Veterinary Parasitology, 4 (1978) 21--27 21 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

POSSIBLE RELATIONSHIP OF LEVELS OF MUCOSAL IgA AND SERUM IgG TO IMMUNE UNRESPONSIVENESS OF LAMBS TO H A E M O N C H U S C O N T O R T US

J.L. DUNCAN, W.D. SMITH* and J.D. DARGIE

Faculty of Veterinary Medicine, University of Glasgow, Glasgow (Great Britain) *Moredun Institute, Edinburgh (Great Britain)

(Received 2 February 1977)

ABSTRACT

Duncan, J.L., Smith, W.D. and Dargie, J.D., 1978. Possible relationship of levels of mucosal IgA and serum IgG to immune unresponsiveness of lambs to Haemonchus contortus. Vet. Parasitol., 4: 21--27.

Abomasal mucus IgA and serum IgG antibodies were studied in adult sheep and young lambs vaccinated with irradiated Haemonchus contortus larvae and subsequently challenged with normal larvae. In the adult sheep protection against challenge was associated with raised levels of these antibodies. Vaccination of the lambs, on the other hand, did not protect against challenge nor did it stimulate either serum IgG or mucus IgA antibodies. The latter remained at levels similar to those of both control lambs given a single challenge infection and worm-free adult sheep.

INTRODUCTION

Despite the well known ability of young lambs to respond satisfactorily to bacterial and viral vaccines and the fact that humoral and cell-mediated re- sponses have been demonstrated in foetal lambs (Sterzl and Silverstein, 1967), the ability of lambs to develop immunity to the abomasal nematode Haernon- chus con tor tus is apparently insignificant. Thus, laboratory investigations have shown that a previous infection with H. con tor tu s in young lambs 2 4 months old confers no protect ion against a subsequent challenge (Manton et al., 1962). Similarly, vaccination of lambs up to 5 months of age with irradiated larvae is also ineffective (Urquhart et al., 1966 a and b). In contrast, immunization of sheep reared worm-free until at least 7 months of age with either normal or irradiated larvae conferred a very high degree of protect ion to subsequent challenge (Jarrett et al., 1961; Manton et al., 1962; Benetez-Usher et al., 1977).

The explanation for this prolonged period of unresponsiveness to H. con- tor tus is unknown. In a recent s tudy involving 9-month-old sheep, it was shown that the resistance to challenge stimulated by vaccination with irradiated

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H. contortus larvae was associated with elevated levels of IgA antibodies in the abomasal mucosa and IgG antibodies in the serum (Smith and Christie, personal communication, 1977). The aim of the present experiment was to determine whether the irradiated vaccine stimulated similar antibody res- ponses in lambs vaccinated at 2 months of age.

MATERIALS AND METHODS

The methods of rearing lambs parasite free together with the parasitological and irradiation techniques were those described by Benitez-Usher et al. (1977).

Larval antigen was prepared from a mixture of third and fourth stage H. contortus larvae (80% L4, 20% L3) which had been cultured in vitro in protein- free medium (Mapes, 1969). Several million of these larvae were homogenized in phosphate buffered saline pH 7.2, and the soluble antigens were then ex- tracted by ultra-centrifugation (Smith, 1977).

Collection o f abomasal mucus

After washing gently to remove any worms and adherent debris, the abo- masum was cut into two halves along the omasal--pyloric axis. The mucosa was then scraped off with a microscope slide. Each gram of scraping was homogenized with 5 ml of buffer (0.1 M Tris, 0.1 M NaC1, pH 8.0, with sodi- um azide added to 0.02%) and clarified by ultra-centrifugation (30 000g). The resultant clear yellowish supernatant was subsequently referred to as abomasal mucus.

Anti-ovine immunoglobulin anti-sera

Specific rabbit anti-sheep IgA and IgG were prepared as described elsewhere (Smith et al., 1975). Anti-sheep IgA was used to estimate the levels of IgA in the mucus and serum by single radial diffusion (Mancini et al., 1965) as de- scribed before (Smith et al., 1975). IgG fractions of anti-IgA and anti-IgG sera were labelled with iodine-125 (Hunter and Greenwood, 1962) and used in the radio-immunoassay.

Radio-immunoassay o f anti-larval antibodies

A double antibody solid-phase radio-immunoassay was used to measure IgG and IgA anti-larval antibodies in the serum and mucus (Smith, 1977). Briefly, small polystyrene tubes were filled with larval antigen (10 pg protein/ml) and held overnight at room temperature. These antigen-coated tubes were washed and, together with an equal number of untreated control tubes, were filled with 1% normal rabbit serum (NRS) and held overnight at room temperature. For the assay, 0.8 ml of diluted test serum (1/1000) or mucus (1/10) was added to duplicates of both antigen-coated and control tubes and left over-

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night at room temperature. The tubes were then washed five times with 1% NRS, filled with labelled anti-globulin and incubated for 1 h at 37 ° C. The labelled anti-serum was then aspirated and each tube washed five times with water before being counted.

Tubes, to which test serum had not been added but which had been in- cubated with labelled anti-globulin, served as blanks. After removing the activity of these blanks, the specific activity due to antibody was obtained by subtracting the activity of the control tubes from that of the antigen- coated tubes.

E X P E R I M E N T A L D E S I G N

A group of five adult (1-year-old) worm-free Blackface sheep (Group 1) to- gether with a group of five, eight-week-old worm-free Blackface lambs (Group 2), were orally inoculated with two doses of 10 000 H. contortus larvae irradi- ated at 60 kilorSntgen (kR); these larvae were given at an interval of 3 weeks.

These two groups received a challenge infection of 10 000 normal larvae 3 weeks after the second inoculation together with a second group of five worm- free lambs (Group 3). All three groups were necropsied 3 weeks later. Serum and abomasal mucus samples were collected from these animals at necropsy. Similar samples were also obtained from a group of six 1-year-old Blackface sheep reared worm-free from birth (Group 4).

R E S U L T S

The individual and mean worm burdens of the challenged animals are shown in Table I.

The mean worm burden of the adult vaccinated sheep (Group 1) was re- duced by 87% compared with that of the young challenge controls (Group 3). The reduction in the mean worm burden of the vaccinated lambs (Group 2) was not significantly different from that of the challenge control group.

Serum IgG antibody

Serum IgG ant ibody was detected in the post-mortem samples from the adult vaccinates (Group 1). In contrast, the levels found in the young vacci-

TABL E I

Haemonchus contor tus w o r m burdens of vaccinated and challenged adult sheep and lambs

Individual w o r m burdens Mean S E.

Group 1 -- Vaccinated adults 0, 0, 350, 700, 1 300 470 * 244 7 Group 2 - - Vaccinated lambs 1 425, 1 775, 2 150; 2 525, 5 750 2 725 ~ 778 3 Group 3 - - Control lambs 600, 2 750, 4 500, 5 000, 5 250 3 630 , 872.2

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25

20

.? 10

5 • + i

1 2 3 4

G rou p

Fig.1. Individual and group mean ± S.E. (+) serum IgG antibody concentrations in shee~ and lambs vaccinated and challenged with Haemonchus contortus larvae and in challenge control lambs and worm-free adult sheep.

nated sheep (Group 2) and in the young challenge controls (Group 3) were similar to the background levels present in samples from adult worm-free sheep (Group 4) (Fig.l).

Mucus IgA antibody

Mucus IgA ant ibody concentrations were significantly increased in the adult vaccinated sheep (Group 1) compared with concentrations of the samples from adult worm-free sheep (Group 4), the young vaccinates (Group 2) and the young challenge controls (Group 3) (Fig.2).

Measurement of total IgA concentrations in the serum and mucus revealed that each adult vaccinated sheep (Group 1) had more IgA in the mucus than in the serum, whereas in the other groups the reverse situation usually held (Table II). IgA concentrations in the mucus of Group 1 were also increased compared with those of all the other groups in which the IgA levels were frequently below 5 mg/100 ml, the detection limit of the assay.

DISCUSSION

The results of this experiment show, as previously reported (Jarrett et al., 1959, 1961; Urquhart et al., 1966 a and b), that an irradiated H. contortus larval vaccine protects adult sheep against a challenge infection but is inef-

25

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40

30

20

10

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2 3 4

Group Fig.2. Individual and group mean -+ S.E. (+) mucus IgA an t i b o d y concen t ra t ions in sheep and lambs vaccinated and challenged wi th Haernonchus con tor tu s larvae and in challenge cont ro l lambs and worm-free adult sheep.

TABLE II

Mean IgA concen t ra t ions (mg/100 ml _+ S.E.) in the abomasal mucus and serum o f sheep and lambs vaccinated and challenged wi th H a e m o n c h u s con tor tu s larvae and of challenge cont ro l lambs and worm-free adul t sheep

Total mucus Total se rum IgA IgA

Group 1 - - Vaccinated adul ts 43.4 _+ 11.4 Group 2 - - Vaccinated lambs <5 Group 3 - - Cont ro l lambs *5.0 -+ 1.0 Group 4 - - Worm-free adults < 5

1 5 . 2 ± 4 . 3 " 1 0 . 2 ± 2 . 2

1 4 . 1 ± 5 . 4 7 . 2 ~ 0 . 8

*Not t rue mean - - at least one sample wi th a value of less than 5 mg/100 ml is included and given a value of four for the purpose of calculating a mean.

fective in young lambs. They also confirm the more recent observations of Smith and Christie (personal communication, 1977) that adult sheep respond to H. contortus vaccination and challenge with the product ion of abomasal mucus IgA and serum IgG antibodies. This was also associated, in the adults, with increased total IgA levels in the abomasal mucosa. In contrast, our observations showed that in young lambs, similarly vaccinated and challenged, mucus IgA and serum IgG anti- bodies remained at extremely low levels, similar, in fact, to those of both worm-free adult sheep and control lambs given a single challenge in- fection. The total abomasal mucus IgA levels in these three groups of animals were also low, the majority being below 5 mg/100 ml, the detect ion limit of the assay.

There is some evidence that the secretory IgA system develops slowly in

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y o u n g roden t s (Bazin, 1976) and the results p re sen ted here suggest t h a t a similar s low m a t u r a t i o n m a y occur in lambs. I t is in teres t ing to specula te t ha t the unrespons iveness of y o u n g l ambs to H. c o n t o r t u s in fec t ion migh t be due, at least in par t , to a def ic ien t p r o d u c t i o n of mucosa l IgA or se rum IgG anti- bodies. However , i t is k n o w n t h a t y o u n g l ambs can r e spond to viral vaccines or in fec t ions of the r e sp i ra to ry t r ac t wi th b o t h types of a n t i b o d y wi th in the first f ew weeks of life (Smi th et al., 1976 a and b) and intes t inal IgA anti- bodies have been de t ec t ed in n e w b o r n l ambs oral ly i m m u n i z e d wi th horse spleen ferr i t in as foe tuses (Hus band and McDowell , 1975) .

A possible exp l ana t i on m a y be t h a t y o u n g l ambs c a n n o t m o u n t a pro- tec t ive i m m u n e response to c o m p l e x n e m a t o d e antigens, or pe rhaps the a b o m a s u m itself is slow, c o m p a r e d to the r e sp i r a to ry t r ac t or the in tes t ine , to deve lop the capac i ty to p r o d u c e IgA. Whatever the reason, the p rospec t s o f p r o d u c i n g a pract ica l vacc ina t ion p r o g r a m m e against this i m p o r t a n t paras i te of sheep are p o o r unt i l the unde r ly ing m e c h a n i s m s of this p h e n o m e n o n are be t t e r u n d e r s t o o d .

ACKNOWLEDGEMENTS

The au tho r s wish to t h a n k Mrs E. J a c k s o n o f the Paras i to logy D e p a r t m e n t , Moredun Ins t i tu te , Edinburgh , for exce l l en t technica l assistance. Financial s u p p o r t fo r this w o r k was p rov ided b y the Minis t ry o f Overseas D e v e l o p m e n t and the Wellcome Trust .

REFERENCES

Bazin, H., 1976. The secretory antibody system. In: Anne Ferguson and R.N.M. MacSween (Editors), Immunological Aspects of the Liver and Gastrointestinal Tract. M.T.P. Press, Lancaster, pp.33--82.

Benitez-Usher, C., Armour, J., Duncan, J.L., Urquhart, G.M. and Gettinby, G., 1977. A study of some factors influencing the immunization of sheep against Haemonchus con- tortus using attenuated larvae. Vet. Parasitol., 3: 327--342.

Hunter, W.M. and Greenwood, F.C., 1962. Preparation of Iodine-131 labelled human growth hormone of high specific activity. Nature (London), 194: 495--496.

Husband, A.J. and McDowell, G.H., 1975. Local and systemic immune responses following oral immunisation of foetal lambs. Immunology, 29: 1019--1028.

Jarrett, W.F.H., Jennings, F.W., McIntyre, W.I.M., Mulligan, W. and Sharp, N.C.C., 1959. Studies on immunity to Haemonchus contortus infection -- vaccination of sheep using a single dose of X-irradiated larvae. Am. J. Vet. Res., 20: 527--531.

Jarrett, W.F.H., Jennings, F.W., McIntyre, W.I.M., Mulligan, W. and Sharp, N.C.C., 1961. Studies on immunity to Haernonchus contortus infection -- double vaccination of sheep with irradiated larvae. Am. J. Vet. Res., 22: 186--188.

Mancini, G., Carbonara, A.O. and Heremans, J.F., 1965. Immunochemical quantitation of antigens by single radial diffusion. Immunochemistry, 2: 235--254.

Manton, V.J.A., Peacock, R., Poynter, D., Silverman, P.H. and Terry, R.J., 1962. The influence of age on naturally acquired resistance to Haemonchus contor tus in lambs. Res. Vet. Sci., 3, 308--314.

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Mapes, C.J., 1969. The development of Haemonchus contortus in vitro I. The effect of pH and pCO 2 on the rate of development to the fourth stage larva. Parasitology, 59: 215-- 231.

Smith, W.D., 1977. Anti-larval antibodies in the serum and abomasal mucus of sheep hyperinfected with Haernonchus contortus. Res. Vet. Sci., 22: 334--338.

Smith, W.D., Dawson, A.McL., Wells, P.W. and Burrells, C., 1975. Immunoglobulin con- centrations in ovine body fluids. Res. Vet. Sci., 19: 189--194.

Smith, W.D., Dawson, A.McL., Wells, P.W. and Burrells, C., 1976 a. Immunoglobulins in the serum and nasal secretions of lambs following vaccination and aerosol challenge with parainfluenza 3 virus. Res. Vet. Sci., 21: 341--348.

Smith, W.D., Wells, P.W., Burrells, C. and Dawson, A.McL., 1976 b. Maternal immuno- globulins and parainfluenza 3 virus inhibitors in the nasal and lachrymal secretions and serum of newborn lambs. Clin. Exp. Immunol., 23: 544--553.

Sterzl, J. and Silverstein, A.M., 1967. Developmental aspects of immunity. Adv. Immunol., 6: 337--459.

Urquhart, G.M., Jarrett, W.F.H., Jennings, F.W., McIntyre, W.I.M., Mulligan, W. and Sharp, N.C.C, 1966 a. Immunity to Haemonchus conto~tus infection: Failure of X-irradiated larvae to immunise young lambs. Am. J. Vet. Res., 27: 1641--1643.

Urquhart, G.M., Jarrett, W.F.H., Jennings, F.W., McIntyre, W.I.M. and Mulligan, W., 1966 b. Immunity to Haemonchus contortus infection: Relationship between age and successful vaccination with irradiated larvae. Am. J. Vet. Res., 27: 1645--1648.