Anthelmintic Effect of Biocompatible Zinc Oxide Nanoparticles (ZnO NPs) on Gigantocotyle explanatum, a Neglected Parasite of Indian Water Buffalo.

Helminth parasites of veterinary importance cause huge revenue losses to agrarian economy worldwide. With the emergence of drug resistance against the current formulations, there is a need to focus on the alternative approaches in order to control this menace. In the present study, biocompatible zinc oxide nanoparticles (ZnO NPs) were used to see their in vitro effect on the biliary amphistomes, Gigantocotyle explanatum, infecting Bubalus bubalis because these nanoparticles are involved in generation of free radicals that induce oxidative stress, resulting in disruption of cellular machinery. The ZnO NPs were synthesized by using egg albumin as a biotemplate and subsequently characterized by Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), X-ray Diffraction and Spectrophotometrical, which showed that ZnO NPs were highly purified wurtzite type polycrystals, with a mean size of 16.7 nm. When the parasites were treated with lower concentrations (0.004% and 0.008%) of the ZnO NPs, the worms mounted a protective response by stimulating the antioxidant system but the treatment of G. explanatum with 0.012% ZnO NPs produced significant inhibition of the antioxidant enzymes like superoxide dismutase (SOD) (p< 0.05) and glutathione S- transferase (GST) (p<0.01), while the level of malondialdehyde (MDA), a lipid peroxidation marker, was significantly (p< 0.01) elevated. SEM and histopathology revealed pronounced tegumental damage showing the disruption of surface papillae and the annulations, particularly in the posterior region near acetabulum. The under expression of a number of polypeptides, loss of worm motility in a time dependent manner, further reflect strong anthelmintic potential of ZnO NPs. It can be concluded that the anthelmintic effect might be due to the production of reactive oxygen species that target a variety of macromolecules such as nucleic acid, protein and lipids which are involved in different cellular processes.

Somatic antigens of tropical liver flukes ameliorate collagen-induced arthritis in wistar rats.

Parasitic helminths polarize immune response of their vertebrate hosts towards anti-inflammatory Th2 type and therefore it is hypothesized that they may suppress the inflammatory conditions in autoimmune disorders. The present study was undertaken to investigate in vivo immunomodulatory and therapeutic potential of somatic antigens (Ag) of liver infecting digenetic trematodes [Fasciola gigantica (Fg) and Gigantocotyle explanatum (Ge)] in collagen-induced arthritic (CIA) Wistar rats. The CIA rats were administered subcutaneously with different doses (50 µg, 100 µg and 150 µg) of somatic antigens of Fg and Ge, daily for 21 days, the time period required to establish infection in natural host (Bubalus bubalis). Thereafter, the control, diseased and treated rats were compared for different parameters viz. hind paw thickness; serum interleukins, IL-4 and IL-10, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ); expression level of matrix metalloproteinases (MMPs) -2, -9, -13 and nitric oxide (NO) in knee joints and patellar morphology. The CIA rats treated with different antigens, Fg-Ag and Ge-Ag, show significant amelioration of the disease by down regulation of serum TNF-α and IFN-γ (p< 0.05) and upregulation of IL-4 and IL-10 cytokines (p< 0.05); inhibition (p< 0.05) of MMPs (-2,-9,-13) and NO in knee joints and improved patellar morphology with decreased synovial hypertrophy and reduced infiltration of ploymorphonuclear cells. The activity of pro as well as active MMPs (-2 and -9) and active MMP-13 in knee joints of CIA rats was very high compared to the control and treatment groups, suggesting the extent of collagen degradation in CIA rats. Interestingly, the highest dose (150 µg) of Ge-Ag almost wiped out MMP-13 expression. The overall findings suggest that the somatic proteins of Ge-Ag appeared to be therapeutically more effective than Fg-Ag, reflecting interspecific molecular differences which could contribute to the ability of these worms to successfully ameliorate the pathology of CIA.

Immunodetection of coproantigens for the diagnosis of amphistomosis in naturally infected Indian Water Buffalo, Bubalus bubalis.

The infection of gastrointestinal helminths in livestock is routinely diagnosed by microscopical examination of faecal samples for the presence of ova/eggs but this approach becomes ineffective for the seasonally egg producing trematodes. Therefore, an alternative approach to detect the coproantigens of liver and rumen amphistomes, Gigantocotyle explanatum and Gastrothylax crumenifer respectively, infecting Indian water buffalo Bubalus bubalis, was undertaken using ELISA, immunodot and countercurrent immunoelectrophoresis (CCIEP). The hyperimmune polyclonal antisera were separately raised in rabbits against excretory/secretory (ES) antigens of both the flukes under study. An overall 70% buffalo faecal samples were tested positive for G. crumenifer and 75% for G. explanatum in Aligarh region. The ELISA results reflected higher infection intensity among individual buffaloes that was also observed at necropsy. Using the respective homologous hyperimmune antiserum, 55% buffaloes tested positive for G. crumenifer and 65% positive for G. explanatum in immunodot assay. Further, the faecal samples with high absorbance values in ELISA and strong immunodot reaction tested positive in CCIEP. The analysis of CCIEP result revealed two and one precipitin bands in G. crumenifer and G. explanatum respectively, indicating prominent antigenic differences in the coproantigens of these two parasites. Taken together, it is suggested that polyclonal antibodies could be conveniently used for the detection of coproantigens by ELISA and immunodot methods, particularly during the non-egg producing phase of the seasonally regulated reproductive cycle of the rumen amphistome G. crumenifer. It is concluded that the coproantigen detection is a good alternative over conventional method for the diagnosis of amphistomosis in livestock; however, further studies are required on a larger sample size of field buffaloes to augment the reproducibility of the present results.

Isolation and partial characterization of excretory/secretory antigens of Gastrothylax crumenifer.

The present study was carried out to identify the excretory/secretory (E/S) antigens of the rumen infecting digenetic trematode Gastrothylax crumenifer that may be useful for the immunodiagnosis of rumen amphistomosis particularly during the pre-monsoon season during which this rumen parasite stops shedding eggs. The in vitro released E/S proteins were purified on a Sephadex G-200 column. The gel filtration profile revealed three distinct fractions F1-F3 where F1 and F3 appeared as sharp peaks while the F2 fraction was dispersed. The antibody titre against each of the purified E/S fractions was determined by ELISA using anti-whole E/S polyclonal antibodies raised in rabbit. Among the three fractions, the antibody titre against F1 was highest (1:12,800) whereas IgG titre was very low (1:50) for fraction F2 and F3 (1:100). Of the total polypeptides resolved on gradient SDS-PAGE, only a few antigenic polypeptides were detected in each fraction with hyperimmune anti-serum as revealed by Western Blot analysis. However, a 33 kDa antigen detected in each fraction appeared to be immunodominant which could be exploited for the diagnosis of the pouched amphistome.

CD166 expression, characterization, and localization in salivary epithelium: implications for function during sialoadenitis.

CD166 is an Ig superfamily molecule that binds homotypically to itself and heterotypically to CD6. Interactions between CD6 and CD166 are important during immune development and in alloreactivity. CD166 is expressed at increased levels in selected cancers and in rheumatoid arthritis synovium. Knowledge that CD166 was expressed in normal human salivary epithelium led to these studies of CD166 and CD6 in diseased mouse salivary glands, that resemble pathology seen in the human disease, Sjögren's syndrome. We showed that in mouse salivary epithelium CD166 was expressed but that expression of CD166 did not necessarily predict its function. Recombinant soluble CD6-Ig bound to CD6 ligands (CD6L) on transformed and freshly isolated salivary epithelial cells. Cross-blocking studies showed that binding of CD6-Ig to salivary epithelium was in part dependent on CD166, but that CD6-Ig binding may also involve additional CD6L. Binding of CD6-Ig was sensitive to trypsin digestion but resistant to digestion by collagenase and sialidase. Anti-CD166 ab precipitated CD166 from salivary epithelium pre- and post-treatment with the pro-inflammatory cytokine IFN-gamma. In contrast CD6-Ig only precipitated CD166 from IFN-gamma treated cells. More extensive colocalization between CD166 and the actin cytoskeleton was observed in sialoadenitis epithelium compared to control. We conclude that during sialoadenitis, CD166 undergoes a gain of function, resulting in closer association with the actin cytoskeleton and increased capacity to bind CD6. We suggest that altered CD166 function may contribute to the pro-inflammatory milieu during sialoadenitis seen in Sjögren's syndrome.

Expression and characterization of a novel CD6 ligand in cells derived from joint and epithelial tissues.

CD6 is a T cell surface glycoprotein that plays an important role in interactions of thymocytes with thymic epithelial cells and in mature T cell interactions with selected nonprofessional tissue APCs. We describe a novel CD6 ligand (CD6L) 3A11 Ag that is distinct from the known CD6L (CD166). The 3A11 protein is expressed on cells derived from human thymus, skin, synovium, and cartilage, and its expression is enhanced by IFN-gamma. mAbs directed against the 3A11 Ag and CD166 exhibit distinct patterns of binding to a panel of cell lines. Confocal microscopy shows that both CD166 and the 3A11 Ag are expressed at the cell surface, and that these proteins colocalize. The 3A11 Ag has a molecular mass of 130 kDa and is immunoprecipitated using either mAb 3A11 or soluble CD6-Ig fusion protein. mAbs directed against individual CD6L were less potent than was soluble CD6-Ig fusion protein in reducing adhesion of T cells to adherent 3A11-positive epithelial cells in vitro, suggesting that these Abs recognize epitopes on the 3A11 Ag and CD166 that are distinct from CD6 binding sites. Finally, transfection of epithelial cells with CD166-specific small interfering RNAs significantly decreased CD166 expression without alteration in 3A11 Ag levels, and thus confirmed that these two CD6L are distinct. Taken together, our data identifies a novel 130-kDa CD6L that may mediate interactions of synovial and epithelial cells with T lymphocytes.