Anti-enzyme antibodies in rabbits and pigs against Hyostrongylus rubidus (Hassall and Stiles, 1892)

Veterinary Parasitology, 4 (1978) 167--174 © Elsevier Scientific Publishing Company, Amsterdam - - Printed in The Netherlands

ANTI-ENZYI~[E ANTIBODIES

167

IN I~ABBITS AND PIGS AGAINST

H Y O S T R O N G Y L U S R UBIDUS ( H A S S A L L A N D S T I L E S , 1 8 9 2 )

S. MASABA* and I.V. HERBERT

Department of Applied Zoology, University College of North Wales, Bangor, Gwynedd (Great Britain) *Present address: Ministry of Health, Division of Vector Borne Diseases, P.O. Box 20750, Nairobi (Kenya) (Received 7 September 1977)

ABSTRACT Masaba, S. and Herbert, LV., 1978. Anti-enzyme antibodies in rabbits and pigs against Hyostrongylus rubidus (Haasall and Stiles, 1892). Vet. Parasitol., 167--174. Ether extracts ofHyostrongylus rubidus adult worms isolated on days 14, 35 and 64 of infection were found to contain two isoenzymes of malic dehydrogenase (MDH) and three of acid phosphatase. Sera from rabbits immunized against these extracts and sera from pigs experimentally infected with H. rubidus were tested for their anti-enzyme activity by two different techniques. Sera from rabbits actively immunized with a Day 14 worm extract contained an antibody which complexed only with a slow migrating isoenzyme of acid phosphatase but not with isoenzymes of MDH or acetylcholinesterase (ACHE). No antibodies against worm acid phosphates or MDH were detectable by this technique in the sera of pigs which were experimentally infected with H. rubidus. A different technique, however, where the worm extracts were incubated at 60 ° C either with rabbit anti-H, rubidus serum, or serum from infected pigs, indicated the presence of anti-AChE globulin in both immunized rabbits and infected pigs. When individual immunoglobulins isolated from infected pigs were incubated with the same worm extract it was seen that activity was associated with IgG 1 but not with IgG2, IgM or IgA. IgG Zprepared from worm-free pigs did not complex with worm ACHE. There was no interaction between the third stage larval AChE and pig IgG 1. Levels of AChE were highest in worms isolated at a period of infection when the hosts immune responses were beginning to manifest themselves and lowest in those worms surviving the population crisis.

INTRODUCTION A number of parasitic nematodes are known to secrete acetyl cholinesterase ( A C H E ) a n d c o n s i d e r a b l e s t u d y h a s b e e n d e v o t e d t o s h o w i n g t h a t t h i s is a n t i g e n i c in t h e h o s t a n i m a l (e.g. O g i l v i e e t al., 1 9 7 3 ; S a n d e r s o n e t al., 1 9 7 6 ) . O f p a r t i c u l a r i n t e r e s t , h o w e v e r , is w h e t h e r o r n o t t h i s e n z y m e is i n v o l v e d in p r o v o k i n g , in t h e h o s t , a p r o t e c t i v e i m m u n o l o g i c a l r e s p o n s e . S t u d i e s o n r a t s i n f e c t e d w i t h Nippostrongylus brasiliensis ( S a n d e r s o n a n d O g i l v i e , 1 9 7 1 ;

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Jones and Ogilvie, 1972; Sanderson et al., 1976); on sheep infected with Trichostrongylus colubriformis (Rothwell et al., 1973), on cattle infected with Oesophagostomum columbianum (Bremner et al., 1973) have shown the presence of antibodies to AChE in the sera of infected animals. Antibodies against AChE in N. brasiliensis (Jones and Ogilvie, 1972) and T. colubriformis (Rothwell and Merritt, 1974) produced in the rat and sheep, respectively, have been found to be IgG~. The present work investigates whether anti-enzyme antibodies are produced against enzymes of Hyostrongylus rubidus in the pig during experimental infections, and in the rabbit following the injection of extracts of these nematodes. MATERIALS AND METHODS

Preparation of antigens Conventionally reared minimal disease (M.D.) growing pigs were orally inoculated with 40 000 infective Hyostrongylus rubidus larvae. Animals were sacrificed 14, 35 or 63 days after infection and the worms were recovered alive b y the technique of Reinecke (1967). All the worms recovered were adults except those obtained 14 days after infection, when a small proportion of Ls larvae were obtained. Worms recovered on each of the above days were submitted to ether extraction (see Soulsby, 1974), and the protein concentration of the extracts assessed. A sonicated suspension of ensheathed third stage larvae was also prepared in 0.85% saline.

Demonstration of enzymes in worm homogenates Enzymes of day 14 and L3 worms were detected following polyacrylamide gel electrophoresis (Shandon Scientific Co., London) and 0.15 ml amounts of day 14 (24 mg P/ml) and L3(5 mg P/ml) were applied to the top of a 7.5% gel. Electrophoresis was done at 2.5 m A per tube for 1 h. Acid phosphatase was visualised by the coupling diazo dye m e t h o d of Barka (1961). Malic dehydrogenase (MDH) was visualised as described by O y a et al. (1970). Cholinesterases were visualised by the m e t h o d of Karnovsky and Roots (1964). AChE was identified following the use of inhibitors incorporated with the staining medium -- BW 284C51 (10 -s M), isoOmpa (10 -s M) and eserine (10 -s M). Three ml of the day 14 worm extract (5 mg protein/ml) was applied to a Sephadex G 75 column, dimensions 2.5 × 45 cm, and eluted with 0.15 M phosphate buffered saline (PBS}, pH 7.4 at 4°C. The flow rate was 20 ml/h and the elution pattern was recorded b y ultraviolet transmission at 280 nm. The various fractions were analysed for ACHE. The AChE activities of day 14, day 35, day 63 and L3 extracts were also assessed b y the colorimetric m e t h o d of EUman et al. (1961), as modified by Hutchinson and Probert (1972) (see Table I).

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TABLE I The relative AChE activities of worm antigens incubated at 37°C for 30 min

Antigen

Concentration

AChE activity in

(mg P/ml)

international units

(iv) Day 14 Day 35 Day 63 L3

1 1 1 1

0.9 0.6 0.55 0.25

Preparation o f antisera Porcine immunoglobulin, IgM, IgG1, IgG2 and IgA were prepared as follows. Serum collected from a pig which had been orally inoculated with 40 000 infected H. rubidus larvae 21 days previously was fractionated on Sephadex G200 (Pharmacia Fine Chemicals AB, Uppsala, Sweden) using PBS (pH 7.4) as eluent. Fractions corresponding to IgM and IgG were collected separately and concentrated. IgG1 and IgG2 were prepared by the technique of Curtis and Bourne (1971) and the purity determined by electrophoresis: IgM, IgG1 and IgG2 concentrations were adjusted to 10 mg P/ml. IgA was isolated following fractionation of colostral whey on Sephadex G200. Fractions obtained from the descending parts of the first and the ascending part of the second peaks were pooled and dialysed against PBS, pH 7.4. The dialysate was applied to a DEAE cellulose column and eluted stepwise with phosphate buffers of differing molarity as described by Porter {1969). The concentration of the IgA was adjusted to 0.2 mg P/ml. Antiserum to day 14 H. rubidus extract was prepared in rabbits. Worm extract was subcutaneously injected with Freunds Complete Adjuvant (Difco Laboratories, Detroit, Michigan, U.S.A.), followed by intravenous and subcutaneous infection 3 and 5 weeks later, respectively. Animals were bled a week after the last injection. Demonstration of anti-enzyme activity in the sera The sera taken from experimentally infected pigs and from vaccinated rabbits were assessed for their antibody activity against MDH, ACHE, and acid phosphatase enzyme obtained from worm extract. Sera from nonsensitised rabbits or pigs were used for control. Several workers have shown that when AChE is complexed to antibody it fails to migrate through polyacrylamide gel though its enzyme activity is unaffected (Jones and Ogilvie, 1972; Rothwell and Merritt, 1974). Anti-AchE antibody activity was assessed by testing the ability of rabbit or pig antibody to inhibit the migration of enzyme antibody complex in polyacrylamide gel

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electrophoresis and to protect the enzyme against heat inactivation (Michaeli et al., 1969). (a) 0.5 ml of the day 14 worm extract (5 mg P/ml) was mixed with 0.5 ml rabbit or pig antiserum which had been heated at 60°C for 10 min to inactivate serum enzymes. After incubation for 1 h at room temperature 0.05 ml of each mixture was submitted to polyacrylamide gel electrophoresis. Control gels contained worm extract and normal serum which had been inactivated by heat. The gels were 'stained' for MDH, AChE and acid phosphatase. (b) 0.5 ml aliquots of day 14 worm extract were mixed with 0.5 ml of rabbit antiserum of dilutions up to 1:2 or with 0.5 ml of immune pig serum. The mixture was incubated at room temperature for 1 h and its AChE activity read both before and after further incubation at 60°C for 5, 10, 20 and 30 min. The AChE activity of the day 35, day 63 and L3 antigens were also assessed following complexing separately with IgGl, IgG2, IgA and IgM prepared from an infected pig. Dilutions of up to 1:25 o f IgG1 were also assessed for their reactivity. RESULTS

Enzymes isolated from the worm homogenates Fractionation of day 14 worm extract on Sephadex G75 produced two peaks of AChE activity, whilst polyacrylamide gel electrophoresis identified 3 of 4 cholinesterase isoenzymes as AChE (Fig. 1). A low enzyme activity was detected in the L3 extract. The relative AChE activities of worm homogenates indicated that the extracts of L3 and older worms contained less enzyme than the day 14 extract (Table I). Two isoenzymes of MDH and three of acid phosphatase were seen following electrophoresis in polyacrylamide gel (Fig. 1).

The anti-enzyme activity of porcine and rabbit sera The technique of Jones and Ogilvie (1972) in which enzyme is complexed with antibody before polyacrylamide gel electrophoresis followed by specific A

B

C

D

E

Fig. 1. Polyacrylamide gel electrophoresis of day 14 worms. A, two isoenzymes of malic dehydrogenase; B, three isoenzymes of acid phosphatase; C, four isoenzymes of cholinesterase, three of which are acetylcholinesterase isoenzymes and which are inhibited in migration by BW 284051 (D) and eserine (E) but not by iso-Ompa.

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staining for MDH, AChE and acid phosphatase produced equivocal results. Only the antibody produced in rabbit against one of the isoenzymes of acid phosphatase appeared to complex and thus be impeded in its migration. Failure to demonstrate antibody against AChE may have been caused by low concentrations of soluble AChE in the reacting mixture. The results of heat inactivation of AChE in worm extracts following incubation with either anti-H, ru b i d u s rabbit serum or immune porcine serum are detailed in Figs 2 and 3. The AChE antibody complex is relatively heat stable whereas the non-complexed AChE is inactivated after heating at 60°C for 30 min. Fig. 2 shows that despite an initial drop in AChE activity following heating of the antigen and rabbit anti-H, r u b i d u s serum, a stable complex was formed which retained 50% or more of its original enzyme activity 30 min later. This was found even where the antiserum was diluted. The AChE in the day 14 worm extract was quickly inactivated when incubated alone as was the same extract when incubated with non-immune pig serum. 100

100

>j--

>F-

>

Q

~50 (.9

b

b

¢

I.IJ

5o

5

L)

10

10

f 0

10

20

TIME(mins)AT 60°C

30

0

10

20

3,3

TIf~iE(mins)AT CG°C

Fig. 2. E f f e c t o f r a b b i t a n t i - w o r m a n t i b o d y o n the inactivation o f A C h E activity o f

Hyostrongylus rubidus. D i l u t i o n s o f r a b b i t a n t i - w o r m serum were i n c u b a t e d at 6 0 ° C w i t h Day 14 w o r m e x t r a c t . N o r m a l pig serum was also i n c u b a t e d w i t h w o r m e x t r a c t . A c t i v i t y at each t i m e is e x p r e s s e d as a percentage o f activity before heating, a, u n d i l u t e d r a b b i t serum + w o r m e x t r a c t ; b, 1 : 1 d i l u t i o n o f r a b b i t s e r u m + w o r m e x t r a c t ; c, 1:2 d i l u t i o n o f r a b b i t s e r u m + w o r m e x t r a c t ; d, u n d i l u t e d rabbit serum alone; e, d a y 14 w o r m extract alone; f, pig serum + w o r m e x t r a c t . Fig. 3. E f f e c t o f i m m u n o g l o b u l i n s o n the inactivation o f Day 14 w o r m A C h E activity at 60 ° C. A c t i v i t y at each t i m e is e x p r e s s e d as a percentage o f activity b e f o r e heating, a, l g G 1 f r o m i n f e c t e d pig serum + w o r m e x t r a c t ; b, l g A f r o m i n f e c t e d pig c o l o s t r u m + w o r m e x t r a c t ; c, l g M f r o m i n f e c t e d pig serum + w o r m e x t r a c t ; d, l g G 2 f r o m i n f e c t e d pig serum + w o r m e x t r a c t ; e, day 14 w o r m e x t r a c t alone.

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Of the porcine immunoglobulin fractions (Fig. 3) which were incubated with day 14 worm extract only IgG1 formed a complex which was stable after heating for 30 min at 60°C. This activity was evident when the IgG1 was diluted (Masaba, 1975). The day 63 worm extract was of similar activity to that of the day 14 worm extract, when incubated with the IgG~ preparation. The IgG1 preparation from non-immunised pig did n o t complex with worm preparations taken on days 14, 35 or 63. The L3 antigens did n o t complex with any of the immunoglobin preparations. DISCUSSION

Two isoenzymes of malic dehydrogenase, three of acid phosphatase, and three isoenzymes of acetyl cholinesterase have been found in ether extracts of adult H. rubidus worms. It is probable that the MDH and acid phosphatase enzymes are intracellular in origin, as is the case for the acid phosphatase of N. brasiliensis {Edwards et al., 1971) and are n o t likely to be secreted during infection. This would explain the finding that only the rabbit anti-H, rubidus serum was capable of complexing with one of the acid phosphatase isomers. It is n o t k n o w n whether MDH and acid phosphatase enzymes play a role in the development of protective immunity to H. rubidus infection in the pig. Rhodes et al. (1964} suggested that MDH in Ascaris suum may be immunogenic b u t offered no p r o o f that this is so. The colorimetric m e t h o d of Ellman et al. (1961) detected AChE even in antigens of low protein concentration. Attempts to hydrolyse acetyl of b u t y r y l thiocholine iodide with the day 14 worm antigen, however, were successful only where the protein concentrations of the antigen was 25 mg/ml or over; even then the b u t y r y l thiocholine iodide was hydrolysed slowly. Despite the demonstration of AChE in worm extract and that antibody produced in immunized rabbits or infected pigs would complex with it, it was n o t possible to demonstrate such complex formation b y the technique described b y Jones and Ogilvie (1972}. This technique has been used successfully to demonstrate antibodies produced against AChE in rats infected with N. brasiliensis (Jones and Ogilvie, 1972), in sheep infected with T. colubriformis (Rothwell et al., 1973; Rothwell and Merritt, 1974) and cattle infected with Oesophagostomum radiatum (Bremner et al., 1973). The reason for the failure of this technique with the H. rubidus extract is n o t k n o w n though it would appear that amounts o f H . rubidus AChE in the extracts were relatively low. Ogilvie et al. (1973) state that this technique requires a very high concentration of soluble enzyme. It is clear, however, that b o t h immunised rabbits and infected pigs produce an anti-AChE antibody which was detectable b y the m e t h o d of Michaeli et al. (1969) and this technique would appear to be more sensitive than the above in this system. Table I shows that extracts of all the stages of H. rubidus analysed contain

173

AChE and it seems probable t h a t the adult worms produce less as t h e y age. The infection following the administration of a single dose of H. rubidus larvae generally becomes patent at or shortly after 14 days. A crisis in population occurs between days 21--25 so that by day 35, and certainly by day 63, the remaining worms represent a residual population of reduced fecundity (Masaba, 1975). The worm population is at its m a x i m u m during the period 14--21 days after infection with m a x i m u m production of eggs 3--4 days after patency. Thus AChE activity is at its highest at the time when the protective immune response is beginning to manifest itself. The only immunoglobulin preparation which unequivocally complexed with all adult worm antigens produced was IgGl. The AChE of the L3 antigens may have complexed to some extent with IgG1 obtained from a sensitised pig b u t this suggestion requires confirmation. There is growing evidence t h a t the anti-AChE antibodies produced against those parasites which secrete AChE belong to the IgG~ fraction (Ogilvie, 1970; Curtain and Anderson, 1971; Crandall and Crandall, 1972; Jones and Ogilvie, 1972; Rothwell and Merritt, 1974; Rothwell et al., 1976), though Rothwell et al. (1976) found that sheep infected with Oesophagostomum colurnbianum produced an anti-AChE antibody in the IgM fraction. Despite this accumulating knowledge and the present finding it is still n o t known that the immune reaction against AChE has any protective value.

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Masaba, S., 1975. Studies on the immune responses of growing pigs to infection with Hyostrongylus rubidus (Hassall and Stiles, 1892). I. The population dynamics of animals receiving single doses of parasites at infection levels ranging from 2500 to 40 000 infective larvae. Ph.D. Thesis, University of Wales, Bangor. Michaeli, D., Pinto, J.D., Benjamin, E. and De Buren, F.P., 1969. Immunoenzymology of acetylcholinesterase. 1. Substrate specificity and heat stability of acetylcholinesterase and of acetylcholinesterase--antibody complex. Immunochemistry, 6: 101--109. Ogilvie, B.M., 1970. Immunoglobulin responses in parasitic infections. J. Parasitol., 56: Sect. 2, Part 3,525--534. Ogilvie, B.M., Rothwell, T.L.W., Bremner, K.C., Schnitzerling, H.J., Nolan, J. and Keith, R.K., 1973. Acetylcholinesterase secretion by parasitic nematodes -- 1. Evidence of secretion of the enzyme by a number of species. Int. J. Parasitol., 3: 589--597. Oya, H., Hayashi, H. and Aoki, T., 1970. Comparison of malate dehydrogenase isoenzymes between adult worms of Schistosoma mansoni and Schistosoma japonicurn. Recent. Advances In Research In Filariasis and Leishmaniasis in Japan, Tokyo. University of Tokyo Press, pp. 393--402. Porter, P., 1969. Transfer of immunoglobulins IgG, IgA and IgM to lacteal secretions in the parturient sow and their absorption by the neonatal piglet. Biochim. Biophys. Acta, ] 81 : 381--392. Reinecke, R.K., 1967. Improved methods for the recovery of parasitic nematodes at autopsy. Onderstepoort J. Vet. Res., 34: 547--562. Rhodes, M.B., Marsh, C.L. and Kelly, G.W., 1964. Studies in helminth enzymology. III. Malic dehydrogenase in Ascaris suum. Exp. Parasitol., 15: 403--409. Rothwell, T.L.W. and Merritt, G.C., 1974. Acetylcholinesterase secretion by parasitic nematodes -- IV. Antibodies against the enzyme in Trichostrongylus colubriformis infected sheep. Int. J. Parasitol., 4: 63--71. Rothwell, T.L.W., Ogilvie, B.M. and Love, R.J., 1973. Acetylcholinesterase secretion by parasitic nematodes -- II. Trichostrongylus spp. Int. J. Parasitol., 3: 599--608. Rothwell, T.L.W., Anderson, N., Bremer, K.C., Dash, K.M., Le Jambre, L.F., Merritt, G.C. and Ng, B.K.Y., 1976. Observations on the occurrence and specificity of antibodies produced by infected hosts against the acetylcholinesterase present in some c o m m o n gastrointestinal nematode parasites. Vet. Parasitol., 62: 367--373. Sanderson, B.F. and Ogilvie, B.M., 1971. A study of acetylcholinesterase throughout the life cycle of Nippostrongylus brasiliensis. Parasitology, 62: 367--373. Sanderson, B.F., Jenkins, D.C. and Phillipson, R.F., 1976. Nippostrongylus brasiliensis: Further studies of the relation between host immunity and worm acetylcholinesterase levels. Int. J. Parasitol., 6: 99--102. Soulsby, E.J.L., 1974. Immunological methods in helminthology. In: D.M. Weir (Editor), Handbook of Experimental Immunology Vol. 3, Blackwell, Oxford.