Fasciola gigantica: the in vitro effects of artesunate as compared to triclabendazole on the 3-weeks-old juvenile

The in vitro effect of artesunate (ATS) on the 3-week-old juveniles of Fasciola gigantica was compared with triclabendazole (TCZ) by incubating the parasites in M-199 medium containing the drugs at concentrations of 20, 40, and 80 μg/ml for 1, 3, 6, 12, and 24h. The anthelmintic activities of these drugs were evaluated based on the relative motility value (RM) and the alterations of the tegument as observed by scanning (SEM) and transmission (TEM) electron microscopy. The RM values of TCZ-treated flukes decreased significantly from 6 to 24h for all dosages. For ATS-treated flukes, RM value decreased markedly from 12 to 24h, but the rates of decline were less than TCZ at the same doses. When observed by SEM, the tegument showed similar sequence of morphological changes after treatments with both drugs, comprising of swelling of tegumental ridges, followed by blebbing and later rupturing of the blebs, leading to erosion and lesion, and disruption of the tegument. When examined by TEM, ultrastructural changes in the tegument and associated structures after treatments with TCZ and ATS were similar which comprised of swelling, blebbing of the tegument, dilation of basal infoldings, and depolymerization of the microtrabecular network. After a longer incubation time, the tegument was completely sloughed off and the tegument cell bodies became necrotic. Additionally, in ATS-treated flukes, mitochondria showed severe swelling, rupturing of outer membrane, and their interior filled with flocculent materials.     

Ultrastructure of the tegument of Metamicrocotyla macracantha (Alexander, 1954) Koratha, 1955 (Monogenea, Microcotylidae).

The ultrastructure of the body tegument of Metamicrocotyla macracantha (Alexander, 1954) Koratha, 1955, parasite of Mugil liza from Brazil, was studied by transmission electron microscopy. The body tegument is composed of an external syncytial layer, musculature, and an inner layer containing tegumental cells. The syncytium consists of a matrix containing three types of body inclusions and mitochondria. The musculature is constituted of several layers of longitudinal and circular muscle fibers. The tegumental cells present a well-developed nucleus, cytoplasm filled with ribosomes, rough endoplasmatic reticulum and mitochondria, and characteristic organelles of tegumental cells.     

东方杯叶吸虫体被的超微结构

扫描电镜显示东方杯叶吸虫体被有许多族生棘,具纤毛乳突和无纤毛窝状乳突,附着器皮层特化为微毛。透射电镜观察表明,皮层是由合胞体、基膜和肌肉层组成,合胞体通过细胞通道与皮层细胞相连。文中详细描述了这些结构,揭示无纤毛窝状乳突为腺乳突,皮层细胞间存在线粒体细胞和附着器皮层形成微毛,探讨了这些结构的生理功能。体外培养成虫皮层结构的某些不正常变化,如皮层细胞的空虚松散,可作为评价吸虫体外培养的一种指标。     

Ultrastructure and cytochemistry of the tegument of Orthocoelium scoliocoelium and Paramphistomum cervi (Trematoda: Digenea)

The tegument of Orthocoelium scoliocoelium and Paramphistomum cervi was examined using histochemical techniques and electron microscopy. On the basis of the distribution of acid and alkaline phosphatase (E.C. 3.1.3.2, E.C. 3.1.3.1), non-specific esterase (E.C. 3.1.1.1), cholinesterase (E.C. 3.1.1.7) and succinate dehydrogenase (E.C. 1.3.99.1) at light microscope level two distinct regions were recognized, an outer and an inner zone. Electron microscopy revealed that the tegument comprises an outer surface syncytium underlain by a thick subsyncytial zone and musculature. Deeper still occur the nucleated "tegumental cells". The latter are in cytoplasmic continuity with the surface syncytium via vacuolated cytoplasmic trabeculae which traverse the muscle layers and the subsyncytial zone. Three types of tegumental cells each lacking mitochondria were observed. The T1 cells synthesize discoid and electron dense T1 bodies while T2 cells produce oval and electron lucent T2 bodies. The third type of tegumental cells apparently produce no secretory bodies and may represent an embryonic cell type. The surface syncytium contains T1 and T2 secretory bodies and is bounded apically by a plasma membrane invested externally by a fuzzy and filamentous glycocalyx. The surface syncytium lacks mitochondria and is traversed by infoldings of the basal plasma membrane. Beneath the surface syncytium the subsyncytial zone is largely comprised of fibrous interstitial material. This zone, which is particularly thick in the amphistomes, is traversed by trabeculae and extensions of underlying parenchymal cells which usually contain mitochondria and lysosomes. The subsyncytial zone overlies numerous circular and longitudinal muscle fibres. The absence of mitochondria and enzymes associated with active transport suggests that the amphistome tegument may be mainly specialized for protection of the worm against mechanical and chemical conditions prevailing in the rumen. Active uptake of nutrients is probably not a primary function.     

扩张莫尼茨绦虫(圆叶目:裸头科)节间腺的超微结构及组织化学

用光学显微镜和透射电子显微镜观察了扩张莫尼茨绦虫节间腺形成过程的精细结构及一些组化变化。结果表明：节间腺是扩张莫尼茨绦虫皮层的特化部分,由节片后缘的皮层及其邻近细胞体向绦虫实质组织中陷入开始其形成过程,随着虫体发育的进行,新的陷入不断形成,原陷入的部分不断脱离皮层形成簇状腺体结构。节间腺的数目随着体节的发育不断增加,幼节中仅有少数几个(6～9个),而远端的孕节中多于100个。电镜下可见腺细胞体由细胞质管与腺皮层相联,簇状腺体结构为一合胞体形态,腺细胞体围绕并开口于椭球体或不规则形状的皮层腔中。离腺皮层远的腺细胞体电子密度高并含有与腺皮层相应的典型分泌颗粒,而靠近腺皮层的腺细胞体电子密度低,所含分泌颗粒较少。扩张莫尼茨绦虫节间腺的组化性质尚不完全清楚。糖与蛋白质等组化结果不稳定,随染液pH值及染色时间的变化等多种因素而改变。基于我们的研究及其他研究者的观察表明,节间腺可能参与外源基质形成虫卵的转运,同时他们可能在虫体节片脱落及虫卵溢出时起作用。     

Fasciola hepatica: tegumental changes induced in vitro by the deacetylated (amine) metabolite of diamphenethide

The effect of the deacetylated (amine) metabolite of diamphenethide (10 mugm/ml) on the tegument of Fasciola hepatica over a 24 hr period in vitro has been determined by means of transmission electron microscopy. In the tegumental syncytium, there is an initial accumulation of T2 secretory bodies at the apical surface (after 6 hr), together with increased exocytosis of secretory bodies and blebbing of the surface membrane. After 9 hr, the two surfaces of the fluke show different tegumental responses to drug treatment with a marked swelling of the basal infolds in the dorsal tegument, while the ventral tegument remains normal. By 18 hr, the swelling in the dorsal tegument is very severe, the entire basal region becoming edematous. In some areas, the tegument becomes detached to expose the basal lamina. The ventral tegument retains a fairly normal morphology, although there is a slight swelling of the basal infolds. The edema spreads internally to the cell bodies, beginning after 9 hr on the dorsal side of the fluke and 18 hr on the ventral side. By 18 hr, the flooding on the dorsal side is very severe and the cells attenuated, retaining few contacts with the surrounding parenchyma. From 9 hr onwards, there are progressive changes in cell structure, including a decrease in amount of granular endoplasmic reticulum and extent of its ribosomal covering, a decrease in numbers of secretory bodies, a swelling of the trans-most Golgi cisternae and disruption of the release of secretory bodies, and a swelling and disorganization of the mitochondria. The results are discussed in relation to the postulated activity of the deacetylated (amine) metabolite of diamphenethide as a Na+ ionophore.     

Antigenic localities in the tissues of Metagonimus yokogawai observed by immunogoldlabeling method]

In order to determine the antigenic localization in the tissues of the adult Metagonimus yokogawai, immunogoldlabeling method was applied using serum immunoglobulins(IgG) of cats which were infected with isolated metacercariae from Plecoglossus altivelis. The sectioned worm tissue was embedded in Lowicryl HM 20 medium and stained with infected serum IgG and protein A gold complex(particle size: 12 nm). It was observed by electron microscopy at each tissue of the worm. The gold particles were observed on the tegumental syncytium as well as cytoplasm of tegumental cells and epithelial lamella of the caecum. The gold particles were not observed on the basal lamina of the tegument, interstitial matrix of the parenchyma, the muscle tissue and mitochondria of the tegument. The gold particles were specifically labeled in the secretory granules in the vitelline cells. They were also labeled on the lumen of bladder and egg shell. The above findings showed that antigenic materials in the tissue of adult worms were specifically concentrated on the tegumental syncytium as well as cytoplasm of tegumental cells and epithelial lamella of the caecum.     

Ultrastructure of the tegument of Saccocoelioides godoyi

The tegument of adult Saccocoelioides godoyi Kohn & Froes, 1986 (Digenea: Haploporidae), specimens of which were collected from the intestine of the freshwater fish, Leporinus friderici (Bloch, 1794) (Anostomidae) from the reservoir of Itaipu Hydroelectric Power Station, Parana State, Brazil, was studied by transmission electron microscopy. The tegument comprises an external anucleate layer, covered by a surface plasma membrane and associated glycocalyx. The surface layer is bound by the basal plasma membrane and contains spines, two types of inclusion bodies and mitochondria. Tegumental cell bodies are located beneath the surface musculature and contain a single nucleus, cytoplasm with rough endoplasmic reticulum, mitochondria, ribosomes, and inclusion bodies similar to those found in the external layer. Cytoplasmic strands connect the cell bodies to the external surface layer, suggesting that the inclusion bodies are produced in these cells and pass up into the syncytium, as is known for other digeneans from experimental evidence.     

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.     

Scanning electron microscopy of in vitro serum-mediated destruction of juvenile Fasciola hepatica

The tegumental surface of immature Fasciola hepatica was damaged when incubated in vitro with serum collected from an experimentally infected calf. Degeneration of the tegumental surface was observed by scanning electron microscopy (SEM) at 4 hr. after incubation. Decomposition was observed 8 to 12 hr after incubation and complete destruction of the tegument occurred by 16 hr. The flukes became inactive after 8 to 12 hr of incubation. None of the above findings were observed for the tegument of flukes incubated in tissue culture media or in media containing normal calf serum and the trematodes remained motile throughout the incubation period. Latex particles were used as an immunological marker for SEM studies to determine if gamma globulin could be responsible for the observed changes and, if so, the site of antibody attachment. The coated latex particles covered the entire surface of flukes recovered from mice 5 days after infection with metacercariae. In contrast, latex particles coated with either normal gamma globulin or gamma globulin from serum of the experimentally infected calf that had been adsorbed with disrupted adult flukes were not attached to the surface of the flukes. Absorption of the serum with disrupted, adult flukes decreased the concentrations of immunoglobulins (Ig)G1 and G2 whereas IgA and IgM were apparently not affected.     

Ultrastructural observations on the tegument and associated structures of the monogenean Cichlidogyrus halli typicus (Price & Kirk, 1967) Paperna, 1979.

The tegument of the ancyrocephaline monogenean gill parasite Cichlidogyrus halli typicus is basically similar to that of other monogeneans. However, there are some tegumental features shared with relatively few other monogeneans, namely the presence in the surface tegument of extensive regions of homogeneous granular cytoplasm, a close association between myofibres and the basal region of the tegument and the production of two different tegumental secretory bodies by the same cyton. It is suggested that the regions of homogeneous granular cytoplasm found in the troughs between the circular muscle fibres provide some kind of support for the tegument, perhaps related to contraction of the tegumental circular muscle fibres and body shape changes. Three kinds of transtegumental sensory structures are recognized, namely a uniciliated bulb, a bulb bearing a non-ciliary process and a non-ciliated bulb. The latter two kinds are reported for the first time in monogenans. Possible functions of these sensory structures are discussed.     

Topography and ultrastructure of the tegument of Lecithochirium musculus (digenea: Hemiuridae), a parasite of the European eel Anguilla anguilla (osteichthyes: Anguillidae)

The tegumental ultrastructure of the stomach fluke Lecithochirium musculus was studied using scanning and transmission electron microscopy. The surface of the tegument was smooth and covered by transverse cytoplasmic ridges. Cobblestone‐like units of the tegument were observed on the ventral surface. Invagination and evagination of the ecsoma induced variations in the tegumental surface. The ultrastructural study revealed that the tegument of L. musculus had a typical syncytial organization with a distal cytoplasm lying over a basal matrix and cytons. Two types of intra‐tegumental sensory structures were observed. Type 1 sensory receptor was a domed‐like fusiform structure consisting of a smooth elevation of the tegument. Four receptors of this type were observed on the anterior dorsal surface of the fluke. Three nerve bulbs filled with electron‐lucent material and mitochondria composed this receptor. Hemispherical electron‐dense collars were observed at the top of the nerve bulbs. Striated rootlets laid just beneath the hemispherical electron‐dense collars. Type 2 sensory receptor presented two morphological variations, i.e., a bulb‐like monolobed structure, and a bulb‐like bilobed structure observed at two different degrees of evagination. For both variations, the nerve bulb enclosed mitochondria, electron‐lucent material, and a conical electron‐dense collar from which extended a striated rootlet. Numerous sensory receptors of this type were observed around the ventral sucker. J. Morphol., 2012. © 2011 Wiley Periodicals, Inc.     

Antigenic localities in the tissues of the young adult worm of Paragonimus westermani using immunogold labeling method

In order to observe the antigenic localization in the tissues of the young adult Paragonimus westermani, immunogold labeling method was applied using serum immunoglobulins(IgG) of the dog which infected with isolated metacercariae from Cambaroides similis. The sectioned worm tissue was embedded in Lowicryl HM 20 medium and stained with infected serum IgG and protein A gold complex (particle size; 12 nm). It was observed by electron microscopy at each tissues of the worm. The gold particles were not observed on the basal lamina of the tegument, interstitial matrix of the parenchyma, the muscle tissue and mitochondria of the tegument. The gold particles were specifically labeled in the secretory granules in the vitelline cells. They were predominantly labeling on the epithelial lamela and lumen of caecum. The above finding showed that antigenic materials in young adult worm tissue were specifically concentrated on the tegumental syncytium as well as cytoplasm of tegumental cells.     

Effects of praziquantel and artesunate on the tegument of adult Schistosoma mekongi harboured in mice

The effects of praziquantel and artesunate on the tegument of adult Schistosoma mekongi harboured in mice were compared using scanning electron microscopy (SEM). Forty-two mice infected with S. mekongi for 49 days were treated intragastrically with either 300 mg/kg praziquantel or 300 mg/kg artesunate. Mice were sacrificed 1 or 3 days post-treatment. Worms were collected by perfusion and examined by SEM. One to 3 days after administration of artesunate, the tegument of S. mekongi showed severe swelling, vacuolization, fusion of the tegumental ridges and loss or shortening of the spines on the trabeculae, collapse and peeling. Praziquantel induced similar tegumental alterations as those observed after administration of artesunate, but they were less severe. Three days post-treatment, there was evidence of recovery only in the case of praziquantel. The results of our study suggest that artesunate is more effective than praziquantel in causing tegumental damage in adult S. mekongi, and provides a basis for subsequent clinical trials.     

鱊头槽绦虫原尾蚴皮层的超微结构观察

利用透射电镜观察了头槽绦虫 (Bothriocephalusacheilognathi)原尾蚴皮层的超微结构。头槽绦虫原尾蚴的皮层为典型的合胞体结构。皮层表面有浓密的微毛与少量的突起。与成虫相比 ,原尾蚴的微毛长而且粗 ,呈现单态性 ,推测在进入终末宿主的过程中有脱落和再生现象。突起分布在头部与体侧 ,含有较少的电子致密颗粒。在原尾蚴皮层细胞质中观察到三种腺细胞的分泌过程 ,即顶分泌、外分泌和微分泌过程。原尾蚴身体后部观察到尾钩和穿刺腺管道的开口。在核周区和纤维层下可见发育较好的环肌与纵肌。本文还对原尾蚴皮层突起的功能进行了探讨。     

Fine structure of the germinal membrane of Echinococcus granulosus cysts.

The ultrastructure of the hydatid cyst germinal membrane was studied. It was divided into 3 regions, the tegument, the tegumental cell region, and the innermost area bordering the cyst cavity. The morphology of tegumental, muscular, flame, duct, and glycogen-containing cells and cells containing lysosomal-like bodies is described. The significance of these findings is discussed in terms of the possible function of these structures and present knowledge on the penetration of macromolecules into hydatid cysts.     

河鲈锚首吸虫体壁的超微结构观察

寄生鳜鳃部的河鲈锚首吸虫的体壁由表皮合胞体、基板、环肌、纵肌和表皮细胞核周体所组成。合胞体顶部质膜起伏形成表皮的嵴纹,基部质膜折叠形成指状突起伸入到合胞体中。合胞体表面覆盖着一层糖萼。河鲈锚首吸虫的表皮中含有四类分泌体,即电子致密的分泌颗粒、中等电子致密的分泌颗粒、有膜包围的电子稀疏的分泌体和多囊体．可见分泌体和合胞体基质通过胞吐作用排到体外,未见吞饮小泡,推测表皮的主要功能在于分泌和渗透压调节而非营养吸收。在外侧头瓣的乳突状结构所在处,合胞层较薄,基板平滑,在实质组织中的一腔体样结构中可见囊状体、电子致密度各异的颗粒体、泡状体和电子致密的基质团,神经突起分布于腔体周围,这类乳突可能代表一类新的非纤毛感受器类型。     

Fasciola hepatica: tegumental alterations in adult flukes following in vitro and in vivo administration of artesunate and artemether

The tegumental changes in adult Fasciola hepatica induced by artemether and artesunate were assessed utilizing scanning electron microscopy (SEM). F. hepatica were incubated with artemether and artesunate for 48h at a concentration of 10microg/ml in the absence or presence of haemin. For the latter experiment both, a triclabendazole-resistant and sensitive F. hepatica isolate were used. For the in vivo studies rats were treated with single 200mg/kg oral doses of artemether and artesunate and flukes recovered from the bile ducts after 24-96h. SEM analysis of the flukes incubated in the presence of the drugs without haemin showed only minor and localized damage of the tegument. In the presence of haemin extensive tegumental damage, including sloughing, blebbing and eruptions, particularly in the ventral and dorsal mid-body and tail region, was evident. No difference in the extent of damage could be observed between artemether and artesunate and between the triclabendazole-resistant and non-resistant flukes. After 24h in vivo disruption of the tegument was evident in the artemether-treated flukes, and the damage increased in severity 48-72h post-treatment. Sloughing, swelling and extensive furrowing of the tegument was observed in several flukes, in particular in the tail region and the ventral apical cone region. In the artesunate treatment, tegumental damage was evident after 72h, but seemed slightly less pronounced when compared to the artemether-treated specimens examined at the same time point. Concluding our experiments confirm that artemether and artesunate are potent fasciocidal drugs and the tegument of adult F. hepatica appears to be a target for the action of these drugs.     

Tegumental changes in adult Schistosoma mansoni harbored in mice treated with artemether

A detailed temporal examination was made of alterations induced by artemether in the tegument of adult Schistosoma mansoni worms using scanning electron microscopy (SEM). Mice infected with S. mansoni cercariae 42 days previously were treated intragastrically with artemether at a single dose of 400 mg/kg. Groups of 3 mice were killed at 24 hr, 72 hr, and 7 days after treatment; the worms were collected by perfusion and examined by SEM. Twenty-four hours after artemether treatment, focal damage to the tubercles on the tegumental surface of male worms was seen. In both male and female worms, there was focal swelling and fusion of tegumental ridges, and sometimes peeling. After 72 hr, the damage to the tegument had increased, especially in female worms, with extensive swelling, fusion, and peeling of the tegumental ridges. In the most severely damaged worms, host leukocytes were seen to be adhered to the damaged tegument. Damage to the oral sucker was also occasionally seen in both male and female worms. Seven days after treatment, the appearance of the tegument had returned to normal in some male and female worms, whereas others still showed apparent damage. The results demonstrate that artemether damages the tegument of adult S. mansoni, and the intensity of damage is more severe in female worms than in males.     

Fasciola hepatica: Surface tegumental responses to in vitro and in vivo treatment with the experimental fasciolicide OZ78

The tegumental alterations in adult Fasciola hepatica induced by the experimental fasciolide OZ78 were investigated utilizing scanning electron microscopy (SEM). Twelve weeks post-infection with F. hepatica, rats were treated with a single 100mg/kg oral dose of OZ78 and flukes were recovered from the bile ducts after 24-72 h. In vitro F. hepatica were incubated with OZ78 for 48 h at a concentration of 10 microg/ml in the absence or presence of haemin. Twenty-four and 48 h post-treatment of rats disruption of the tegument of F. hepatica as blebbing, swelling and furrowing was evident. The recovery of flukes 72 h post-treatment was low. Flukes examined at this time point showed an increasing severity of tegumental damage as sloughing and absence of spines, in particular in the tail region. SEM analysis of F. hepatica incubated in the presence of OZ78 without haemin showed only minor and localized damage of the tegument. In the presence of haemin extensive tegumental damage, including sloughing or blebbing, in particular in the anterior part, was observed. In conclusion, our experiments confirm the interesting fasciocidal properties of OZ78. The tegument of adult F. hepatica might play a role in the action of this drug.