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.

Analysis of potential antigens of protoscoleces isolated from pulmonary and hepatic hydatid cysts of Bubalus bubalis.

The aqueous soluble proteins of the protoscoleces of Echinococcus granulosus isolated from the pulmonary and hepatic hydatid cysts of Bubalus bubalis were partially purified on Sephadex G-200 column. The isolated fractions were tested for their antigenicity by immunodot using the sera collected from experimentally infected puppies during the prepatent period of infection. Six protein profiles (F1-F6) were recovered from both the isolates but the polypeptide recovery of each fraction of the two isolates were some what different, particularly protein concentration of F5 in the liver isolate was greater than the lung isolate. Contrary to this, the concentration of F1 polypeptides of the lung origin protoscoleces is greater as compared to that of the liver. In addition, some antigenic dissimilarity has also been observed in the two isolates. A weak IgG response against F1, F2 and F6 polypeptides was observed in the 4th day post infection (p.i.) sera. With the age of infection the response against these antigens increased as revealed by the intensity of the spot in immunodot analysis. Our studies show that the differences in the elution profile and antigenic profile of the lung and liver isolates, as revealed by gel filtration and immunodot analysis, might be either due to the influence of the microhabitat or these may be two different strains. Further studies are certainly required in this direction.

Partial purification and characterization of Gastrothylax crumenifer somatic antigens.

The soluble extracts of Gastrothylax crumenifer isolated from the rumen of buffalo (Bubalus bubalis) were fractionated on a Sephadex G-200 column. A total of eight major fractions (F1, F2, F3, F4, F5, F6, F7, and F8) were separated from the whole homogenate of G. crumenifer, and each of these fractions was tested for their antigenicity by ELISA against rabbit hyperimmune sera. It was observed that F1, F2, F3 and F4 were highly antigenic, F6 and F7 were moderately antigenic and F5 and F8 were poorly antigenic. The individual fractions analysed after SDS-PAGE and Western blotting indicated that the antigenic fractions of G. crumenifer are of low molecular weight, in the range of <14-50kDa, and predominant antigenic components which were evident in most of the Sephadex profiles were of Mr 15, 18, 19, 23-24 and 28-32kDa.

Analysis of excretory-secretory and somatic antigens of Gastrothylax crumenifer.

The excretory/secretory (ES) metabolic products released by Gastrothylax crumenifer (Trematoda: Digenea) during in vitro incubations and the somatic extract of the adult parasite were analysed using polyacrylamide gel electro-phoresis (PAGE). Immunogenicity of ES and somatic extracts were evaluated by immunoblotting and ELISA using sera raised against ES and somatic antigens in rabbits. The electropherograms of ES and somatic extracts have been resolved into 38 and 41 polypeptides, respectively. The apparent molecular weights of these polypeptides range from <29 to > 205 kDa. A total of 14 polypeptides were found to be common to both of the samples. The analysis of immunoblot results revealed 22 and 27 antigenic polypeptides in ES and somatic extracts respectively. Only 11 and 13 antigenic polypeptides were found specific to ES and tissue extract respectively. The molecular weights of these specific polypeptides were calculated to be <14.4, 16, 20, 25, 33, 42, 119, 125 and > 205 kDa for metabolic products and <14.4, 25, 30, 35, 78, 84 and > 205 kDa for the tissue extracts, respectively. Analysis of ELISA results revealed that a dilution of up to 1:3200 of the test sera could react with the ES product. Further, when the ES antigens were allowed to react with antisomatic extracts in hyperimmune sera the titre of IgG increased up to a dilution of 1:12800. The potential importance of these antigens in the immunodiagnosis of amphistomiasis is discussed.

Monoamine oxidase in amphistomes and its role in worm motility.

The quantitative assay of mitochondrial monoamine oxidase (MAO) activity revealed a higher enzyme level in Explanatum explanatum than Gastrothylax crumenifer. The specific MAO inhibitors, chlorgyline, pargyline, deprenyl and nialamide produced different degrees of interspecific inhibition. The differential effects on enzyme activity of chlorgyline and deprenyl suggests the possible existence of polymorphic forms of the enzyme, MAO-A and MAO-B, in amphistomes. These specific inhibitors also had a differential influence on the in vitro motility of amphistomes, further indicating the involvement of different forms of MAO in the oxidative deamination of biogenic monoamines which might be partly responsible for neuromuscular coordination in amphistomes. The experimental procedures used in this study could be conveniently used for quick screening and evaluation of some of the qualitative effects of anthelmintic drugs under in vitro conditions.

Coproantigens: early detection and suitability of an immunodiagnostic method for echinococcosis in dogs.

In the present study immunodiagnostic tests for Echinococcus granulosus-specific coproantigens have been evaluated. The techniques are based on polyclonal antisera collected from dogs experimentally infected with E. granulosus protoscoleces. ELISA, Countercurrent immunoelectrophoresis (CCIEP) and Immunodot were evaluated for their application in coprodiagnosis. The level of coproantigens was detectable on day 7 post-infection and reached a maximum level on day 56 as detected by ELISA. Very high O.D values were obtained when faecal samples of 7-8 weeks post-infection were used. Besides this, immunodot and CCIEP were also tested and the 4th week post-infection samples gave significant reactions. It is evident from the results that ELISA was more sensitive and detected the coproantigens as early as the first week post-infection whereas immunodot and CCIEP detected coproantigens later. The latter methods are rapid, cost-effective and can be used to diagnose suspect cases of echinococcosis under field conditions. Thus, it is suggested that future studies should be aimed at early detection of echinococcosis by a rapid and cost-effective immunodot test.

[3H]-amino acid uptake and metabolic studies on Gigantocotyle explanatum and Gastrothylax crumenifer (Digenea: Paramphistomidae).

The amphistomes Gigantocotyle explanatum and Gastrothylax crumenifer utilize leucine, alanine, proline and methionine during in vitro incubations. Autoradiography on sections of these flukes reveal a time-dependent differential incorporation of tritium-labelled amino acids in various tissues. The tegument appears to be the primary surface through which amino acids are absorbed. Following absorption, the reappearance of [3H]-leucine and [3H]-alanine on the tegumental surface during late chase periods indicates their possible involvement in tegumental secretion. A combination of diffusion and carrier-mediated uptake, possibly involving gamma-glutamyl transpeptidase, is indicated. The transport loci show differences in carrier-affinity (Kt) and maximum uptake velocities (Vmax) for amino acids under study, which suggest multiple transport molecules. Metabolic studies reveal that aspartate, alanine, ornithine, proline, leucine and methionine undergo transamination through 2-oxoglutarate-linked transaminases, distributed in the cytosolic and mitochondrial fractions of G. explanatum and G. crumenifer. With the exception of alanine transaminase, the enzyme levels in the cytosolic fraction were higher than the mitochondrial fraction of the two amphistomes. Predominantly cytosolic glutamate dehydrogenase which was comparatively higher in G. explanatum, catalyse amination of alpha-ketoglutarate. A high level of cytosolic arginase alone does not indicate a functional urea cycle. A tentative pathway of amino acid metabolism in these amphistomes is proposed.

Ultrastructure and histochemistry of the tegument of juvenile paramphistomes during migration in Indian ruminants.

The tegument of juvenile Paramphistomum epiclitum and Fischoederius elongatus (Paramphistomidae: Digenea) resembles those of other digeneans. Seven types of papillae were observed, mostly on the oral and acetabular surfaces, and increase in number during migration. Also evident are two types of secretory body (T1 and T2) which are synthesized separately in tegumental cytons underlying the syncytium. Exocytosis of T2 bodies occurs at the apical membrane and appears to contribute to a fibrous glycocalyx. The tegumental syncytium lining the pharynx and acetabulum is thinner and has a higher capacity for vacuolation than the general tegument. These may represent important sites for osmoregulation. The absence of mitochondria from the tegument in migrating juveniles suggests limited involvement in energy demanding processes. Pigmentation of the subtegument is first evident in mature cercariae and is progressively eliminated during migration.

Ultrastructure and histochemistry of the digestive tract of juvenile Paramphistomum epiclitum (Paramphistomidae: Digenea) during migration in Indian ruminants.

The digestive tract of juvenile Paramphistomum epiclitum consists of a foregut with a highly muscular terminal pharynx and an oesophagus, which leads to a pair of unbranched and blind-ending intestinal caeca. A syncytium lining the foregut is continuous with the external tegument and displays similar sensory papillae and secretory bodies (T1 and T2). A third type of secretory body (T3) is confined to the oesophageal cytons of newly excysted juveniles and is first evident in the syncytium by day 14 of migration. An epithelium lining the caeca is composed of a single layer of morphologically uniform cells whose apical surface is amplified by microvilli. Dense secretions synthesized in the caecal epithelium of mature cercariae are released during migration by a mechanism resembling modified apocrine discharge. The caecal epithelium of migrating juveniles undergoes a 10-fold increase in surface amplification (irrespective of growth) during its transition from a primarily secretory tissue to one apparently specialized for absorption.

Ultrastructure and histochemistry of the protonephridial system of juvenile Paramphistomum epiclitum and Fischoederius elongatus (Paramphistomidae: Digenea) during migration in Indian ruminants.

The protonephridial system of juvenile Paramphistomum epiclitum and Fischoederius elongatus consists of a bilaterally symmetrical arrangement of primary, secondary and tertiary ducts which connect individual flame cells with a simple common bladder. Primary and secondary ducts are formed from columns of adjoining cells which provide an epithelial lining, whose luminal surface is elaborated with either short tubercles or lamellae. Groups of cilia project from the luminal surface at frequent intervals along secondary ducts. By contrast, the tertiary ducts and bladder are lined with a nucleated syncytium which ends at a junctional complex formed with the terminal canal. The latter is continuous with the tegumental syncytium and opens at a nephridiopore on the postero-dorsal surface. Tertiary ducts of mature cercariae contain concretions which are voided by migrating juveniles in whose tertiary ducts lipids are progressively accumulated. Evidence for the role of protonephridia in excretion and possibly in osmoregulation and ionic balance is currently examined.

Ultrastructure and histochemistry of the lymph system and parenchyma of juvenile paramphistomes (Paramphistomidae: Digenea) during migration in Indian ruminants.

The lymph system of juvenile Paramphistomum epiclitum and Fischoederius elongatus consists of a single pair of longitudinal primary vessels from which sub-dividing branches extend laterally to associate with most major tissues and organs. The system originates shortly after excystation in the definitive host and is fully developed in day 14 juveniles. Lymph vessels are syncytial and membrane limited, with a matrix which contains autophagic-like inclusions, clusters of SER and free nuclei. Similar organelles are evident in the matrix of parenchyma and specialized cells juxta-posing the pharynx (JP cells). These tissues are intimately associated and perhaps functionally integrated. Parenchyma represents a major site for carbohydrate storage and turnover, whilst the lymph appears to perform a similar role for proteins. The JP cells of juveniles display prolific autophagic-like activity only during migration, which coincides with the depletion of carbohydrate reserves in parenchyma. Key mitochondrial enzymes were histochemically demonstrated in the lymph despite the apparent absence of mitochondria from this system in post-day 14 juveniles. Succinate dehydrogenase activity was cytolocalized in mitochondria, whilst attempts to perform a similar localization of this enzyme in lymph were unsuccessful. The possibility of non-enzymatic interference in the histochemical demonstration of dehydrogenase is examined.

Ultrastructural observations on the redial tegument of Paramphistomum epiclitum from the planorbid snail, Indoplanorbis exustus.

The morphology of the tegument in the redia of Paramphistomum epiclitum (Digenea: Paramphistomidae) resembles that shown by most larval and adult digeneans; an outer surface syncytium is in continuity with the cytoplasm of in-sunken, nucleated cytons. Although tegumental cytons usually contain a single nucleus, some display up to six nuclei. The tegumental syncytium lining the pharynx of P. epiclitum rediae lack underlying cytons. The apical membrane of the tegument is elaborated by folds and microvilli, which presumably facilitate uptake of nutrients and/or exchange of ions involved in osmoregulation. A single type of secretory body, resulting from the fusion of smaller vesicles produced at Golgi complexes in the cytons, occurs throughout the tegument. Uniciliate sensory receptors occur in the surface syncytium particularly around the oral opening.

Studies on the ultrastructure and histochemistry of the lymph system of Gastrodiscoides hominis (Paramphistoma: Digenea).

The lymphatic system of the paramphistome, Gastrodiscoides hominis consists of numerous fluid-filled branches embedded in parenchyma and surrounded by extracellular material and is closely associated with the major organ systems of the fluke. The lymph matrix consists of a cytoplasmic syncytium within which nuclei, mitochondria and various sized granules and membranous structures occur. The granules found throughout the lymph system morphologically resemble autophagosomes and lysosomes. The lymph system provides a storage site for proteins which can be broken down to amino acids via autophagy, for subsequent mobilization and transport to tissues undergoing active protein synthesis. Many branches of the lymph system are surrounded by specialized parenchymal cells referred to as juxta-lymphatic cells. These cells are apparently associated with autophagic degradation of sequestered lymph cytoplasm, which may serve as an additional mechanism for the mobilization and transport of precursor molecules throughout the fluke via the parenchymal network.

Ultrastructural and histochemical observations on the tegument of Gastrodiscoides hominis (Paramphistoma: Digenea).

The tegument of the paramphistome, Gastrodiscoides hominis, is basically similar to that of other digeneans. It is folded into concentrically arranged furrows and ridges bearing numerous tightly packed tubercules, and extends into the oral cavity. An area of specialized tegument is present on the ventral surface, anterior to the disc region. Mitochondria are absent from the tegumental syncytium and underlying tegumental cells, suggesting that the tegument may serve principally as a protective layer rather than in active uptake phenomena. However, extensions of the lymph and parenchyma systems are closely associated with the base of the tegumental syncytium and may provide ATP for active processes. Ciliated and non-ciliated sensory papillae are present, particularly around the oral opening. Numerous lymph channels are present in the sub-tegument and may be involved in osmoregulation.

Biochemical changes during the development of the miracidium of Gigantocotyle explanatum.

Analysis of various biochemical components during the development of the miracidium of G. explanatum showed marked changes, particularly in glycogen, protein and DNA levels. Though the total lipids remained more or less unchanged, alterations in the levels of cholesterol, triglycerides, free fatty acids, phospholipids and phospholipid fractions were also recorded. Such changes could be intrinsically programmed for the cellular differentiation and organogenesis in larval amphistomes.

The effects of age, sex and breed of chicken on susceptibility to Clinostomum complanatum (Digenea: Clinostomatidae).

Susceptibility and development of Clinostomum complanatum in the chicken (Gallus gallus domesticus) have been studied. The progenetic metacercariae of C. complanatum developed into ovigerous worms in 3 days in the buccal cavity of the chicken and survived only 6-7 days in this experimental host. The susceptibility is influenced by the age, sex and breed/type of the host. Both percent susceptibility and recovery of the worms decreased with increase in the age of the host. The egg laying chickens were found to be least susceptible to the infection. Mature male chickens were more susceptible than the females. The single comb white Leghorn (SCWL) was found to be susceptible while the local type (LT) and the cross breed (SCWLxLT) were not susceptible to C. complanatum. The possible reasons for these variations have been discussed.

A comparative study of the protein content of some helminths and the suitability of assay methods.

The protein content of fresh homogenates and their corresponding TCA precipitated fractions of 10 different species of helminths was estimated by the methods of Lowry et al. and Spector using the Folin phenol reagent and Coomassie brilliant blue G-250 respectively. The former method gives exaggerated values as compared to the latter method. The parasite phenols, phenolic proteins and catecholamines could be responsible for interference in the Lowry's procedure. The TCA noln-precipitable moieties also give colour only with the Folin phenol assay. The pronounced intra-specific differences in the total protein content of helminths reflect their metabolic variations and adaptations. Habitat does not appear to influence the protein content of parasites, however, the effect of host variation was evident in the pouched amphistome G. crumenifer. It is concluded that the dye binding method gives more consistent results and it can be conveniently applied to crude tissue homogenates of helminths.

Isoparorchis hypselobagri: ultrastructure of the tegument and associated tissues in a digenean inhabiting a gaseous aerobic environment.

Adult I. hypselobagri live in the swim bladder of the Indian catfish Wallago attu, a gaseous environment with a relatively high oxygen content. The ventral tegument, which in life is applied close to the swim bladder wall, is relatively unspecialized, showing typical ultrastructural features of the digenean surface. The dorsal tegument, which is exposed to the oxygen-rich surroundings, has numerous pyriform extensions of superficial parenchymal cells closely applied to the base of the surface syncytium. These extensions bear numerous mitochondria and send finger-like processes deep into the basal cytoplasm of the syncytium where they interdigitate with corresponding infolds of the basal tegument membrane. The pyriform parenchymal extensions are connected with underlying nucleated cell bodies via irregular glycogen-filled tubular processes, many of which end blindly in the interstitial tissue or expand into glycogen-filled bulbs beneath the cell bodies. These superficial parenchymal processes associate at gap junctions with ramifications of a distinct deeper parenchymal tissue which contains lipid, residual bodies and glycogen. The dorsal tegument and associated structures may constitute a respiratory organ, taking advantage of molecular oxygen diffusing across the surface syncytium to carry out aerobic energy transduction in the superficial parenchymal extensions. ATP so generated may diffuse inwards for distribution throughout the body in the deep parenchymal tissue. The extensive network of ramifying cytoplasmic tubules is supported by a fibrous matrix of interstitial tissue.

Biochemical variations in Gigantocotyle explanatum and Gastrothylax crumenifer with respect to their seasonal reproduction.

Biochemical components, glycogen, protein, nucleic acids and lipid fractions were analysed every month from January to December 1986, in the liver and rumen amphistomes Gigantocotyle explanatum and Gastrothylax crumenifer, respectively. The results reveal a considerable seasonal variation. In the rumen amphistome the components reach their maximum level only once a year, whereas in the liver amphistome, more than one peak is observed in a year. In G. crumenifer the content of glycogen and nucleic acids increases before the onset of egg production while protein and lipids reach their maximum level during the egg production phase. The variations in biochemical components were associated with the reproductive cycles and gonad recrudescence of these parasites.

Biochemical characterization of Taenia hydatigena cysticerci from goats and pigs.

Analysis of the major biochemical components of Taenia hydatigena cysticerci collected from goats and pigs showed marked differences, particularly in glycogen, protein, lipid and DNA levels. Differences were also detected in the levels of cholesterol, triglycerides, free fatty acids and phospholipids. Furthermore, the profile of phospholipid fractions revealed quantitative differences between the two species. It is concluded that the cysticerci of goat and pig origin probably represent two different strains and possibly follow the same pattern of speciation as reported in the related taeniid, Echinococcus granulosus.