Identification of circulating antibodies in fasciolosis and localization of 66 kDa antigenic target using monoclonal antibodies.

We identified three specific circulating antibodies in serum of cattle naturally infected with Fasciola gigantica. Two of the antibodies were found to react specifically to 97 and 66 kDa antigenic molecules of adult worm tegumental membrane extract. The third antibody was identified by the reaction with 26-28 kDa molecule of the excretory/secretory antigens. Monoclonal antibody against 66 kDa protein was developed and used for localization of its antigenic target in adult worm frozen sections. The experiment demonstrated that 66 kDa protein is a component on the outer surface membrane and on the membrane lining of the caecal epithelial of adult worm. The 66 kDa antigen was considered as a promising candidate for immunodiagnosis and vaccine.

Fasciola gigantica: studies of the tegument as a basis for the developments of immunodiagnosis and vaccine.

The tegument of bile-dwelling Fasciola gigantica is the interfacing layer that helps the parasite to maintain its homeostasis, and evade the hostile environment, including the host's immune attacks. The tegument is a syncytial layer about 10 mm thick, that is formed by the fusion of cytoplasmic processes of tegument cells, whose soma lie underneath the two muscle layers. The surface of the tegument is highly folded and invaginated into numerous ridges, pits and spines, which help to increase the surface area of the tegument for the absorption and exchanging of molecules, as well as for attachment. The outer membrane covering the tegument is a trilaminate sheet about 12 nm thick, and coated with a carbohydrate-rich glycocalyx layer that also bears high negative charges. Some host molecules may also be adsorbed onto this layer. These unique characteristics enable the parasite to evade the antibody-dependent cell-mediated cytotoxicity (ADCC) reaction exerted by the host. The outer membrane and glycocalyx is continuously replaced by the reserved membrane synthesized and stored in secretory granules of tegument cells, that are transported via cell processes towards the tegument by microtubules. The basal membrane of the tegument is trilaminate and invaginated to form membrane infoldings with closely aligned mitochondria. The tegument cytoskeleton is composed of a highly cross-linked network of 4-6 nm knobby microtrabecular fibers, bundles of intermediate filaments, microtubules that splay out from the tegument cells' processes. Major proteins of the cytoskeleton are actin, paramyosin and tubulin. The flukes' antigens that can elicit strong immunological responses in animal hosts are synthesized and released mainly from the tegument and the cecum. The majority of antigens derived from the surface membrane and the tegument are of MW 97, 66, 58, 54, 47 and 14 kDa, while those released from the cecum are cysteine proteases of MW 27, 26 kDa. Monoclonal antibodies have been raised against some of these antigens, and have been employed in immunodiagnosis of the infection. From the protection conferred to animal models and the in vitro killing assays of young parasites by specific antibodies, candidate vaccines could be selected from these antigens, such as, an antioxidant enzyme, glutathione-S-transferase, the digestive enzyme cysteine proteases, the surface-tegument proteins, such as fatty acid binding protein (14 kDa), membrane proteins (at 66 kDa), as well as muscle protein paramyosin, and hemoprotein. Ongoing research have been directed at deciphering the genetic codes and the syntheses of some of these antigens by recombinant DNA technology.

The potential alveolar hazard of carbon dioxide laser-induced smoke.

Carbon dioxide laser is a continuous wave laser, it is well known for its capacity of tremendous smoke production while contact with tissue. Smoke may cause nausea, vomiting, headache and airway irritation. Smoke particles 0.5-2 micrometers in diameter usually travel down the tracheobronchial tree and lodge in the alveoli posing a health hazard. The objectives of this study were to evaluate possible health hazards of carbon dioxide laser smoke in the operating room environment, by determining the size and density of smoke particles also determine the efficacy of surgical masks as a smoke protectant. Ten fresh specimens of papillomatous tissue obtained from the patients were lased by carbon dioxide laser in a continuous mode. The plume generated was collected by 0.45 micrometers pore size microfilter which was attached to the tip of a suction hose connecting the smoke evacuator. The effectiveness of 2 types of commonly used surgical masks were also determined by trapping the smoke after passing through each mask using the same model. Smoke particles were evaluated by scanning electron microscope. The smoke particle density of microfilter that directly trap plume averaged 6 particles/mm2, particles ranging in size from 0.5-27 micrometers, of which 70 per cent were 0.8 micrometers. For the particles trapped after passing through both cotton and paper surgical mask, the size were ranging from 1.6-37 micrometers where 65 per cent were 3.7 micrometers and the particle density average 2.7/mm2. We concluded that the smoke particles derived from carbon dioxide laser application are within the alveolar hazard zone. The conventional surgical masks may not be an effective tool against laser smoke hazard.

Scanning electron microscopic study of microfilaria and the third stage larva of Wuchereria bancrofti.

The surface structures of microfilaria and of the third stage larva of Wuchereria bancrofti were studied by scanning electron microscopy. Distinct features were observed that could be used for differentiating species of this parasite. Specifically, the sheath of microfilariae of W. bancrofti projected beyond the head. The head region of the microfilaria was composed of a cephalic cap with hook, mouth and amphidial opening, and its cuticle showed annulation. Spines were absent at the first transverse annulation, and the tail end showed a slight constriction. In the infective stage larva, characters which are used for differentiating species, such as the two bubble-like ventro-lateral papillae and one dorso-terminal papilla were rather similar to each other in size, but the grooves seen around the base were absent. A previously unreported feature of the third stage larva of W. bancrofti that was discovered in this study is a papilliform process on the left side of the posterior region, between the anus and the tail end.

Diagnosis of cattle fasciolosis by the detection of a circulating antigen using a monoclonal antibody.

A monoclonal antibody (MoAb) 1C12 that reacts with a 66 kDa surface tegumental (ST) antigen of adult worms of Fasciola gigantica was used to detect circulating antigen in sera of experimentally and naturally infected cattle. A combination of rabbit anti ST-antigens and MoAb 1C12 were used to capture and detect the circulating antigen in sandwich ELISA. The dilutions of 1:1,000 of rabbit anti ST-antigens and 1:100 for MoAb 1C12 were used to reduce cross-reactivity with other trematodes' antigens. The circulating antigen of F. gigantica was demonstrated in sera of all experimentally infected animals as early as the first week after the infection, and it remained detectable until the experiment was terminated at week 32 after the infection. Of the 97 serum samples from naturally infected cattle, the sensitivity of 86.6% was observed when the cut-off point was calculated from 32 serum specimens from uninfected control calves. The sensitivity increased to 100% when the commercial fetal calf and trematode-free baby calves sera were used for calculation of the control cut-off point. Based on these results, the combination of rabbit anti ST-antigens and MoAb 1C12 sandwich ELISA appeared to be sensitive, specific, and applicable in the immunodiagnosis of fasciolosis in cattle for epidemiological study and monitoring of chemotherapeutic efficacy.

Development and characterization of monoclonal antibodies against excretory-secretory antigens of Fasciola gigantica.

Monoclonal antibodies (MAbs) directed against Fasciola gigantica excretory-secretory (ES) antigens were developed from BALB/c mice. Four were selected for further study, from the panel of hybridomas. The antigen specificities of these MAbs were characterized and localized by enzyme-linked immunoeletrotransfer blot (EITB) and immunoperoxidase technique. The target epitopes of these MAbs are 66 kDa protein (MAb 2D10), 66 and 27-26 kDa proteins (MAbs 5D10 and 4F5) and 27-26 kDa protein (MAb 2D9). MAb 2D9 reacted to the antigenic components of the luminal content and epithelial cell lining the cecum, whereas MAb 2D10 reacted specifically to the antigens of the tegument and surface membrane. It was found that all MAbs cross-reacted to various degrees with the antigens extracted from Schistosoma mansoni, S. mekongi, S. spindale and Paramphistomum spp. However, when MAbs were diluted to 1:100 or 1:400 significant reduction of their cross-reactivities was observed.

Opisthorchis viverrini: effect of praziquantel on the adult tegument.

Ultrastructural changes of the tegument of adult liver flukes, Opisthorchis viverrini, after in vitro incubation in Minimal Essential Medium containing 0, 0.1, 1.0 and 10.0 micrograms/ml of anthelminthic praziquantel for 5, 15, 30, 45 and 60 minutes were investigated by scanning (SEM) and transmission (TEM) electron microscopy. SEM observations showed that the surface damage was composed of blebbing due to the swelling of microvilli, followed later by the disruption of these structures to form lesions that caused the erosion and desquamation of the surface. Sensory papillae, by contrast, appeared relatively unaffected. The surface changes could be observed at all doses but the extent of damage increased with increasing duration of incubation and concentration of the drug. The ventral as well as the dorsal surfaces exhibited similar change, whereas the anterior part tended to be damaged less than the posterior part. Under TEM observations, the earliest sign of changes was the depolymerization of the microtrabecular network in scattered foci, which resulted in the formation of non-membrane-bound vacuoles under microvilli. The basal infoldings also became dilated, and some turned into membrane-bound vacuoles in the basal zone. Subsequently, microvilli became enlarged, and eventually formed blebs that later rupture to form lesion spots as observed in the SEM. Finally, the microtrabecular network in all regions broke down, creating vacuoles of various sizes throughout the tegument, leading to its total disintegration and detachment. The sequence of morphological changes was generally similar at all doses; however, the changes occurred faster at the higher doses and the longer incubation times. In addition, at the longer durations myofilaments in most muscle cells also became depolymerized, while microtubules were unchanged by the drug. Therefore, it is possible that praziquantel, through its induction of Ca2+ influx, causes depolymerization of the microtrabecular network that leads to the vacuolization, swelling, blebbing, and eventually the disruption and detachment of the tegument, and the breakdown of myofilaments in the muscle cells.

Opisthorchis viverrini: the effects of colchicine and cytochalasin B on the adult tegument.

The roles of the tegumental cytoskeleton were tested by treating adult flukes with colchicine and cytochalasin B. Following a short incubation period (10-20 minutes), colchicine disrupted microtubules in the tegumental cells' processes which, in turn, affected the transport of dense granules from the cells' soma to the tegument; as a result some of these granules were fused together to form membrane-bound vacuoles. In addition, at many spots microtrabeculae were also depolymerized, which resulted in the formation of non-membrane-bound vacuoles and the distension of microvilli to form blebs, some of which were disrupted. After prolonged incubation (120 minutes), general breakdown of the tegumental cytoskeleton occurred, and parts of it were sloughed off. In cytochalasin B treatment, the responses were similar to those of colchicine but with less severity. After a short incubation period (10-20 minutes), the microtrabeculae were depolymerized which led to the formation of non-membrane-bound vacuoles in the apical and middle zones of the tegument. Later, the tegumental microvilli were distended to form blebs but no evidence of tegumental sloughing occurred even in prolonged incubation. From these observations, it was concluded that microtubules played a role in the translocation of granules from the tegumental cells to the tegument which modulated the synthesis of membrane and glycocalyx, while microtrabeculae were involved in the maintenance of the structure and integrity of the tegument.

Monoclonal antibodies against Schistosoma mekongi surface tegumental antigens.

Monoclonal antibodies were produced from naturally infected BALB/c mice. Thirteen hybridomas which were found to produce monoclonal antibodies against surface tegumental antigens of Schistosoma mekongi by ELISA assay were used in this study. The antigen specificities of hybridomas reactive with surface tegumental antigens were characterized and localized by immunoblotting analysis and Avidin-Biotin method. Of the 13 hybridomas, only three produced monoclonal antibodies to the single epitopes in the surface tegumental antigens. These epitopes (125 kDa, 97 kDa and 38 kDa) have been found to be the major antigenic components of the surface tegument of S. mekongi. The 38 kDa antigen was found to associate with the surface tegumental layers, the muscular layers lying just beneath the tegument, as well as in the gut surface. The 97 and 125 kDa antigens were detectable only in the surface tegumental area. The biochemical identity of these proteins or glycoproteins is unknown. However, these antigens have also been described in S. japonicum and S. mansoni.

Characterization of two monoclonal antibodies against teguments of adult and schistosomula of Schistosoma japonicum.

Two monoclonal antibodies (McAbs) were produced from BALB/c mice hyperimmunized with tegumental extract of Schistosoma japonicum (Chinese strain). The two McAbs were characterized with regard to antibody isotype, antigen binding specificity and parasite stage specificity. One McAb, 8G9-5, was identified as IgM, whereas the other McAb, 9E7, was determined to be IgG2a. Immunoblotting assay indicated that McAb 8G9-5 binds strongly to the band of tegumental antigens of Mr 64 kDa but also binds weakly to other bands at 116, 105, 97, 54, 50, 47 and 45 kDa, whereas 9E7 McAb reacts specifically at Mr 54 kDa. Anatomical localization of the antigens in the adult worm by indirect immunofluorescence assay indicated that the target epitopes of McAb 8G9-5 are in the intra-tegumental structure, whereas the McAb 9E7 epitope is on the surface membrane. The two McAbs also react at similar sites within the teguments of schistosomula and lung worms.

Alterations of the surface tegument of Opisthorchis viverrini exposed to praziquantel in vitro and in vivo.

The in vitro and in vivo effects of praziquantel on the ultrastructural surface of Opisthorchis viverrini were investigated using scanning electronmicroscopy. For the in vitro study, adult flukes were collected from experimentally infected hamsters, and were incubated for various time intervals at 37 degrees C in Earle's basal medium containing praziquantel at final concentrations of 0.01-100 micrograms/ml. For the in vivo study, flukes were collected from the biliary system of experimentally infected hamsters that had been treated 4 hours previously with 350 mg of praziquantel per kg body weight (mg/kg). Flukes were also obtained from the feces of a patient with opisthorchiasis who had been given praziquantel once at a dose of 40 mg/kg 4-6 hours previously and from the bile of a patient at the time of operation 24 hours after praziquantel treatment. Scanning electronmicroscopic analyses of the surface teguments of flukes exposed to praziquantel either in vitro or in vivo showed similar changes. Tegumental bubbles of different sizes appeared on the surface; they later ruptured and resulted in the formation of crater-like lesions. These lesions might be so extensive as to result in the peeling of the entire areas. On occasions, "micronodules" appeared later in these areas and those at the periphery of the lesions; these micronodules may represent an attempt by the worm to regenerate new tegument. The possibility that these ultrastructural changes may represent a generalized response of the tegumental surface to an obnoxious agent was discussed.

Microtopography of the surface of adult Schistosoma japonicum-like (Malaysian) as observed by scanning electron microscopy.

The surface of adult Schistosoma japonicum-like (Malaysian) was studied by scanning electron microscopy. The basic pattern of surface microtopography is similar to other strains of S. japonicum as previously reported. However, among male member there are some unique differences in the types, number and distribution of surface papillae and morphology of ridges. Three kinds of papillae were observed: (1) the large fungiform papillae (3.5-4 micron in diameter, most without cilia) are more numerous than in other strains of S. japonicum, they concentrate on the lateral aspect of the anterior and middle parts close to the edge of the gynecophoral canal, and on the dorso-lateral aspect of the posterior part towards the tail tip; (2) the small hemispherical papillae (1.5-2 micron in diameter, all bearing cilia) are especially numerous in the suckers, the gynecophoral canal and parts of the tegument around the suckers and close to the tail tip; on the rest of the surface they are evenly distributed; (3) the cratered papillae (3-4 micron in diameter, about half having cilia) are more numerous than on other strains, they concentrate on the lateral aspect of the middle part and on the edges of the gynecophoral canal. The surface ridges (about 0.2-0.3 micron in width) are tall, highly branching and perforated; they are most developed in the middle part. Spines were observed only in the suckers and the gynecophoral canals. In contrast to the male, the female has numerous spines on all parts of the surface except the most anterior, where a large number of long cilia were observed. All three kinds of papillae were present; fungiform papillae are more numerous than in females of other strains; they concentrate on the latero-dorsal aspect of middle and posterior parts, and around the excretory pore. Ridges are much less developed than in the male and are prominent only in the middle part.