Vaccination against the nematode Trichostrongylus colubriformis—II. Attempts to protect guinea-pigs with worm acetylcholinesterase

Inmnofional

Journal

for

Parasitology.

1975.Vol.5. pp. 453-%%.

Pergarnon

Press.

Printed

in Great

Briroin

VACCINATION AGAINST THE NEMATODE TRICHOSTRONGYLUS COLUBRIFORMIS-II. A7M’EMPTS TO PROTECT GUINEA-PIGS WITH WORM ACETYLCHOLINESTERASE T. L. W. ROTHWELL and G. C. MERRITT C.S.I.R.O. Division of Animal Health, McMaster Laboratory,

Glebe, N.S.W. 2037, Australia

(Received 6 August 1974) Abstract-RoT~wm. T. L. W. and MERRITTG. C. 1975. Vaccination against the nematode Trickostrongylas colubriformis-II. Attempts to protect guinea-pigs with worm acetylcholinesterase. International Journal for Parasitology 5: 453-460. Soluble material extracted from T. colubriformis fourth stage larvae was fractionated by membrane ultrafiltration, gel filtration and ion-exchange chromatography. The acetylcholinesterase (AChE) peak obtained by gel filtration protected guinea-pigs against infection but it was contaminated by worm allergen. However, there was no relationship between the AChE content of fractions obtained by membrane ultrafiltration and ion-exchange chromatography and their ability to stimulate protective immunity. Purified T. colubriformis AChE, prepared by ionexchange chromatography and free from demonstrable allergen did not stimulate protective immunity whereas another fraction, containing less than one-thousandth the amount of AChE, was effective in doing so. INDEX KEY WORDS: Acetylcholinesterase; strongylus colubriformis; vaccination.

antigens; guinea-pigs;

INTRODUCTION LITTLE is known about the antigens of parasitic nematodes responsible for stimulating protective immunity in their hosts. This appears to be partly due to the common failure of vaccination with nonliving nematode material to stimulate protective immunity. However, in the first paper of this series it was shown that a single injection into guinea-pigs of a small quantity of a crude homogenate of Trichostrong& coiubriformis fourth-stage larvae stimulated siguificant immunity against a subsequent challenge’infection, and it was concluded that this host-parasite system is well suited to the study of

nematode protective antigens (Rothwell & Love, 1974). Work with a number of nematode parasites, notably Nippostrongylus brasiliensis, suggests that their acetylcholinesterase (EC 3.1.1.7, AChE) might play an important role in the pathology and immunology of infection with these parasites (Lee, 1970; Edwards, Burt & Ogilvie, 1971; Sanderson & Ogilvie, 1971; Jones & Ogilvie, 1972; Ogilvie, Rothwell, Bremner, Schnitzerling, Nolan & Keith, 1973). In addition, AChE is present in the subventral excretory glands and dorsal oesophageal gland of T. colubriformis, and antibodies against the enzyme occur in the serum of infected sheep and guinea-pigs (Rothwell, Ogilvie & Love, 1973;

immunity;

nematode;

Tricko-

Rothwell & Merritt, 1974; McLaren, Burt & Ogilvie, 1974). The studies referred to above, all suggest that nematode AChE might be a protective antigen, and this paper describes attempts to establish whether this is so in T. colubriformis infections in guinea-pigs. In the present experiments, the soluble material obtained from homogenized T. colubriformis fourthstage larvae was fractionated by membrane ultrafiltration, gel filtration and ion-exchange chromatography. The capacity of the AChE-rich fractions to stimulate protective immunity was then determined. Another antigen which has been implicated in protective immunity in this host-parasite system is worm allergen (Rothwell, Dineen & Love, 1971; Rothwell & Dineen, 1972; Rothwell, Prichard & Love, 1974). The allergen(s) of T. colubriformis are distinct from AChE (Hogarth-Scott, Watt, Ogilvie & Rothwell, 1973) and so the AChE-rich fractions prepared in the present experiments were also tested for contamination with worm allergen. The results of the experiments described suggest that worm AChEper se does not stimulate protective immunity in guinea-pigs. MATERIALS AND METHODS Details of the experimental animals, the techniques used in the collection of T. colubriformis larvae, the 453

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administration of infective larvae to guinea-pigs and the method of performing worm counts have been described (Rothwell & Love, 1974).

Rosebrough, Farr & Randall (1951). This was done to monitor the fractionation procedures and does not imply that the protective antigen(s) are protein.

Extraction

AChE determinations

of soluble material from T. colubriformis Fourth-stage T. colubriformis larvae were blotted with fine filter paper then homogenized with 0.1 M pH 7.0 phosphate buffer with an ice-cooled Potter-Elvehjem tissue homogenizer. The crude homogenate was then centrifuged at 25,000 g for 30 min and the supernatant retained. The pellet was again homogenized in buffer, pooled with the original supernatant and centrifuged at 109,000 g for 1 h. Lipid droplets were then removed from the supernantant by passage through a 0.22u pore size millipore filter. Approximately 2.5-4 mg protein were obtained from the larvae collected from each sheep. The final volume of the extract varied from 5 to 10 ml. Membrane

ultrafiltration

A membrane ultrafiltration system (Diaflo-Amicon Corporation, Lexington, Mass., U.S.A.) was used to prepare fractions of the soluble material extracted from T. colubriformis with varying AChE content. A 65 ml cell and 43 mm dia membranes with molecular weight retention limits of 100,000 (XM 1OOA)or 50,000 (XM 50) were used. In each case a volume of 0.1 M pH 7.0 phosphate buffer equivalent to at least 10 times that of the starting material was used to wash small molecules through the filter membrane. The final filtrate was concentrated on a membrane with a molecular weight retention limit of 1000 (UM 2). Membrane ultrafiltration was also used as a preliminary purification procedure prior to gel filtration and to concentrate pooled fractions obtained by both gel filtration and ion-exchange chromatography. Membranes with molecular weight retention limits of 300,000 (XM 300), 50,000 (XM 50) and 10,000 (PM 10) were used in different experiments, as described in the results. Gelfiltration

Membrane ultrafiltration prior to gel filtration was done with 0.1 M pH 8.0 Tris-HCl buffer in 1 M-NaCl. The washed and concentrated T. colubriformis extract was then fractionated on a G-200 Senhadex column, 2.5 cm wide and 100 cm long using O.lL~ pH 8.0 TrisHCI buffer in 1 M-NaCl. The flow rate was adiusted to give 8.0 ml fractions every 40 min. All operations were done at 4°C. Ion-exchange

chromatography

Prior to fractionation by ion-exchanee chromatography, the T. colubriformis extract w’;s repeatedly washed with 0.02 M pH 8.0 phosphate buffer over a 10,000 molecular weight (PM 10) ultrafiltration membrane. The extract was-then applied to a DEAE Sephadex column, 2.5 cm wide with an effective length of approximately 40 cm. A gradient elution system consisting of an initiai0.02 M pH 3.0 phosphate buher and limit b&er of 0.3 M nH 8.0 was used in the first two chambers of a Tech&on Autograd. The flow rate was adjusted to 20 ml/h and the elution pattern was monitored by U.V. transmission at 254 mu. Protein determinations

The protein content of worm homogenates and fractions was determined by the method of Lowry,

AChE activity was determined by the calorimetric method of Ellman, Courtney, Andres & Featherstone (1961), using acetylthiocholine iodide as substrate and a reaction temperature of 37.5”C. AChE activity of fractions obtained by gel filtration and ion-exchange chromatography was compared by determining the optical density (at 412 mu) of the colour developed by 0.1 ml aliquots of the fractions. Enzyme activity is expressed in the results as umoles acetylthiocholine hydrolyzed/h/mg protein or 0.1 ml. Passive cutaneous anaphylaxis

Some worm fractions were tested for the presence of allergens by homologous passive cutaneous anaphylaxis. White guinea-pigs were injected intradermally with 0.1 ml of doubling dimtions of pooled serum from T. colubriformis-infected and worm free guinea-pigs and were challenged seven or eight days later by the intravenous injection of the test fractions with 0.25 ml of 1 per cent Evan’s blue dye. The presence of anti-T. colubriformis IgE-type antibodies in the serum pool was confirmed by giving positive control guinea-pigs lO-1OOug protein from an homogenate of T. colubriformis third stage larvae. Tests were done in duplicate. Detection of anti-AChE antibodies

Globulins prepared from some sera by sodium sulphate precipitation were tested for their ability to inhibit the inactivation of T. colubriformis AChE at 60°C as described previously (Rothwell & Merritt, 1974). Design of experiments

Guinea-pigs were given a single subcutaneous injection of the material being tested generally at 0.1 ml/lOOg body weight, challenged with 2000 T. colubriformis infective larvae 21 days later and killed for worm counts after a further 14 days. The efficacy of vaccination was then determined by comparing worm counts in unvaccinated control and vaccinated groups. Statistical analysis of worm count data

Prior to analvsis, logarithmic transformation of worm count data was perTormed. This transformation is variance-stabilizing and normalizes such data. The data are therefore pres&ted in the results as means of log worm counts and as geometric means (antilog of mean log worm count). RESULTS Vaccination with decreasing doses of the soluble material from fourth-stage larvae In order to confirm the presence of protective antigen in the soluble material extracted from fourth-stage T. colubriformis larvae and to obtain information on the dose-response relationship with this material, groups of 10 guinea-pigs were injected with 100, 10 or 1 ug protein/lOOg body weight,

Vaccination

I.J.P. VOL. 5. 1975 TABLE ~-WORM MATERIALFROM

COUNTS

455

against Trichostrongylus colubriformis

IN GUINEA-PIGS VACCINATED WITH DECREASINGDOSESOFTHESOLUBLE FOURTH-STAGELARVAE,THENCHALLENGEDWITH T.colubriformis

T.colubriformis

Dose (pg protein/lOOg body wt) -

Group Controls Vaccinated

100 10 1

Geometric mean worm count

Mean log worm count 2.78”* 2.23’ 2.50b 2.68ab

601 169 313 482

Pooled estimate of standard error (log worm counts) = 0.07. * Means bearing the same superscript are not significantly different at the 5 per cent level by Duncan’s (1955) ‘new multiple-range’ test. Means bearing different superscripts are significantly different ai the 3 per cent level. y challenged

with T. colubriformis,

and then killed for

wormcounts.

These worm counts are summarized in Table 1 and show that both 100 and 10 pg soluble protein/ IOOg body weight stimulated significant protective immunity. Vaccination with fractions prepared from T. colubrifourth-stage larvae by membrane ultrafiltration For this experiment the soluble material extracted from T. colubriformis fourth-stage larvae (32.8 mg protein) was passed in turn through membranes with molecular weight retention limits of 100,000 and 50,000. The retentate on the 100,000 mol. wt membrane is referred to as fraction I. The filtrate was applied to a 50,000 mol. wt membrane and the retentate so obtained is referred to as fraction II. The filtrate from the 50,000 mol. wt membrane, which was concentrated on a 1000 mol. wt membrane is referred to as fraction III. Details of the amount of protein and AChE in these fractions are given in Table 2. formis

During the preparation of fraction I, a precipitate formed. This was removed by centrifugation and probably explains the loss of approximately 75 per TABLE ~-PROTEIN

cent of the original

II III

% of total

Retained by 100,000 mol. wt membrane Filtrate from 100,000 mol. wt membrane and retained by 50,000 mol. wt membrane Filtrate from 50,000 mol. wt membrane and retained by 1000 mol. wt membrane

* Total protein receovered Total AChE recovered 7 pmoles Acetylthiocholine

32.8

fractionation.

Further attempts were made to fractionate T. colubriformis using membrane ultrafiltration. On each occasion new membranes were used and the distribution of proteins in the fractions was similar to that summarized in Table 2. However, the fractions corresponding to fraction III hydrolyzed 0.1,

AND ACETYLCHOLINESTERASE IN FRACTIONS PREPARED FROM BY MEMBRANE ULTRAFILTRATION

Description

during

These worm counts show that significant protective immunity was stimulated only in guinea-pigs given fraction II. Guinea-pigs given the AChE-rich fraction I received approximately 63 times as much AChE as those given fraction II, and although their mean worm count was considerably less than that of the controls, the difference was not statistically significant.

T. colubriformis

FOURTH-STAGE

LARVAE

Acetylcholinesterase

Protein Fraction No. Before filtration I

protein

Of the protein recovered in the fractions, 57.8 per cent was present in fraction I, 10.5 per cent in fraction II and 31.7 per cent in fraction III. Groups of guinea-pigs were therefore vaccinated with 100 1.18 protein per 1OOg body weight of the material before fractionation and 57.8, 10.5 and 31.7 pg/lOOg body weight of fractions I, II and III respectively. Worm counts in these animals after challenge with T. colubriformis are summarized in Table 3.

% of* recovered

% of total

% of* recovered

314.3

loo

-

pmoles/t mg protein

loo

-

4.6

14.1

57.8

1856.6

83.4

98.4

0.8

2.6

10.5

157.6

1.3

1.5

2.5

7.7

31.7

2.9

0.1

0.1

=24.4 per cent. = 84.8 per cent. hydrolyzed/mg protein/h.

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TABLE ~-WORM COUNTSIN GUINEA-PIGSVACCINATEDWITH FRACTIONSPREPAREDFROM T. colubriformis STAGELARVAEBYMEMBRANEULTRAFILTRATIONTHENCHALLENGEDWITH T. colubriformis Dose of protein Group _ Controls Vaccinated

Fraction Before fractionation I II III

(;:!y?:

Dose of AChE*

FOURTH-

Number of animals in group

Mean log worm count

Standard error

7

2.87at

0.13

739

4 5 5 5

2.62” 2.54” 2.01b 2.66”

0.18 0.16 0.16 0.16

419 347 103 457

100 57.8 10.5 31.7

5. 1975

31.4 107.3 1.7 0.1

Geometric mean worm ccunt

* umoles Acetylthiocholine hydrolyzed/h/lOOg body weight. t Means bearing the same superscript are not significantly different at the 5 per cent level by Duncan’s (1955) ‘new multiple-range’ test. Means bearing different superscripts are significantly different at the 5 per cent level. 17.1 and 169.1 umoles acetylthiocholine/mg protein/h respectively (compared with 2.9 in Table 2). The results with this technique were therefore too inconsistent to warrant further work with it, and, in addition, passive cutaneous anaphylaxis tests using 0.5 mg protein revealed that all three fractions contained worm allergen. However, the results suggest that the capacity of the worm fractions to protect guinea-pigs against infection is not related to their AChE content. Fractionation of T. colubriformis fourth-stage larvae by Sephadex G-200 gel filtration Prior to fractionation by gel filtration, an attempt was made to increase the proportion of AChE in the material applied to the column by preliminary membrane ultrafiltration. In fractionation A (see Table 4) the soluble material from T. colubriformis fourth-stage larvae which passed through a 300,000 mol. wt membrane but was retained by a 50,000 mol. wt membrane was applied to the column. In frac-

tionation B (see Table 4) the material retained by a 50,000 mol. wt membrane was used. The elution profiles for protein and AChE for each fractionation were similar, and those from one are shown in Fig. 1. The AChE peak from each fractionation was concentrated on a 50,000 mol. wt membrane and protein and AChE content determined. Details of these fractionations are given in Table 4. The capacity of the AChE peaks from each fractionation to protect guinea-pigs against infection was tested by injecting 10 ug of protein per 1OOg body weight from the peaks into groups of guinea-pigs and then challenging them with T. colubriformis. The worm counts from these experiments are summarized in Table 5. The counts in the two groups of controls emphasize the difference in susceptibility of different groups of guinea-pigs to T. colubriformis infection. Nevertheless, they show that both AChE peaks stimulated significant protective immunity. The AChE peak from a third fractionation was

0.02

0.01

.i 0.0

1

a’ 0.005

t 0 Tube number

FIG. 1. Elution profile from G-200 fractionation of the soluble material from T. colubriformis fourthstage larvae. Solid line-protein (mg/ml). Broken line-acetylcholinesterase (optical density at 412 mu when 0.1 ml aliquots tested using the method of Ellman et a/., 1961). AChE peak tubes 15-22 (inclusive).

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OF T. colubriformis FOURTH-STAGE LARVAE BY SEPHADEX FILTRATION. PROTEINAND ACETYLCHOLINESTERASE (AChE) CONTENTS

TABLE &-FRACTIONATION

G2Ml

GEL

Fractionation A Soluble material obtained from T. colubriformis fourth-stage larvae by homogenization and centrifugation Material after preliminary membrane ultrafiltration-applied to column AChE peak

Protein (mg) AChE (pmoles*/ mg protein) Protein (mg) AChE @moles*/ mg protein) Protein (mg) AChE (pmoles*/ mg protein)

* pmoles Acetylthiocholine

B

5.9

14.4

607.7 2.1

374.6 9.4

868.0

469.2 0.44

0.37 2563.7

4217.4

hydrolyzed/h.

TABLE5-WORMCOUNTSIN

GUINEA-PIGS VACCINATED WITH ~OH~PROTEIN PER 1OOg BODY WEIGHTFROMTHE CHOLINESTERASEPEAKOBTAINEDFROM T. colubriformis FOURTH-STAGELARVAEBYSEPHADEXG~OOGELFILTRATION

Group Control Vaccinated with AChE peak from fractionation A Control Vaccinated with AChE peak from fractionation B * pm&s

Acetylthiocholine

Dose of AChE* -

5

Mean ‘og worm count 2.76

No. of animals grip

Standard error 0.30

Geometric mean worm count

ACETYL-

Significance of difference between mean count of control and vaccinated groups P=

579 0.02

25.6 -

4

1.40

0.39

25

10

2.25

0.22

180

42.2

10

0.76

0.22

6

0~00014

hydrolyzed/h/lOOg

body weight.

tested for the presence of allergen by means of passive cutaneous anaphylaxis. Allergen was present, but its content appeared low because 100 or 500 pg of the peak gave weak reactions with only undiluted and 1:2 dilution of the positive serum, whereas both 100 and 10 pg of an homogenate of T. colubriformis third stage larvae gave intense positive reactions up to and including the 1:8 dilution. These results show that the AChE peak obtained by gel filtration of T. colubriformis fourth-stage larvae contains protective antigen(s). However, because the peak is not free from worm allergen, the contribution the AChE makes to the stimulation of protective immunity is uncertain. Fractionation of T. colubriformis fourth-stage larvae by DEAE-Sephadex chromatography T. colubriformis larvae for fractionation by ion-

exchange chromatography were collected on a Baermann apparatus as described previously (Rothwell & Love, 1974), except that the tube into which the larvae fell was kept iced. This procedure was found to reduce substantially the rate these larvae released AChE and to increase the AChE content of the soluble material extracted from them. No preliminary membrane ultrafiltration was performed. Two fractionations using this technique were

performed. The elution patterns for protein and AChE for each fractionation were similar, and those from the second are shown in Fig. 2. The AChE peak from the first fractionation was concentrated on a 10,000 mol. wt membrane and tested for the presence of allergen by passive cutaneous anaphylaxis. Doses of 200 and 100 pg protein gave negative reactions, whereas control animals tested with 100, 50 and 10 pg of T. colubriformis third larval stage protein gave strong positive reactions up to the 1:32 dilution. The individual tubes from the second fractionation were pooled into three fractions with the aim of obtaining worm material containing very little AChE on the one hand and purified worm AChE on the other. Fraction I comprised tubes containing very little AChE. Fraction II comprised tubes in the ascending part of the AChE peak and fraction III the upper part of the peak itself (see Fig. 2). The fractions were concentrated on 10,000 mol. wt membranes. Their protein and AChE contents are given in Table 6. Groups of guinea-pigs were vaccinated with 10 pg protein/lOOg body weight of the three fractions, then challenged with T. colubriformis. The worm counts are summarized in Table 7. They show that

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ROTHWELL and G. C. MERRITT

Fraction I

L

Fraction

II

38

1

Fraction 111

-1

1

59

13

I

-c

u

40

80

60

Tube number FIG. 2. Elution profile from DEAE Sephadex fractionation of the soluble material from T. cohbriformis fourth-stage larvae. Solid line- % transmission at 254 mp. Broken line-acetylcholinesterase

(optical density at 412 rnp when 0.1 ml aliquots tested using the method of Ellman et al., 1961). TABLE &-FRACTIONATION OF T. colubriformis FOURTH-STAGELARVAE BY DEAE-SEPHADEX CHROMATOGRAPHY.PROTEIN AND ACETYLCHOLINESTERASECONTENT OF FRACTIONS

Fraction

Protein (mg)

AChE @moles*/ mg protein)

Soluble material obtained from

T. colubriformis fourth-stage apphed to column Fraction I Fraction II Fraction III

* pmoles Acetylthiocholine

larvae14.0 2.3 0.8 0.25

1204.5 6.8 517.4 7175.3

hydrolyzed/h.

TABLE ~-WORM COUNTS IN GUINEA-PIGSVACCINATED WITH 10 wg PROTEINPER 1OOg BODY WEIGHT OF FRACTIONS OBTAINEDFROM T. colubriformis FOURTH-STAGELARVAEBY DEAE-SEPHADEX CHROMATOGRAPHY

-

Dose of

AChE* -

Group Controls

Vaccinated

Before fractionation material Fraction I Fraction II Fraction III

12.0 0.07 5.2 71.8

No. of animals

Mean log

in group 13

worm count 2.61”t

9 9 9 6

2.35” 1.81b 2.48” 2.78”

Geometric mean

Standard error 0.15 0.18 0.18 0.18 0.22

worm count 410 225 64 305 607

* pmoles Acetylthiocholine hydrolyzed/h/lOOg body weight. t Means bearing the same superscript are not significantly different at the 5 per cent level by Duncan’s (1955) ‘new multiple-range’ test. Means bearing different superscripts are significantly different at the 5 per cent level. 10 Ng of fraction I stimulated significant immunity against infection, whereas 10 pg of fractions II and III, which contained 76 times and 1055 times respectively as much AChE as fraction I were ineffective.

Serum was collected from five guinea-pigs from each group after vaccination and prior to challenge with T. colubriformis, and again when the animals were killed for worm counts. Antibodies against AChE were not detected in any of the sera and all

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against Trichostrongylus colubriformis

failed to give passive cutaneous anaphylactic reactions with T. colubriformis third larval stage antigen. DISCUSSION There are good reasons for suspecting that AChE might be a protective antigen in some host-parasite systems. Thus, in N. brasiliensis infections, the enzyme is present in glands opening to the exterior of the worm (Lee, 1970); the enzyme is probably released in situ in the host small intestine (Sanderson & Ogilvie, 1971; Jones & Ogilvie, 1972); the host responds to infection by producing anti-AChE antibodies, and there is evidence that these in turn modulate AChE production by the worm (Jones & Ogilvie, 1972; Sanderson, Jenkins & Phillipson, 1972). AChE is also present in the glands of a number of other gastro-intestinal nematode parasites and in some of these there is evidence for a host immune response against the enzyme (Ogilvie et al., 1973; Bremner, Ogilvie, Keith & Berrie, 1973; Rothwell & Merritt, 1974). The present results clearly show that the capacity of T. colubriformis fractions to stimulate protective immunity is not related to their AChE content (see Tables 3 & 7) and that a highly purified preparation of AChE is ineffective (see Table 7). The latter conclusion would have been strengthened by the detection of antibodies against the enzyme in guineapigs vaccinated with the purified AChE. However, in view of the small dose of protein without adjuvant injected, the failure to detect antibodies is hardly surprising, and when the results are considered as a whole, the absence of direct evidence for an immune response against the injected enzyme does not seriously weaken either conclusion. Comparison of the results summarized in Tables I, 3, 5 and 7 shows that a dose of 10 pg protein of some worm fractions was more immunogenic than the same dose of unfractionated soluble material from T. colubriformis fourth-stage larvae. For example, the difference between mean worm counts in control and 10 pg vaccinated groups was 0.28 and 0.26 logs for the unfractionated material (see Tables 1 and 7 respectively). However, the difference for membrane ultrafiltration fraction II was 0.86 logs, for the G200 AChE peaks 1.36 and 1.49 logs, and for the DEAE fraction I 0.80 logs. Clearly, substantial purification of the protective antigen(s) was achieved, and the results suggest that further work might lead to their identification. Previous studies (Hogarth-Scott et al., 1973) have suggested that T. colubriformis AChE has a molecular weight of approximately 65,000. It was surprising then, that over 98 per cent of the enzyme was retained by ultrafiltration through a membrane with a molecular weight retention limit of 100,000. However, these retention limits apply to globular vroteins and the shave of T. colubriformis AChE

is unknown.

Other possible explanations for this finding include the tendency of the enzyme to polymerize and to associate with other molecules. The results do not allow any firm conclusion on the possible role of worm allergen in stimulating protective immunity. Nevertheless, it was of interest that the G-200 AChE peak, which was highly immunogenic, contained detectable allergen. Current work is directed towards purification of worm allergen and the study of its ability to stimulate protective immunity. In conclusion, the present work, although suggesting that worm AChE per se is not responsible for stimulating protective immunity, leayes the question of its significance in the relationship between host and parasite unanswered. wish to thank Dr. D. A. Griffiths for advice on statistical analysis of the data, Mrs. Wendy Cottee, Mrs. Michelle Veness, Mr. P. Marshall and Mr. J. Weaver for able technical assistance, and

Acknowledgements-We

Mr. I. Roper for preparation

of the figures.

REFERENCES BREMNERK. C., OGILVIE B. M., KEITH R. K. & BERRIE D. A. 1973. Acetylcholinesterase secretion by parasitic nematodes. III. Oesophagostomes. InternationalJournal for Parasitology 3: 609-618. DUNCAND. B. 1955. Multiple range and multiple F tests. Biometrics 11: l-42. EDWARDSA. J., BURT J. S. & OGILVIE B. M. 1971. The effect of immunity upon some enzymes of the parasitic nematode, Nippostrongylus brasiliensis. Parasitology 62: 339-347. ELLMAN G. L., COURTNEYK. D., ANDRESV. & FEATHERSTONER. M. 1961. A new and rapid calorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology 7: 88-95. HOGARTH-SCOTTR. S., WAN B. J., OGILVIE B. M. & ROTHWELL T. L. W. 1973. The molecular size of nematode acetylcholinesterases and their separation from nematode allergens. International Journal for Parasitology 3: 735-741. JONES V. E. & OGILVIE B. M. 1972. Protective immunity to Nippostrongylus brasiliensis in the rat. Il. Modulation of worm acetylcholinesterase by antibodies. Immunology 22: 119-129. LEE D. L. 1970. The fine structure of the excretory system in adult Nippostrongylus brasiliensis (Nematoda) and a suggested function for the “excretory glands”. Tissue and Cell 2 : 225-23 1. LOWRY 0. H., ROSEBROUGH N. J., FARR A. L. & RANDALL R. J. 1951. Protein measurement with the folin phenol reagent. Journal of Biological Chemistry 193: 265-275. MCLAREN D. J., BURT J. S. & OGILVIE B. M. 1974. The anterior glands of Necator americanus (Nematoda: II. Cytochemical and functional Strongyloidea). studies. International Journalfor Parasitology 4: 39-46. OGILVIE B. M., ROTHWELL T. L. W., BREMNER K. C., SCHNITZERLINGH. J., NOLAN J. & KEITH R. K. 1973. Acetylcholinesterase secretion by parasitic nematodes. I. Evidence for secretion of the enzyme by a number of 3: species. International Journal for Parasitology 589-597.

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ROTHWELL T. L. W. & DINEEN J. K. 1972. Cellular reactions in guinea-pigs following primary and challenge infection with Trichostrongylus colubriforrnis with special reference to the roles played by eosinophils and basophils in rejection of the parasite. Zmmunology 22: 733-745. ROTHWELLT. L. W., DINEENJ. K. & LOVE R. J. 1971. The role of pharmacologically active amines in resistance to Trichostrongylus colubriformis in the guinea-pig. Immunology 21: 925-938. ROTHWELL T. L. W. & LOVE R. J. 1974. Vaccination against the nematode Trichostrongylus colubriformis. I. Vaccination of guinea-pigs with worm homogenates and soluble products released during in virro maintenance. International Journal for Parasitology 4: 293-299. ROTHWELL T. L. W. & MERRITT G. C. 1974. Acetylcholinesterase secretion by parasitic nematodes. IV. Antibodies against the enzyme in Trichostrongylus

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