Faeces, FACS, and functional assays--preparation of Isospora suis oocyst antigen and representative controls for immunoassays.

SUMMARY: Highly purified antigen and appropriate controls are essential for antigen-specific immunoassays. In the case of Isospora suis, the causative agent of neonatal porcine coccidiosis, the only current source of antigen is oocysts isolated from faeces. The aim of this study was to develop a procedure for high-grade purification of I. suis oocysts from piglet faeces to obtain both antigen and representative controls suitable for in vitro re-stimulation of lymphocytes. This was achieved by use of filtration, density-gradient centrifugation and fluorescence-activated cell sorting (FACS). The feasibility for immunological studies was demonstrated with IFN-gamma ELISPOT assays after in vitro re-stimulation of lymphocytes from previously infected swine using the obtained antigen. The developed method allowed the production of highly purified antigen and representative controls from faeces with an oocyst recovery rate of 14%. Regarding the application of the obtained material it could be shown that lymphocytes from I. suis-infected pigs react in an antigen-specific manner in terms of an in vitro recall response by the production of IFN-gamma. This demonstrates the suitability of the developed method for the production of antigen and controls for sensitive immunological readout systems. Moreover, the detected specific IFN-gamma response encourages further functional studies on the cellular immune response to I. suis.

Cytosolic glutathione S-transferases of Oesophagostomum dentatum.

Oesophagostomum dentatum stages were investigated for glutathione S-transferase (GST) expression at the protein and mRNA levels. GST activity was detected in all stages (infectious and parasitic stages including third- and fourth-stage larvae of different ages as well as males and females) and could be dose-dependently inhibited with sulfobromophthalein (SBP). Addition of SBP to in vitro larval cultures reversibly inhibited development from third- to fourth-stage larvae. Two glutathione-affinity purified proteins (23 and 25 kDa) were detected in lysates of exsheathed third-stage larvae by SDS-PAGE. PCR-primers were designed based on peptide sequences and conserved GST sequences of other nematodes for complete cDNA sequences (621 and 624 nt) of 2 isoforms, Od-GST1 and Od-GST2, with 72% nucleotide similarity and 75% for the deduced proteins. Genomic sequences consisted of 7 exons and 6 introns spanning 1296 bp for Od-GST1 and 1579 and 1606 bp for Od-GST2. Quantitative real-time-PCR revealed considerably elevated levels of Od-GST1 in the early parasitic stages and slightly reduced levels of Od-GST2 in male worms. Both Od-GSTs were most similar to GST of Ancylostoma caninum (nucleotides: 73 and 70%; amino acids: 80 and 73%). The first three exons (75 amino acids) corresponded to a synthetic prostaglandin D2 synthase (53% similarity). O. dentatum GSTs might be involved in intrinsic metabolic pathways which could play a role both in nematode physiology and in host-parasite interactions.

Ecdysis of Oesophagostomum: possible involvement of eicosanoids and development of a bioassay.

Bioassays were developed and applied to test the role of eicosanoids and pH changes in ecdysis of Oesophagostomum dentatum. Exsheathment (80-100%) was achieved by subjecting third-stage larvae (L3) either to chlorine (hypochlorite assay) for 5 min or by incubating them in HCl followed by addition of NaHCO3 (pH-change assay) with subsequent cultivation at 38.5 degrees C/10% CO2 for 1 week. Addition of the lipoxygenase (LOX) inhibitor diethylcarbamacine (DEC) to the larvae resulted in a reduction of the exsheathment rates which could be restored by the addition of leukotrienes (LT)B4, LTC4, LTD4 and LTE4. Addition of the cyclooxygenase (COX) inhibitor acetylsalicylic acid (ASA) also resulted in decreased exsheathment rates both in the hypochlorite and in the pH-change assays in a dose-dependent manner. However, the primary COX products (prostaglandins) were not able to reverse this effect, in contrast to LTC4. It was concluded that: (1) both tests are suitable for bioassaying the effect of substances on exsheathment, and (2) eicosanoids involved in the control of exsheathment of L3 of O. dentatum are primarily LT.

Oesophagostomum dentatum: expression patterns of enzymes involved in eicosanoid production.

Cytosolic and membrane-bound proteins of various stages of Oesophagostomum dentatum, the nodular worm of pigs, were investigated for the presence of lipoxygenases (LOX) and cyclooxygenases (COX) using polyclonal and monoclonal antibodies. Putative 12-LOX and 15-LOX, but not 5-LOX, were detected in both fractions of all developmental stages in the expected size range of 75 kDa, with an isoelectric point of 6.0-6.5. The protein could be precipitated with 50% ammonium sulfate, as described for mammalian LOX. An antibody directed against both COX isoforms and one against mammalian COX-2 detected proteins of approximately 70 kDa with an isoelectric point of 6.0-6.5 in the membrane-bound fractions of third-stage larvae and adults, but not in the fourth-stage larvae. Anti-COX-1 or more specific anti-COX-2 antibodies failed to detect proteins. The constitutive LOX expression supports the assumption that the metabolites of this enzyme previously detected in O. dentatum serve intrinsic functions, while the production of anti-inflammatory COX-products in the invasive and luminal stages of the parasite implies a possible role in host-parasite interactions.

Characterisation of stage-specific proteins of Oesophagostomum dentatum by preparative isoelectric focusing and lectin blotting.

The nodular worm of pigs, Oesophagostomum dentatum, has previously been shown to undergo distinct biochemical changes during its life cycle. This phenomenon was studied in more detail for the early parasitic stages. Differences between infective third-stage larvae (L3), parasitic fourth-stage larvae cultivated in vitro (L4c), and pre-adult larvae recovered from the intestinal contents of pigs (L4p) were compared with respect to their protein and glycoprotein patterns by solubility-based protein fractionation and preparative isoelectric focusing followed by SDS-PAGE or by Western blotting with various lectins. While differences between the L4 were only minor (only three bands were specific for either L4c or L4p), L3 displayed distinctly different protein patterns with four L3-specific and nine L4-specific bands. Concanavalin A bound to a variety of glycoproteins, partly in a stage-specific manner, while Ricinus communis Agglutinin 120, Wheat Germ Agglutinin, Peanut Agglutinin and Soybean Agglutinin bound to fewer, partly stage-specific, molecules.

Comparative studies on the development of Oesophagostomum dentatum in vitro and in vivo.

Although in vitro cultivation of Oesophagostomum dentatum provides defined material of third- (L3) and fourth- (L4) stage larvae, these are morphologically and biochemically different from larvae recovered ex vivo. The development of pre-cultivated larvae was investigated by rectal transplantation into worm-free pigs with subsequent recovery of worms from intestinal contents after different time periods and determination of worm burdens and sizes. Additionally, the in vitro maintenance of L4 and adults recovered from intestinal contents of orally infected pigs in different media was investigated. Although growth and development rates of cultivated L4 are lower than those of larvae recovered from intestinal contents after oral infection, pre-cultured L4 are able to develop into egg-laying adults in the large intestines (without nodule formation) within 9-14 days in rates comparable with those after oral infection. In contrast, rectally transplanted L3 only establish in low numbers without egg excretion. L4 and adult worms recovered from intestinal contents cannot be maintained in cultivation medium for more than 1 week, although most L4 grow and moult during the first 3 days. Although the standard cultivation conditions for mass production of L4 are not suitable for development or maintenance of preadult and adult stages, L4 recovered from cultures have the ability to establish in vivo as fertile adults, indicating that the basic biological functions are retained in vitro.

PCR-based differentiation of three porcine Eimeria species and Isospora suis.

Isospora suis and Eimeria are frequent coccidian parasites of pigs. The unsporulated oocysts of Eimeria species and of I. suis are difficult to differentiate. Therefore, a species-specific PCR was developed. PCR products were amplified from Eimeria polita, Eimeria porci, and Eimeria scabra using primers from the conserved 18S rRNA regions and were subsequently sequenced. Based on variable sequence regions, primers were constructed for the differentiation of the three Eimeria species and I. suis. Using a combination of PCRs detecting one or two species, all four coccidian species were detected (theoretical lower detection level: DNA content of 250 oocysts of each Eimeria species or 25 oocysts of Isospora in 1microl) and differentiated. The PCR-based differentiation of the above mentioned species provides a useful alternative to microscopy.

Changing surface antigen and carbohydrate patterns during the development of Oesophagostomum dentatum.

Living and fixed specimen of Oesophagostomum dentatum were labelled in situ with serum antibodies or a panel of biotin-labelled lectins. Specific binding of antibodies was observed in all parasitic stages--freshly exsheathed 3rd-stage larvae (L3), 3rd- and 4th-stage (L4) larvae cultured in vitro and L3 and L4 and adults isolated from pig intestines. The shedding of the stained layer by motile larvae was inhibited by levamisole-induced paralysis. Larvae cultured in vitro exposed serum-derived proteins on their surface which could be labelled with secondary antibody directed against the respective serum donor species. While freshly exsheathed larvae were recognized by O. dentatum-positive serum only, older larvae and adults cross-reacted with serum from pigs infected with O. quadrispinulatum, a closely related species. Lectin binding varied considerably between stages. While binding was not observed in pre-parasitic stages, Concanavalin A, Soybean Agglutinin, Wheat Germ Agglutinin, Ricinus communis Agglutinin and Peanut Agglutinin bound to developing larvae in varying degrees. Dolichos biflorus Agglutinin only bound to advanced (luminal) larval stages, while adults generally displayed only weak or partial lectin binding (except with Concanavalin A and Wheat Germ Agglutinin). Ulex europaeus Agglutinin only labelled larvae derived from cultures containing 10% pig serum. Cleavage of the carbohydrate residues by sodium periodate treatment resulted in reduction of antibody binding to cultured larvae, but not to freshly exsheathed L3. Concanavalin A, Soybean Agglutinin, and Peanut Agglutinin binding was also reduced by periodate treatment, while binding of Wheat Germ Agglutinin and Ricinus communis Agglutinin was inhibited only in early L3, but not in older stages. The different lectin labelling patterns are related to the different stages of the nematode--infective, invasive, histotropic, and luminal--and may serve as a mode of adaptation for the parasite against the host's immune attack by surface glycoprotein variation, together with antigen shedding (as demonstrated by labelling of motile larvae) and a possible acquisition of host molecules at the parasite's surface. Furthermore, a possible role of this developmental variation in surface carbohydrates in parasite-parasite interactions is discussed.

Identification, isolation, and characterization of a species-specific 30-kDa antigen of Oesophagostomum dentatum.

In the search for a serology tool for the diagnosis of nonpatent as well as patent infections with Oesophagostomum dentatum in pigs a water-soluble, unglycosilated antigen of about 30 kDa specific for the third-stage larvae of the parasite was purified by ion-exchange chromatography. In Western blots, the antigen was first detected by antibodies at day 7 postinfection. Cross-reactivity with O. quadrispinulatum, Ascaris suum, or Trichuris suis was not detected. It is suggested that this protein is a suitable tool for the species-specific serodiagnosis of O. dentatum infection in pigs.

Modulation of migration of Oesophagostomum dentatum larvae by inhibitors and products of eicosanoid metabolism.

The effects of eicosanoids and of inhibitors of eicosanoid synthesis on the migration of third-stage larvae (L3) of Oesophagostomum dentatum were studied in an in vitro migration assay procedure. The L3 were incubated with diethylcarbamazine (DEC), indomethacin (INDO) or acetylsalicylic acid (ASA). Incubation with these inhibitors of eicosanoid metabolism resulted in a dose-dependent reversible inhibition of migration. The antimigratory effect of DEC could be completely reversed by treatment of the L3 with the lipoxygenase (LOX)-products leukotriene (LT) B4 or LTC4. LTD4 had a less distinct but similar effect, while LTE4 failed to reverse migration inhibition. Treatment with combinations of cyclooxygenase (COX)-products (prostaglandin, PG) partially restored the migration ability of ASA-treated L3, while PGD2, PGE2, PGF2 alpha or PGI2 exerted no distinct effect on ASA-treated L3 when given separately. The suppression of L3 migration by compounds that are known as antagonists of eicosanoid synthesis and the stimulation of migration of inhibitor-treated L3 by simultaneous application of eicosanoids indicate that these lipid mediators may play a significant role in physiological processes that interact with worm motility.

Changes in antigen and glycoprotein patterns during the development of Oesophagostomum dentatum.

During its development from free-living infectious third-stage larvae to the adult worms in the large intestines of pigs, Oesophagostomum dentatum experiences several environmental changes. Differences in protein patterns can reflect such changes. Somatic and ES antigens and glycoproteins of pre-parasitic, histotropic and intestinal stages were compared by single-dimension SDS-PAGE and stage-specific proteins were defined. Furthermore, fourth-stage larvae derived from different sources--in-vitro cultivation and intestinal contents--were compared and also found to be different. It is hypothesised that O. dentatum reacts to environmental stimuli by differential expression of specific proteins as a possible mode of adaptation to the host.