The immunogenicity of the acetylcholinesterases of the cattle lungworm Dictyocaulus viviparus

InrernarionulJuurnalfor Parasrrolog~.Vol. 24, No.4,pp. 501-510,1994 Copyright(I"lYY4 AustralianSociety for Parasitology Elsevier Science Ltd Printed in Great Britain. All nghts reserved Ml20-75lY;Y4$700+ 000

Pergamon

OOZO-7519(93)E0044-A

THE IMMUNOGENICITY OF THE ACETYLCHOLINESTERASES THE CATTLE LUNGWORM DZCTYOCA ULUS VIVIPARUS J. B. MCKEAND,*

D. P. KNOX,?

J. L. DUNCAN*

OF

and M. W. KENNEDY:

*Department $Wellcome

of Veterinary Parasitology, University of Glasgow, Bearsden Road, Glasgow G61 IQH, U.K. tMoredun Research Institute, 408 Gilmerton Road, Edinburgh EH17, U.K. Laboratories for Experimental Parasitology, University of Glasgow, Bearsden Road, Glasgow G61 lQH, U.K. (Received 4 October 1993; accepted 10 December 1993)

Abstract-MCKEAND J. B., KNOX D. P., DUNCAN J. L. and KENNEDY M. W. 1994. The immunogenicity of the acetylcholinesterases of the cattle lungworm Dictyocaulus viviparus. International Journal for Parasitology 24: 501-510. Somatic extracts and excretory/secretory (ES) products of the adult stage of the cattle nematode, Dictyocaulus viviparus, were examined for acetylcholinesterase (AChE) activity. Both were found to contain activity which had an optimum pH of 9.5, however, the adult ES products contained over 200 times more AChE activity per unit protein. Gel electrophoresis and specific enzyme staining revealed 5 migratory isoforms of AChE which were common to adult ES products and adult homogenates. Comparison of L3 with L4 and adult extracts indicated that the AChE were only produced by later developmental stages ofthis parasite. The antigenicity of D. viviparus AChE was demonstrated by binding to serum IgG from naturally and experimentally infected calves but the enzymes were not recognized by calves vaccinated twice with 400 Gy-irradiated larvae. This is the first report of helminth AChE release by a parasitic nematode in a pulmonary location. The presence of these enzymes in such high amounts in the ES products, along with their immunogenicity, suggests that they might have an important role to play in the immunobiology of D. viviparus in the lungs.

INDEX immune

KEY WORDS: recognition.

Dictyocaulus

viviparus; excretory/secretory

INTRODUCTION INFECTION

parasitic

with bronchitis,

Dictyocaulus a serious

viviparus disease

results

of grazing

products;

acetylcholinesterases;

parasitic nematode species (Lee, 1970; Ogilvie, Rothwell, Bremner, Schnitzerling, Nolan & Keith, 1973; Rothwell, Ogilvie & Love, 1973; Bremner, Ogilvie, Keith & Berrie, 1973; Burt & Ogilvie, 1972; Rhoads, 1981; Rathaur, Robertson, Selkirk & Maizels, 1987; Pritchard, Leggett, Rogan, McKean & Brown, 1991). The neuronal function of nematode AChE has been well documented (reviewed by Opperman & Chang, 1992), but the biological significance of the released forms remains to be ascertained. It has been postulated that secretory AChE may contribute to immune evasion by inhibiting functions known to be stimulated by acetylcholine (ACh); for example, gut peristalsis, mucus secretion, neutrophil-mediated antibody dependent cellular cytotoxicity (ADCC) and mast cell degranulation (reviewed by Rhoads, 1984). This work aimed to determine whether D. viviparus parasites secrete such enzymes in the lungs and whether these enzymes are immunogenic. Evidence for AChE release by gastrointestinal nematodes in vivo

in cattle

in temperate areas. Natural infection stimulates a strong acquired immunity and the infection is unique in that a radiation-attenuated larval vaccine (‘Dictol’) has been used for almost 30 years for the prevention of this disease. Despite, and perhaps because of, the success of the vaccine, there has been little progress towards the definition of protective antigens of D. viviparus, nor of the parasite-derived factors which contribute to the pathology of this disease. In order to address these issues, and to improve our understanding of the biology of nematodes inhabiting the lungs, we have aimed to define components of D. viviparus which may be essential to the survival of parasites in the lungs and determine host responses which may result in their elimination. Acetylcholinesterases (AChE, EC 3.1.1.7) have been characterised in the ES products of several 501

502

J. B. MCKEAND ef al.

has been provided by several studies in which antibodies against these enzymes were demonstrated in the serum of naturally or experimentally infected hosts (Jones & Ogilvie, 1972; Bremner et al., 1973; Rothwell & Merritt, 1974; Rothwell, Anderson, Bremner, Dash, Le Jambre, Merritt & Ng, 1976; Beaver & Dobson, 1978). The significance of antibodies against AChE in protective responses has not been assessed in any detail, although immunisation with purified Trichostrongylus colubriformis AChE failed to induce significant protective immunity in guinea pigs (Rothwell & Merritt, 1975). There have been no reports of AChE secretion by parasites which occupy a lung environment, despite the fact that inactivation of some of the host effector mechanisms mentioned above might be of benefit to the parasites. Here, we report that adult stages of D. viviparus release several isoforms of AChE which are targets of circulating antibody responses in animals rendered immune by natural or experimental infection. MATERIALS AND METHODS Animals andparasites. Third stage larvae (L3) and 400Gy (40 krad)-irradiated L3 were obtained from Pitman-Moore, Cambridgeshire, and maintained in PBS, pH 7.2, at 4°C. To provide adult parasites for ES products, 6-month-old male Friesian calves were obtained locally and reared indoors under conditions designed to prevent exposure to D. viviparus. They were infected orally at a dose rate of 20 L3/kg body weight and necropsied between days 28 and 35 of infection. The lungs were removed and the respiratory passages were perfused (Inderbitzen, R., unpublished, thesis, University of Zurich, 1976). Adults obtained were immediately placed into sterile PBS at 37°C. To obtain fourth stage larvae for L4 ES production, calves were infected at a dose rate of 200 L3/kg, necropsied on day 14 of infection and parasites obtained from the lungs as described for the adult stages. ESproducts andparasite extracts. Live adult parasites were immediately washed in sterile PBS, cultured for 3 days in supplemented RPM1 1640 medium (Kennedy & Qureshi, 1986). The parasites were cultured at approximately 6 parasites per ml and maintained in a humid, 5% CO, incubator at 37’C for 24 h. The culture medium was removed at 24 h and replaced with fresh medium and the parasites cultured for a further 24 h. This process was repeated at 48h and 72 h, after which the parasites were discarded. The media harvested from each day were maintained separately and passed through 0.22 pm sterile filters (Millex GV, Millipore SA, Molshiem, France), dialysed and concentrated approximately 20-fold with Amicon Centricon 10 devices (Amicon Div., W. R. Grace and Co., Danvers, MA) and stored at - 7o’C until use. ES material from I_4 stages was produced in a similar manner except that the parasites were cultured at a concentration of 20 parasites per ml of culture medium. L3 ES was obtained by culturing approximately 300

L3 per ml of supplemented RPM1 and the ES products harvested at day 5 of culture without a change in medium, after which they were sterilised, dialysed and concentrated as for the adult ES products. A PBS-soluble extract of adult parasites was obtained using approximately 100 live adult worms taken from the lungs. The parasites were washed 3 times in ice cold PBS and homogenised in 1 ml PBS on ice in a glass tissue-homogeniser. The extract was centrifuged at 12,000 g for 30 min, at 4°C and the supernatant stored at -70°C. L4 and L3 somatic extracts were obtained by sonication. Approximately 1 x 105 L3 or 1 x lo4 L4 were spin-washed 3 times in PBS, resuspended in 0.5 ml and added to the same volume of PBS. The parasites were sonicated on ice for approximately 10 min in a MSE Soniprep 150 ultrasonic disintegrator (MSE, Scientific Instruments, Crawley, England), at 16 pm amplitude. The sonicates were spun at 12,000 8 for 30 min, at 4°C and the supernatants stored at -7o’C. Protein estimations of ES products and somatic extracts were estimated using a Coomassie Blue-based assay (Pierce Chemical Co., Rockford, IL, cat no. 23200) according to manufacturer’s instructions. Antisera. Natural D. viviparus infections at pasture: serum was obtained from 9 calves which were grazed from May until September on D. viviparus-infected pastures at Glasgow University Veterinary School. The calves were approximately 6 months at turnout and serum was obtained from each calf prior to going to pasture and then after 60 days. By day 35 after turn-out, the calves were deemed positive for D. viviparus infections following Baermannisation of faecal samples and examination for first stage larvae (Henriksen, 1965). Experimental D. viviparus infections: 4 6-month-old male Friesian calves were infected with 5000 D. viviparus L3 and were bled on days 0 and 28 of infection. Experimental vaccination with 400 Gy-irradiated D. viviparus L3: 3 6month-old male Friesian calves were infected on two occasions with 5000, 400 Gy-irradiated D. viviparus. The calves were vaccinated 28 days apart and were bled on day 0 and on day 28 of each vaccination. All bleeds were by jugular venepuncture and the serum obtained was stored at - 20°C. IgG from experimentally infected calves. Four calves were infected twice with 1000 L3 and once with 10,000 L3 and the sera obtained on day 0 and on day 21 of tertiary infection. The sera from the calves were pooled after each bleed and IgG was affinity-purified using Protein G as previously described (B&ton, Knox, Canto, Urquhart & Kennedy, 1993) and was donated by Dr C Britton, Wellcome Laboratories for Experimental Parasitology, University of Glasgow. The protein concentration of the purified IgG was estimated to be approximately 5 mg/ml. Test-tube assay for acetylcholinesterase activity. The AChE content of D. viviparus adult ES and adult homogenate was determined using the calorimetric assay of Ellman, Courtney, Andres & Featherstone (1961) using acetylthiocholine iodide (ATCI, Sigma Chemical Co., Dorset, UK, cat. no. A 5751) as substrate. Five microlitres of each antigen sample were mixed with 300 pl of reagent and the change in absorbance between 5 and 65 s at 412 nm, at 37”C, monitored using a

Immunogenicity

of D. viviparus

Multistat III microcentrifugal analyser (Instrument Laboratory, Warrington, UK). The samples were referenced relative to the absorbance of a reaction containing distilled water in place of test material. The protein concentration of the analysed samples were 0.30 mg/ml for adult ES and 3.4 mg/ ml for adult homogenate. All adult ES samples were diluted 1: 10 in PBS. The effect of pH on parasite AChE activity was measured over a range of pH 411, using 0.1 M-acetatephosphate buffer (pH 4.0-6.0) 0.1 M-phosphate buffer (pH 6.0-8.0) 0.1 M-Tris-HCl buffer (pH 8.0-9.5) and 0.1 Mcarbonate-bicarbonate buffer (pH 9.5-l 1).

0.4

Change

Immunogenicity of D. viviparus AChE. Antibody binding to parasite AChE was detected in an electrophoresis migration assay in which serum from non-infected and infected or vaccinated calves was incubated with adult ES products prior to electrophoresis and staining for esterase activity. Briefly, 5 ~1 of adult ES were incubated with 5 ~1 of each serum sample and incubated at room temperature for 1 h. The incubation products were then subjected to electrophoresis on non-denaturing, 10% minigels and stained for esterase activity as outlined above. Five ~1 of IgG purified from non-infected and D. viviparus-infected calves were also incubated with adult ES products and the incubation products analysed for esterase activity in a similar manner. RESULTS of adu!t D. viviparus AChE AChE activity was assayed quantitatively at a range of pH using the Ellman assay (Fig. 1.). Two peaks of AChE activity were apparent in the ES produced by adult parasites, one at approximately pH 7.5 and the Detection

of pH optima

I” absorbance

1

4

Characterisation of AChE isoforms by polyacrylamide gel electrophoresis. Electrophoretic separations of adult, L4 and L3 preparations were carried out in non-denaturing, 10% polyacrylamide gels (70 x 80 x 0.5 mm) using a Mini Protean II Dual Slab Cell (Bio-rad, Herts, UK). Samples (10 ~1) were mixed with 10 d of sample loading buffer (0.5 M-Tris, pH 7.5,20% glycerol v/v and 0.01% w/v bromophenol blue), applied to the gel and electrophoresis performed at 4°C at a constant voltage of 200 V for approximately 45 min with 25 mM-Tris, 0.18 t+glycine as electrode buffer. In order to determine non-specific esterase activity, gels were stained, following electrophoresis, by the method of Grunder, Sartori & Stormont (1965) using naphthyl acetate as substrate. The reaction was stopped after approximately 5 min by rinsing the gels in distilled water. To distinguish if the esterase isoforms present were AChE and not pseudocholinesterases, specific gel tracks were pre-incubated in 20 ml of 1 mMeserine sulphate (Sigma, E8625) for 60 min (Pearse, 1972) prior to staining by the method described above. In addition, tracks were stained specifically for AChE activity following the method of Karnovsky & Roots (1964). For protein visualisation, gels were stained with 0.1% Coomassie Brilliant Blue R-250 (Sigma) in 25% methanol, 10% acetic acid and 1% glycerol for l-2 h and destained by rocking in the solvent until the background was clear. All gells were then photographed and dried on a slab gel drier at 60°C.

503

acetylcholinesterases

5

66.5

77.5

88.5

99.5

1011

pH

FIG. 1. The effect of pH on D. viviparus AChE activity. Adult ES materials, diluted 1: 10 in PBS (closed squares) and somatic extracts (open squares) were examined for AChE activity over a range of pH using the method of Ellman et al. (1961). The results are expressed as the change in absorbance (412 nm) measured between 5 and 65 s after the start of the reaction. The protein concentrations of the samples examined were: adult ES, 0.3 mg/ml and adult homogenate, 3.4 mg/ml. The means of 2 observations are shown, with error bars representing the SD. of these values.

other at pH 9.5. Activity was much lower in the homogenate extract and a peak of activity was only distinguishable at pH 9.5. On a per unit protein basis, the adult ES products contained approximately 200 times more activity than did the somatic extract. In order to determine whether the AChE activity comprised more than 1 isoform, ES products from adult worms were subject to electrophoresis, the gel cut into strips and the tracks stained separately for protein, non-specific esterase or AChE activity (Fig. 2). Coomassie Biue staining of the adult ES (Track A) revealed at least 10 major protein bands, the relative molecular weights of which could not be estimated as the sample was run under non-denaturing conditions. In the track stained for esterase activity (Track B), 5 zones of activity were detected in the lower half of the gel. These were sensitive to 1 mM-eserine (Track C) and could be visualised using the method of Karnovsky & Roots (1964, Track D) and were consequently classified as AChE. The enzymes ran close together in Track D but 5 distinct bands of activity were apparent on the original gel. The zones of AChE activity could not be correlated with any major components on the Coomassie Blue stained track. Somatic extracts of adult parasites were also examined for esterase activity (Track E) and a similar pattern was observed. Staining was much less intense, however, which was consistent with the results obtained by Ellman assay. To establish whether earlier developmental stages of D. viviparus release AChE, L4 were obtained from a calf on day 14 of infection and the somatic extracts and ES products of L4 stages were examined for esterase

504

J. 8. MCKEAND et al.

A

B

C

D

E

Fro. 2. Characterisation of the AChE of adult D. viviparus. Ten microlitres of adult ES products (Tracks A to D) were run under non-denaturing conditions on a 10% polyacrylamide minigel. Adult homogenate extract was run in one track (Track E). The gel was then cut’into tracks which were then stained with Coomassie Blue for protein (Track A); naphthyl acetate for esterase activity (Tracks B and E). For specific AChE activity, one track was pre-incubated for 1 h in 20 ml, lmt+eserine prior to staining with naphthyl acetate (Track C) and another stained following the method of Karnovsky & Roots (1964, Track D).

as before and compared with that from adult and L3 stages (Fig. 3). The L4 ES material was found to contain a pattern of AChE activity similar to that of adult ES products, but staining was considerably less intense. AChE activity was not detected in the L3 preparations. activity

I~~u~oge~ic~ty of D. viviparus AChE in ~~turul~~v and e~~erimentaliy infected calves

Host responses to the various isoforms of AChE were examined in a gel retardation assay in which adult ES material was incubated with serum from naturally and experimentally-infected calves. Esterase activity present in the serum was found to interfere with the quantitative Ellman assay so the specificity of host antibody for parasite AChE was therefore estimated using this gel assay. Staining for esterases following the method of Gunder et al. (1965) was found to be the clearest for this purpose and was used for all subsequent experiments. The anti-AChE serum responses of calves infected at grass are shown in Fig. 4. The parasite-derived AChE activities can be identified with reference to the control ES track (Track ‘ES’). Slower-migrating bands are either due to enzyme activity endogenous to the bovine sera (Track ‘S’) or due to specific antibody-mediated retardation

FIG. 3. Comparison of AChE release by different developmental stages of L). viviparus. Ten microlitres each of D. viviparus L3 homogenate (Track Al), L3 ES (Track A2), L4 homogenate (Track Bl), L4 ES (Track B2) and adult ES (Track C) were run on non-denaturing 10% minigels and stained for esterase activity. All tracks were stained for several hours in order to detect any esterase activity present in the samples. The adult ES track is, therefore, over-stained. The protein con~ntrations of the extracts were: adult ES, 0.30 mg/ml; L3 homogenate, 0.84 mg/ml; L3 ES, 0.10 mg/ml; L4 homogenate, 0.30 mg/ml; L4 ES, 0.33 mg/ml.

of the parasite enzymes at the stack/resolving gel interface. When the ES products were pre-incubated with serum from uninfected calves (Tracks 1a, 2a, 3a, etc.), there was little reduction in staining intensity of the parasite esterases. In contrast, when the ES products were pre-incubated with serum from infected calves (Tracks lb, 2b, 3b, etc.), qualitative differences were observed in the parasite esterase patterns. For example, serum antibody from calf 1 (Track lb) appeared to recognise the lowermost AChE isoforms in that these disappeared from their usual position on the gel (compare with Track la and ‘ES’). Increased levels of activity were apparent at the top of this track indicating that the parasite AChE were complexed and unable to enter the resolving gel. By 2 months at grass, antibody from calves 4,5 and 7 (Tracks 4b, 5b and 7b) appeared to recognise all parasite AChE isoforms and there was no increase in intensity at the top of these tracks indicating inhibition of parasite AChE activity. Thus, post-infection sera from all of the calves reduced the intensity of parasite esterase staining, however, the pattern of recognition differed amongst the individual animals. To examine whether heterogeneity in production of anti-AChE antibody was exposure-related, 4 calves were experimentally infected with 5000 D. viviparus L3

Immunogenicity

505

of D. viviparus acetylcholinesterases

ES

S

la

lb

2a

2b

3a

3b

4a 4b

5a

5b

6a

6b

7a

7b

8a

8b

9a 9b

FIG. 4. Recognition of D. viviparus adult AChE by field-infected calves. Sera were sampled from 9 calves (l-9) before and 60 days following turn-out to D. viviparus-infected pasture. Five microlitres of adult ES were incubated with 5 ~1 ofeach bovine serum sample and incubated at room temperature for 1 hand the incubation products subjected to electrophoresis on non-denaturing, 10% minigels and stained for esterase activity as outlined in Materials and Methods. The pre-infection serum samples for each animal were run in tracks labelled ‘a’ (Tracks la, 2a, etc.) and the post-infection samples in those labelled ‘b’ (Tracks lb, 2b, etc.). The samples are compared with activity present in adult ES products (‘ES’) and normal bovine serum (3’) run on their own.

and their serum responses to the parasite AChE monitored over time. The responses at day 28 of infection are shown in Fig. 5 and the samples are compared with esterase activity present in adult ES (‘ES’) and with ES pre-incubated with pre-infection serum from the individual animals (Tracks la, 2a, 3a, 4a). Again, little reduction in staining intensity was evident in the tracks in which ES had been preincubated with serum from non-infected calves. In

contrast, when ES was pre-incubated with serum from calves taken at day 28 of infection (Tracks lb, 2b, 3b, 4b), all 5 parasite AChE isoforms disappeared from the gels. Complexing and retardation up the gel did not seem to have occurred with these samples. IgG complexing of D. viviparus AChE Confirmation that the retardation of enzyme activity was due, at least in part, to antibody was

506

J. B. MCKEANDet al.

ES

S

la

lb

2a 2b

3a

3b

4a 4b

FIG. 5. Recognition of D. viviparus adult AChE by experimentallyinfected calves. Sera were sampled from 4 calves (14) before and 28 days following a single infection with 5000 D. viviparus L3. The samples were incubated and analysed as described in Fig. 4. The pre-infection serum samples were run in tracks labelled ‘a’ (Tracks la, 2a, etc.) and the post-infection samples in those labelled ‘b’ (Tracks lb, 2b, etc.). The samples are compared with activity present in adult ES products (‘ES’) and normal bovine serum (‘S’) run on their own. sought by repeating the assay using Protein G-purified IgG from the serum of infected calves (Fig. 6). In the track which contained preparations which had been pre-incubated with IgG from D. viviparus-infected calves (Track B), there were diminished levels of free enzyme in the lower half of the gel but an intense band of esterase staining was present at the stack/resolving gel interface (arrowed on Fig. 6). Equivalent bands were not evident in the sample which had been preincubated with IgG affinity-purified from normal bovine serum (Track A). Responses

of vaccinated calves to adult D. viviparus AChE The calves used in the above experiment had been exposed to adult parasites, but this should not be true of vaccinated animals because larvae irradiated to 400 Gy rarely develop to mature adult stages following vaccination (Jarrett, Jennings, McIntyre, Mulligan & Urquhart, 1958). Figure 7 shows the effect of serum from 3 calves vaccinated twice with 5000 irradiated L3. Qualitative changes in parasite esterase patterns were not evident following pre-incubation with postvaccination sera after both primary and secondary vaccination. DISCUSSION

This study has demonstrated that the adult stages of the bovine lungworm, D. viviparus, like many gastrointestinal nematodes, release AChE in vitro. In addition, in vivo release was inferred by the finding that sera from naturally and experimentally infected calves

*

contained antibody which specifically recognised these enzymes. This was demonstrated in polyacrylamide gels by the formation of enzymically-active AChE/ antibody complexes at the top of the gels or by the inhibition of parasite AChE activity. Somatic extracts and ES products from the adult stage D. viviparus showed AChE activity over a range of pH from 6 to 11 and a biphasic pH profile was evident in the adult ES products but not the somatic extract. The biphasic profile might indicate different optimal activities of the various AChE isoforms and it may be that the low levels of AChE activity present in the somatic extract did not enable us to detect activity at the lower pH. Per unit protein, the adult ES products contained 200 times more AChE activity than did the somatic extract suggesting that the enzymes are secreted rapidly after synthesis and not stored in an active form. Polyacrylamide gel analysis showed 5 isoforms of AChE in both the ES products and somatic extracts of the adult stage. These isoforms did not appear to be released or contained within L3 stages, but were present in the L4 preparations although the total activity was substantially lower (Fig. 3). These results are similar to those obtained for Nippostrongylus brasiliensis by Sanderson & Ogilvie (1971), Necator americanus by Burt & Ogilvie (1972) and Trichostrongylus spp. by Rothwell et al. (1973) in which low levels of esterase activity were demonstrated in free-living compared with parasitic stages. In agreement with the results obtained in this study, it was found that calves vaccinated with 400 Gy-irradiated L3 did not appear

Immunogenicity

A

of D. viviparus acetylcholinesterases

B

FIG. 6. Complexing of D. viviparus adult AChE by IgG from experimentally-infected calves. Four calves were infected twice with 1000 D. viviparus L3 and once with 10,OOOinfective L3. Sera were taken 21 days after final infection and pooled and IgG affinity-purified from the pool and from preinfection serum as described in Materials and Methods. The samples were incubated and analysed as described in Fig. 4. Adult ES products pre-incubated with IgG from noninfected calves was run in Track A and those incubated with IgG from the experimentally-infected calves in Track B. The increase intensity in esterase staining at the top of Track B is indicated by the arrow. to produce substantial antibody responses against the parasite AChE following 2 vaccinations (Fig. 7). Previous workers have shown that larvae irradiated to this degree have a reduced lifespan resulting in the development of fewer mature stages in the lungs (Jarrett & Sharp, 1963). It is probable that the vaccinated calves examined here were exposed to few adult stages and thus were unlikely to mount immune responses against antigens, such as AChE, which are released in higher amounts by these later parasite stages. Antibodies to parasite AChE were detected in serum from naturally-infected calves 60 days after turnout to D. viviparus-infected pastures. Although sera from some calves completely inhibited parasite AChE activity on polyacrylamide gels, other calves appeared to produce antibodies which complexed the enzymes whilst enabling them to retain their activity near the top of the gel. Purified IgG from infected calves bound the parasite AChE confirming the role of antibody.

507

Results obtained by Ellman assay (not shown) also indicated that, although parasite AChE was bound by antibody from infected calves, not all calves produced antibodies which inhibited enzyme activity. From these results, we can hypothesize that epitopes outside the active site may be recognised by some calves, whilst other calves produce antibodies specific for epitopes within, or close to, the active site and thereby ablate enzyme activity. The differential recognition of the different regions of AChE which was exhibited by this group of calves may be a quantitative phenomenon resulting from varying levels of parasite, and therefore antigen, exposure amongst the different field-infected animals. Alternatively, the differential epitope recognition may be a result of host genetic effects, for which there has been preliminary evidence in calves and in the guinea pig laboratory model (Britton, Canto, Urquhart & Kennedy, 1992; McKeand, Knox, Duncan and Kennedy, in press a). Complexing of parasite AChE by antibody, without inhibition of enzyme activity, has been observed in T. colubriformisinfected sheep (Rothwell et al., 1973, Rothwell & Merritt, 1974) and Oesophagostomum species in ruminants (Bremner et aI., 1973). However, other nematode species appear to stimulate antibodies which can inhibit parasite AChE activity, for example Ostertagia species in ruminants (Rothwell et al., 1976). In studies with the above nematodes, it is possible that the different reactivities against parasite esterases were a result of genetic control of responses to different epitopes on the enzyme molecules. Antibody from all 4 calves experimentally-infected with D. viviparus appeared to inhibit all AChE isoforms by day 28 of infection. This may have been due to a dose-related response to the parasite AChE in that these calves were probably exposed to more parasite antigen following the single large challenge as compared with the trickle infection encountered during field exposure. The fact that the experimentally-infected calves recognised the parasite AChE within 28 days of primary infection further suggests that these enzymes are actually released in vivo because it is unlikely that parasite death, and therefore exposure to internal parasite antigens, would have occurred by this point in infection. Although binding by antibody might have direct inhibitory effects on AChE activity, it might also act to complex and retard diffusion of the enzyme away from the parasite, thereby preventing its functions at peripheral sites of action. The potential relevance of antibody to protective immunity in dictyocaulosis has been inferred by the successful passive immunisation of calves using serum from recovered field cases (Jarrett, Jennings, McIn-

508

J. B. MCKEANDet al.

ES

S

1A 1B 1C

2A 2B 2C

3A

3B

3C

FIG. 7. Absence of anti-AChE antibody in calves vaccinated twice with 400 Gy-irradiated Lf. vivipurus L3. Sera were sampled from 3 calves (l-3) before 2 vaccinations with 5000,400 Gyirradiated L3 and then 28 days after each vaccination. The samples were incubated and analysed as described in Fig. 4. The pre-infection serum samples were run in Tracks IA, 2A and 3A, the samples taken after primary vaccination in Tracks 1B, 2B and 3B, after secondary vaccination, Tracks IC, 2C and 3C. The samples are compared with activity present in adult ES products (‘ES) and normal bovine serum (‘St run on their own.

tyre, Mulligan & Urquhart, 1955; Rubin & Weber, 1955) and of guinea pigs using serum raised against experimental infections with normal L3 (Canto, 1990, thesis cited above). We have observed that significant levels of protection can be obtained in guinea pigs immunised by passive transfer of serum raised against adult ES products (McKeand, Knox, Duncan & Kennedy, in press b). Moreover, we have found that serum antibody recognition of parasite AChE correlates with protective immunity amongst different strains of ES-immunised guinea pigs (McKeand, ef af., in press a). It is possible, therefore, that antibodymediated inactivation of parasite AChE may play a part in this immunity. AChE released by D. viviparus in the lungs might have several functions, including the avoidance of host immune effector mechanisms. For example, large numbers of mast cells have been observed in the lungs of L). viviparus-infected calves (McKeand, Miller and Kennedy, unpublished) and chemical mediators released by these cell types are known to be stimulated by ACh (Kaliner, Orange & Austen, 1972; Tauber, Kaliner, Stechschulte & Austen, 1973; Kaliner & Austen, 1975). Furthe~ore, pulmonary neutrophilia occurs in the lungs of calves with parasitic bronchitis (Simpson, Wade, Dennis & Swanson, 1957) and ADCC mediated by these granulocytes is also known to be activated by ACh (Gale & Zhigelboim, 1974), although it is not yet known whether neutrophil-

mediated ADCC is effective against D. viviparus adult parasites. We can postulate that inactivation of these processes by parasite AChE might assist survival of the parasites in the lungs. The potential importance of AChE’s role in immunity against D. v~vjpara~ was further emphasised recently when we obtained significant levels of protective immunity in immunised guinea pigs using partially-purified preparations of adult parasite AChE (McKeand, 1992, thesis, University of Glasgow). Given the quantities of AChE which appear to be released by D. viviparus into its pulmonary environment, there is clearly a need to further our understanding of its biochemical activity and biological role. To this end, we are presently attempting to affinity purify AChE from D. viviparus for direct examination of its role in the immunobiology infection.

and

pathology

of this

Acknowledgements--This work was supported by a Wellcome Veterinary Research Training Scholarship to J.B.M. and a Scottish Office Agriculture and Fisheries Department grant to D.P.K. We are also indebted to Pitman-Moore for the provision of infective I). viviparus and to Dr G. J. Canto for some of the calf sera and Dr C. B&ton for the purified IgG. We also thank Alan May for photographic work. REFERENCES

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levels to the immune

immuno~enicity

of D. vivip~r~s acetylcholinesterases

response in rats. International Journal for Parasitology 8: 9-13.

BREMNERK. C., OGILVIEB. M., KEITHR. K.&BERRIED. A. 1973. A~etylcholinesteras~ secretion by parasitic nematodes. III. ~esop~lagosiomum spp. International Journalfor Parasitology 3: 609-6 18.

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