. Later, the patient was splenectomized. It's not clear why only the spleen was involved and why the anti-E. granulosus serum levels of were increased only a little. The man, in case 4, was admitted with chest pain and electrocardiographic findings of myocardial anterior ischemia. He underwent surgical treatment of three hepatic cysts by E. granulosus, during the previous year. Two-dimensional echocardiography, transesophageal echocardiography, and cardiac magnetic resonance revealed a round cystic mass, 6 x 6 mm, located in the middle interventricular septum. The cardiac isoenzymes were in the normal ranges, but the anti-E. granulosus immunoglobulins were positive 1:5120 (n.v. < 64). The patient was treated with Albendazole. This caused the almost simultaneous disappearance of the circular cystic and clinical and electrocardiographic findings of myocardial ischemia. A cardiac hydatid cyst is an uncommon lesion, occurring in about 0.4-2% of patients with echinococcosis. In conclusion, Cystic echinococcosis is a problem in Mediterranean regions because of the high population of stray dogs, favourable conditions created by man and, above all, the illegal slaughtering.     

Experimental infection of the baboon (Papio cynocephalus) with Echinococcus granulosus of camel, cattle, sheep and goat origin from Kenya

In different areas of the world, strains of Echinococcus granulosus have been described which appear to vary in their infectivity, and laboratory primates have been used as indicators of their infectivity to man. This phenomenon was evaluated in Kenya for hydatid material of human, camel, cattle, sheep and goat origin. Viable eggs, produced by experimental infections in dogs with larval material from all the above intermediate hosts, were fed separately to four baboons (Papio cynocephalus) in each case. Baboons were autopsied between 373 and 501 days following infection and the liver, lungs, heart, spleen and kidneys were thoroughly inspected. Hydatid cysts were recovered from two baboons in each of the camel, sheep and goat groups, three baboons in the cattle group and none of the baboons in the human group. Fertile cysts were found in the cattle and goat groups and it is suggested that the baboon could be used as an experimental model for this parasite.     

A glycosylation-deficient endothelial cell mutant with modified responses to transforming growth factor-beta and other growth inhibitory cytokines: evidence for multiple growth inhibitory signal transduction pathways.

An endothelial cell line (M40) resistant to growth inhibition by transforming growth factor-beta type 1 (TGF beta 1) was isolated by chemical mutagenesis and growth in the presence of TGF beta 1. Like normal endothelial cells, this mutant is characterized by high expression of type II TGF beta receptor and low expression of type I TGF beta receptor. However, the mutant cells display a type II TGF beta receptor of reduced molecular weight as a result of a general defect in N-glycosylation of proteins. The alteration does not impair TGF beta 1 binding to cell surface receptors or the ability of TGF beta 1 to induce fibronectin or plasminogen activator inhibitor-type I production. M40 cells were also resistant to growth inhibition by tumor necrosis factor alpha (TNF alpha) and interleukin-1 alpha (IL-1 alpha) but were inhibited by interferon-gamma (IFN gamma) and heparin. These results imply that TGF beta 1, TNF alpha, and IL-1 alpha act through signal transducing pathways that are separate from pathways for IFN gamma and heparin. Basic fibroblast growth factor was still mitogenic for M40, further suggesting that TGF beta 1, TNF alpha, and IL-1 alpha act by direct inhibition of cell growth rather than by interfering with growth stimulatory pathways.     

Echinococcus multilocularis: inhibition of murine neutrophil and macrophage chemotaxis

Resident peritoneal neutrophils and macrophages from mice infected with 50 +/- 5 cysts of Echinococcus multilocularis were collected at 2, 4, 6, 8, 10, and 16 weeks postinfection. Their ability to respond and migrate to purified parasite larval antigens or endotoxin-activated mouse serum (EAMS) in comparison to normal peritoneal cells from uninfected mice was tested in vitro using Boyden chambers. Early in the infection, both cell types responded to the specific and nonspecific chemoattractants as the control group. However, at 8 and 10 weeks postinfection, the neutrophils and macrophages lost their response to parasite antigens but retained their ability to migrate to EAMS. Toward the 12th and 16th week postinfection, both cell types lost their ability to migrate to the specific as well as the nonspecific factors. The data presented suggest that the cellular mechanisms of recognition and chemotaxis in mice infected with alveolar hydatid cysts are impaired.     

Morphological variability and degenerative evolution of human hepatic hydatid cysts

The findings are presented of a macro and microscopic investigation of 89 hydatid hepatic cysts removed intact from 59 patients by total pericystectomy. Detailed analysis revealed significant morphostructural variability and cysts grouped into 10 types were characterized, providing useful clinical indications. Only 30 cysts resulted fertile (33.7%), probably due to mean age of sample; 7 of these were "classic" cysts, 1 "septated" and 22 "multivesicular" packed with daughter cysts (DC), of varying turgidity or collapsed. Among the remaining 59 sterile cysts, 52 were degenerated and classified as "hyperlaminated" cysts due to the presence of large convoluted sheets of laminar tissue (SLT) surrounded by varying amounts of caseous (40 specimens), granular (6) or gelatinous (6) matrix. Moreover, "multivesicular", "acephalocyst", "caseous" and "serous" cysts were also recovered among the sterile specimens. Some "multivesicular" cysts (14) appeared as "transitional forms" towards the various types of "hyperlaminated" cysts containing all different forms of DC and large SLT intermingled with a variously degenerated matrix. The comprehensive study allows to hypothesize the train of events leading, over the years, to the gradual transformation and degeneration of the larval form Echinococcus granulosus in the human liver.     

Observations on the suitability and importance of the domestic intermediate hosts of Echinococcus granulosus in Uttah Pradesh, India

The present study investigated the suitability and importance of buffaloes, camels, sheep, goats and pigs in maintaining the life-cycle of Echinococcus granulosus in Aligarh, India. A total of 565 (36%) of 1556 buffaloes, 20 (2%) of 1208 goats, 5 (1%) of 559 pigs, 6 (6%) of 109 sheep and two of three camels were found to harbour hydatid cysts. The frequency distribution of the hydatid cysts in each intermediate host species was over-dispersed and in buffaloes cyst fertility increased with increasing cyst size. Of 2171, 95 and four buffalo, goat, and camel cysts examined 327 (15%), two (2%) and three cysts respectively were fertile. No pig or sheep cysts were found to contain protoscoleces. The unfenced buffalo abattoir and the large number of dogs allowed access to the abattoir coupled to the number of buffalo slaughtered in comparison to the other potential hosts, indicates that the buffalo is the most significant host for maintaining the life-cycle of the parasite in this area of India. Applicable control measures for the region are suggested.     

Hydatidosis in camels (Camelus dromedarius) and their potential role in the epidemiology of Echinococcus granulosus in Iran

Hydatid cysts were recovered from 35.2% (233/661) of camels (Camelus dromedarius) slaughtered in five different regions of Iran. The degree of prevalence between males (34.4%) and females (36.6%) was not statistically significant. The highest rate of infection (59.3%) was found in the Isfahan region (in the central part of Iran) while the lowest (25.7%) was found in Kerman province. The organ distribution of cysts was 49.4% in lungs alone, 30.0% in both liver and lungs, 14.6% in liver only and 6.0% in other organs. Therefore, the lungs were the predominant sites of the hydatid cyst. The range in the number of cysts was 1-48 in infected animals. The majority of the camels had 1-5 cysts, with 21.9%, 11.6% and 5.6% of infected camels having 6-10, 11-20 and 21 or more cysts respectively. There was a direct relationship between the rate and intensity of infection and host age. The fertility rate of lung cysts (69.7%) was higher than that of liver cysts (58.7%) and other organs (50.0%) whilst the viability rate of protoscoleces of liver fertile cysts (80.3%) was significantly higher than that of lung cysts (55.8%) and other organs (57.1%). The role of camels in the epidemiology of Echinococcus granulosus in Iran is discussed.     

Occurrence of the G3 Indian buffalo strain of Echinococcus granulosus in cattle

During a survey carried out to define the occurrence of Echinococcus granulosus in cattle bred in the province of Rieti (Central Italy), molecular diagnostics (PCR amplification and sequencing of a partial region of the mitochondrial CO1 gene) showed that 6/10 positive bovines harboured hydatid cysts (No.=16) genetically identical (95.8-100%) to the Indian buffalo genotype G3. As far the location of the 16 cysts, 11 of them were found in the lungs of three animals, whereas 5 cysts were in the liver of three parasitized hosts. The occurrence of genotype G3 in 60% of parasitized bovines living in an area never studied before provides more definite evidence about the existence of the strain in this region, and proves that cattle have to be considered a non-accidental host.     

Cold stress-induced modulation of inflammatory responses and intracerebral cytokine mRNA expression in acute murine toxoplasmosis

The effects of a physical stressor, cold water stress (CWS), within the central nervous system were investigated in the acute phase of infection with Toxoplasma gondii. Female BALB/c mice were subjected to CWS for 5 min each day for 8 days prior to oral infection with 20 cysts of the low virulent ME 49 strain. Animals were killed at 10-day intervals to detect inflammation, gliosis, and expression of intracerebral cytokine mRNAs. Zones of inflammation were detected by Nissl staining and gliosis by immunoreactivity to glial fibrillary acidic protein. Larger zones of inflammation and reactive astrogliosis were consistently observed in mice subjected to CWS and infected (CWS +INF) compared to control infected (INF) mice. Expression of interleukin (IL)-1beta, IL-2, IL-12, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and inducible nitric oxide synthase (iNOS) were decreased in CWS+INF mice at 10 days postinoculation (PI), followed by a gradual increase after day 20 PI. This was in contrast to increased expression of these cytokines at 10 days PI in INF mice with a gradual decline thereafter. Inflammation and astrogliosis in CWS+INF mice were associated with an increased expression of IL-1beta, IL-6, IL-12, and TNF-alpha between 20 and 30 days PI. These findings correlated with the continuous gene expression of tachyzoite surface antigen (SAG)-1 mRNA in CWS+INF mice compared to its sharp decline in INF mice after 20 days PI. These results suggest that CWS delays regulation and control of intracerebral Toxoplasma gondii during acute infection in BALB/c mice by decreasing the early expression of IFN-gamma, IL-2, TNF-alpha, iNOS, IL-1beta, and IL-12, while increasing the expression of IL-6, a counterregulatory cytokine.     

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.     

Comparative cytotoxicity of secondary hydatid cysts, protoscoleces, and in vitro developed microcysts of Echinococcus granulosus.

Infection with the metacestode of Echinococcus granulosus is characterized by a concomitant immunity. Survival of established and developing hydatid cysts in the intermediate host implies a mechanism to modulate its immunological reactions. In order to investigate this mechanism, secondary hydatid cysts were isolated from intraperitoneally infected laboratory white mice (strain NMRI) 12 months p.i. A number of hydatid cysts were freed from the surrounding host adventitial tissue. Monolayer cultures of non-stimulated peritoneal macrophages of NMRI mice were prepared and incubated in the presence of the hydatid cysts. By means of a trypan blue exclusion test and by measuring the incorporation of tritium labelled uridine, it was found that the presence of hydatid cysts reduced the viability of the macrophages in vitro. Toxic substances are probably secreted since the medium of cultured hydatid cysts also displayed cytotoxic activity. Hydatid cysts with adventitia, as well as culture medium of those cysts, were less toxic. When toxins, partially purified from hydatid cyst fluid, were previously incubated on a collagen coated surface, a reduced level of toxicity was found, suggesting that collagen of the host adventitia may play a role in controlling the liberation of toxins by the hydatid cyst. Virtually no toxicity was exerted by protoscoleces or by the medium of cultured protoscoleces, in contrast to in vitro vesiculated protoscoleces (so called microcysts). The results reveal a novel feature of hydatid cysts that may play a role in the survival of the parasite in the immunized host.     

Leishmania major: low infection dose causes short-lived hyperalgesia and cytokines upregulation in mice

Neural involvement was traditionally associated with leprosy. However, more recent studies have shown the presence of a persistent hyperalgesia in cutaneous leishmaniasis caused by the infection of BALB/c mice with a high dose of Leishmania major. In this study, we report the presence of hyperalgesia within the first two weeks of infection caused by a low dose of the parasite. Using BALB/c mice, we demonstrate the presence of hyperalgesia during the first 10 days of infection as assessed by thermal pain tests. After 10 days these decreased pain thresholds start to recover resulting in similar levels to those in uninfected controls during the third week of infection. This hyperalgesia is accompanied by a sustained upregulation of interleukin-1beta (IL-1beta) and an early upregulation of interleukin-6 (IL-6) which is restored to normal levels after five days of infection. In conclusion, this study shows that, during early infection, the low dose of L. major causes hyperalgesia accompanied by an upregulation of IL-1beta and IL-6 and that these effects are reversed within the first two weeks of infection.     

Clustering of hydatid infection in macropodids

The introduced parasite, Echinococcus granulosus, has been reported in numerous macropodid species in various areas of Australia, but no extensive studies investigating the prevalence and risk factors for infection in wild macropodids have been reported. In this study, 2998 macropodid carcasses were examined following commercial culling on 21 properties in southern Queensland. Of the 71 infected animals, all had cysts in the lung tissue, while two also had cysts in the pleural cavity and one animal had a liver cyst. The number of cysts in infected animals ranged from 1 to 17, with the majority of animals (n=64) having one to three cysts. Estimated total cyst volume varied from 0.2 to 1075 cm3. Some animals had a total lung cyst volume likely to have impacted significantly upon respiratory function and cyst degeneration was only seen in approximately one-third of infected animals. Multilevel models were used to investigate putative risk factors at both kangaroo and property levels. At the kangaroo level, females were twice as likely to be infected as males. After adjusting for sex, no property-level risk factors were significantly associated with the presence of hydatid infection. Prevalence varied substantially between properties (range 0-12%) and this high degree of clustering of infection was reflected in a high intra-class correlation co-efficient in the final model (0.333). These results have important implications for both public health and conservation strategies, and suggest that there are important unidentified risk factors for hydatid infection associated with properties. They also demonstrate that spatial clustering should be considered when analysing hydatid infection data in macropodids, particularly when assessing area-level risk factors.