Egbert Geyer

Taenia crassiceps Metacestode Vesicular Fluid Antigens Shared with the Taenia solium Larval Stage and Reactive with Serum Antibodies from Patients with Neurocysticercosis

After removal of host (mouse) serum proteins (albumin, transferrin, IgG and another five unidentified proteins) by immunoaffinity chromatography, the vesicular fluid of T. crassiceps metacestodes (TcVF) was immunoelectrophoretically examined for antigens recognized by rabbit antiserum to aqueous crude extract of T. solium cysticerci. Based on a precipitate pattern developed in electroimmunodiffusion, nine cross-reactive antigens could be demonstrated. In the ELISA, TcVF was shown to be a potent antigen for the demonstration of IgG antibodies in the sera of Mexican patients (n = 14) with confirmed neurocysticercosis (mean E490 values +/- SD: 0.39 +/- 0.38) although it was less sensitive when compared to T. solium VF (0.95 +/- 0.54). Sensitivity was much higher using cross-reactive TcVF antigens selected by immunoaffinity chromatography with rabbit IgG antibodies to larval T. solium crude extract (0.87 +/- 0.57). SDS-PAGE fluorograph of cross-reactive, radioiodinated TcVF protein antigens and selected by antibodies of individual neurocysticercosis sera (n = 13), exhibited six to nine bands depending on the serum tested. Altogether ten 125I-labelled proteins (Mr range from about 20,000 to 200,000) were recognized by neurocysticerosis antibodies. Four proteins (Mr about 22,000, 25,000, 32,000 and 45,000) were detected by all sera.

Comparative serological reactivity of Taenia crassiceps, Taenia solium and Taenia saginata metacestode neutral glycolipids to infection serum from Taenia crassiceps-infected mice

A comparative survey was undertaken of the neutral fraction glycolipids from the metacestodes of 3 taeniid species, Taenia crassiceps, Taenia solium and Taenia saginata, to determine their chemical and serological staining patterns on separation by thin-layer chromatography. The orcinol-positive patterns of T. solium and T. saginata metacestodes exhibited a closer superficial resemblance to each other than to T. crassiceps or T. saginata adults. A comparison of component migration properties against standards of known structure indicated the main oligosaccharide chains to be mono-, di-, tri- and tetrasaccharides; however, in T. solium this was extended to at least a heptasaccharide. The multiple banding characteristic of each component is a consequence of lipid moiety heterogeneity. Serologically, the patterns of the 3 taeniid species neutral fraction glycolipids showed virtually the same immunological reactivity towards mouse normal serum, infection serum and a monospecific, polyclonal antibody directed against the trisaccharide component of T. crassiceps. The latter antibody was isolated from mouse infection serum by affinity chromatography on a column of glycolipid-bound octyl-Sepharose CL-4B. Immunochemically, the major common epitope expressed by the neutral fraction glycolipids of the 3 taeniid species is the same or very similar to the glycosphingolipid, neogalatriaosyl ceramide derived from the marine mollusc Turbo cornutus (Gal(beta 1-6) Gal(beta 1-6) Gal(beta 1-1)Cer). Host tissue neutral fraction glycolipids, porcine muscle and bovine muscle, as well as human spleen, were not immunoreactive.

Litomosoides carinii: macrofilariae‐derived glycolipids—chromatography, serology and potential in the evaluation of anthelminthic efficacy

A preliminary characterization of the glycolipids of Litomosoides carinii macrofilariae, resolved according to their chromatographic, chemical and serological properties, has been performed. Emphasis has been placed on the neutral fraction glycolipids. These are separable on thinlayer chromatography into two groups of fast and slow migrating band components, that differ in their migration, differential chemical staining and serological traits, respectively. Serological analyses have been accomplished by thin‐layer chromatography immunostaining and ELISA. Only components of the slow migrating band group react with infection serum from Litomosoides carinii‐infected Mastomys coucha. Cross‐reactivity experiments with homologous and heterologous infection sera of various helminthiases indicate that, epitopes bound to the neutral glycolipid fraction show structural similarity within the Nematoda, but not to the Cestoda or Trematoda. The dynamic development of specific Ig‐, IgG‐ and IgM‐anti‐neutral glycolipid fraction antibody levels were correlated with the different progression of L. carinii and Brugia malayi infections in the multimammate rat, Mastomys coucha. The reduction in the dynamics of IgG‐ and IgM‐antibody levels on chemotherapeutic treatment with the filaricides flubendazole and CGP 20376 has been related to their macrofilaricide‐activity.

Immunological recognition of larvalTaenia crassiceps glycolipids by sera from parasite-infected mice

The isolation and purification of a neutral glycolipid fraction fromTaenia crassiceps metacestodes (KBS strain), harvested from both male and female NMRI mice at 70–80 days following intraperitoneal infection, revealed 24 thin-layer chromatography-designated glycolipid bands. The glycolipids were defined as ceramide mono-(n=3), di-(n=3), tri-(n=4), tetra-(n=5), and >tetrasaccharides (n=9) according to their running properties as defined by thin-layer chromatography against standards of known structure. The defined glycolipids were tested for immunoreactivity with sera from noninfected andT. crassiceps-infected NMRI mice (intraperitoneal injection or implantation of 15 larvae/animal) using the enzyme-linked immunosorbent assay (ELISA) until day 33 p.i. (IgM and IgG reaction) and high-performance thin-layer chromatography (HPTLC) combined with immunostaining (IgG reaction) until day 7 p.i. ELISA-determined IgM and IgG titres were significantly elevated from day 5 p.i. Immunostaining revealed early reactivity for certain ceramide tetra- and >tetrasaccharides (n=6) on day 3 p.i. From day 5 p.i. onwards, nearly all glycolipids, including ceramide mono-and disaccharides, were recognized by the sera from metacestode-challenged mice. On day 7 p.i., a total of 22 bands were serologically active; of these, a considerable number (n=10) showed increased staining intensity. Remarkably, in many cases (10 of 20), 3 glycolipids (tetra-and >tetrasaccharides) were weakly recognized by mouse sera taken before infection.

Experimental dicrocoeliasis: The humoral immune response of golden hamsters and rabbits to primary infection withDicrocoelium dendriticum

The results presented deal with the humoral immune response of golden hamsters to primary experimental infection withD. dendriticum. The development of serum antibodies has been comparatively investigated with three hamster groups (n=43) harbouring different burdens of adult flukes. The mean numbers of parasites were 11, 30, or 130 per animal. Serum antibody response was studied during an observation period of at least 331 and up to 496 days postinfection. For antibody detection the sensitivities of precipitation tests (PTs) (double diffusion test, immuno- and counterimmunoelectrophoresis), of the indirect haemagglutination test (IHAT), the complement fixation test (CFT), and the enzyme linked immuno sorbent assay (ELISA) were compared using aqueous crude fluke antigen and crude egg antigen.CFT and ELISA were most sensitive for the early detection of initial response. Thereafter all the tests employed revealed increasing antibody titres, which in general remained at constant levels and persisted until the end of the observation period with the exception of CF-antibodies. In general fluke antigen was found to be more sensitive than egg antigen. However, in CFT this antigen occasionally has been associated with unspecific inhibition of haemolysis. Comparison of the results shows that ELISA using crude fluke antigen gave the most realistic picture of the actual fluke burden. Also preliminary results on the precipitin response of rabbits (n=3) after primary experimental exposure to different numbers of metacercariae (500, 1,000, and 3,000 per animal respectively) are reported. Employing the above mentioned PTs a persisting antibody response could be demonstrated only after exposure to at least 3,000 infective larvae. The initial response was found on day 63, the observation period was 550 days.

Immunoelectrophoretic analyses of antigens shared by the vesicular fluid and cyst wall of Taenia crassiceps and Taenia saginata metacestodes

In this study, the antigenic mosaic of the vesicular fluid (VF) and hydrosoluble cyst-wall extract (CWE) ofT. crassiceps (Tc; harvested from mice) andT. saginata (Ts) metacestodes was analyzed by combined precipitation maps developed in single and bidimensional immunoelectrophorsis against the respective rabbit antiserum. Hostserum proteins demonstrated by immunodiffusion within TcVF (albumin, transferrin, IgG, and another five noncharacterized proteins), TcCWE (albumin, IgG, and six additional unknown proteins), TsVF (albumin) and TsCWE (albumin and IgG) were removed by immunoaffinity chromatography prior to immunoelectrophoretic analysis. Neither TcVF nor TcCWE contained demonstrable amounts of mouse IgM and IgA. In TcVF a total of 18 and in TcCWE a total of 36 parasitic antigens were recognized by the corresponding antiserum. In the case of TsVF and TsCWE, antiserum to the crude extract ofT. saginata larvae developed a total of 26 and 30 precipitates, respectively. Examination of the precipitation maps developed by the respective heterologous antiserum (vice-versa testing) showed that both TcVF and TsVF contained ten antigens sharing identity. For TcCWE and TsCWE, nearly the same number of shared antigens (20 and 18, respectively) could be demonstrated. For screening of IgG antibodies againstT. saginata metacestodes from heavily and moderately infected calves (n=6) by ELISA, VF and CWE antigens of bothTaenia species were found to be potent reagents; TsVF was the most sensitive antigen.

Elektrophoretische Analysen an Fasciola hepatica-Totalextrakten

ZusammenfassungMitgeteilt werden die Befunde elektrophoretischer Analysen an Extrakten aus Fasciola-Totalhomogenaten. Die mit verschiedenen Techniken dargestellten Extraktkomponenten sowie die in der Immuno-Elektrophorese gegen homologe Kaninchen-Antiseren gebildeten Immunpräzipitate (max. 23) wurden auf Färbereaktion mit Protein-, Lipid-und Kohlenhydrat-Indikatoren geprüft. Neben ungebundenen Lipiden und Kohlenhydraten (Papier-Elektrophorese) konnten bis zu 14 reine Protein-, 10 Lipoprotein- und 6 Glykoprotein-Fraktionen (PAA-Disc-Elektrophorese) festgestellt werden.Präzipitin-Kreuzreaktionen mit Fasciola-Adulten-Totalextrakt-Antiseren zeigten Adulten-Totalextrakte von Dicrocoelium lanceolatum (4), Schistosoma mansoni (3) und Ascaris lumbricoides suis (1). Hydatiden-Flüssigkeit von Echinococcus cysticus und Cysticercus tenuicollis ergaben keine Reaktion. CRP-Rheumaserum reagierte mit Fasciola-Totalextrakten mit der Ausbildung eines Präzipitats. Fasciola-Adulten-Totalextrakt-Antiseren wiesen CRP-Titer auf. Die Befunde werden diskutiert.SummaryAnalytical studies on extracts of total adult liver fluke (Fasciola hepatica) homogenates were carried out by the use of different electrophoresis techniques. Differential staining procedures of PAA-electrophoresis columns revealed the presence of a maximal number of 14 protein, 10 lipoprotein and 6 glycoprotein fractions. Furthermore the extracts contain free lipid and carbohydrate material as observed in paper-electrophoresis patterns. In immunoelectrophoresis of extracts and homologous rabbit antiserum a maximal number of 23 precipitates was found. Extracts of adult Dicrocoelium lanceolatum, Schistosoma mansoni and Ascaris lumbricoides suis homogenates formed, 4, 3 and 1 precipitates respectively as opposed to Fasciola-extract antiserum. No precipitates were formed using hydatid fluid of Echinococcus cysticus and Cysticercus tenuicollis. Immunodiffusion of fluke extracts and human serum containing C-reactive protein (CRP) resulted in the formation of one precipitate. C-reactive protein could be demonstrated in Fasciola-extract antisera. The results are discussed.

Inhibition of in vitro and in vivo Mast Cell Degranulation by Taenia crassiceps Metacestodes in vitro Incubation Products

In vitro released products of T. crassiceps metacestodes (TcIP) harvested from the peritoneal cavity of NMRI mice were tested for inhibitory effects on the in vitro degranulation of peritoneal mast cells (MCs) of normal mice (NMRI) and rats (Wistar) and on the in vivo degranulation of rat (Wistar) skin MCs (PCA-assay). In vitro degranulation was elicited chemically (compound 48/80, polymyxin B or the bee venom peptide, mellitin). In vivo degranulation was triggered immunologically (anaphylactic systems ovalbumin/anti-ovalbumin or Fasciola hepatica crude fluke extract antigen/serum of fluke-infected rats (Wistar]. In vitro degranulation of murine peritoneal MCs or the in vitro histamine release of rat peritoneal MCs normally induced chemically was significantly inhibited when the MCs were preincubated with the TcIP or with serum of T. crassiceps-infected NMRI mice from day 35 post infection and thereafter. In vitro degranulation of peritoneal MCs of infected mice was strongly inhibited beginning on day 10 after infection. Also in vivo degranulation of the IgE-sensitized rat skin MCs was significantly reduced by intradermal injection of the TcIP before (6, 3 and 1 h) antigen challenge and by preinjection (1 h) of serum from infected mice (day 80 p.i.)-The inhibitory effect was also demonstrated after immunoadsorption of mouse serum proteins naturally contaminating the TcIP. Heating (100 degrees C/15 min), even in the presence of 0.25 M HCl, did not suppress the inhibitory activity.

Bovine cysticercosis: demonstration in experimentally infected calves of serum IgG antibodies reactive with neutral glycolipids ofTaenia saginata andT. crassiceps metacestodes

The immunoreactivity ofTaenia saginata andT. crassiceps metacestode neutral glyco(sphingo)lipids towards IgG antibodies derived from the sera of calves with experimental cysticercosis has been established. The glyco(sphingo)lipids are separable by normal-phase HPTLC (high-performance thin-layer chromatography) into groups of increasing sugar-chain length (lipid/ceramide mono-, di-, tri-, tetra- and >tetrasaccharides), with those corresponding to three and four hexoses being the main immunoreactive components (HPTLC immunostaining). In ELISA (enzyme-linked immunosorbent assay), reverse-phase HPTLC-isolatedT. crassiceps metacestode glyco(sphingo)lipids equivalent to tri- and tetrahexoside allowed a discrimination between non-infected and infected calves (at least 80 metacestodes recovered). The formation of IgG antibodies was correlated with the infection, not with other non-specific inducing factors, as seen by the differential humoral response detected in experimentally infected (T. saginata) calves before and after Praziquantel treatment (HPTLC immunostaining and ELISA).

Isolation of antigen fromLitomosoides carinii macrofilariae detecting serum antibodies due toOnchocerca volvulus

Crude aqueousLitomosoides carinii adult worm extract was used as antigen for the detection of antibodies in sera from African patients with proven onchocerciasis (n=45) resident in rural endemic areas of Togo and Sierra Leone. In 71% of cases this extract was found to produce 1 to 5 precipitation arcs in immunoelectrophoresis. Using a crude aqueous extract from adultOnchocerca volvulus, precipitation tests were positive in 75% of cases.The complexity of theL. carinii curde extract was shown by PAG-disc electrophoresis, PAG-electrofocusing, immunoelectrophoresis and crossed immunoelectrophoresis with the appropriate rabbit-antiserum. An antigen detecting onchocercal antibodies was isolated by two step preparative flat bed electrofocusing in granulated gel (PEGG). The antigen (pI 6.55, molecular weight 55 to 60 kd as estimated by SDS-PAG electrophoresis) was very suitable for antibody demonstration in double diffusion test and immunoelectrophoresis. Preliminary controls for specificity were performed by diffusing the antigen against sera from human and animal helminthoses including filarial infections. In contrast to the crudeL. carinii extract no reaction was observed with sera from helminthic infections others than filariasis.