Production and characterization of a monoclonal antibody specific to 16 kDa antigen of Paramphistomum gracile.

A number of monoclonal antibodies (MoAbs) against the 16 kDa antigen of Paramphistomum gracile (16 kDaAgPg) were produced in vitro by hybridoma technique. Reactivity and specificity of these MoAbs were evaluated by ELISA and immunoblotting assays. Seven MoAb clones were selected from the stable hybridoma clones, namely 1D10, 2D7, 3B10, 3D9, 4F1, 4G4, and 5G12. It was found to be IgM and kappa light chain isotypes. By immunoblotting and ELISA, all MoAbs reacted with purified 16 kDaAgPg at molecular weight (MW) of 16 kDa and with the native 16 kDa antigen at MW of 16 kDa in the whole body (WB) and excretory-secretory (ES) fractions, but not with tegumental antigens (TA) of adult fluke. All of these MoAbs showed no cross-reactions with antigens of other parasites commonly found in ruminants, including Eurytrema pancreaticum, Gigantocotyle explanatum, Schistosoma spindale, Moniezia benedeni, Avitellina centripunctata, Haemonchus placei, Trichuris sp., and Setaria labiato-papillosa. Localization and distribution of the native 16 kDaAg in adult P. gracile by immunohistochemistry, using MoAbs as probes, showed that the native 16 kDaAg was present in high concentration in the cytoplasm of vitelline cells, eggshell globules, and the shells of eggs, but not in the tegument, muscle, parenchymal cells, and cecum of adult fluke. This finding indicated that the 16 kDaAg is a copiously expressed parasite protein that is released into the ES; thus, 16 kDaAg and its MoAb could be a good candidate for immunodiagnosis of paramphistomosis in ruminants.

Antigenic profile, isolation and characterization of whole body extract of Paramphistomum gracile.

An antigenic component of adult Paramphistomum gracile was characterized by means of indirect enzyme-linked immunosorbent assay (indirect ELISA), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using sera from cattle naturally infected with P. gracile, Eurytrema pancreaticum, Fasciola gigantica, Moniezia benedeni, strongylids, Trichuris sp. and Strongyloides sp. The whole body (WB) extracts of P. gracile were fractionated by gel filtration chromatography in a Sephadex G-200 column. It was found that the WB extract fractions, F1-F3 were highly antigenic, F5 was moderately antigenic and F4 was poorly antigenic. For SDS-PAGE and immunoblotting, the antigenic molecules of WB extract and all five fractions were mostly at molecular weights (MW) ranging from 12 to 150 kDa. One antigenic protein of 16 kDa detected in WB extract and F1-F3 was found to give a consistent reaction with sera from infected cattle. The antigenicity of the purified 16 kDa protein was confirmed by immunoblotting and indirect ELISA using a pool of sera and individual serum samples from infected cattle (at 1 : 78 125 dilution) and hyperimmunized rabbit (at 1 : 390 625 dilution). This finding suggests that the 16 kDa protein may be a potential antigen for the immunodiagnosis of cattle paramphistomosis caused by P. gracile.

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.

Evaluation of antibody response to various developmental stage specific somatic antigens of Paramphistomum epiclitum in goats.

Electrophoretic analysis of various developmental stage specific somatic antigens of Paramphistomum epiclitum (Digenea: Paramphistomidae), namely, metacercariae (McAg), immature intestinal flukes (ImIAg), immature ruminal flukes (ImRAg), and adult flukes (AAg), was done by native polyacrylamide gel electrophoresis. Result revealed presence of 3 (range 15.2-40.3 kDa), 13 (9.3-121.2 kDa), 14 (9.3-169.3 kDa), and 15 (8.0-169.3 kDa) polypeptides in McAg, ImIAg, ImRAg, and AAg, respectively. With an aim to identify a suitable immunodiagnostic antigen for early diagnosis of amphistomosis, the IgG antibody response to various developmental stage antigens in goats experimentally infected with metacercariae of P. epiclitum was evaluated by ELISA. The highest OD values were recorded with ImIAg which ranged between 0.23 and 0.55 with a significant increase from the 2nd week till 8th week of infection with a peak at 6th week. The analysis of statistical significance using a one-way analysis of variance with multiple pair wise comparisons revealed that IgG response was significantly higher with all antigens (P < 0.01) except McAg (P > 0.05) with a maximum mean difference of 0.1838 in comparison to control with ImIAg, thus, indicating that ImIAg which could be further exploited for its potential is a candidate for immunodiagnostic antigen for early diagnosis of amphistomosis.

Evaluation of Gastrothylax crumenifer antigenic preparation in serodiagnosis of paramphistomiasis in sheep.

An evaluation of Gastrothylax crumenifer crude antigen preparation viz., Somatic Antigen (SAg), Excretory Secretory Antigen (ESAg) and Egg Antigen (EAg) in serodiagnosis of disease was undertaken. Test sera samples were obtained from 30 Paramphistomiasis Positive and 30 Gastrothylax free sheep slaughtered at Hazratbal Kashmir. The referral antigenic preparation were evaluated against Paramphistomiasis positive sera, via., control negative sera, using double immunodiffusion test (DID), (IEP) Immunoelectrophoretic assay and ELISA. The performance of referral antigens, as assessed from percent sensitivity and specificity, revealed an increasing trend from DID (Double immunodiffusion-An immunological technique used in the detection, identification and quantification of antibodies and antigens) to IEP (immunoelectrophoresis-A general name for a number of biochemical methods for separation and characterization of proteins based on electrophoresis and reaction with antibodies), followed by ELISA, detecting higher number of sheep positive for paramphistomiasis. In ELISA the ESAg and SAg were evaluated as most reactive antigens with no significant difference and EAg was the least antigenic. In IEP, EAg had the higher sensitivity (60%) and analogous specificity of SAg and ESAg. The formation of the preceptin lines in the proximity to EAg containing wells (cathode end) in IEP was suggestive of higher molecular weight of G. crumenifer specific protein molecules with slower rate of migration. Purification and characterization of G. crumenifer and identification of specific antigenic molecules, particularly in EAg has been suggested for qualitative improvement of diagnostic value of the antigens in the tests used here in.

Identification of the rumen fluke, Calicophoron daubneyi, in GB livestock: possible implications for liver fluke diagnosis.

The liver fluke, Fasciola hepatica, is common in many parts of Great Britain. To detect liver fluke infection and to assess whether fasciolicide treatment has been successful, the faecal egg count (FEC) and faecal egg count reduction test (FECRT) are widely used. Rumen fluke is also increasingly reported from Great Britain, but its species identity is yet to be determined. Liver fluke and rumen fluke eggs are morphologically similar, which may lead to erroneous diagnoses of liver fluke infection or treatment failure. As an alternative to FEC, a coproantigen ELISA (cELISA) can be used. The potential for this test to cross-react with rumen fluke species from Great Britain has not been evaluated. Rumen fluke specimens from cattle and sheep in Scotland were identified to species level using DNA sequencing of the ITS-2 region. Subsequently, rumen and liver fluke obtained from naturally co-infected sheep were subjected to immunohistochemistry using antibodies from a commercially available cELISA kit for F. hepatica. Finally, faecal samples from naturally co-infected sheep flocks were examined by FEC and cELISA. Rumen fluke from imported and home-bred cattle and sheep in Scotland belonged to the species Calicophoron daubneyi, rather than Paramphistomum cervi, the species presumed to be most common in Great Britain. Intense staining of the gastrodermis was observed in F. hepatica but cross-reactivity with C. daubneyi was not seen. Faecal samples that contained rumen fluke eggs but not liver fluke eggs were all negative by cELISA. We conclude that C. daubneyi is the most common rumen fluke of domestic ruminants in Scotland and that cELISA reduction testing may be a valuable alternative to FECRT in herds or flocks that are co-infected with liver and rumen fluke.

Antigenic components, isolation and partial characterization of excretion-secretion fraction of Paramphistomum cervi.

The immunogenic components of adult Paramphistomum cervi excretion-secretion (ES) fraction were revealed by SDS-PAGE and immunoblotting technique using sera from cattle naturally infected with P. cervi, Fasciola gigantica, strongylids, Trichuris sp., and Strongyloides sp. By SDS-PAGE, it was found that the ES fraction comprised 13 distinct protein bands. Immunoblotting analysis of these proteins exhibited nine prominent antigenic bands which were recognized by paramphistomosis antisera. These antigenic proteins had molecular weights ranging from 10-170 kDa. One antigenic protein band of 40 kDa was found to give a consistent reaction with sera from all infected cattle. Its diagnostic sensitivity, specificity and accuracy using this test were 100%, 98.9% and 99.3%, respectively. The positive and negative predictive values were 98% and 100%, respectively. The 40 kDa antigen was partially purified by gel filtration and ion-exchange chromatography. The antigenicity of 40 kDa protein for diagnosis of P. cervi infection was confirmed by immunoblotting and indirect ELISA (at 1:78,125 dilution) using a pool of sera and individual serum samples from infected cattle. The present findings suggest that the 40 kDa protein may be used as a diagnostic antigen for paramphistomosis.

Specificity of a coproantigen ELISA test for fasciolosis: lack of cross-reactivity with Paramphistomum cervi and Taenia hydatigena.

A commercial coproantigen ELISA test for fasciolosis, based on the use of MM3 monoclonal antibody for antigen capture, was investigated for possible cross-reactivity with Paramphistomum cervi, a trematode that commonly infects cattle and sheep grazing in fluke-infested pasture in Ireland. Histological sections of adult and immature Fasciola hepatica and P cervi were incubated with MM3 monoclonal antibody, and its binding to tissue-localised coproantigen was subsequently visualised by immunocytochemistry. In a related study, the soluble antigenic fractions derived from homogenates of P cervi adults and Taenia hydatigena metacestodes were tested for cross-reactivity with MM3 monoclonal antibody in an antigen-capture ELISA, using known F hepatica-positive and F hepatica-negative ovine faecal samples as natural controls. It was found that, while intense immunocytochemical labelling was located over the gastrodermis and gut contents of adult and immature F hepatica, sections of adult and immature P cervi were unlabelled. In the ELISA tests, the soluble fractions of F hepatica reacted strongly with MM3 monoclonal antibody, but those of P cervi and T hydatigena gave negative results. These findings support the specificity of the coproantigen ELISA test for fasciolosis in areas where paramphistomosis and cysticercosis are liable to occur singly or as coinfections with F hepatica.

The hemoglobins of the trematodes Fasciola hepatica and Paramphistomum epiclitum: a molecular biological, physico-chemical, kinetic, and vaccination study.

The trematode Fasciola hepatica (Fa.he.) is a common parasite of human and livestock. The hemoglobin (Hb) of Fa.he., a potential immunogen, was chosen for characterization in the search for an effective vaccine. Characterization of trematode Hbs show that they are intracellular single-domain globins with the following remarkable features: (1) Fa.he. expresses two Hb isoforms that differ at two amino acid sites (F1: 119Y/123Q; F2: 119F/123L). Both isoforms are monoacetylated at their N-termini; (2) the genes coding for Fa.he. and Paramphistomum epiclitum (Pa.ep.) Hbs are interrupted by two introns at the conserved positions B12.2 and G7.0.; (3) UV/VIS and resonance Raman spectroscopy identify the recombinant Fa.he. HbF2 as a pentacoordinated high-spin ferrous Hb; (4) electron paramagnetic resonance spectroscopy of cyano-met Fa.he. HbF2 proves that the endogenously bound imidazole has no imidazolate character; (5) the major structural determinants of the globin fold are present, they contain a TyrB10/TyrE7 residue pair on the distal side. Although such distal-site pair is a signature for high oxygen affinity, as shown for Pa.ep. Hb, the oxygen-binding rate parameters for Fa.he. Hb are intermediate between those of myoglobin and those of other trematode Hbs; (6) the three-dimensional structure of recombinant Fa.he. HbF2 from this study closely resembles the three-dimensional structure of Pa.ep. determined earlier. The set of distal-site polar interactions observed in Pa.ep. Hb is matched with small but significant structural adjustments; (7) despite the potential immunogenic character of the fluke Hb, vaccination of calves with recombinant Fa.he. HbF2 failed to promote protection against parasitic infection.

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.

Analysis of the IgG antibody response against Paramphistomidae trematoda in naturally infected cattle. Application to serological surveys.

The IgG antibody response to Calicophoron daubneyi (Digenea: Paramphistomidae) excretory/secretory antigens was evaluated in naturally infected cattle from Lugo (Galicia, NW Spain) by using an ELISA procedure. Two studies were conducted, first a survey in 524 cattle separated into three groups according to age, G-1 (0-2 years old), G-2 (3-5 years old) and G-3 (> 6 years old). In the second study, three groups of cattle were employed: G-I, naturally infected; G-T, naturally infected and treated with oxyclozanide plus levamisole (Nilzan Plus); G-C, cattle maintained in a farm where C. daubneyi has never diagnosed. Variations on egg-output and haematic parameters (erythrocytes, haematocrite, leukocytes and lymphocytes) were also analyzed. The ELISA procedure showed that 61.2% of the cattle in the first study had been exposed to the trematode, but only 10.1% passed eggs in the feces. Age-association with egg-output was shown but not with the IgG values. In the second experiment, the administration of the anthelmintic reduced significantly the IgG kinetic levels and the C. daubneyi-egg-output was suppressed during 12 weeks in the G-T group. The values of red cells, haematocrite, leukocytes and lymphocytes increased significantly in the treated cattle 5 weeks after chemotherapy; however, new reduction after week 5 was recorded, as results of the challenge of these cattle. This is the first investigation in which evaluation of the IgG humoral response against C. daubneyi in cattle has been carried out. We proved that a notable IgG response in naturally infected cattle is induced, and can be detected by using an ELISA procedure. The IgG antibodies did not increase after challenge infection. Our results proved an important percentage of cattle were exposed to this trematode in the area of study and suitable measures for preventing this relationship must be considered.

Characterization of specific and cross-reacting antigens of Fasciola gigantica by immunoblotting.

Somatic antigens of F. gigantica, G. explanatum, S. spindale and hydatid cyst ingredients were analysed to identify the cross-reactive antigens among them using Western blot technique. When probed with F. gigantica infected cattle sera, the immunodominant 156 kDa and 28 kDa proteins of F. gigantica was found common amongst the antigens prepared from hydatid cysts ingredients like germinal layer, fertile and sterile, hydatid fluid, fertile and sterile, while another protein of 34 kDa was shared between F. gigantica and antigen prepared from protoscolices. In F. gigantica-buffalo system the proteins of 34 kDa and 28 kDa were found reactive with most of the antigens tested. Immunoaffinity chromatography using, F. gigantica infected rabbit immunoglobulins as legands isolated the immunodominant 34 kDa and 28 kDa proteins in dimer form and the same were found immunodominant in F. gigantica-cattle, F. gigantica-buffalo and F. gigantica-sheep system. No cross-reaction was noted with the sera of goats experimentally infected with Paramphistomum epiclitum. ELISA with the immunodominant proteins of 34 kDa and 28 kDa could be a feasible diagnostic tool for the early detection of bovine fasciolosis.

Partial purification and characterization of Gigantocotyle explanatum somatic antigens.

Soluble extracts of Gigantocotyle explanatum, isolated from the liver of buffalo Bubalus bubalis were fractionated on Sephadex G-200 columns. Nine major fractions referred to as F1, F2, F3, F4, F5, F6, F7, F8 and F9 were separated. Each fraction was tested by ELISA for antigenicity using sera from G. explanatum-infected field buffaloes. Fractions F1 and F2 were highly antigenic, F3, F4, F6 and F7 were moderately antigenic and F5, F8 and F9 were poorly antigenic. Analyses by SDS-PAGE revealed that each fraction comprised several polypeptide(s) in the molecular weight range of <29 to >205 kDa. Results of Western blotting indicated that not all polypeptides which appeared in the SDS-PAGE were antigenic. The antigenic molecules of each fraction were mostly in the low molecular weight range of <14 to >94 kDa with the polypeptides in the range of >14, 14, 18, 21-25 and 34-36 kDa.

Time-course analysis of antibody response by EITB and ELISA before and after chemotherapy in sheep infected with Fasciola gigantica.

The time-course analysis of the antibody response to Fascicola gigantica infection in sheep was studied by enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunoelectrotransfer blot (EITB). Sera from sheep experimentally infected with F. gigantica were reacted with excretory-secretory antigens of the worm before and after chemotherapy with oxyclozanide. In ELISA, there was a significant increase in anti-Fasciola antibody by 2 weeks after infection and there was a sharp decrease in antibody titer by 4 weeks after treatment. By EITB, the infected sheep sera recognised four polypeptides in the range of 43-75 kDa as early as 2 weeks after infection, with more polypeptides being recognised as the infection progressed. Recognition of an 87 kDa antigen was lost by 2 weeks after treatment and is therefore a good marker for treatment efficacy. Comparative immunoblotting with sheep anti-Paramphistomum, anti-Dicrocoelium and anti-Fasciola sera revealed that the 17 kDa, 21 kDa, 57 kDa and 69 kDa proteins are specific to fasciolosis and are good antigens for early and specific immunodiagnosis of F. gigantica infection in sheep.

Detection of stable diagnostic antigen from bile and feces of Fasciola hepatica infected cattle.

Diagnostic antigens in bile and feces from Fasciola hepatica infected cattle were detected and characterized by enzyme-linked immunotransfer blot (EITB) techniques. As sources of antigen, samples of bile, intestinal contents and feces were collected from five uninfected calves and from 10 calves with known Fasciola hepatica burdens. A band detected by EITB using a densitometer in the area corresponding to 26 kDa reacted with rabbit anti-fresh fluke antigen and infected cattle sera but not with fluke-negative rabbit sera, rabbit anti-Fasciola hepatica egg sera, Fascioloides magna positive or negative cattle sera. This band was not detected by Coomassie blue in sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels or by Ponceau-S stained nitrocellulose strips. Band groups located at 104-66, 66-42, 42-26 and 25-16 kDa reacted inconsistently with the above sera. Sera from mice hyperimmunized with Fasciola hepatica excretory-secretory (ES) products detected only the 26 kDa band by EITB, without cross-reactivity with bands in the other molecular weight (MW) ranges. The results suggest that the 26 kDa antigen may consist of a stable component of ES products and/or tegument-related worm antigen. Diagnosis of Fasciola hepatica through detection of specific, stable antigens in feces of infected animals offers potential advantages over serum-based tests of better sample accessibility, discrimination between previous and current infections, and possible semi-quantitation of fluke burdens.

[Antigenic structure of Dicrocoelium dendriticum (Rudolphi, 1819)]. / Badania struktury antygenowej Dicrocoelium dendriticum (Rudolphi, 1819).

The purpose of this paper was to study the antigenic structure of Dicrocoelium dendriticum with a particular consideration of its antigenic relationship between F. hepatica and Paramphistomum sp. Studies of the antigenic composition of somatic extract of D. dendriticum and its four fractions obtained by gel separation chromatography on Sephadex G 75 were carried out by using double diffusion reaction with homologous (SDd) and heterogeneous (SFh, SPs) rabbit sera as well as sera of sheep naturally infected with liver fluke. The highest seroantigenic activity was observed for the somatic extract and its fraction I. A distinct antigenic relationship was found between the fluke species studied, a closer relationship between D. dendriticum and F. hepatica than between D. dendriticum and Paramphistomum sp. The studied sera of 7 sheep naturally infected with liver fluke were inactive in double diffusion reaction with D. dendriticum extract and its fractions.

[Immunodiffusion studies of the complete somatic antigens of Paramphistomum sp. and Fasciola hepatica]. / Imunodifuzionni izsledvaniia na pulni somatichni antigeni ot Paramphistomum sp. i Fasciola hepatica.

Investigations were carried out through the agar gel immunodiffusion test of complete somatic antigens of Paramphistomum sp. and Fasciola hepatica, using the respective homologic and heterologic hyperimmune rabbit sera. Differences were established in the spectrum and number of the produced precipitation lines - 6 to Paramphistomum and 4 to Fasciola. With the identity test by the scheme in three after Zilber and Abelev (1962) no precipitation arcs were formed against the heterologic hyperimmune rabbit sera. This pointed the fact that there was no antigenic relation between the helminth species studies.