Paramphistomum cervi: antigenic profile of adults as recognized by infected cattle sera

An antigenic profile of adult Paramphistomum cervi was revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using sera from cattle naturally infected with P. cervi, Fasciola gigantica and strongylids. SDS-PAGE of whole worm extracts exhibited 26 distinct protein bands. Immunoblotting analysis of these proteins showed five major antigenic bands which were recognized by serum of individual cattle naturally infected with P. cervi. These antigenic proteins had molecular weights ranging from 23 to 116kDa. One antigenic protein with a molecular weight of 52kDa exhibited a consistent reaction with sera from all infected cattle. It's diagnostic sensitivity, specificity and accuracy using this test were 100%, 98% and 98.9%, respectively. The positive and negative predictive values were 97.6% and 100%, respectively. This finding suggests that the 52kDa protein may be a diagnostic antigen for paramphistomosis.     

Nicotinic receptor stimulation enhances enkephalin-like peptides processing in chromaffin cells

Acute stimulation of chromaffin cells in cultures with acetylcholine (ACh), 1,1-dimethyl-4-phenylpiperazinium (DMPP), or high potassium gave rise to a significant increase in the release of [Met5]-enkephalin immunoreactive material (ME-IRM) into the assay medium. The cellular content of ME-IRM following the actual release induced by these secretagogues remained constant suggesting the replenishment of the cellular peptides. The repletion of the peptides may occur through an enhancement of the processing rate of the proenkephalin precursor. Furthermore, the increase in secretion as well as the repletion of the cellular ME-IRM were calcium-dependent and were inhibited by the nicotinic receptor antagonist, hexamethonium, but not by atropine. These results indicate that secretion and repletion of the peptides are tightly coupled and activated by nicotinic receptor stimulation.     

Immunodiagnosis of Fasciola gigantica Infection Using Monoclonal Antibody-Based Sandwich ELISA and Immunochromatographic Assay for Detection of Circulating Cathepsin L1 Protease

Background

Tropical fasciolosis caused by Fasciola gigantica infection is one of the major diseases infecting ruminants in the tropical regions of Africa and Asia including Thailand. Parasitological diagnosis of fasciolosis is often unreliable and possesses low sensitivity. Therefore, the detection of circulating parasite antigens is thought to be a better alternative for diagnosis of fasciolosis, as it reflects the real parasite burden.

Methods

In this study, we have produced a monoclonal antibody (MoAb) against recombinant F. gigantica cathepsin L1 (rFgCatL1), and developed both sandwich enzyme-linked immunosorbent assay (sandwich ELISA) and immunochromatographic (IC) test for rapid detection of circulating cathepsin L1 protease (CatL1) in the sera from mice experimentally and cattle naturally infected with Fasciola gigantica. MoAb 4E3 and biotinylated rabbit anti-recombinant CatL1 antibody were selected due to their high reactivities and specificities.

Results

The lower detection limits of sandwich ELISA and IC test were 3 pg/ml and 0.256 ng/ml, respectively. Sandwich ELISA and IC test could detect F. gigantica infection from day 1 to 35 post infection. In experimental mice, the sensitivity, specificity and accuracy were 95%, 100% and 98.6% (for sandwich ELISA), and 93%, 100% and 98.2% (for IC test), while in natural cattle they were 98.3%, 100% and 99.5% (for sandwich ELISA), and 96.7%, 100% and 99.1% (for IC test).

Conclusions

These two assay methods showed high efficiencies and precisions for diagnosis of fasciolosis by F. gigantica.     

Fischoederius cobboldi: A scanning electron microscopy investigation of surface morphology of adult rumen fluke

Adults Fischoederius cobboldi are conical-shaped, concave ventrally and convex dorsally, measures about 8-10mm in length and 4-6mm in width across the mid section. Scanning electron microscopy (SEM) of entire body showed that the tegumental surface exhibits highly corrugation and transverse folds alternating with grooves and without spines. At higher magnification, the surface of each fold is further increased with a meshwork of ridges separated by irregular-sized pits. The ventral surface has more complex corrugations and invaginations than those of the dorsal surface of the body. Both anterior and posterior suckers have thick edges covered with transverse folds and appear spineless. The genital pore is located at the anterior one-third of the body. There are two types of sensory papillae on the surface: type 1 is bulbous in shape and nipple-like tips, measuring 10-15 μm in diameter at the base, and also type 2 is a similar shape and has short cilia on tips. These sensory papillae occur in large clusters, each having between 7 and 25 units depending on the region of the body. Clusters of papillae on the ventral surface and around the anterior suckers tend to be more abundant and larger in size. The dorsal side of the body exhibit similar surface features, but papillae appear less numerous and are smaller. Corrugations and invaginations of the dorsal aspect are also less extensive than those on the ventral surface of the body.     

Fasciola gigantica: Production and characterization of a monoclonal antibody against recombinant cathepsin B3

A number of monoclonal antibodies (MoAbs) against a recombinant cathepsin B3 (rCatB3) of Fasciola gigantica were produced in BALB/c mice. Reactivity and specificity of these MoAbs were assessed by indirect ELISA and immunoblotting techniques. Six stable clones, namely 1C4, 1E9, 2E5, 2F9, 5B4, 5D7 were obtained. All MoAbs reacted with rCatB3 at molecular weight (MW) 37 kDa as well as the glycosylated peptide at 55–75 kDa and with the native CatB3 at MW 37 kDa in WB extracts of metacercariae (Met) and newly excysted juveniles (NEJ). It was found to be IgG₁ and λ light chain isotypes. Immunolocalization of CatB3 in metacercariae, NEJ, 4-week-old juvenile and adult F. gigantica performed by immunoperoxidase technique by using these MoAbs as probes indicated that CatB3 was present in high concentration in the caecal epithelium and caecal lumen of the Met and NEJ, but not in the 4-week-old juvenile and adult fluke. The MoAbs show no cross-reactions with antigens of other parasites including Gigantocotyl explanatum, Eurytrema pancreaticum, Paramphistomum cervi, Schistosoma spindale, S. mansoni, Haemonchus placei and Setaria labiato-papillosa. Thus, it is possible that these MoAbs could be a good candidate for immunodiagnosis of fasciolosis.     

Distribution and changes of serotonin and dopamine levels in the central nervous system and ovary of the Pacific white shrimp, <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>, during ovarian maturation cycle

We investigated changes in serotonin (5-HT) and dopamine (DA) levels and in their distribution patterns in the central nervous system (CNS) and ovary during the ovarian maturation cycle in the Pacific white shrimp, Litopenaeus vannamei. The concentrations of these two neurotransmitters were determined by using high performance liquid chromatography with electrochemical detection. The 5-HT concentration exhibited a gradual increase in the brain and thoracic ganglia during early ovarian stages I, II, and III, reaching a maximum at the mature ovarian stage IV, whereas DA showed its highest concentration at ovarian stage II in the brain and thoracic ganglia and then declined to its lowest concentration at ovarian stage IV. In the ovaries, 5-HT was lowest at ovarian stage I and gradually increased to a peak at ovarian stage IV. Conversely, the concentration of DA was highest at ovarian stages I and II and lowest at ovarian stage IV. In the brain, 5-HT immunoreactivity (−ir) from stage IV and DA-ir from stage II were distributed extensively in neurons of clusters 6, 11, and 17, in fibers, and in the anterior and posterior medial protocerebral, olfactory, antenna II, and tegumentary neuropils. In the circumesophageal, subesophageal, thoracic, and abdominal ganglia, both 5-HT-ir and DA-ir were detected in neuropils and surrounding neurons and fibers. 5-HT-ir and DA-ir were more intense in the thoracic ganglia than in other parts of the CNS. In the ovary, 5-HT-ir exhibited high intensity in late oocytes, whereas DA-ir was more intense in early oocytes. Thus, opposing changes occur in the levels of these two neurotransmitters and in their specific localizations in the CNS and ovary during ovarian maturation, indicating their important involvement in female reproduction.     

Fasciola gigantica: Immunolocalization of 28.5 kDa antigen in the tegument of metacercaria and juvenile fluke

Specific monoclonal antibody (MoAb) to 28.5 kDa tegumental antigen (TA) was used to localize this antigen in the tissues of metacercariae, newly excysted juvenile (NEJ), 1, 3, 5, and 7-week-old juveniles of Fasciola gigantica by using indirect immunofluorescence, immunoperoxidase and immunogold techniques. Both indirect immunofluorescence and immunoperoxidase detections showed that this antigen was concentrated in the tegument particularly in its outer rim, tegumental cells and their processes as well as epithelial linings of the oral sucker. Unlike adult F. gigantica, it was not detected in spermatogenic cells in the testes, cells of Mehlis’gland, oocytes within the ovary, and ovum within the egg of parasites. At the ultrastructural level, the immunogold labeling showed deposit of gold particles specifically in G₂ tegumental granules and on the surface membrane. Thus, this 28.5 kDa antigen is expressed in the tegument and associated structures of juvenile parasites, and it could be a major component of the G₂ granules which are shown to fuse with the surface membrane and contribute material to replace the casted-off membrane. This process is the replenishment and turnover of the surface membrane to prevent the attachment of the host immune effector cells.     

Characterization of 1,1-dimethyl-4-phenylpiperazinium induced increased proenkephalin processing in bovine chromaffin cells

Acute stimulation of bovine adrenal chromaffin cells in culture with 1,1-dimethyl-4-phenylpiperazinium (DMPP) gives rise to a significant increase in secretion of [Met5]-enkephalin immunoreactive material (ME-IRM) into the culture medium (1). Following this secretion the cellular ME-IRM levels do not decrease, suggesting the replenishment of the peptides. The repletion of the cellular ME-IRM appears to result from an increase in processing of large molecular weight peptides containing [Met5]-enkephalin and [Leu5]-enkephalin. Gel filtration chromatography on Bio-Gel P-10 was used to fractionate the enkephalin-like peptides (ELPs) present in the culture media and chromaffin cell extracts. Fractionation was done for samples before and after nicotinic receptor stimulation by DMPP to demonstrate the secretion and repletion of the ELPs. Gel chromatographic profiles of ELPs present in the culture media after DMPP stimulation revealed the presence of 4 peaks, representing different molecular forms of these peptides (Peaks 1-4), with a selective increase in secretion of Peaks 3 and 4. The chromatograms of ELPs extracted from cultured chromaffin cells showed similar patterns to those obtained from ELPs present in the culture medium after stimulation. Analyses of individual peaks after fractionation of cell culture extracts showed an increase in the amount of immunoreactive material found in Peak 4 with a concomitant decrease in the immunoreactivity found in the higher molecular weight peaks (Peaks 1-3). Further purification of Peak 4 from cell extracts on reversed-phase HPLC (RP-HPLC) showed a significant amount of ELPs existed as the sulfoxide derivative of [Met5]-enkephalin. The content of [Met5]-enkephalin sulfoxide (ME-O-enk) did not decrease following DMPP stimulation. We conclude that acute stimulation of nicotinic receptors in the chromaffin cells enhances the processing of proenkephalin precursors to keep pace with the secretion of low molecular weight peptides.     

Existence and distribution of gonadotropin-releasing hormone-like peptides in the central nervous system and ovary of the Pacific white shrimp, <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

We used antibodies against octopus gonadotropin-releasing hormone (octGnRH) and tunicate GnRH (tGnRH-I) in order to investigate the existence and distribution of GnRH-like peptides in the central nervous system (CNS) and in the ovary during various stages of the ovarian cycle of the white shrimp, Litopenaeus vannamei. OctGnRH-immunoreactive and tGnRH-I-immunoreactive neurons and fibers were present in several regions of the supraesophageal ganglion (brain), subesophageal ganglion (SEG), thoracic ganglia, and abdominal ganglia. In the brain, both octGnRH immunoreactivity (ir) and tGnRH-I-ir were detected in neurons of clusters 6, 11, 17, and associated fibers, and the anterior medial protocerebral, posterior medial protocerebral, olfactory, and tegumentary neuropils. In the SEG and thoracic ganglia, octGnRH-immunoreactive and tGnRH-I-immunoreactive neurons and fibers were present in dorsolateral and ventromedial cell clusters and in surrounding fibers. Only immunoreactive fibers were detected in the abdominal ganglia. In the ovary, both octGnRH and tGnRH-I were detected at medium intensity in the cytoplasm of early step oocytes (Oc2) and, at high intensity, in Oc3. Furthermore, octGnRH-ir and tGnRH-I-ir were intense in follicular cells surrounding Oc2 and Oc3. The presence of GnRH-ir in the CNS and ovary indicates that GnRH-like peptides occur in the white shrimp, and that GnRHs are involved in the reproductive process, especially ovarian maturation and the differentiation of oocytes, as reported in other species.