Ultrastructural aspects of fertile and sterile cysts of Echinococcus granulosus developed in hosts of different species

Bortoletti G. and Ferretti G. 1978. Ultrastructural aspects of fertile and sterile cysts of Echinococcus granulosus developed in hosts of different species. International Journal for Parasitology8: 421–431. Research was carried out on fertile and sterile cysts of Echinococcus granulosus taken from hosts of different species. Since the animals were bom and bred in Sardinia and had undergone spontaneous infection, this entails two reservations: firstly the stunted development of cysts in certain hosts, i.e. bovines, may be peculiar to a possible ‘Sardinian strain’ of E. granulosus; secondly, certain alterations in the germinal membrane may be due to parasite ageing. In some cases, the parasite may be dead despite the cyst appearing macroscopically normal because of the presence of the laminar layer.The germinal membrane ‘thrives’ to remarkably different degrees which do not, however, correlate with cyst fertility. The most thriving conditions are to be found in human cysts which are fertile; the germinal membrane may be thriving in pig cysts which are sterile; in sheep, the germinal membrane develops quite well and cysts are generally fertile; bovine cysts are generally sterile with stunted germinal membranes.There seems to be a direct correlation between cyst development and laminar layer thickness, whereas no correlations emerged with the organ in which cysts are located.     

Observations on the microtriches and stages in their development and emergence in Caryophyllaeus laticeps (Caryophyllidea: Cestoda)

Richards K.S. and Arme C. 1981. Observations on the microtriches and stages in their development and emergence in Caryophyllaeus laticeps (Caryophyllidea: Cestoda). International Journal for Parasitology11: 369–375. Fully developed microtriches of the posterior two-thirds of the body consist of three regions. The long, proximal shaft is ovoid in transverse section and has a peripheral electron-dense support and a central lucent core. The tapering, recurved, electron-dense spine lacks substructure and is ovoid at its base. The membrane-bound distal extension is in continuity with the proximal regions and its cytoplasm is coarsely granular. It is frequently observed in a reflexed position lying parallel to the shaft.In developing microtriches the ovoid spine lies parallel to the worm surface whereas the shaft support and core are perpendicular to the syncytial surface; the distal extension is not present. During microthrix emergence the spine comes to lie in an erect position above the apical membrane, then the shaft support and core begin to emerge and the distal extension develops.     

Morphological studies on the early development of Taenia taeniaeformis larvae in susceptible mice

Bortoletti G. and Ferretti G. 1985. Morphological studies on the early development of Taenia taeniaeformis larvae in susceptible mice. International Journal for Parasitology15: 365–375. Taenia taeniaeformis larvae which develop into infective strobilocerci in C3H mice have been studied from the 5th to the 15th day of development (L5–L15), both at light and electron microscope level. The L5 were initially compact, without a central cavity but then become vacuolized. Until stages L7–L8 they were surrounded by a perilarval amorphous layer (PAL) made up of a finely granular material which prevented the host cells from making contact with the larval tegument. The larval volume increased considerably between stages L6 and L8, remained unchanged from L9 to L13, but continued to increase thereafter. The larval cellular layer, which appeared as a single, large ‘syncitial system’, grow until stages L14–L15 when the scolex anlagen began to form. The tegument was initially incompletely organized and was covered by microvilli. These were completely replaced by microtriches from stage L8 onward. Sometimes both microvilli and microtriches were together observed in stage L7. Microvilli fragments, sometimes beaded, could be observed at L5 within the damaged cytoplasm of host cell debris. Very often they were branched at different heights, especially in stages L5–L7. In L10–L15 all undamaged microtriches increased in density and formed bundles which invaded the host cells. In stages L5–L8 and in some L9, muscular bundles started to become organized inside the tegumental distal cytoplasm (TDC), and after become independent in the subtegumental cellular layer (SCL). Until L8–L9 the larvae were surrounded by host cells debris. From stages L8–L10 onwards the adjacent host cells were less damaged though the larval microtriches penetrated them deeply. In stages L5–L7 neutrophils together with macrophages and some damaged hepatocytes were detected, while eosinophils were present only from L8 onward. In the other stages neutrophils clearly diminished in numbers, whereas macrophages had increased. No mastcells and few plasma cells were observed.     

Effects of juvenile hormone on polysome integrity

Juvenile hormone inhibits protein and RNA synthesis in cell cultures from Trichoplusia ni and in the testicular germinal cysts of Hyalophora cecropia pupae in vitro. Sucrose gradient analyses revealed that the polysomes of both the T. ni cells and the germinal cysts were disaggregated almost immediately after the addition of juvenile hormone in vitro with a corresponding dose-dependent increase in monosomes. It is suggested that previous reports revealing juvenile hormone inhibition of ecdysone stimulated RNA and protein synthesis may be due to polysome disaggregation. Further studies demonstrated that the effect is not restricted to insect cells and can be elicited by several other lipids devoid of juvenile hormone morphogenetic activity. Experiments with broken cell preparations and isolated polysomes suggest the necessity of cell membrane integrity for the effect on the polysomes. Several probing studies utilizing cycloheximide, ribonuclease, and high K⁺ concentrations were conducted on the means by which juvenile hormone and other lipids may elicit polysome disaggregation.     

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.     

Molecular Characterization of Echinococcus granulosus Sensu Lato from Farm Animals in Egypt

Little is known on the diversity and public health significance of Echinococcus species in livestock in Egypt. In this study, 37 individual hydatid cysts were collected from dromedary camels (n=28), sheep (n=7) and buffalos (n=2). DNA was extracted from protoscoleces/germinal layer of individual cysts and amplified by PCR targeting nuclear (actin II) and mitochondrial (COX1 and NAD1) genes. Direct sequencing of amplicons indicated the presence of Echinococcus canadenesis (G6 genotype) in 26 of 28 camel cysts, 3 of 7 sheep cysts and the 2 buffalo derived cysts. In contrast, Echinococcus granulosus sensu stricto (G1 genotype) was detected in one cyst from a camel and 4 of 7 cysts from sheep, whereas Echinococcus ortleppi (G5 genotype) was detected in one cyst from a camel. This is the first identification of E. ortleppi in Egypt.     

A study of the microtriches of adult Echinococcus granulosus by scanning electron microscopy

Thompson R. C. A., Houghton A. and Zaman V. 1982. A study of the microtriches of adult Echinococcus granulosus by scanning electron microscopy. International Journal for Parasitology12: 579–583. The microtriches in different regions of the scolex and strobila of sexually mature Echinococcus granulosus were examined using the scanning electron microscope. E. granulosus was found to possess two morphologically distinct types of microthrix. A cylindrical, slender filamentous type of microthrix which appeared to be flexible was restricted to the scolex. On the strobila, the only type of microthrix observed was a flattened blade-like form which appeared to be rigid for most of its length. The occurrence of two distinct types of microthrix in separate regions of the body of E. granulosus suggests that they may be involved in different functional activities.     

Ultrastructure of the cirrus sac of echinophallid tapeworms (Cestoda, Bothriocephalidea) and the terminology of cirrus hard structures

Transmission (TEM) and scanning (SEM) electron microscope methods were used to study the fine structure of the cirrus, cirrus sac, internal seminal vesicle, ejaculatory duct, prostate glands and cirrus armature of Echinophallus wageneri (Monticelli, 1890) and Paraechinophallus japonicus (Yamaguti, 1934) (Bothriocephallidea: Echinophallidae). The cirrus sac of these species has two unique ultrastructural features: a thick wall with two bands of muscles and prominent, rooted hard structures. Rare traits echinophallids share with diphyllobothriideans are microtriches on the ejaculatory duct and with spathebothriideans, well-developed unicellular prostate glands outside the cirrus sac. Because there is a similarity of cirrus armature and rostellar hooks in having a tegumental localisation and in having a heterogenous structure of the blade and root, a cortex, a central pulp region and a recurved apex, these structures are named “modified hooks” instead of spines. They also have a spiral arrangement; no base plate was observed. True spines, as found in trematodes, are between the surface and basal plasma membrane of the external syncytial layer of the tegument, rest on the basal plasma membrane of the distal epithelial cytoplasm, show a homogeneous electron-dark crystalline appearance and are covered by the surface plasma membrane. Aside from the characteristic hooks on the scolex of various cestodes, we see no evidence that would preclude the development of still other specialised structures, such as these modified hooks, from microtriches. In spite of the absence of studies on the development of modified hooks from the cirrus of echinophallids and/or its consideration as derived from microtriches, we assume that like microtriches, formation of modified hooks is from tegumental bodies and therefore they are derivative structures of the cestode tegument.     

Quantitative microscopical studies of the mouse peritoneal macrophage following stimulation in vivo

Summary The results of an objective two- and three-dimensional analysis of the morphological features of normal and triolein-induced mouse peritoneal macrophages are reported. An equivalent circle technique for resolving the effects of volume and surface area on volume-to-surface parameters is described. The method is a simple comparative one which does not require the actual determination of cell volume.Macrophage stimulation promoted increases in mean cell size, cytoplasmic granularity and volume-to-surface ratio. In addition, a reduction in nuclear volume-to-surface ratio accompanied in vivo stimulation. Nucleocytoplasmic ratio remained constant. The equivalent circle procedure showed that the increase in cellular volume-to-surface ratio was due largely to the increase in cell volume; the decrease in nuclear volume-to-surface ratio was primarily the result of a substantial increase in nuclear membrane surface area. Stereological estimations suggest that interiorized cell membrane (in the form of triolein-containing phagosomes) is replaced by newly reconstructed surface membrane.     

Surface ultrastructure of the elasmobranchia parasitizing <Emphasis Type="Italic">Grillotiella exilis</Emphasis> and <Emphasis Type="Italic">Pseudonybelinia odontacantha</Emphasis> (Trypanorhyncha,Cestoda)

The surface ultrastructure of two monotypic trypanorhynch genera is described based on new material of Grillotiella exilis (Linton, 1909) and type material of Pseudonybelinia odontacantha Dollfus 1966. In G. exilis, spiniform microtriches cover the bothrial surfaces and the anterior part of the pars vaginalis posterior to the bothria. Bifurcate microtriches adorn the bothrial margins, filiform microtriches the scolex peduncle, and capilliform microtriches the posterior scolex end. This microthrix pattern resembles that found in, e.g., Grillotia erinaceus (van Beneden, 1858), with the difference that the anterior part of the pars vaginalis is covered with a collar of multidigitate palmate microtriches. The position of Grillotiella within the Grillotiinae, Lacistorhynchidae is supported based on these data. The bothria and scolex peduncle of P. odontacantha are covered with acerosate and unciniform microtriches on the distal bothrial surface and capilliform microtriches on the scolex peduncle. Short filiform microtriches cover the appendix. The microthrix pattern resembles that of the Tentaculariidae but with unciniform and acerosate microtriches densely covering the entire distal bothrial surface. Tegumental grooves are present on the posterior bothrial margin. They can be distinguished from bothrial pits in otobothrioid trypanorhynchs in having similar unciniform microtriches compared to the other parts of the bothrial surface and in lacking any spiniform microtriches. With the absence of bothrial pits as characteristic for the otobothrioids and its characteristic microthrix pattern, P. odontacantha together with Paranybelinia otobothrioides Dollfus 1966, both belonging to the Paranybeliniidae change their position in the most recent system from the Otobothrioidea into the Tentacularioidea.     

The growth performance,intestinal digestive and absorptive capabilities in piglets with different lengths of small intestines

The small intestine is an important digestive organ and plays a vital role in the life of a pig. We tested the hypothesis that the length of the small intestine is related to growth performance and intestinal functions of piglets. A total of 60 piglets (Duroc × Landrace × Yorkshire), weaned at day 21, were fed an identical diet during a 28-day trial. At the end of the study, all piglets were sacrificed, dissected and grouped according to small intestine lengths (SILs), either short small intestine (SSI), middle small intestine (MSI) or long small intestine (LSI), respectively. Positive relationships between SIL and BW, average daily gain (ADG), average daily feed intake (ADFI) and gain-to-feed ratios (G : F) were observed. Final BW, ADG, ADFI and G : F significantly increased (P < 0.05) in MSI and LSI piglets compared with SSI piglets. Short small intestine and MSI had greater jejunal mucosa sucrase and alkaline phosphatase activities (P < 0.05) than LSI piglets. The mRNA level of solute carrier family 2 member 2 (Slc2a2) in the jejunal mucosa of SSI piglets was the greatest. The MSI piglets had a greater (P < 0.05) ileal villus height than other piglets and greater (P < 0.05) villus height-to-crypt depth ratios than LSI piglets. However, the LSI piglets had a greater (P < 0.05) ileal crypt depth than SSI piglets. No significant differences in duodenal, jejunal, caecal and colonic morphologies were detected among the groups. Moreover, luminal acetate, propionate, butyrate and total short-chain fatty acid contents were greater (P < 0.05) in SSI and MSI piglets than those in LSI piglets. In addition, there was greater serum glucose concentration in MSI piglets than other piglets. Serum albumin concentration in SSI piglets was the lowest. In conclusion, these results indicate that SIL was significantly positively associated with growth performance, and in terms of intestinal morphology and mucosal digestive enzyme activity, the piglets with a medium length of small intestine have better digestion and absorption properties.     

Spin label studies of microsomal membranes from Acanthamoeba castellanii in different states of differentiation

Two fatty acid spin labels—[I(1,14)], stearic acid bearing a paramagnetic nitroxide group on carbon 16, and [I(12,3)], stearic acid bearing a paramagnetic nitroxide group on carbon 5—have been used to compare the physical properties of lipid in rough and smooth microsomal membranes from trophozoites and cysts of Acanthamoeba castellanii. Arrhenius plots of rotational correlation times (τ_c) calculated from the spectra for I(1,14) showed an abrupt discontinuity in slope for membranes from both trophozoites and cysts. This occurred at temperatures ranging from ?3 to 1 °C for smooth microsomes and from 8 to 11 °C for rough microsomes for both cysts and amoebae. The value of τ_c at 29 °C, the culturing temperature, in effect scores fluidity of the membrane matrix, and did not show any significant difference for either rough or smooth microsomes during the transition from exponential to stationary phase growth. However, smooth microsomes from cysts showed a 14% increase in fluidity relative to trophozoites, and the fluidity of rough microsomes from cysts tended to be lower. An order parameter (S) calculated from spectra for I(12,3) did not change as a function of encystment for the smooth membranes and increased only slightly for rough microsomes. The activation energy (E_a) for Arrhenius plots of τ_c above the inflection temperature increased as a result of encystment, indicating a greater degree of molecular interaction within the cyst membranes. Moreover, the τ_c plots for both rough and smooth microsomal membranes from trophozoites tended to converge at 29 °C, the growth temperature, whereas plots for cyst membranes were virtually parallel, bracketing those for the trophozoite membranes. This suggests that the trophozoite is able to regulate its membrane fluidity and that cysts, which are resting cells, have lost this regulatory capacity.     

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.     

Comparative morphology of surface ultrastructure of diphyllobothriidean tapeworms (Cestoda: Diphyllobothriidea)

Microtriches on the scolices and adjacent strobila of seven species of diphyllobothriidean cestodes (Bothridium pithonis, Cephalochlamys namaquensis, Dibothriocephalus latus, Duthiersia expansa, D. fimbriata, Ligula intestinalis, and Schistocephalus solidus) from different hosts (frogs, snakes, lizards, birds, and mammals) and biogeographic areas were examined using scanning and transmission electron microscopy. The basic structure of the tegument of the seven species studied does not differ markedly from that found in other cestodes. The main characteristic is the presence of electron‐dense bodies and vesicles in the distal cytoplasm. However, this study has shown differences in the morphology of microtriches even among species of the same family. Two different types of microtriches were found, filitriches and spinitriches, with the latter represented by two forms. Our study reveals that capilliform filitriches are most commonly found in Diphyllobothriidea. They were observed mainly on the strobila and the scolices of all but one studied species; individuals of L. intestinalis bore only coniform spinitriches on their surface. The same type of microtriches was found on the cirrus in D. latus. Gladiate spinitriches covered the scolex in both species of Duthiersia, and gladiate spinitriches interspersed with capilliform filitriches were observed on the anterior part of the strobila in D. fimbriata and the posterior part of the scolex in B. pithonis. Individuals of C. namaquensis were covered only by small acicular filitriches. No obvious pattern in the type and distribution of microtriches was observed among species that belong to different families and parasitize distantly related definitive hosts.     

Different life cycle strategies of the dinoflagellates Fragilidium duplocampanaeforme and its prey Dinophysis acuminata may explain their different susceptibilities to the infection by the parasite Parvilucifera infectans

Some marine dinoflagellates form ecdysal cyst (=temporary cysts) as part of their life cycle or under unfavorable growth conditions. Whether the dinoflagellates form ecdysal cysts or not may influence susceptibility to parasitism. In this study, parasite prevalence relative to inoculum size of the parasitoid Parvilucifera infectans zoospores for two dinoflagellate hosts (i.e., Fragilidium duplocampanaeforme and Dinophysis acuminata), which have different life cycle strategies, was examined. Further, susceptibility of cysts to parasitism, encystment signal, duration of encystments, and effects of induced encystment on diel periodicity, using ecdysal cyst-forming F. duplocampanaeforme were explored. The percent hosts infected by P. infectans plotted as a function of inoculum size showed a sharp increase to a maximum in D. acuminata, but a gradual linear rise in F. duplocampanaeforme: while the parasite prevalence in D. acuminata increased to a maximum of 78.8 (±2.4%) by a zoospore:host ratio of 20:1, it in F. duplocampanaeforme only reached 8.9 (±0.3%), even at a zoospore:host ratio of 120:1. In F. duplocampanaeforme, infections were observed only in the vegetative cells and not observed in ecdysal cysts. When exposed to live, frozen, and sonicated zoospores and zoospore filtrate, F. duplocampanaeforme formed ecdysal cysts only when exposed to live zoospores, suggesting that temporary cyst formation in the dinoflagellate resulted from direct contact with zoospores. When the Parvilucifera zoospores attacked and struggled to penetrate F. duplocampanaeforme through its flagellar pore, the Fragilidium cell shed all thecal plates, forming a ‘thecal cloud layer’, in which the zoospores were caught and immobilized and thus could not penetrate anymore. The duration (35 ± 1.8 h) of ecdysal cysts induced with addition of zoospores was significantly longer than that (15 ± 0.8 h) of normally formed cysts (i.e., without addition of zoospores), thereby resulting in delayed growth as well as influencing the pattern of diel periodicity. The results from this study suggest that in addition to the classical predator-prey interaction and allelopathic interaction, parasitism and its accompanying defense can make the food web dynamics much more complicated than previously thought.     

Genetic Diversity of Echinococcus granulosus in Center of Iran

Hydatid cyst caused by Echinococcus granulosus is one of the most important parasitic diseases around the world and many countries in Asia, including Iran, are involved with this infection. This disease can cause high mortality in humans as well as economic losses in livestock. To date, several molecular methods have been used to determine the genetic diversity of E. granulosus. So far, identification of E. granulosus using real-time PCR fluorescence-based quantitative assays has not been studied worldwide, also in Iran. Therefore, the aim of this study was to investigate the genetic diversity of E. granulosus from center of Iran using real-time PCR method. A total of 71 hydatid cysts were collected from infected sheep, goat, and cattle slaughtered in Isfahan, Iran during 2013. DNA was extracted from protoscolices and/or germinal layers from each individual cyst and used as template to amplify the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) (420 bp). Five cattle isolates out of 71 isolates were sterile and excluded from further investigation. Overall, of 66 isolates, partial sequences of the cox1 gene of E. granulosus indicated the presence of genotypes G1 in 49 isolates (74.2%), G3 in 15 isolates (22.7%), and G6 in 2 isolates (3.0%) in infected intermediate hosts. Sixteen sequences of G1 genotype had microgenetic variants, and they were compared to the original sequence of cox1. However, isolates identified as G3 and G6 genotypes were completely consistent with original sequences. G1 genotype in livestock was the dominant genotype in Isfahan region, Iran.     

Echinococcus granulosus: penetration of macromolecules and their localization on the parasite membranes of cysts

Host IgG was revealed in the concentric lines of the laminated membranes of mouse, human, gerbil, and sheep hydatid cysts by the indirect fluorescent antibody test on cryostat and paraffin embedded sections. The penetration of these immunoglobulins into the laminated membrane was demonstrated by transplantation studies. Peroxidase (MW 40,000) and fluorescein-labeled rabbit IgG (MW 150,000) were also shown to reach the germinal membrane by in vitro studies. The results are discussed in terms of their immunological and pharmacological implications, present knowledge on the penetration of macromolecules into hydatid cysts and the structure of the laminated membrane.     

Echinococcus granulosus: Protoscolicidal effect of high intensity focused ultrasound

High intensity focused ultrasound (HIFU) is a new non-invasive technique which can cause cell death and tissue necrosis by focusing high-energy ultrasonic waves on a single location. The aim of our work is to investigate the damaging effect of HIFU on Echinococcus granulosus protoscolices, as well as its inhibitory effect on growth of hydatid cysts derived from protoscolices. The damaging effect of HIFU on protoscolices was investigated by following parasite mortality after irradiation, while the inhibitory effect was investigated by infection experiments in vivo. The results demonstrated that HIFU was able to damage protoscolices and the protoscolicidal effect was dose-dependent and showed late-onset. The growth of protoscolices that survived the exposure to HIFU was obviously suppressed in vitro, and the mean weight of hydatid cysts resulting from such protoscolices in the experimental group was less than that in controls. Evidences including the protoscolicidal effect, fragmentized protoscolices and low post exposure temperatures, suggest that cavitation may contribute to the protoscolicidal effect of HIFU. In addition, the structure of the germinal membrane in cysts developing from the irradiated protoscolices was not as normal or intact as that from non-irradiated ones, and morphological changes related to degeneration were observed, suggesting that HIFU could prevent protoscolices from developing normal germinal membrane and consequently stop the proliferation of secondary hydatid cysts. HIFU demonstrated damaging effect on protoscolices, inhibited the growth of protoscolices in vitro and in vivo, and could be a possible therapeutic option for cystic echinococcosis.