: Dr P. Geldhof, Department of Parasitology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B9820 Merelbeke, Belgium (e-mail: firstname.lastname@example.org).
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.
Ostertagia ostertagi is a parasitic nematode which infects the abomasum of cattle in temperate parts of the world. Bovine ostertagiosis is of major economic importance and an enormous amount of money is spent every year for the anthelmintic treatment of animals against this parasite. The potential threat of drug resistance and increasing consumer awareness of the possibility of chemical residues of the anthelmintics in meat, milk and the environment has led to a search for immunological means of control. One possibility is control by vaccination. However, data on vaccination trials with O. ostertagi are scarce. The use of either infection with irradiated larvae (1,2) or immunization with crude somatic or excretory–secretory (ES) products of the parasites (3,4) have not been successful. Recently, Smith et al. (5) reported a moderate reduction in egg output (30–50%) by vaccination with gut membrane glycoproteins of O. ostertagi. No effect was seen on the number of worms.
Proteinases in ES products of the parasite can provide an alternative source of protective antigens. Proteinases are released during in vitro culture of many parasitic helminths (6), including O. ostertagi (7). It was shown that the Ostertagia L3, L4 and adult stages released proteases which degraded a variety of protein substrates in a pH dependent and stage-specific manner. This indicates that some of these enzymes may have very specific roles to play in parasite maintenance within the abomasum. One of these enzymes was a cysteine proteinase present in adult ES material with cathepsin L-like activity. It showed high proteolytic activity against mucin, which is a normal constituent of gastrointestinal mucus (7). It can be anticipated that this proteinase is important for penetration and feeding in the abomasal mucus. Cysteine proteinases have already been demonstrated to be protective against other helminth parasites. In vitro released enzymes from Fasciola hepatica were shown to induce high levels of protection in sheep (8) and cattle (9). A 35-kDa cysteine proteinase isolated from glycerol extracts of Haemonchus contortus protected lambs against challenge infection with the same parasite (10,11). However, it was not clearly established whether the proteinase was an ES component or a gut-associated enzyme. More recently, Knox et al. (12) reported several trials where the protective efficacy of cysteine proteinase-enriched fractions from adult Haemonchus was analysed. The water-soluble and membrane-associated fractions were without effect. However, the gut membrane fraction induced a substantial reduction in faecal egg output and final worm burdens. This fraction contained the gut-associated cysteine proteinases.
In the present study, we prepared Ostertagia protein extracts enriched for cysteine proteinase activity from adult membrane protein extract and complete adult ES material and assessed their efficacy as protective antigens against an Ostertagia infection.
Materials and Methods
Preparation of antigens
Adult O. ostertagi parasites were obtained as described previously (7). Successive phosphate-buffered saline soluble (S1), Tween 20 soluble membrane-associated (S2) and Triton X-100 soluble membrane-bound (S3) protein extracts were prepared from adult parasites as described by Smith et al. (13). Adult ES-products were produced as previously described (7).
Chromatography on thiol-sepharose
S3 and total ES were preincubated with a final concentration of 2·5 mm dithiothreitol (DTT) for 30 min at 37°C prior to chromatography. Excess DTT was removed by passage through a 10 × 2·6 cm Sephadex G-25 (Pharmacia Biotech, Uppsala, Sweden) column and eluted with 10 mm Tris, 0·5 m NaCl, pH 7·4 at 5 ml/min. An activated thiol-sepharose 4B (Sigma, St Louis, MO, USA) column, 5 ml bed volume, was equilibrated in 10 mm Tris, 0·5 m NaCl, pH 7·4. Protein samples (10 mg/run) were applied to the thiol-sepharose 4B column at a flow rate of 5 ml/h. Unbound material was eluted by washing the column with equilibration buffer (10 mm Tris, 0·5 m NaCl, pH 7·4) till the OD280 had returned to a steady baseline. Bound material was eluted with equilibration buffer containing 50 mm DTT at a flow rate of 5 ml/h. The peak fractions were pooled. DTT was removed from the eluted proteins by passage, at 5 mL/minute, through a Sephadex G-25 (Pharmacia) column in 10 mm Tris pH 7·4. The peak fractions were again pooled and protein content determined by the BCA method (Pierce Chemical Co., Rockford, IL, USA). Both purifications, S3- and ES-thiol, had a yield between 10% and 15%. Aliquots of the S3- and ES-thiol fractions were removed for SDS-PAGE and substrate gel analysis. The remainder of the eluates were then stored at −70°C until required.
Peptide and proteinase composition
The peptide components of S3- and ES-thiol fractions were visualized by Coomassie Blue staining (0·1% Coomassie Blue R-250 in 30% methanol and 10% acetic acid) following fractionation of 10 µg of protein sample on a 10–20% gradient gel (Biorad, Nazareth-Eke, Belgium) under reducing conditions. Proteinase activity associated with both thiol-fractions was monitored by gelatin-substrate gel analysis under nonreducing conditions (7) to allow the definition of individual proteases. For inhibition experiments, thiol-fractions were incubated with l-transepoxysuccinyl-l-leucylamido-(4-guanidino)-butane (E64, 100 µm) as previously described by Geldhof et al. (7).
Animals and vaccination trial
Twenty-one male MontBéliard calves, aged 7 months of age at the start of the experiment, were randomized over three groups of seven animals. The animals were randomly housed in individual pens in the same stable in conditions excluding accidental infection with nematode parasites. The calves were fed corn silage ad libitum and 1 kg/day of concentrate and had free access to drinking water. All animals were immunized three times by intramuscular injection in the neck at 3-week intervals. Immunogens (S3-thiol, ES-thiol and adjuvant alone) were randomly assigned to the treatment groups. S3- and ES-thiol fractions were diluted with 10 mm Tris pH 7·4 and mixed with QuilA (Superfos Biosector, Frederickssund, Denmark) so that each calf received 100 µg protein and 750 µg adjuvant at each immunization. Control immunogen was prepared identically, except that Tris buffer was substituted for antigen. The challenge infection with O. ostertagi consisted of 25 000 L3 larvae per animal given over 25 days (1000 L3/day, 5 days/week) and started the same day as the final immunization. The animals were observed daily for adverse reactions to the immunizations and for clinical signs of ostertagiosis.
All animals were bled at weekly intervals. Faecal egg counts were performed three times a week from 20 days after the first infection until the calves were killed 24 days after the last infection. The faecal egg output was determined using a modified McMaster technique (14) with a sensitivity of 25 eggs per gram (EPG). Cumulative faecal egg counts were calculated for each animal as described by Vercruysse et al. (15). Necropsy, abomasal washings and abomasal digests (HCl-pepsin) were performed according to standard techniques (16,17). Two percent of the abomasal worm burden was counted. Geometric mean egg and worm counts were calculated after transformation of the individual counts to ln(count + 1). Adult O. ostertagi worm lengths were measured (n = 50 per animal) and the arithmetric mean length of female and male worms was calculated per animal. Twenty female worms from each calf were subsequently examined under a light microscope to count the eggs in the uterus. The geometric mean of the number of eggs per female worm was calculated per calf. All parasitological techniques were performed blindly (i.e. without knowledge of the treatment group that the animal belonged to).
Mucus was collected by gently scraping the mucosal surface of the abomasum with a glass microscope slide. Mucus scrapings were homogenized with an equal weight of phosphate-buffered saline (0·05 m, pH 7·2), using an Ultra-turrax homogeniser (IKA-labortechnik, Staufen, Germany) (13 000 r.p.m., 3 × 1 min). The homogenates were centrifuged at 20 000 g for 30 min. The supernatant was stored at −70°C prior to analysis by enzyme-linked immunosorbent assay (ELISA).
The serum antibody responses of the calves to the immunizations were evaluated by Western blotting using sera harvested 1 week after the second immunization. Five µg of S3- and ES-thiol were fractionated using 10% SDS-PAGE under reducing conditions and then blot transferred onto a PVDF membrane. The blot sections were cut into strips and blocked overnight in 10% horse serum in PBS Tween 20 (PBST). After 2 h of probing with pooled sera (diluted 1 : 400 in 2% horse serum in PBST) from the different groups, the conjugate (Rabbit antibovine-HPRO, Sigma, 1 : 8000 in 2% horse serum in PBST) was added for 1 h. Recognized antigens were visualized by adding 0·05% 3,3 diaminobenzidine tetrachloride in PBS containing 0·01% H2O2 (v/v).
Abomasal immunoglobulin (Ig)G1, IgG2, IgA and IgM levels against Ostertagia were determined by ELISA. Crude adult Ostertagia antigen extracts were coated in carbonated buffer (pH 9·6) at a concentration of 4 µg/ml. One hundred µg mucus extract from all animals was administered in PBS in duplicate. Monoclonal antibodies against bovine IgG1 (1 : 2500), IgG2 (1 : 25 000), IgA (1 : 5000) and IgM (1 : 2500) were administrated in PBS buffer. All monoclonal antibodies were derived from the ILRI-institute (Nairobi, Kenia). Goat anti-mouse IgG coupled to horseradish-peroxidase (Sigma) was used as a conjugate (1 : 4000 in PBS for IgG1, IgG2 and IgM; 1 : 8750 for IgA). O-phenylenediamine 0·1% in citrate buffer (pH 5·0) served as substrate. Optical density was measured at 492 nm.
Mast cell and eosinophil counts
Mucosal tissue for histological examination was taken from the abomasal fundus at necropsy, before washing of the abomasum. The tissue for eosinophil staining was fixed with 4% paraformaldehyde in PBS at pH 7·4 for 6 h. The tissue for mast cell counts was fixed in Carnoy’s fluid for 4 h. After fixation, the tissues were dehydrated and embedded in paraffin. Sections were cut at a thickness of 6 µm. For the eosinophil counts, the sections were stained with carbol chromotrope (18). Sections for mast cell counts were stained with 0·25% toluidine blue. The eosinophils and mast cells were counted in the epithelium and lamina propria mucosae of the abomasum on 100 graticule fields with a total surface of 5·26 mm2 at × 400 magnification under the light microscope.
Data are presented as arithmetic means ± SEM) or geometric means (+ range). At necropsy, indicators of worm fitness (cumulative faecal egg counts, total worm burden, length of adult worms and number of eggs per female worm) were expected to be lower in the vaccinated animals, while indicators of hosts’ immune responses (mucosal antibody levels, mast cell counts and eosinophil counts) were expected to be increased. The significance of differences between the three groups was assessed using a Kruskal–Wallis one-way analysis of variance, followed by a one-tailed Mann–Whitney U-test for pairwise comparison of each vaccinated group with the adjuvant controls. A correlation between different components of the hosts’ immune response and different parameters of worm fitness was investigated by Spearman’s correlation test. P < 0·05 was considered statistically significant.
Peptide composition of S3- and ES-thiol
Protein profiles obtained after reducing SDS-PAGE fractionation of S3- and ES-thiol are shown in Figure 1. ES-thiol comprised a prominent band around 30 kDa as well as three lower molecular bands and approximately six peptides in the size range from 45 to 92 kDa (Figure 1). A more complex banding pattern was visible in S3-thiol. It comprised numerous peptides in the size range from 20 to > 200 kDa with two particularly prominent proteins at 52 and 32 kDa (Figure 1).
Cysteine proteinase activity in S3- and ES-thiol
The gelatin-substrate gels indicated that passage of the S3-extract and complete ES material over the thiol-sepharose column resulted in more intense proteolytic bands in both the S3- and ES-thiol fractions (Figure 2). A smear of proteolytic activity was visible in S3-thiol with some stronger activity around 85 and 40 kDa (Figure 2). In ES-thiol, three prominent zones of proteolysis at approximately 82, 56 and 40 kDa were vizualized (Figure 2a). All proteolytic activities in S3- and ES-thiol were abolished by the cysteine protease inhibitor E64 (Figure 2b).
One calf from the control group was euthanized during the immunization period due to a femur fracture. No adverse reactions to the immunizations and no clinical signs of ostertagiosis were observed in any animal.
Faecal egg counts during the vaccination trial are plotted in Figure 3 as geometric means. The geometric mean egg counts for the ES-thiol group were lower than the control and S3-thiol group throughout the experiment. Geometric mean cumulative egg counts in the ES-thiol group were reduced by 60% compared to the control group (P < 0·002) (Table 1). In contrast, no reduction in egg output was observed in the S3-thiol group (Figure 3, Table 1).
Table 1. Number of animals per group (n), geometric mean cumulative egg counts (EPG), geometric mean total worm counts, % L4 stage, geometric mean worm lengths and geometric mean number of eggs per female worm at necropsy
No. of worms
Eggs per worm
*** P < 0·002, ** P < 0·01, * P < 0·05. F, female; M, male.
Calves immunized with ES-thiol had a significantly higher percentage of inhibited L4 larvae (9·8%) and had in total 18% less worms, although this reduction was not statistically significant (Table 1). Both the female and male adult worms were significantly smaller in the ES-thiol group than in the control group (P < 0·001) (Table 1). 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 (Table 1). Number of worms, size of the adult worms and number of eggs per female worm were not significantly different between the S3-thiol group and the control group (Table 1).
Antibody responses of immunized calves
The serum antibody responses from all groups to the vaccinations was monitored by Western blotting against both S3- and ES-thiol fractions (Figure 4). The control animals showed some minor background recognition of a few peptides in both S3- and ES-thiol (Figure 4). There was some cross-recognition of peptides between the S3- and the ES-thiol group against both protein samples (Figure 4). However, some peptides were strongly and almost specifically recognized by one group. A 32 and a 52-kDa protein in S3-thiol were strongly recognized by antibodies from the S3-thiol group (Figure 4a). The ES-thiol group strongly recognized a region around 30 kDa and some peptides around 52 kDa from ES-thiol (Figure 4b). However these 52 kDa peptides were also recognized by the S3-thiol group (Figure 4b).
Both the Ostertagia-specific IgG1 and IgG2 levels in the abomasal mucus were significantly higher in the S3- and ES-thiol group compared to the control-group (P < 0·05) (Figure 5). Only the ES-thiol group showed a significantly higher local Ostertagia-specific IgA-level than the control group (P < 0·05) (Figure 5). There was no statistically significant difference in the Ostertagia-specific IgM level between the groups (Figure 5). 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 (Table 2). Only IgA levels were significantly correlated with a reduction in worm counts (Table 2).
Table 2. Correlations between immunological parameters measured in the mucosal tissue after necropsy and parasitological parameters of worm fitness
The numbers of mast cells and eosinophils are presented in Figure 5. The ES-thiol group showed the highest levels for both mast cells and eosinophils. However, only the level of eosinophils in this group was significantly higher compared to the control group (P < 0·05). Mast cell counts were negatively correlated with the length of female and male Ostertagia, while eosinophil numbers showed a negative correlation with the size of female worms only (Table 2).
The present study demonstrated that vaccination with adult ES antigens, enriched for cysteine proteinases, can induce a protective immune response against Ostertagia. This response was reflected in a significant reduction of 60% in faecal egg counts, a significant higher percentage of inhibited L4 larvae, a reduction of 18% in total worm numbers and a significant reduction in worm length of both female and male parasites. Based on published studies, these results can be considered as the highest level of protection induced against O. ostertagi in cattle by vaccination.
ES cysteine proteinases have already shown their protective capacity against trematodes (6); however, equivalent proteinases from gastrointestinal nematodes have not been tested to date. These enzymes have been used in vaccination trials with H. contortus (11,12). All these trials were performed with extract-derived proteinases, not those from ES. Knox et al. (12) suggested that ES proteinases could be inappropriate vaccine targets because S1/S2 material from H. contortus only induced a marginal, not significant reduction in egg output. The S1/S2 fraction would be expected to contain proteinases which are released from the parasites (19,20). However, the present data indicate that ES-proteinases are more appropriate vaccine targets then the gut-associated proteinases for a vaccine against Ostertagia. In addition, the S1/S2 extract from Ostertagia showed a completely different proteinase profile on substrate-gels than the ES products (results not shown).
Although the proteinases are the interesting vaccine targets, it should be noted that the ES-thiol preparation contained other proteins next to the proteinases. It cannot be ruled out that one of these other proteins also contributed to the partial protection against Ostertagia. It is tempting to focus on proteins which are strongly and almost specifically recognized by antibodies from the ES-thiol group, such as the 30 kDa band (Figure 4b, arrow). Recognition of protein bands by antibodies from immune animals has been shown to be an effective means of identifying protective antigens of H. contortus (21,22) and recognition of specific antigens of Teladorsagia circumcincta by sheep antibodies was strongly correlated with reduced worm fecundity (23). Further fractionation and characterization of the ES-thiol fraction to identify the components that are responsible for the protective immune response is in progress.
Immunization of the calves with S3-thiol antigens did not produce any levels of protection against the O. ostertagi challenge infection. This fraction contains adult gut membrane proteins. Vaccination with gut antigens afforded a high degree of protection against the blood-sucking abomasal nematode H. contortus in sheep (24), but was less effective against O. ostertagi (5). According to Smith et al. (5), the difference in susceptibility to this type of vaccination between these two closely related parasites could be explained by the fact that Ostertagia does not ingest adequate quantities of bovine immunoglobulin to be sufficiently compromised by a circulating antibody response. This hypothesis seems to be supported by the work of Siefker and Rickard (25,26). Vaccination with Haemonchus placei intestinal homogenate conferred protection against a homologous challenge infection with H. placei in calves (25), but showed no protective capacity against a heterologous infection with O. ostertagi (26), although both parasites share some of their intestinal antigens (27).
A reduction in worm fecundity and stunted growth of the parasites, as observed in the ES-thiol group, are the first manifestations of acquired immunity against O. ostertagi in calves (28). It is unclear which immunological mechanisms were responsible for the reduced worm fitness in the ES-thiol vaccinated animals. Parasite-specific local IgA responses have been correlated with reduced fecundity of T. circumcincta in sheep (29) and O. ostertagi in calves (30). Mucosal Ostertagia-specific IgA levels were significantly higher in the ES-thiol group compared to the control group, but the correlation between IgA levels and worm fecundity was rather weak. However, the importance of the local antibody responses may have been underestimated because the ELISA results measure the antibody response to a variety of antigens, some of which may be irrelevant to protective immunity (29). On the other hand, the results indicate that the difference between a protective and a nonprotective mucosal immune response may essentially be quantitative (i.e. depending on the level of antibodies rather than their specificity). Indeed, IgG1, IgG2, mast cell and eosinophil numbers were all elevated in the ES-thiol group compared to the S3-thiol group and the control group. In any case, our ELISA results show that, by systemic immunization with QuilA as adjuvant, we induced a significant rise in Ostertagia-specific antibody levels in the abomasal mucosa. It is promising that we can alter the local immune response in the abomasum by systemic vaccination.
In most vaccination experiments, the animals are given a single challenge infection (5,21,22,25,26). In the present experiment, the challenge infection was spread over 25 days to mimic a natural challenge infection. The calves were not killed until 2 months after the start of the challenge infections to evaluate the reduction of faecal egg output during that period. In naturally infected first grazing season calves, the peak egg output occurs approximately 2 months after turnout and determines the pasture infection level in the second half of the grazing season (31). A reduction of the mean faecal egg output by 60% during the first 2 months of infection, as obtained in the ES-thiol vaccinated calves in the present trial, would therefore result in a dramatic reduction in pasture infection. A similar reduction of egg shedding during the initial months of grazing, obtained by preventive anthelmintic treatment, has been shown to substantially reduce the pasture contamination, to prevent parasitic gastroenteritis and to increase weight gain in treated calves (32). It can be anticipated that a vaccine that reduces the mean faecal egg output by approximately 60% during the first 2 months after turnout would sufficiently protect calves against parasitic gastroenteritis during their first grazing season and allow them to develop a natural immunity without production loss.
We thank N. Dierickx and I. Peelaers for their excellent technical assistance. This work was supported by grants from Ghent University (BOF 01108301) and the European Community (QLK2-CT-1999-00565).