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.     

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.     

Cellular organization and appearance of differentiated structures in developing stages of the parasitic platyhelminth Echinococcus granulosus

Echinococcus granulosus is the causative agent of hydatidosis, a major zoonoses that affects humans and herbivorous domestic animals. The disease is caused by the pressure exerted on viscera by hydatid cysts that are formed upon ingestion of E. granulosus eggs excreted by canine. Protoscoleces, larval forms infective to canine, develop asynchronously and clonally from the germinal layer (GL) of hydatid cysts. In this report, we describe the cellular organization and the appearance of differentiated structures both in nascent buds and developed protoscoleces attached to the GL. Early protoscolex morphogenesis is a highly complex and dynamic process starting from the constitution of a foramen in the early bud, around which nuclei are distributed mainly at the lateral and apical regions. Similarly, distribution of nuclei in mature protoscoleces is not homogenous but underlies three cellular territories: the suckers, the rostellar pad, and the body, that surrounds the foramen. Several nuclei are associated to calcareous corpuscles (Cc), differentiated structures that are absent in the earlier bud stages. The number of nuclei is similar from the grown, elongated bud stage to the mature protoscolex attached to the GL, strongly suggesting that there is no significant cellular proliferation during final protoscolex development. The amount of DNA per nucleus is in the same range to the one described for most other platyhelminthes. Our results point to a sequential series of events involving cell proliferation, spatial cell organization, and differentiation, starting in early buds at the GL of fertile hydatid cysts leading to mature protoscoleces infective to canine.     

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.     

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.     

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.     

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.     

Proteomic analysis of the larval stage of the parasite Echinococcus granulosus: causative agent of cystic hydatid disease

We describe the preparation of Echinococcus granulosus metacestode protein extracts for two-dimensional electrophoresis (2-DE). Protoscoleces and hydatid fluid were prepared by precipitation using trichloroacetic acid (TCA) to remove nonprotein contaminants. Compared to the untreated control, TCA precipitation improved the 2-DE gel profile of the protoscoleces proteins. Comparison of 2-DE gels from insoluble and soluble fractions of the protoscoleces protein extract showed that most proteins are insoluble after lysis by sonication. Host serum proteins, especially albumin and globulins, caused horizontal streaking problems on the hydatid fluid 2-DE gels due to their high content in this sample. Even after the preparation of a hydatid fluid parasite enriched fraction, the high amount of bovine serum albumin and globulins made parasite-specific proteins difficult to detect by 2-DE. Despite the absence of an E. granulosus genome sequencing or expressed sequence tag (EST) projects, it was possible to identify 15 prominent protein spots from a whole protein protoscoleces 2-DE gel by peptide mass fingerprinting. These include actins, tropomyosin, paramyosin, thioredoxin reductase, antigen P-29, cyclophilin, and the heat shock proteins hsp70 and hsp20. This work demonstrates that 2-DE and PMF are important tools to identify proteins from the hydatid fluid and protoscoleces and for the comparative analysis of cysts from different hosts or between active and resting cysts.     

Immunohistological localisation of two hydatid antigens (antigen 5 and antigen b) in the cyst wall, brood capsules and protoscoleces of Echinococcus granulosus (ovine and equine) and E. multilocularis using immunoperoxidase methods.

Cyst wall, brood capsules and evaginated protoscoleces of E. granulosus (ovine and equine) and E. multilocularis were fixed in 10% formol-saline, embedded in paraffin and cut at 8 micrometer. Specific rabbit antisera to antigen 5 and antigen B of hydatid cyst fluid were used with immunoperoxidase methods to localise the antigens in the histological sections. Antigen 5 was found in all parasites and was associated with cells of the subtegumental area of the protoscolex, the brood capsule wall and the germinal membrane. The labelled antigen appeared as distinct granules in all areas. It is suggested that antigen 5 may be synthesised in all of these sites and that a source of the antigen in cyst fluid may be the germinal and brood capsule membranes. The laminated membranes of E. granulosus (ovine and equine) were, except for the superficial layers, free from antigen 5. Antigen B was present in all parasites. It was distributed diffusely throughout the laminated membrane, germinal membrane and brood capsule wall. There were areas of densely labelled antigen B on the surface of the distal cytoplasm of the protoscolex tegument and the surface of calcareous corpuscles. The distribution of antigen B in relation to PAS positive material and possible complement activating substances is discussed. The laminated membrane of E. granulosus was apparently more permeable to antigen B than to antigen 5. It is suggested that differences in the diffusion of these antigens through the laminated membranes of hydatid cysts in the same or different host species may account for variable serological responses during infection.     

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.     

Echinococcus granulosus: Distribution of hydatid fluid antigens in tissues of the larval stage: I. Localization of the specific antigen of hydatid fluid (antigen 5)

The indirect immunofluorescent test employing a monospecific antiserum has been used to detect the tissue localization of Echinococcus granulosus specific antigen “5.”The antigen was revealed in the inner portion of the germinal “membrane” and in the parenchyma of the protoscoleces. In these stages, it was also demonstrated fixed to the walls of some collecting ducts.It is postulated that the synthesis of the antigen “5” may occur in specialized cells of both the germinal “membrane” and the protoscoleces of the hydatid cysts.The osmoregulatory system of E. granulosus larvae seems to be involved in the transfer of the substance to the cystic cavity.     

Viability of Echinococcus granulosus cysts in mice following cultivation in vitro.

The viability of hydatid cysts developed in vitro for 90 days was assessed by implantation into mice. Cysts removed from mice at 270 days post-infection (p.i.) increased their size 13.5-fold and contained several brood capsules containing protoscoleces. Thus, cysts remain viable after prolonged in vitro culture. The implantation in mice of 15 cysts developed in vitro yielded an average of 10 cysts per mouse, which is indicative of a high survival rate in these experimental infections. The ultrastructural study of cysts recovered from mice 270 days p.i. showed that the germinal membrane was more compact than before implantation and several layers of tegumental cells had developed. Observations of cysts removed from mice indicated that the plasma membrane surrounding microtriches had prolongations opening into the laminated layer.     

The prevalence and fertility of hydatid cysts in buffaloes from Iran

Cystic echinococcosis caused by Echinococcus granulosus is considered to be an important parasitic infection in livestock. In the present study, which aimed to determine the epidemiology of hydatidosis in buffalo in Iran, slaughterhouses of West Azerbaijan (Urmia), East Azerbaijan (Tabriz), Ardabil (Ardabil), Gilan (Rasht and Hashtpar) and Khuzestan (Ahvaz) were inspected. Age, sex and infected organs were recorded separately, and the observed cysts were examined for fertility and viability. Our results showed that 344 (9%) of 3832 inspected buffaloes were infected with hydatid cysts. The maximum and minimum infection rates occurred in Khuzestan (9.9%) and Ardabil (8%) provinces, respectively. There was no significant difference in the rate of infection in all provinces. Of 344 infected buffaloes, the rate of fertility was 7.3% and the rate of viability in fertile cysts was 78.75%. Hydatid cysts were more prevalent in female compared with male buffaloes (P < 0.05). There was a positive correlation between the age and number of infected hosts in all provinces except East Azerbaijan. The prevalence of infection in lungs was significantly higher than that in the livers of buffaloes in the provinces studied (P < 0.001). In conclusion, the fertility of hydatid cysts in buffaloes was low, as previously demonstrated in cattle, and this animal may play a minor role in the epidemiology of hydatidosis in Iran.     

Potential biological role of poly (ADP-ribose) polymerase (PARP) in male gametes

Maintaining the integrity of sperm DNA is vital to reproduction and male fertility. Sperm contain a number of molecules and pathways for the repair of base excision, base mismatches and DNA strand breaks. The presence of Poly (ADP-ribose) polymerase (PARP), a DNA repair enzyme, and its homologues has recently been shown in male germ cells, specifically during stage VII of spermatogenesis. High PARP expression has been reported in mature spermatozoa and in proven fertile men. Whenever there are strand breaks in sperm DNA due to oxidative stress, chromatin remodeling or cell death, PARP is activated. However, the cleavage of PARP by caspase-3 inactivates it and inhibits PARP's DNA-repairing abilities. Therefore, cleaved PARP (cPARP) may be considered a marker of apoptosis. The presence of higher levels of cPARP in sperm of infertile men adds a new proof for the correlation between apoptosis and male infertility. This review describes the possible biological significance of PARP in mammalian cells with the focus on male reproduction. The review elaborates on the role played by PARP during spermatogenesis, sperm maturation in ejaculated spermatozoa and the potential role of PARP as new marker of sperm damage. PARP could provide new strategies to preserve fertility in cancer patients subjected to genotoxic stresses and may be a key to better male reproductive health.     

Free amino acid concentration in hydatid cyst fluids from fertile and infertile human and animal Echinococcus granulosus.

The aim of this study was to search and compare free amino acid composition of fertile and infertile cyst fluids obtained from humans and animals infected naturally with Echinococcus granulosus, by using automated analysis based on cation-exchange chromatography with post-column ninhydrin derivatization system. 11 free amino acids from fertile (sheep origin), nine from infertile (cattle origin), 13 from infertile (human origin) hydatid cyst fluids and 19 amino acids from sera of patients with hydatid infection were detected. The levels of glycine, alanine, valine and lyrosine in fertile and infertile hydatid cysts fluids were significantly higher than in sera from patients with hydatid cysts. Glycine level in the fertile hydatid cyst fluids (sheep origin) was significantly higher than those of infertile cysts fluids (cattle and human origin) and sera with hydatid patients. Glycine level in fertile hydatid cyst fluids was about two times more concentrated in infertile cattle cyst fluids, 10 times more concentrated in infertile human hydatid cyst fluids and 13 times more concentrated in sera with hydatid patients. On the other hand, alanine and valine concentration in the fertile and infertile cyst fluids were at similar level with the exception that valine level in fertile cyst fluids was 12 times more concentrated in infertile human cyst fluids. The levels of tyrosine, citrulline, leucine, isoleucine and lysine amino acids in fertile and infertile hydatid cyst fluids were similar. Our findings with respect to fertile and infertile cysts fluids showed that free amino acids concentrations in cyst fluids were significantly, higher in sera from patients with hydatid cyst. Total amount of free amino acids content in fertile and infertile cyst fluids was three to eight times higher from that of human sera with hydatid patients.     

Proteomic characterisation of Echinococcus granulosus hydatid cyst fluid from sheep, cattle and humans

The hydatid cyst fluid (HCF) of Echinococcus granulosus is a complex biological mixture containing a wide range of proteins of both parasite and host origin. Using a combination of in- and off-gel protein fractionation techniques and tandem mass spectrometry 130 HCF proteins were identified from fertile cysts of sheep and human origin and infertile cysts from cattle. Forty-eight proteins were of parasite origin including Antigen 5 and Antigen B--the most abundant parasite proteins, thioredoxin, low-density lipoprotein receptors, cyclophilin and ferritin. Across the three host species the identified HCF proteins were broadly similar although, based on spectral counts, three proteins, including an antigen B isoform, were more abundant in sheep HCF compared with the fluids of cattle and human origin. Eighty-two host proteins were identified in HCF from the three species. Host plasma proteins were the most abundant, although approximately thirty of the host proteins that were identified are not considered constituents of plasma. The identification of parasite heat shock proteins and annexin A13 exclusively in infertile cysts, along with an increased spectral count for cathepsin B, supports the hypothesis of increased cellular stress and apoptosis as the cause of their infertility.     

Epidemiology of Echinococcus granulosus in Arbil province, northern Iraq, 1990-1998

During the period 1990-1998, 99 cases of human cystic hydatidosis (12.4 cases per year) were surgically treated at the two main hospitals in Arbil province, northern Iraq, and from this the human occurence for the province was estimated to be 2 per 100,000 inhabitants. In the same area, 1270 sheep, 550 goats and 320 cattle were examined at slaughter for hydatid cysts and prevalence rates were found to be 15.0%, 6.2% and 10.9%, respectively. A decreasing tendency in livestock prevalences was found towards the end of the study period. As in humans, most of the hydatid cysts in livestock were located in the liver. Fertility of sheep cysts, i.e. those containing protoscoleces, was found to be significantly higher (64%) than that of goats (35.7%) and cattle (29.8%). The percentage of fertile cysts containing viable protoscoleces varied between 63 and 82% in the livers and between 72 and 79% in the lungs of the different animal species. A total of 97 stray dogs were examined post-mortem in the years 1991, 1992 and 1998, and Echinococcus granulosus worms were found in the intestines of 48 dogs (49.5%). High worm burdens (> 1000) were observed in 37% of the dogs, medium worm burdens (200-1000) in 41%, and low worm burdens (< 200) in 22%. In 1998, the prevalence of canine echinococcosis (24.3%) was found to be significantly lower than in 1991 (70.4%) and 1992 (60.6%). The prevalence of human hydatidosis did not differ significantly over the years, but the study confirmed that hydatidosis is endemic in northern Iraq, and that housewives, labourers and farmers appear to be at the greatest risk of infection.     

Spermatozoal sensitive biomarkers to defective protaminosis and fragmented DNA

Human sperm DNA damage may have adverse effects on reproductive outcome. Infertile men possess substantially more spermatozoa with damaged DNA compared to fertile donors. Although the extent of this abnormality is closely related to sperm function, the underlying etiology of ensuing male infertility is still largely controversial. Both intra-testicular and post-testicular events have been postulated and different mechanisms have been proposed to explain the presence of damaged DNA in human spermatozoa. Three among them, i.e. abnormal chromatin packaging, oxidative stress and apoptosis, are the most studied and discussed in the present review. Furthermore, results from numerous investigations are presented, including our own findings on these pathological conditions, as well as the techniques applied for their evaluation. The crucial points of each methodology on the successful detection of DNA damage and their validity on the appraisal of infertile patients are also discussed. Along with the conventional parameters examined in the standard semen analysis, evaluation of damaged sperm DNA seems to complement the investigation of factors affecting male fertility and may prove an efficient diagnostic tool in the prediction of pregnancy outcome.     

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.