Setaria Cervi Research Articles

Purification and characterization of glutathione reductase (E.C. 1.8.1.7) from bovine filarial worms Setaria cervi

Antioxidant enzymes are the parasite's premier resource to defend themselves against reactive oxygen species generated by macrophages, neutrophils and eosinophils of the host. These enzymes may be particularly important for parasites involved in chronic infections, such as parasitic helminths. Glutathione (GSH) and glutathione reductase (GR) are parts of the GSH redox cycle, which protects cells against damage by oxidants. Both GSH and GR are present in significant amounts in Setaria cervi female worms. GR has a central role in glutathione metabolism and as such is a potential target for chemotherapy. The aim of the work was to purify and characterize GR from S. cervi and to compare the properties of the helminth enzyme with its mammalian counterpart. GR was purified from filarial parasites S. cervi and preliminary steady state kinetics was performed. The purified protein was observed to be a dimer of 55kDa subunit as evident from SDS-PAGE analysis. Kinetic studies revealed significant differences in the properties of S. cervi GR from its mammalian counterpart which may be exploited in chemotherapy of filariasis. Filarial GR is thus proposed as a potential drug target.

Molecular evidence of curcumin-induced apoptosis in the filarial worm Setaria cervi

Curcumin (diferuloyl methane) is a major curcuminoid from Curcuma longa that exhibits various pharmacological effects and has shown multiple beneficial activities. Our understanding of its anticarcinogenic and other activities occurring through curcumin-induced apoptosis in several cancer cells has greatly expanded in recent years. Lymphatic filariasis is a worldwide health problem causing global disability in humans and is caused by filarial nematodes. Development of efficient strategies to promote programmed cell death in filarial worms remains a key challenge for anti-filarial drug developing research and a crucial unmet medical need. In this study, we have taken molecular and biochemical approaches toward understanding the molecular basis for curcumin-mediated anti-filarial activity in the filarial nematode Setaria cervi. Results of MTT assay showed that curcumin causes a significant reduction in viability of Mf and adults and thus acts as a potent macro- and micro-filaricidal agent. Hoechst staining, TUNEL staining, showed several apoptotic nuclei in different parts of curcumin-treated adults. At 25 μM concentration it showed chromosomal DNA fragmentation in adult worms. Our results indicate that curcumin decreases protein and mRNA expression levels of anti-apoptotic gene ced-9 and enhances both the levels of pro-apoptotic genes ced-3 and ced-4 in a dose-dependent manner. All these observations ascertained the apoptogenicity of curcumin at a minimum concentration of 50 μM in this filarial worm. Furthermore, we showed that curcumin causes depletion of parasitic glutathione level, enhances the activities of glutathione S-transferase and superoxide dismutase and stimulates rapid generation of reactive oxygen species (ROS). Here, we present molecular evidence on curcumin-induced apoptosis in the filarial nematode S. cervi with probable involvement of ROS in a caspase-dependent manner.

Effect of ferulic acid from Hibiscus mutabilis on filarial parasite Setaria cervi: Molecular and biochemical approaches

In the reported work the in vitro activity of a methanolic extract of leaves of Hibiscus mutabilis (Malvaceae) against bovine Setaria cervi worms has been investigated. Bioassay-guided fractionation led to isolation of ferulic acid from ethyl acetate fraction. The crude extract and ferulic acid, the active molecule, showed significant microfilaricidal as well as macrofilaricidal activities against the microfilaria (L1) and adult of S. cervi by both a worm motility and MTT reduction assay. The findings thus provide a new lead for development of a filaricidal drug from natural products. To examine the possible mechanism of action of ferulic acid, the involvement of apoptosis in adult worms of S. cervi was investigated. We found extreme cellular disturbances in ferulic acid-treated adult worms characterized by chromatin condensation, in situ DNA fragmentation and nucleosomal DNA laddering. In this work we are reporting for the first time that ferulic acid exerts its antifilarial effect through induction of apoptosis and by downregulating and altering the level of some key antioxidants (GSH, GST and SOD) of the filarial nematode S. cervi. Our results have provided experimental evidence supporting that ferulic acid causes an increased proapoptotic gene expression and decreased expression of anti-apoptotic genes simultaneously with an elevated level of ROS and gradual dose dependent decline of parasitic GSH level. We also observed a gradual dose dependent elevation of GST and SOD activity in the ferulic acid treated worms.

Synthesis and Antifilarial Evaluation of 7-O-Acetamidyl-4-alkyl-2H-1-benzopyran-2-ones

A series of 7-O-acetamidyl-4-alkyl-2H-1-benzopyran-2-ones (5-23) has been synthesized by amidation of 7-O-(carbethoxymethyl)-4-alkyl-2H-1-benzopyran-2-ones (2a, 2b) with different primary and secondary amines in fair to good yield. The resulting compounds were screened for their filarial DNA topoisomerase inhibitory activity under in vivo condition in Setaria cervi. The compounds were tested in vitro against Brugia malayi. A few of the compounds possess promising antifilarial activity.

Observations on in vitro and in vivo antimicrofilarial effects of Bishop’s weed (Trachispermum ammi)

The antimicrofilarial efficacy of Trachispermum ammi extacts in vitro and in vivo using Setaria cervi as a model, was investigated. T. ammi seed extracts were prepared using different solvents (with increasing order of polarity of the solvent) including petroleum ether, diethyl ether, chloroform, ethyl acetate, acetone, ethanol, and methanol. The extracts were tested for in vitro antimicrofilarial activity. The ethanolic and the methanolic extracts showed maximum activity in causing flaccidity in the microfilariae. The extracts were potent even at concentrations as low as 5μl/ml. When orally administered to experimentally infected rats, the extracts eliminated circulating microfilariae within 2weeks. It is inferred that the antimicrofilarial molecule(s), are polar in nature. They induce flaccidity in the microfilariae, by possibly inhibiting monoamine oxidase. This communication supplements the ethnopharmacological information for the use of T. ammi as an antihelminthic, and indicates that T. ammi could be used as a potential source of antimicrofilarial drugs.

Effect of diethylcarbamazine, butylated hydroxy anisole and methyl substituted chalcone on filarial parasite Setaria cervi: Proteomic and biochemical approaches

For survival, parasite exerts several lines of defense of which drug neutralization is one of the major phenomena. Lack of phase I cytochrome P450 in some of the nematode render them depend on the phase II detoxification system involving GST as a major detoxifying enzymes. In present study, the antifilarial DEC, phenolic compound BHA and methyl chalcone have been evaluated for proteomic and biochemical studies in Setaria cervi. BHA and methyl chalcone showed cytotoxic effect leading to irreversible inhibition in motility and viability of parasites. These drugs showed marked alteration in proteomic profile of S. cervi at 100 μM concentration with 10.82, 8.52 and 6.75% downregulated (<0.5) and 7.64, 31.78 and 24.32% upregulated (>1.5) in DEC, BHA and methyl chalcone treatment respectively. Significant depletion in GSH level with increase in NO production was observed. Amongst these compounds, methyl chalcone demonstrated significant inhibitory effect (p<0.05) on GST, PGHS and PTP activity leading to loss of metabolic homeostasis and parasite death. The cytotoxic response and altered expression profile of major enzymes under drug exposure suggested the oxidative stress induced apoptosis as a major cause of parasite killing which was further supported by DNA fragmentation in BHA and methyl chalcone.

Setaria cervi, a test organism for screening antifilarial agents.

Setaria cervi, a test organism for screening antifilarial agents.

Albendazole induces apoptosis in adults and microfilariae of Setaria cervi

Setaria cervi, a filarial nematode of cattle, inhabits in the peritoneal cavity and has been used as a suitable model for screening antifilarial agents. Albendazole (ABZ), a tubulin-disrupting benzimidazole (BZ) and a potent microfilaricide binds to β-tubulin, is causing structural impairment of cytoskeleton and worm death. Our present study has revealed that exposure of microfilaria (Mf) and adult to gradually increasing concentration of ABZ leads to a dose-dependent gradual impairment of their motility followed by early death in vitro. We found extreme cellular disturbances in ABZ-treated worms characterized by nucleosomal DNA laddering and chromatin condensation. However, in the treated Mf no nucleosomal DNA laddering was found although presence of TUNEL reactive DNA was evident, thus indicating an apoptotic pathway independent of DNA fragmentation. We present data from molecular studies to provide evidence for ABZ-induced apoptosis in Mf and adult worms of S. cervi.

Identification of major antigenic peptide of filarial glutathione-S-transferase

In our earlier report, a 26kDa Setaria cervi glutathione-S-transferase showed significant protection (82%) in jirds infected with L3 larvae of Brugia malayi. In the present study we have identified the major antigenic epitopes in ScGST. Carboxypeptidase B has been used to digest the ScGST in to smaller fragments. The digested products were separated as four protein bands on SDS-PAGE. The smallest fragment of 6kDa (P4) from ScGST was identified as major antigenic epitope because of its significant reactivity with jird anti ScGST sera and human filarial sera in immunoblotting. The MALDI-LC/MS sequencing of ScGST P4 peptide (5KLTYFSIRGRGLAEPIRL20, 22KVPDDQQFLDDLISR36 and 47VFHFGQGPHHGPPR62) suggested that this protein band has a fragment of 5–62 residues long that matched with the N-terminal end of filarial GST. The antigenicity plot of ScGST was compared with BmGST model and both exhibited three immunogenic peaks within the first 60 residues towards N-terminal. In BmGST the N-terminal region was also detected with N-glycosylation signal peptide NAS adding to its high immunogenic property. Further, P4 showed strong reactivity with IgG1 and IL-4 response in endemic normal sera suggested its role in Th2 response which in turn is correlated with antibody dependent cell mediated cytotoxicity. Thus taking these results into account we propose 5–62 residues long N-terminal peptide of GST as a potential target for further vaccination studies against filarial infection.

Setaria cervidual specific phosphatase: characterization and its effect on eosinophil degranulation

Setaria cervi, a bovine filarial parasite contains significant acid phosphatase (AcP) activity in its various life stages. Two forms of AcP were separated from somatic extract of adult female parasite using cation exchange, gel filtration and concavalin affinity chromatography. One form having a molecular mass of 79 kDa was characterized as dual specific protein tyrosine phosphatase (ScDSP) based on substrate specificity and inhibition studies. With various substrates tested, it showed significant activity in the order of phospho-L-tyrosine>pNPP>ADP>phospho-L-serine. Inhibition by orthovanadate, fluoride, molybdate, and zinc ions further confirms protein tyrosine phosphatase nature of the enzyme. Km and Vmax determined with various substrates were found to be 16.66 mM, 25.0 microM/ml/min with pNPP; 20.0 mM, 40.0 microM/ml/min with phospho-L-tyrosine and 27.0 mM, 25.0 microM/ml/min with phospho-L-serine. KI with pNPP and sodium orthovanadate (IC50 33.0 microM) was calculated to be 50.0 mM. Inhibition with pHMB, silver nitrate, DEPC and EDAC suggested the presence of cysteine, histidine and carboxylate residues at its active site. Cross-reactivity with W. bancrofti-infected sera was demonstrated by Western blotting. ScDSP showed elevated levels of IgE in chronic filarial sera using ELISA. Under in vitro conditions, ScDSP resulted in increased effector function of human eosinophils when stimulated by IgG, which showed a further decrease with increasing enzyme concentration. Results presented here suggest that S. cervi DSP should be further studied to determine its role in pathogenesis and the persistence of filarial parasite.

Antifilarial Activity of 1,3-Diarylpropen-1-One: Effect on Glutathione-S-Transferase, a Phase II Detoxification Enzyme

Chalcone derivatives were evaluated for their antifilarial activity on Setaria cervi using glutathione-S-transferase (GST) as a drug target. The compounds 1-(4-benzotriazol-1-yl-phenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (5), and 3-(4-methoxyphenyl)-1-(4-pyrrolidin-1-yl-phenyl) prop-2-en-1-one (7) showed a significant suppression (P < 0.01) in GST activity of adult female parasite extract at 3 microM concentration in vitro. However, GST activity was detected along with depletion in GSH level. Except Compounds 1 and 2, all exhibited a significant effect on the motility and viability of adult parasites. Compounds 3-(4-chlorophenyl)-1-(4-piperidin-1-yl-phenyl)prop-2-en-1-one (3), 1-(4-benzotriazol-1-yl-phenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (5), and 3-(4-methoxyphenyl)-1-(4-pyrrolidin-1-yl-phenyl) prop-2-en-1-one (7) exhibited major irreversible effects on viability and resulted in parasite death and also inhibited the GST activity by 84-100% in vitro. We report for the first time the antifilarial activity of chalcones on GST of adult parasites. This study also strengthens our previous findings where GST is reported as a potential drug target for antifilarials.

Evidence for the presence of prostaglandin H synthase like enzyme in female Setaria cervi and its inhibition by diethylcarbamazine

Experimental evidence has shown that Setaria cervi a bovine filarial parasite contains significant amount of prostaglandin H synthase like activity in the somatic extract of its different life stages. A protein with characteristics of prostaglandin H synthase was purified to homogeneity from female somatic extract using a combination of affinity and gel filtration chromatography. Molecular weight of purified enzyme was 70 kDa as determined by SDS-PAGE. Purified enzyme showed high activity with arachidonic acid and TMPD substrates suggests the presence of both cyclooxygenase and peroxidase activity in enzyme. Fluorescence spectroscopy and hemin-associated peroxidase activity confirmed presence of heme in purified enzyme. The K m and V max values using arachidonic acid were determined to be 79 ± 1.5 μM and 0.165 ± 0.2 U/ml, respectively. Further, indomethacin and aspirin, specific inhibitors for PGHS, significantly inhibited the enzyme activity. Diethylcarbamazine, an antifilarial drug inhibited the microfilarial PGHS like activity as well as their motility. Here we are reporting for the first time PGHS like activity in filarial parasite and its inhibition with DEC which provide that this enzyme could be used as a drug target.

Screening of different classes of proteases in microfilarial and adult stages of Setaria cervi

Many of the filarial proteases involved in critical physiological functions are expressed in stage-specific manner and belong to various mechanistic classes. Setaria cervi, a bovine filarial parasite express different classes of proteases. This parasite shows strong antigenic cross-reactivity with human filarial parasites Wuchereria bancrofti and Brugia malayi. Somatic extracts of S. cervi microfilariae (mf) and adult stages as well as their excretory-secretory (ES) products were screened for the presence of different classes of proteases using general (casein, bovine hemoglobin) and class specific substrates. Detergent-soluble extracts of male and female worms were also screened. Significant enzyme activity was detected in ES products both at pH 5.0 and 7.0 with casein. Cathepsin B-like activity was found to be much higher in membrane-bound extract than in the crude-soluble extract. However, it was also found to be actively secreted by both mf and adult worms. Cathepsin D-like activity assayed at pH 3.0 was very low both in somatic extract as well as in ES products. Collagenase activity at neutral pH showed higher levels, both in somatic extract and ES products. Cathepsin L-like activity was detected only in crude-soluble extract but was below detectable limit in ES products. Leucine aminopeptidase activity was significant both in crude-soluble extract and ES products. This study, thus, might be helpful for a better understanding of host-parasite interaction and identification of appropriate virulence factors that may be targeted as vaccine and/or drug targets against lymphatic filariasis.

Screening of fruit of Diospyros montana for anti-filarial activity.

Objective: To screen the potential antifilarial activity of petroleum and alcoholic extracts prepared from fruit of Diospyros montana . Materials and Methods: In vitro study was carried out to see the effect of extracts on both the whole worm(w.w). preparation and nerve muscle (n.m) preparation of Setaria cervi , on the survival of microfilariae. Results: Petroleum extract produced initial stimulation followed by reversible paralysis in whole worm The initial stimulation is not seen with petroleum extract on nerve muscle preparation. The alcoholic extract caused reversible paralysis in whole worm whereas irreversible in nerve muscle preparation. Conclusion: Fruit of D. montana was found to possess potential antifilarial activity.

Synthesis of 4-amino-5-cyano-2, 6-Disubstituted Pyrimidines as a Potential Antifilarial DNA Topoisomerase II Inhibitors

A novel series of 4-amino-5-cyano-2, 6-disubstituted pyrimidines have been synthesized and evaluated for their in vitro antifilarial DNA topoisomerase II activity against filarial parasite Setaria Cervi. In particular compounds bearing 4-chloro-phenyl substitutent at position-6, exhibited strong inhibition at 40 microg/mL and 5 microg/mL concentration. The present study based on the biological results obtained, suggests that the nature of substitutent at position-4 in the phenyl ring directly affects DNA topoisomerase II inhibitory activity. Most of the compounds have shown better topoisomerase II inhibitory activity than the standard antifilarial drug (DEC) and the topoisomerase II inhibitors (Novobiocin, Nalidixic acid).

Die Endoparasiten des Sikawildes (Cervus nippon) in Österreich

The endoparasite fauna of 108 sika deer (42 calves <1 year, 20 approximately 1 year old animals, 46 animals >1 year) originating from the two free-living sika deer populations in Austria (Ostrong, 35 animals; Tullner Donauauen, 73 animals) was studied. The deer were shot during the hunting seasons 2003-2005. In all, at least four species of protozoa (Eimeria austriaca, Eimeria robusta, Eimeria sordida; Sarcocystis spp.), two species each of cestodes (Moniezia benedeni, larval cyst of Taenia hydatigena) and trematodes (Dicrocoelium chinensis?, Fasciola hepatica) and 16 species of nematodes were identified including 14 species recovered from the gastro-intestinal tracts and one species each isolated from the lungs (Dictyocaulus eckerti) and the abdominal cavity (Setaria cervi). Endoparasites were recovered from all 108 deer with prevalences of 44% for Sarcocystis spp., 14.8% for Eimeria spp., 4.6% for Fasciola hepatica, 27.6% for Dicrocoelium chinensis?, 3.1% for Dictyocaulus eckerti, 3.7% for Moniezia benedeni and 98.1% for gastro-intestinal nematodes. The burden of gastro-intestinal nematodes ranged from zero to 1089 with a geometric mean of 149 worms. The abomasums, small and large intestines harboured 81%, 14% and 5% of the total gastro-intestinal nematode burden. Spiculopteragia houdemeri (93.5%), Oesophagostomum sikae (87.9%), Oesophagostomum venulosum (51.4%), Cooperia pectinata (42.1%), Spiculopteragia böhmi (23.4%) and Ostertagia leptospicularis (16.8%) were the most prevalent nematode species of the gastro-intestinal tracts. Spiculopteragia houdemeri and Rinadia andreevae were new records for Austria.

Biochemical composition and metabolic pathways of filarial worms Setaria cervi: search for new antifilarial agents

The main problem regarding the chemotherapy of filariasis is that no safe and effective drug is available yet to combat the adult human filarial worms. Setaria cervi, the causal organism of setariasis and lumbar paralysis in cattle, is routinely employed as a model organism for conducting biochemical and enzymatic studies on filarial parasites. In view of the practical difficulties in procuring human strains of Wuchereria bancrofti and Brugia malayi for drug screening, the bovine filarial parasite S. cervi, resembling the human species in having microfilarial periodicity and chemotherapeutic response to known antifilarial agents, is widely used as a model in such studies. For a rational approach to antifilarial chemotherapy, knowledge of the biochemical composition and metabolic pathways of this helminth parasite may be of paramount importance, so that more potent antifilarial agents based on specific drug targets can be identified in drug discovery programmes. The present review provides an update on the biochemistry of the important metabolic pathways functioning within this potentially important bovine parasite, that have so far been studied, and on those that need to be investigated further so as to identify novel drug targets that can be exploited for designing new antifilarial drugs.

Vaccination with Setaria cervi 175 kDa collagenase induces high level of protection against Brugia malayi infection in jirds

A zinc containing metalloprotease, 175 kDa collagenase, purified from adult female Setaria cervi showed strong cross-reactivity with sera from putatively immune (PI) individuals (unpublished observation) and induced cytotoxicity to B. malayi L3 larvae and microfilariae by ADCC mechanism [Srivastava Y, Bhandari YP, Reddy MVR, Harinath BC, Rathaur S. An adult 175 kDa collagenase antigen of Setaria cervi in immunoprophylaxis against Brugia malayi. J Helminth 2004;78:347–52]. These preliminary observations suggested the immunoprotective nature of collagenase. To confirm the vaccine potential of this protease, a vaccine trial was conducted in jirds ( Meriones unguiculatus) against human filarial parasite B. malayi. The vaccination resulted into a mean protection level of 75.86% and produced high level of protease neutralizing antibodies. Cytokine analysis in immune jirds sera suggested a mixed Th1/Th2 type cellular immune response whereas ELISA, immunoblotting and enzyme antibody inhibition assay revealed the presence of specific anti-collagenase antibodies. Taken together, all these results suggest that S. cervi 175 kDa collagenase could form the basis of an effective molecular vaccine against human lymphatic filariasis.

Tissue localization of collagenase and leucine aminopeptidase in the bovine filarial parasite Setaria cervi

BackgroundLike other helminth proteases, filarial proteases have also been shown to require for parasite survival inside the host and mediate various physiologic processes such as tissue invasion, feeding, embryogenesis and host immune evasion. Many of these proteases have shown potential for vaccines and chemotherapeutic agents against active filarial infections. Setaria cervi is a bovine filarial parasite and serves as a good parasite model for the studies in lymphatic filariasis. Recently, a 175 kDa collagenase and leucine aminopeptidase (LAP) have been purified and characterized from the bovine filarial parasite S. cervi and shown to be potential vaccine candidate and diagnostic marker, respectively for human lymphatic filariasis. However, their tissue localizations and putative roles in the parasite biology have not yet been examined and thus remain unclear. Therefore, the current study attempts to localize and explore the putative roles of these two enzymes in S. cervi.MethodsThe tissue distributions of 175 kDa collagenase and leucine aminopeptidase in S. cervi were examined by immunohistochemical and histochemical methods, respectively. Immune sera obtained from the jirds immunized with collagenase served as primary antibody, rabbit anti-mouse IgG-HRP conjugate as secondary antibody and DAB as the substrate for the immunostaining of collagenase. Leu-βNA was used as the substrate for the histochemical staining of LAP.ResultsBoth the collagenase and LAP were present in the body wall; however, they differ in their distribution pattern in different layers of body wall. Collagenase was mainly localized in epicuticle, cuticle, syncytial hypodermis and the nerve cord region whereas LAP was more concentrated in epicuticle, longitudinal muscle layers and almost absent or very faintly stained in syncytial hypodermis and nerve cord region. Both collagenase and LAP showed their common distributions in intestine, uterus and mature eggs, growing embryos and mf. Very strong immunostaining of collagenase in the outer body surface of the parasite indicates its major role in host-parasite relationship whereas the presence of LAP in muscular region suggests its role in tissue remodeling. The common presences of collagenase and LAP in the S. cervi intestine, ovary, uterus, eggs and mf suggest that they also have collaborative roles in molting, nutrition and embryogenesis. The data obtained on their immunological characterizations and their presence in important parasite organs give strong indication that they are critical for the survival of filarial parasite and thus can be good vaccine candidates and/or diagnostic markers for human lymphatic filariasis.ConclusionThe manuscript reports for the first time the tissue distribution of collagenase and LAP in the bovine filarial parasite S. cervi and discuss their putative roles in vivo. Our findings also open the avenue to examine the roles of these two proteases in vivo, which will require further experiments like using their natural substrates and/or specific inhibitors in each tissues.

Setaria cervi: Kinetic studies of filarial glutathione synthetase by high performance liquid chromatography

The bovine filarial worm Setaria cervi was found to have abundance of glutathione synthetase (GS; EC 6.3.2.3) activity, the enzyme being involved in catalysing the final step of glutathione (GSH) biosynthesis. A RP-HPLC method involving precolumn derivatization with o-phthalaldehyde has been followed for the estimation of GS activity in crude filarial preparations. Subcellular fractionation of the enzyme was undertaken and it was confirmed to be a soluble protein residing mainly in cytosolic fraction. Attempts to determine the K m value for l-γ-glutamyl- l-cysteine gave a distinctly nonlinear double-reciprocal plot in which data obtained at relatively high dipeptide concentrations (>1 mM) extrapolate to a K m value of about 400 μM whereas data obtained at lower concentrations (<0.1 mM) extrapolate to a value of about 33 μM. K m was determined to be around 950 and 410 μM for ATP and glycine, respectively. The effect of various amino acids was studied on enzyme activity at 1 mM concentration. l-Cystine caused a significant enzyme inhibition of 11%. Preincubation with N-ethylmaleimide also resulted in significant inhibition of GS activity.