Chaitip Wanichanon

Fasciola gigantica: immunodiagnosis of fasciolosis by detection of circulating 28.5 kDa tegumental antigen.

A monoclonal antibody (MoAb)-based sandwich ELISA was developed for the detection of circulating 28.5 kDa tegumental antigen (28.5 kDa TA) in the sera from mice experimentally infected with Fasciola gigantica. The MoAb was immobilized on a microtiter plate, and the antigen in the serum was captured and detected with biotinylated polyclonal rabbit anti TA antibody. The test could detect 28.5 kDa in the extracts of tegument (TA), whole body (WB) and excretory-secretory (ES) fractions at the concentrations of these crude antigens as low as 600 pg/ml, 16 and 60 ng/ml, respectively. This sandwich ELISA assay could detect the infection from day 1 to 35 post infection and showed that circulating level of 28.5 kDa TA peaked at day 1 post infection. In contrast, the antibody detection by indirect ELISA could only demonstrate the antibody level from 35 days post infection. The reliability of the assay method was evaluated using sera from mice infected with F. gigantica or Schistosoma mansoni, and hamsters infected with Opisthorchis viverrini, as well as healthy mice and hamsters. The sandwich ELISA exhibited a sensitivity and specificity at 94.55% and 100%, respectively, and with a positive predictive value of 100%, a negative predictive value of 97.39%, false positive rate of 0%, false negative rate of 5.50% and an accuracy of 98.2%. Thus, this detection method exhibited high specificity and sensitivity as well as could be used for early diagnosis of fasciolosis by F. gigantica.

Production and characterization of a monoclonal antibody against 28.5 kDa tegument antigen of Fasciola gigantica.

A monoclonal antibody (MoAb) against the 28.5 kDa tegumental antigen of Fasciola gigantica was produced by the hybridoma technique using spleen cells from BALB/c mice immunized with the tegumental extract from adult F. gigantica. This MoAb was found to be of the isotype IgG(1), kappa-light chain, and shown by immunoblotting to specifically react with the 28.5 kDa antigen present in the tegument, excretion-secretion material of the adult, whole-body extracts of newly excysted juveniles, 5-week-old juvenile and adult parasites. It did not cross-react with antigens from other trematode parasites, including Schistosoma mansoni, Eurytrema pancreaticum and Paramphistomum spp. Immunolocalization of this antigen by indirect immunofluorescence indicated that it was present as a major component of the adult tegument, particularly in its outer rim, tegumental cells, and their processes. Furthermore, the epithelium linings of the oral sucker, buccal tube, pharynx, caecal bifurcation, both male and female genital canals, which were the continuation of the tegumental-type epithelium, were also positively stained with this MoAb. A similar pattern of immunolocalization, but with weaker staining intensity, was observed in newly excysted, 5- and 7-week-old juveniles. Thus this antigen is expressed in all developmental stages of the parasite, and it could be a strong candidate for immunodiagnosis and vaccine development.

Vaccine potential of recombinant cathepsin B against Fasciola gigantica.

In Fasciola gigantica, cathepsin Bs, especially cathepsin B2 and B3 are expressed in early juvenile stages, and are proposed to mediate the invasion of host tissues. Thus they are thought to be the target vaccine candidates that can block the invasion and migration of the juvenile parasite. To evaluate their vaccine potential, the recombinant cathepsin B2 (rFgCatB2) and cathepsin B3 (rFgCatB3) were expressed in yeast, Pichia pastoris, and used to immunize mice in combination with Freunds adjuvant to evaluate the protection against the infection by F. gigantica metacercariae, and the induction of immune responses. Mice immunized with both recombinant proteins exhibited high percent of parasite reduction at 60% for rFgCatB2 and 66% for rFgCatB3. Immunization by both antigens induced continuously increasing levels of IgG1 and IgG2a with a higher level of IgG1 isotype, indicating the mixed Th1/Th2 responses with Th2 predominating. When examined individually, the higher levels of IgG1 and IgG2a were correlated with the lower numbers of worm recoveries. Thus, both cathepsin B2 and cathepsin B3 are plausible vaccine candidates whose potential should be further tested in large economic animals.

Stereo architecture of the connective tissue cores of the lingual papillae in the treeshrew (Tupaia glis)

SummaryThe stereo architecture of the lingual connective tissue cores (CTC) in the treeshrew (Tupaia glis) (which has the primitive characteristics of primates) was observed by scanning electron microscopy, and compared to that of other animal orders. The tongue of the treeshrew has three vallate papillae which are situated in the posterior part of the tongue, while some macaques have several vallate papillae. Among numerous filiform papillae, fungiform papillae are sporadically distributed. A filiform papilla consists of a bundle of several slender spine-like processes arranged in a circle at the basal margin. After removal of the epithelium, the CTC of the filiform papilla looks like a human hand raised with the palm facing towards the tongue tip. The fungiform CTC in the threeshrew is columnar in shape (rather similar to that of Insectivora and Rodentia) and at the top there are several round depressions for taste buds. In the treeshrew several large rod-shaped processes are derived from the postero-lateral margin of the tongue, as in Carnivora (dogs and cats), where foliate papillae are located in many other animal species. The treeshrew has numerous characteristics similar to those of the crab-eating macaque (Primates), but at the same time it has some characteristics similar to those of Insectivora, Rodentia, Carnivora and Artiodactyla.

Fine structure of the dorsal lingual epithelium of the juvenile hawksbill turtle, Eretmochelys imbricata bissa

Various species of turtles are adapted to different environments, such as freshwater, seawater, and terrestrial habitats. Comparisons of histological and ultrastructural features of the tongue of the juvenile Hawksbill turtle, Eretmochelys imbricata bissa, with those of freshwater turtles should reveal some aspects of the relationship between the structure of the lingual epithelium and the environment.

Fasciola gigantica: surface topography of the adult tegument.

Adult Fasciola gigantica are leaf-shaped with tapered anterior and posterior ends and measure about 35 mm in length and 15 mm in width across the mid section. Under the scanning electron microscope its surface appears rough due to the presence of numerous spines and surface foldings. Both oral and ventral suckers have thick rims covered with transverse folds and appear spineless. On the anterior part of the ventral surface of the body, the spines are small and closely-spaced. Each spine has a serrated edge with 16 to 20 sharp points, and measures about 20 microm in width and 30 microm in height. In the mid-region the spines increase in size (up to 54 microm in width and 58 microm in height) and number, especially towards the lateral aspect of the body. Towards the posterior end the spines progressively decrease in both size and number. The tegumental surface between the spines appears highly corrugated with transverse folds alternating with grooves. At higher magnifications the surface of each fold is further increased with a meshwork of small ridges separated by variable-sized pits or slits. There are three types of sensory papillae on the surface. Types 1 and 2 are bulbous, measuring 4-6 microm in diameter at the base with nipple-like tips, and the type 2 also have short cilia. Type 3 papillae are also bulbous and of similar size but with a smooth surface. These sensory papillae usually occur in clusters, each having between 2 and 15 units depending on the region of the body. Clusters of papillae on the lateral aspect (usually types 1 and 2) and around the suckers (type 3) tend to be more numerous and larger in size. The dorsal side of the body exhibits similar surface features, but the spines and papillae appear less numerous and are smaller. Corrugation and invaginations of the surface are also less extensive than on the ventral side of the body.

Gastric mucosal lesions in streptozotocin-diabetic rats

Formation of gastric mucosal lesions by streptozotocin-induced diabetes was investigated in rats. A single intravenous administration of streptozotocin in a dose of 65 mg/kg effectively produced hyperglycemia and damaged the gastric mucosa. Incidence and severity of mucosal lesions were progressively increased with time, from one to six weeks posttreatment. Microscopic lesions of the mucosa included hyperemia, desquamation of the surface epithelium with diffuse hemorrhage, and severe hemorrhage with localized erosion. Concurrent to the hyperglycemia, the histamine stimulated gastric H+-secretion was significantly decreased whereas pepsin secretion was not affected. Both soluble mucus and surface mucus gel were increased. The result suggests that the early lesion of gastric mucosa may be associated with the direct action of streptozotocin, the severity of which may be further aggravated by diabetic state.

The anthelmintic effect of plumbagin on Schistosoma mansoni

The anthelmintic effects of plumbagin (PB, 5-hydroxy-2-methyl-1,4-naphthoquinone) and praziquantel (PZQ) against adult Schistosoma mansoni in vitro were compared by estimating the relative motility (RM) values, survival indices (SI) and alterations of the tegument of flukes incubated in M-199 medium containing 1, 10 and 100 μg/ml of the drugs, at 1, 3, 6, 12 and 24 h. For the parasites incubated in 10 μg/ml, the RM values of the PB-treated group decreased at a more rapid rate than the PZQ-treated group. When incubated in 100 μg/ml all PB-treated flukes appeared immobile from 1 to 24 h when 91-100% died, while in the PZQ-treated group RM values were higher than that of PB and most flukes were still alive at 1-12 h, and at 24 h only 21% of flukes were killed. Furthermore, male parasites were more affected by PZQ than females as their RM values were significantly less than that of females at all doses. While in PB treatment, males and females showed less difference in response to the drug as their RM values were closer than those treated with PZQ. When observed by SEM, the tegument of untreated S. mansoni displayed no alteration, while in PB treated parasites it exhibited swelling, blebbing, loss of spines, disruption of tubercles and ridges, leading to erosion and lesion, exposure of the basal lamina, and sloughing of the tegument. PZQ induced similar tegumental changes as those observed in PB treatment but at longer incubation time and higher doses. These data indicated that PB had more anthelmintic effect on both sexes of adult S. mansoni than PZQ.

The existence of gonadotropin-releasing hormone-like peptides in the neural ganglia and ovary of the abalone, Haliotis asinina L.

Gonadotropin-releasing hormone (GnRH) is a neuropeptide that is conserved in both vertebrate and invertebrate species. In this study, we have demonstrated the presence and distribution of two isoforms of GnRH-like peptides in neural ganglia and ovary of reproductively mature female abalone, Haliotis asinina, using immunohistochemistry. We found significant immunoreactivities (ir) of anti-lamprey(l) GnRH-III and anti-tunicate(t) GnRH, but with variation of labeling intensity by each anti-GnRH type. lGnRH-III-ir was detected in numerous type 1 neurosecretory cells (NS1) throughout the cerebral and pleuropedal ganglia, whereas tGnRH-I-ir was detected in only a few NS1 cells in the dorsal region of cerebral and pleuropedal ganglia. In addition, a small number of type 2 neurosecretory cells (NS2) in cerebral ganglion showed lGnRH-III-ir. Long nerve fibers in the neuropil of ventral regions of the cerebral and pluropedal ganglia showed strong tGnRH-I-ir. In the ovary, lGnRH-III-ir was found primarily in oogonia and stage I oocytes, whereas tGnRH-ir was observed in stage I oocytes and some stage II oocytes. These results indicate that GnRH produced in neural ganglia may act in neural signaling. Alternatively, GnRH may also be synthesized locally in the ovary where it could induce oocyte development.

Histological and ultrastructural study of the lingual epithelium of the juvenile Pacific ridley turtle, Lepidochelys olivacea (Chelonia, Cheloniidae)

Histological and ultrastructural studies ot the dorsal lingual epithelium of the juvenile Pacific ridley turtle, Lepidochelys olivacea, were performed by light and electron microscopy, and the results were compared to those of freshwater turtles in order to clarify the relationship between the histological and cellular differences of the lingual epithelium and the habitat of the turtles. The tongue of the juvenile Pacific ridley turtle is triangular with a round apex when viewed from above, but it appears flattened in lateral view. Scanning electron microscopy (SEM) revealed no lingual papillae on the dorsal surface of the tongue. Instead, transverse plicae are found on the surface of the body and the radix. The surface of the apex is smooth. Microridge-like structures are present on the surfaces of the cells, and the cell margins are thickened. The mucosal epithelium is keratinized, stratified squamous with a relatively thick layer of desquamating cells. Cells of the basal and deep intermediate layers appear elliptical in shape; and their nuclei are elliptical and centrally located. Numerous desmosomes join the processes of adjacent cells; and hemidesmosomes anchor the basal cells to the basal lamina. The cytoplasm of these cells contains mitochondria, free ribosomes, rough endoplasmic reticulum, vacuoles, and bundles of tonofilaments. Cells and their nuclei in the intermediate layer display gradual flattening. In the shallow intermediate layer, the cells are significantly flattened, with nuclei condensed or absent. The cytoplasm contains many tonofibrils or bundles of tonofilaments, free ribosomes and keratohyalin granules, with numerous ribosomes attached to their surfaces. A few collapsed mitochondria are visible. Cell membranes of the shallow intermediate cells are smooth and attached to those of adjacent cells by desmosomes. The keratinized layer is located on top of the shallow intermediate layer, and consists of significantly flattened cells lacking nuclei and filled with keratin fibers. Very fine cellular processes joined by desmosomes are visible. The desquamating cells located on top of the keratinized layer contain keratin fibers that are somewhat thicker than tonofibrils and tonofilaments, and clearly distinguishable individually. The microridge-like structures visible by SEM could be attributed to the persistence of cells formed in underlying layer. In conclusion, the histology of the lingual epithelium of the juvenile Pacific ridley turtle differs significantly from that of the adult freshwater turtle in spite of the similarity of the gross morphology of their tongues.