Isabel Vercauteren

An Arabidopsis thaliana Pectin Acetylesterase Gene Is Upregulated in Nematode Feeding Sites Induced by Root-knot and Cyst Nematodes

By using differential display, gene expression was investigated in Arabidopsis thaliana roots shortly after nematode infection, and a putative pectin acetylesterase (PAE) homolog (DiDi 9C-12) was found to be upregulated. PAEs catalyze the deacetylation of pectin, a major compound of primary cell walls. mRNA in situ hybridization experiments showed that the expression of DiDi 9C-12 was enhanced very early after infection in initiating giant-cells and in cells surrounding the nematodes. Later on, the level of DiDi 9C-12 mRNA was lower in giant-cells and transcripts were mainly found in parenchyma, endodermis, and pericycle cells of the root gall. Twenty days after infection, DiDi 9C-12 transcripts could no longer be detected. DiDi 9C-12 transcripts were also found in young syncytia and in the cells surrounding the expanding syncytium. Our results suggest that plant parasitic nematodes can modulate the rapid growth of the feeding cells and the expansion of the root gall by triggering the expression of DiDi 9C-12. PAEs, which probably act together with a range of other pectin-degrading enzymes, could be involved in softening and loosening the primary cell wall in nematode-infected plant roots.

Vaccination of calves against Ostertagia ostertagi with cysteine proteinase enriched protein fractions

Cysteine proteinase enriched fractions obtained by thiol‐sepharose chromatography of Ostertagia ostertagi membrane‐bound protein extract (S3‐thiol) or total adult excretory–secretory (ES‐thiol) products were tested in a vaccination experiment to evaluate their protective efficacy against O. ostertagi in cattle. Calves were vaccinated three times and subsequently challenged with a trickled infection of 25 000 infective larvae in total over 25 days (1000 L3/day, 5 days/week). Geometric mean cumulative egg counts in the ES‐thiol group were reduced by 60% during the 2‐month period between the first challenge infection and necropsy, compared to the control group (P < 0·002). No reduction in egg output was observed in the S3‐thiol group. At necropsy, calves immunized with ES‐thiol had a significantly higher percentage of inhibited L4 larvae (9·8%) and had in total 18% less worms than the control calves, but this reduction was not statistically significant. Both the female and male adult worms were significantly smaller in the ES‐thiol group than in the control group. Although no significant difference was observed in the number of eggs per female worm between the groups, there was a trend to less eggs per female worm in the ES‐thiol group. Number of worms, size of adult worms and number of eggs per female worm were not significantly different between the S3‐thiol group and the control group. Systemic immunization with QuilA as adjuvant induced a significant rise in Ostertagia‐specific antibody levels in the abomasal mucosa. Ostertagia‐specific local antibody levels showed a significant negative correlation with the size of the adult worms, the number of eggs per female worm and the cumulative faecal egg counts. However, these correlations were quite weak and did not appear to be isotype‐specific.

Arabidopsis thaliana genes expressed in the early compatible interaction with root-knot nematodes.

In the compatible interaction between Arabidopsis thaliana and the endoparasitic nematode Meloidogyne incognita, galls are formed on the roots of the host plant. Differential display was used to identify alterations of gene expression in young A. thaliana root galls caused by M. incognita. Six genes were confirmed as plant genes by DNA gel blot hybridizations. Significant homology was found with a trypsin inhibitor, peroxidase, mitochondrial uncoupling protein, endomembrane protein, 20S proteasome alpha-subunit, and diaminopimelate decarboxylase. The cellular and temporal expression of each of the six genes was analyzed by mRNA in situ hybridizations.

Identification of excretory-secretory products of larval and adult Ostertagia ostertagi by immunoscreening of cDNA libraries

Excretory-secretory (ES) products of Ostertagia ostertagi, an abomasal nematode of cattle, are considered to be important for the development and survival of the parasite within the host. To gain insight in the composition of these ES products of both larval (L3, L4) and adult life stages of Ostertagia cDNA libraries of the parasite were immunoscreened with polyclonal rabbit serum raised against these ES products. This approach led to the identification of 41 proteins, amongst which are structural proteins such as actin, kinesin and vitellogenin, housekeeping proteins such as those involved in protein folding, different metabolic pathways or mitochondrial functioning and proteins associated with stress (heat shock protein) or antioxidantia (thioredoxin peroxidase). A large number of the isolated proteins were similar to hypothetical proteins of the model nematode Caenorhabditis elegans. Because somatic proteins can be non-specifically released during in vitro culturing as nematodes deteriorate, it was checked if the isolated proteins are genuinely secreted. The amino acid sequences of the translated cDNAs were investigated for signal peptides and monospecific antibodies against the isolated proteins were purified and used to develop Western blots of ES and somatic extracts. In this manner it could be proven that 15 cDNAs code for genuine secreted proteins. The identification of these ES antigens allows to select proteins with potential protective capacities, which are targets for vaccine development.

Vaccination with an Ostertagia ostertagi Polyprotein Allergen Protects Calves against Homologous Challenge Infection

ABSTRACT As an alternative to antihelminthic drugs, we are exploiting vaccination to control infections with the abomasal nematode Ostertagia ostertagi in cattle. Our focus for vaccine targets is excretory-secretory (ES) products of this parasite. One of the most abundant antigens in larval and adult Ostertagia ES products is a protein homologous to nematode polyprotein allergens. We found that the Ostertagia polyprotein allergen (OPA) is encoded by a single-copy gene. OPA comprises three or more repeated units, and only the 15-kDa subunits are found in ES products. The native antigen is localized in the intestinal cells of third-stage larvae and in the hypodermis and cuticle of fourth-stage larvae and adult parasites. Vaccination of cattle with native OPA (nOPA) in combination with QuilA resulted in protection against Ostertagia challenge infections. The geometric mean cumulative fecal egg counts in the nOPA-vaccinated animals were reduced by 60% compared to the counts in the control group during the 2-month course of the experiment. Both male and female adult worms in nOPA-vaccinated animals were significantly shorter than the worms in the control animals. In the abomasal mucus of vaccinated animals the nOPA-specific immunoglobulin G1 (IgG1) and IgG2 levels were significantly elevated compared to the levels in the control animals. Reductions in the Ostertagia egg output and the length of the adult parasites were significantly correlated with IgG1 levels. IgG2 titers were only negatively associated with adult worm length. Protected animals showed no accumulation of effector cells (mast cells, globular leukocytes, and eosinophils) in the mucosa. In contrast to the native antigen, recombinant OPA expressed in Escherichia coli did not stimulate any protection.

Cytokine responses in immunized and non-immunized calves after Ostertagia ostertagi infection

The objective of this study was to evaluate abomasal cytokine responses in helminth‐naive calves and calves vaccinated with protective antigen fractions from Ostertagia ostertagi after an experimental challenge infection with infective third stage (L3) larvae. Abomasal lymph nodes and/or abomasal mucosa were collected and messenger RNA for the Th1 cytokines (IFN‐γ, IL‐2, IL‐12 p40 subunit), the Th2 cytokines (IL‐4, IL‐5, IL‐6, IL‐10, IL‐13, IL‐15) and the Th3/Tr cytokine TGF‐β was quantified by real‐time RT‐PCR. Vaccination had no effect on cytokine profiles in either the abomasal lymph nodes or the abomasal mucosa. However, following infection all calves showed a significant decrease in the Th1 cytokines, IFN‐γ and IL‐12 p40, and a significant increase in the Th2 cytokines, IL‐4, IL‐5, IL‐10 and IL‐13 in the lymph nodes, compared to non‐infected calves. No correlation between the Th2 response and protection induced by vaccination could be demonstrated. In contrast, a Th2 pattern was not observed in the mucosa of the infected calves, which exhibited an increase in IFN‐γ as well as in the Th2 cytokines IL‐4, IL‐5 and IL‐10 mRNA. No significant association was observed in the abomasal mucosa between any examined cytokine mRNA level and immune effector responses such as parasite‐specific antibodies or the number of mucosal mast cells or eosinophils.

Validation of the protective Ostertagia ostertagi ES-thiol antigens with different adjuvantia

Intramuscular immunization of calves with an excretory–secretory antigen fraction enriched for cysteine proteinase activity (ES‐thiol) and QuilA as adjuvant induces a protective immune response against the abomasal nematode Ostertagia ostertagi. The objectives of the present study were to confirm the protective capacity of ES‐thiol in combination with QuilA, to test Al(OH)3 as adjuvant for vaccination against O. ostertagi and to look for correlations between protection and immunological effector responses. Calves(seven animals/group) were vaccinated three times intramuscularly with 100 µg antigen and/or adjuvant (ES‐thiol with QuilA, ES‐thiol with Al(OH)3, QuilA alone and Al(OH)3 alone) and subsequently challenged with a trickled oral infection of 25 000 infective larvae in total over 25 days. Faecal egg counts in the ES‐thiol QuilA group were reduced by 56% during the two‐month period of the trial compared to the QuilA control group (P < 0·002). Calves immunized with ES‐thiol QuilA had significantly smaller adult worms (P < 0·002) and less eggs/female worm (P < 0·05) compared to the QuilA control group. No differences in egg output, worm counts or parameters of worm fitness were observed in the ES‐thiol Al(OH)3 group compared to the Al(OH)3 control group. Although the protective immune mechanism against O. ostertagi remains unknown, protection in the ES‐thiol QuilA group was associated with high levels of parasite‐specific antibodies in the abomasal mucosa.

An SXP/RAL-2 protein produced by the subventral pharyngeal glands in the plant parasitic root-knot nematode Meloidogyne incognita

Meloidogyne incognita is a major parasite of numerous plant families, including many crop species. Upon infection of the plant root, it induces several multinucleate giant cells by the injection of pharyngeal gland secretions into the root cells. In order to obtain a better understanding of the nematode-plant interaction, characterization of the pharyngeal gland secretions is a necessity. By differential display, a nematode gene was identified that encodes a new member of the SXP/RAL-2 protein family. The gene is specifically expressed in the subventral pharyngeal glands and the protein is most likely secreted.

Protein disulphide isomerase of Ostertagia ostertagi: an excretory-secretory product of L4 and adult worms?

A pepstatin A-agarose column was used in an attempt to purify a previously described antibody-degrading aspartyl proteinase from excretory-secretory material from the L4 and the adult stages of the bovine abomasal nematode Ostertagia ostertagi. However, no aspartyl proteinase activity was detected in the eluted fractions (L4Pepst and AdPepst). Screening of cDNA libraries with polyclonal antibodies raised against L4Pepst and AdPepst showed that a protein disulphide isomerase (Ost-PDI2) was present in both antigen fractions. This multifunctional enzyme was detected in extracts of L3, L4 and adult parasites and, interestingly, also in excretory-secretory material of L4 and adult O. ostertagi. By immunohistochemistry, the Ost-PDI2 enzyme was localised in some parts of the hypodermis of L4 and adult worms and in the intestinal cells of all three parasitic life stages. Two-dimensional Western blot analysis indicated that Ost-PDI2 is recognised by calves during a natural O. ostertagi infection, which suggests that Ost-PDI2 could be used for immunological control of ostertagiosis.

Hairy roots to test for transgenic nematode resistance: think twice

Hairy roots induced by Agrobacterium rhizogenes have been proposed as a versatile, easy and reproducible system for testing nematode resistance in crop plants. Here, A. rhizogenes was used to induce transgenic hairy roots on tomato (Lycopersicon esculentum) containing the LEMMI9 cDNA in sense and/or antisense orientation under control of a nematode responsive promoter. The purpose was to inhibit the expression of this gene that is strongly activated in the nematode-induced feeding sites in order to block their development and, thus, make the plants resistant. Several LEMMI9 transgenic lines produced fewer Meloidogyne incognita second-stage juveniles than the control lines, but a large variation in progeny was also seen among the control lines. Therefore, we recommend that several independent wild-type hairy root lines are generated and tested to obtain a solid control group for evaluating putative resistance constructs.