Serum antibody responses of Texel sheep experimentally infected with Haemonchus contortus

Research in Veterinary Science 1994, 57, 63-68

Serum antibody responses of Texel sheep experimentally infected with Haemonchus contortus H. D. F. H. SCHALLIG, M. A. W. VAN LEEUWEN, W. E. BERNADINA, W. M. L. HENDRIKX, Utrecht University, Institute of lnfectious Diseases and Immunology, Departments of Parasitology and Tropical Veterinary Medicine and Immunology, PO Box 80.165, 3508 TD Utrecht, The Netherlands

less than six months old are unable to acquire natural immunity against H contortus (Urquhart et al 1966, Neilson 1975); the cause of their unresponsiveness is not known. Recently, good progress has been made by immunising lambs with a microvillar integral membrane protein, designated H l l , isolated from the gut of H contortus (Smith 1993, Munn et al 1993, Smith et al 1993). Vaccination with Hl l resulted in the significant protection of young lambs and sheep against H contortus (Tavernor et al 1992b, Smith 1993, Munn et al 1993, Smith et al 1993). However, the type of immunity stimulated by H l l is artificial and quite different from naturally acquired immunity (Smith and Smith 1993). After continuous or seasonal exposure to infective larvae older lambs and sheep can acquire a degree of protective immunity against gastrointestinal nematodes (Barger et al 1985). The mechanisms underlying this naturally acquired immunity are not completely understood. More insight into these mechanisms might provide a basis for the development of natural immunological control methods which might also be applicable to young lambs. In the present study 10-month-old Texel sheep were experimentally infected and challenged with H contortus and the kinetics of their primary and secondary IgA, IgG 1, IgG 2 and IgM serum antibody responses were monitored.

The primary and secondary serum antibody responses of Texel sheep to infective larvae (L3) and adult worms of H a e m o n c h u s contortus were studied. Ten-month-old sheep were infected with 20,000 H contortus L3, treated with ivermectin seven weeks later and, after four weeks, reinfected with 10,000 L3 once a week for six weeks. Faecal egg counts were significantly lower during the secondary infection than during the primary infection, but both infections induced antibody responses, as demonstrated by an enzyme-linked immunosorbant assay (ELISA). The primary antibody response developed rather slowly, but the secondary response developed more rapidly and the IgA responses against L3 antigens and the IgG 1 and IgG 2 responses against adult antigens were twice those observed during the primary infection. These accelerated and enhanced responses after the reinfection suggest an immunological memory for H contortus antigens. THE blood-feeding nematode Haemonchus contortus is a major pathogen which infects the abomasum of sheep and other ruminants worldwide. The strategies used to control the parasite are largely dependent on pasture management combined with the use of anthelmintics, but such strategies have two obvious restrictions. First, clean pastures are not easily available under intensive grazing conditions, and secondly although modern drugs are in general effective, the development of anthelminthic resistance is increasing (Boersema et al 1987, Borgsteede and Duyn 1989). These restrictions could be overcome by the development of immunological methods to control H contortus. However, a problem with the application of such methods is the fact that lambs

Materials and methods Parasites A benzimidazole-sensitive strain of H contortus, originally obtained from the Moredun Research Institute (Edinburgh) was used. Infective 63

64

H. D. F. H. Schallig, M. A. W. Van Leeuwen, W. E. Bernadina, W. M. L. Hendrikx

L3 were cultured from the faeces of donor sheep and were harvested by the Baermann technique. Adult worms were obtained from experimentally infected donor sheep. Sheep

Ten-month-old female Texel sheep, which had been reared under worm-free conditions, were used. The animals were offered a pelleted diet, and hay and water ad libitum. Experimental design

Five sheep were infected intraruminally with 20,000 H contortus L3, treated with 0.2 mg kg-1 iverrnectin seven weeks later and from week 11 reinfected orally with 10,000 L3 weekly for six weeks. Three uninfected sheep were used as controls throughout the experiment. The control animals were given ivermectin at the same time as the infected sheep. Samples of faeces and serum were collected weekly from all the sheep from one week before they were infected until 18 weeks after. The serum samples were stored at -20°C before use. Faecal egg counts were determined by the McMaster technique and expressed in terms of the mean number of eggs per gram faeces (epg). Antigens

The L3 were washed three times with deionised water and centrifuged for 10 minutes at 800 g. Water soluble proteins were isolated from approximately 2 x 106 L3 larvae which were homogenised and subsequently extracted overnight at 4°C in 30 ml phosphate buffered saline pH 7.2 (PBS) containing 2 mM phenylmethylsulphonylfluoride, 2 mM Nc~-p-tosyl-L-lysine chloromethylketone, 2 mM N-tosyl-L-phenylalanine chloromethylketone and 1 mM ethylenediamine tetraacetic acid to prevent proteolytic activity. After centrifugation at 48,000 g for 60 minutes at 4°C, the supernatant was collected and stored at -80°C. Approximately 500 adult worms were washed three times with PBS and homogenised. The water soluble proteins of the adult worms were obtained as described for the L3 antigens. Protein concentrations were determined by the method of Bradford (1976).

Enzyme-linked immunosorbant assay

(EL1SA)

A checkerboard analysis was applied before the experimental ELISASin order to determine the optimal antigen concentrations and sheep serum dilutions. The ELISA plates were coated by incubating them overnight with the appropriate antigen at a concentration of 5 pg m1-1 dissolved in a 0.025 M morpholinoethane sulphonic acid buffer pH 6.0, with 0.1 per cent N-(3-dimethylaminopropyl)-N'ethylcarbodiimidhydrochloride (100 gl per well). The plates were then washed for 30 seconds with tap water containing 0.05 per cent Tween-20 and tapped dry. The plates were blocked with 5 per cent skimmed milk in PBS (SM-PBS, 100 gl per well) and incubated for one hour at 37°C. The plates were then washed and serial dilutions of sheep serum made in SM-PBS (100 pl) were added to the wells. The serum dilutions used included: 1:10, 1:20, 1:40 and 1:80 for IgA; 1:160, 1:320, 1:640 and 1:1280 for IgG 1, and 1:40, 1:80, 1:160 and 1:320 for IgG 2 and IgM. All the samples were run in triplicate. The plates were incubated for one-and-a-half hours and washed as above. Monospecific rabbit-anti-sheep IgA, IgM or IgG 1 or goat-anti-sheep IgG 2 antisera (Goudswaard et al 1980) diluted 1:600 in SM-PBS were added to the appropriate wells and incubated for one-and-a-half hours. The plates were washed and incubated for one-and-a-half hours with swine-anti-rabbit or rabbit-anti-goat immunoglobulins conjugated to horseradish peroxidase (Dako) at a dilution of 1:1500 in SM-PBS. After washing 100 gl of substrate (1 mM 2,2'-azino-bis[3-ethylbenzthiazoline6-sulphonic acid] in 100 mM citrate/200 mM phosphate buffer [pH 5.0] with 0.01 per cent hydrogen peroxide) was added to each well. The colour was allowed to develop for 40 minutes and the optical density was measured at 405 mm in a Microplate reader Model 3550 (Bio-Rad, Richmond, USA). The extinction value of each of the four serum dilutions was in transformed and the mean of these In values was calculated and retransformed to give the extinction value used for the statistical analysis. The statistical significance of the differences between the infected and control groups was assessed by using a univariate measures analysis with the Huynh-Feldt correction and software from Systat (Evanston, USA). Differences between the groups were considered significant at P<0.05.

65

Sheep serum antibody responses to H contortus 1

Iv

1

1

2

12

11 10 e

g ~

4 3 2

-"

0

I

2

3

4

5

6 week

7

e

9

101112131,41516171e

decreased further and reached control levels. A comparison of the serum antibody responses revealed that between two and six weeks after infection the IgG 1 and IgG 2 antibody levels in the infected animals were significantly greater than those observed in the uninfected sheep. The IgA antibody levels were significantly higher between two and three weeks after infection. After the secondary infections the levels of antibodies increased more rapidly. The secondary IgG 1 and IgG 2 responses reached the same levels as observed during the primary infection, but the secondary infections resulted in a twofold increase

of experiment

F I G 1: M e a n (_+ SD) e g g c o u n t s a f t e r p r i m a r y a n d c h a l l e n g e inf e c t i o n s o f T e x e l s h e e p w i t h H contortus. 1 P r i m a r y i n f e c t i o n , Iv Treatment with ivermectin, 2 Period of secondary infections

1,40 1

Results Faecal egg counts

The mean epg values are shown in Fig 1. The infection became patent after three weeks and the epg reached its maximum value after six weeks. Thereafter the epg decreased and became undetectable after the sheep were treated with ivermectin. Three weeks after the administration of the first secondary infection the epg started to rise, but the maximum mean count was approximately eight times lower than after the primary infection and declined to zero within four weeks. The epg of the three control sheep remained zero throughout the experiment.

IV

12o! <~

0.80

--

0.60

1

2

,

'I~,

0.40 0.20 i 0.00 -

i 0

i r i i i , , , , , , , , , , , , 1 2 3 4 5 6 7 e g 10111213141516171B

E

c

14°fi

1,2o

0 ~-

l

1

IV

2 i

i

1.oo

_

o6o

Serum antibody responses evaluated by ELISA <~

The time courses of the serum IgA, IgG I and IgG 2 antibody responses of the infected and control sheep to the larval and adult water soluble antigens are presented in Figs 2 and 3, respectively. The results of IgM testing are not shown because the sera of both the infected and control sheep showed high extinction values in the ELISA throughout the experiment. Two weeks after the primary infection the serum antibody levels of all three isotypes against the L3 antigens had increased. The serum IgA response to the larval antigens was much lower than that observed for either IgG 1 and IgG 2. After three to four weeks the levels of antibodies reacting with the L3 antigens started to decline, and after treatment with ivermectin at week 7 they

-10

1 2 3 4 5 6 7 e cJlO111213141516171B

1.4o

1

IV

1.20

1

1

2 ~,

i

1"00i OJ

0,80

(~

0,60 0.40 0.20 -101

2 3 4 5 S 7 B 9 t0111213141516171B

Week

of

experiment

F I G 2: M e a n (+ SD) s e r u m I g A , IgG1 a n d I g G 2 a n t i b o d y r e s p o n s e s of experimentally infected sheep (-e-) or uninfected con-

trol sheep (-o-) to soluble L3 antigens of H contortus. 1 Primary infection, IV Treatment with ivermectin, 2 Period of secondary infections

66

H. D. F. H. Schallig, M. A. W. Van Leeuwen, W. E. Bernadina, W. M. L. Hendrikx 1.40

1

1.20

|

iv

2 "'

1.00

< ol

0.60 0.60 0.40 0.20 0.t30 --

LO O

i

i

i

i

L

i

i

=

i

0

1

2

3

4

5

6

7

B

i

L

f

i

i

i

~

i

i

¢J ~ 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8

1` 2 0

I

I

1.00

(D U E _Q

b if)

0.60

I/

o.o "

.y~/,

Discussion

t

T-l'~--~

_o

<

-10

1

2

3

4

5

6

'"°tl 1

7

8

9101112131415161716

IV

-2

1` 2 0

I

P

1,00

C~J 0.80 0.60 040 0.20

Y

0.00 -10

1

2

3

4

5

Week

S

7

of

B

g

ondary infections IgG 1 and IgG 2 levels increased significantly with responses equivalent to those previously observed for the L3 antigens. The IgA antibody to adult antigens also increased during the secondary infections, but the increase was not statistically significant. Moreover, the IgA response never reached the level observed against the L3 antigens or the level of the IgG 1 and the IgG 2 responses to the adult antigens. During the experimental period the serum antibody level of the uninfected sheep remained low and never exceeded those of the infected sheep, although they tended to increase towards the end of the experiment.

I01112131415161718

experiment

FIG :3: Mean (+ SD) serum IgA, IgG 1 and IgG 2 antibody responses of experimentally infected sheep (-II-) or uninfected control sheep (-o-) to soluble adult antigens of H contortus. 1 Primary infection, IV Treatment with ivermeatin, 2 Period of secondary infections

in the mean IgA response against L3 antigens. Compared with the control sheep, the anti-L3 antibody levels of the infected sheep were significantly raised from one week after the secondary infection. The antibody responses to the adult antigens showed a different pattern. After the primary infection the IgA anti-adult antibody levels were low and did not differ from those of the uninfected sheep. The mean IgG 1 and IgG 2 levels increased after the primary infection, but the responses were lower than those recorded with the L3 antigens. After treatment with ivermectin the levels dropped to those in the control sheep but during the sec-

After the primary infection the faecal egg counts became high, but after the secondary infections the epg, after an initial rise, declined and became zero. This reduction in epg was not due to a residual effect of the ivermectin because the secondary infections were administered later than the retention time of the drug (McKenna 1986, Zajac et al 1992). As faecal egg counts can be taken as a criterion for immunity (Adams et al 1990, Gill 1991, Adams 1993, Gill et al 1993), it can be assumed that the observed reduction in epg was a result of an effective immune response of the sheep against H contortus. Both the primary and secondary infections induced serum antibody responses against the larval and adult H contortus antigens. In contrast to the primary response which developed rather slowly, the rise in antibody levels after the secondary infection developed rapidly. Moreover, the levels of IgA against the L3 antigens and the levels of IgG 1 and IgG 2 against the adult antigens were twice those recorded during the primary infection. Such rapid and enhanced responses after the reinfection can be explained by an immunological memory for H contortus-antigens. However, it is possible that the antibody responses after the secondary infection were due to the repeated administration of the larvae as a trickle infection, rather than to an increased response due to immunological memory. The authors favour the hypothesis of an immunological memory because Smith (1977a) showed that primary trickleinfections with H contortus resulted in a slow development of the antibody response which declined as soon as the sheep stopped receiving

Sheep serum antibody responses to H contortus

larvae. In the present study, the secondary infection resulted in an immediate increase in serum antibody levels which remained high for at least two weeks after the animals had the last dose of larvae. The isotype specificity of the response was broadly similar to that observed by others who found increased IgG and/or IgA antibodies in the sera of immune sheep after a secondary infection (Duncan et al 1978, Smith and Christie 1978, Charley-Poulain et al 1984, Gill 1991). In the present study, IgG 1 and IgG 2 were the dominant antibody isotypes in serum. In contrast, Gill et al (1993) demonstrated that the IgG 2 response in genetically resistant Merino sheep was smaller than that observed in the Texel sheep in the present study; the differences between the breeds may contribute to this discrepancy. The role of serum IgA in the response against H contortus, as measured by the ELISA, seemed to be tess important than that of IgG 1 and IgG 2. However, several studies have shown that IgA may play an important role in the mucosal response (Charley-Poulain et al 1984, Smith et al 1983, 1984, 1986, Gill et al 1992) and because serum IgA levels are in general a poor reflection of those found in abomasal mucus (Smith 1977b, Goudswaard et'al 1980), they are probably unsatisfactory for monitoring the total IgA response against gastrointestinal nematodes. The present study demonstrated that 10-monthold Texel sheep are capable of mounting effective immune responses against H contortus. Their serum antibody responses to challenge showed characteristics of immunological memory and were associated with a significant reduction of the output of H contortus eggs. Acknowledgements The authors are grateful to Professor A. W. C. A. Comelissen for critically reading and discussing the manuscript. They would also like to thank Mr J. van den Brock for his advice and help with the statistical analysis, and Mr W. van der Aar and the animal keepers for taking part in the experiments. References ADAMS, D. B. (1993) Systemic responses to challenge infection with Haemonchus contortus in immune Merino sheep. Veterinary Research Communications 17, 25-35

67

ADAMS, D. B., LYNCH, J. J., ANDERSON, B. H., FELL, L. R., HINCH, G. N. & MUNRO, R. K. (1990) The intensity of resistance by mature Merino ewes against Haemonchus contortus and Trichostrongylus colubriformis in single-species and combinedspecies infection. Australian Veterinary Journal 67, 443-445 BARGER, I. A., LE JAMBRE, L. F., GEORGI, J. R. & DAVIS, H. I. (1985) Regulation of Haemonchus eontortus populations in sheep exposed to continuous infection. International Journal for Parasitology 15, 529-533 BOERSEMA, J. H., BORGSTEEDE, F. H. M., EYSKER, M , HENDRIKX, W. M. L., JANSEN, J. & SMITH-BUYS, C. M. C. (1987) The prevalence of benzimidazole resistance of nematodes in sheep in the Netherlands. Research in Veterinary Science 43, 18-23 BORGSTEEDE, F. H. M. & DUYN, S. P. J. (1989) Lack of reversion of a benzimidazole resistant strain of Haemonehus contortus after six years of Levamisole usage. Research in Veterinary Science 47, 270274 BRADFORD, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248-254 CHARLEY-POULAIN, J., LUFFAU, G. & PERY, P. (1984) Serum and abomasal antibody responses of sheep to infections with Haemonchus contortus. Veterinary Parasitology 14, 129-141 DUNCAN, J. L., SMITH, W. D. & DARGIE, J. D. (1978) Possible relationship of levels of mucosai IgA and serum IgG to immune unresponsiveness of lambs to Haemonchus contortus. Veterinary Parasitology 4, 21-27 GILL, H. S. (1991) Genetic control of acquired resistance to haemonchosis in Merino lambs. Parasite Immunology 13, 617-628 GILL, H. S., GRAY, G. D., WATSON, D. L. & HUSBAND, A. J. (1993) Isotype-specific antibody responses to Haemonchus contortus in genetically resistant sheep. Parasite Immunology 15, 61-67 GILL, H. S., HUSBAND, A. J. & WATSON, D. L. (1992) Localization of immunoglobulin-containing ceils in the abomasum of sheep following infection with Haemonchus contortus. Veterinary Immunology and lmmunopathology 31, 179-187 GOUDSWAARD, J., VERDOUW-CHAMALAUN, C. V. M. & NOORDZIJ, A. (1980) Quantitation of immunoglobulins in ovine sera and secretions by laser nephelometry. Comparison with the radial immunodiffusion (RID) technique. Veterinary Immunology and lmmunopathology 1, 163-177 McKENNA, P. B. (1986) The persistence of the anthelmintic activity of ivermectin in sheep. New Zealand Veterinary Journal 34, 94-96 MUNN, E. A., SMITH, T. S., GRAHAM, M., TAVERNOR, A. S. & GREENWOOD, C. A. (1993) The potential value of integral membrane proteins in the vaccination of lambs against Haemonchus contortus. International Journal for Parasitology 23, 261-269 NEILSON, J. T. M. (1975) Failure to vaccinate lambs against Haemonchus contortus with functional metabolic antigens identified by immunoelectrophoresis. International Journal for Parasitology 5, 427-430 SMITH, W. D. (1977a) Anti-larval antibodies in the serum and abomasal mucus of sheep hyperinfected with Haemonchus contortus. Research in Veterinary Science 22, 334-238 SMITH, W. D. (1977b) Serum and mucus antibodies in sheep immunised with larval antigens of Haemonchus contortus. Research in Veterinary Science 22, 128-129 SMITH, W. D. (1993) Protection in lambs immunised with Haemonchus contortus gut membrane proteins. Research in Veterinary Science 54, 94-101 SMITH, W. D. & CHRISTIE, M. G. (1978) Haemonchus contortus: local and serum antibodies in sheep immunised with irradiated larvae. International Journal for Parasitology 8, 219-223 SMITH, W. D., JACKSON, F., JACKSON, E. & WILLIAMS, J. T. (1983) Studies on the local immune response of the lactating ewe infected with Ostertagia circumcincta. Journal of Comparative Pathology 93, 295-305 SMITH, W. D., JACKSON, F., JACKSON, E., WILLIAMS, J. T. & MILLER, H. R. P. (1984) Manifestations of resistance to ovine ostertagiasis associated with immunological responses in the gastric lymph. Journal of Comparative Pathology 94, 591-601

68

H. D. F. H. Schallig, M. A. W. Van Leeuwen, W. E. Bernadina, W. M. L. Hendrikx

SMITH, W. D., JACKSON, F., JACKSON, E., WILLIAMS, J. T., WlLLADSEN, S. M. & FEHILLY, C. B. (1986) Transfer of immunity to Ostertagia circumcincta and IgA memory between identical sheep by lymphocytes collected from gastric lymph. Research in Veterinary Science 41,300-306 SMITH, T. S., MUNN, E. A., GRAHAM, M., TAVERNOR, A. S. & GREENWOOD, C. A. (1993) Purification and evaluation of the integral membrane protein H l l as a protective antigen against Haemonchus contortus. International Journalfor Parasitology 23, 271-280 SMITH, W. D. & SMITH, S. K. (1993) Evaluation of aspects of the protection afforded to sheep immunised with a gut membrane protein of Haemonchus contortus. Research in Veterinary Science 55, 1-9 TAVERNOR, A. S., SMITH, T. S., LANGFORD, C. F., GRAHAM, M. & MUNN, E. A. (1992a) Immune response of Clun Forest sheep to vaccination with membrane glycoproteins from Haemonchus contortus. Parasite Immunology 14, 671-657

TAVERNOR, A. S., SMITH, T. S., LANGFORD, C. F., MUNN, E. A. & GRAHAM, M. (1992b) Vaccination of young Dorset lambs against haemonchosis. Parasite Immunology 14, 645-655 URQUHART, G. M., JARRETr, W. F. H., JENNINGS, F. W., MACINTYRE, W. I. M. & MULLIGAN, W. (1966) Immunity to Haemonchus contortus infection. Relationship between age and successful vaccination with irradiated larvae. American Journal of Veterinary Research 27, 1645-1648 ZAJAC, A. M., THATCHER, C. D., BROCK, R. A., UMBERGER, S. H. & NOTI'ER, D. R. (I992) Comparison of ivermecfin formulations in an ovine parasite control programme. Veterinary Record 130, 353-354

Received July 26,1993 Accepted December 20, 1993