l'~,terinap:vParasitology, 38 ( 1991 ) 131-143 Elsevier Science Publishers B.V., A m s t e r d a m

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Serum IgG response of Manchego lambs to infections with Haemonchus contortus and preliminary characterization of adult antigens M. Cuquerella, Maria T. G6mez-Mufioz and J.M. Alunda* Departamento de Patologia Animal L Facultad de Veterinaria, Universidad ('omplutense, 28040 Madrid (Spain) (Accepted for publication 17 September 1990)

ABSTRACT Cuquerella, M., G6mez-Mufioz, M.T. and Alunda, J.M., 1991. Serum IgG response of Manchego lambs to infections with Haemonchus contortus and preliminary characterization of adult antigens. Vet. Parasitol., 38: 131-143. Manchego lambs ( 16-18 weeks old ) were infected with 2500 infective larvae (L 3 ) of Haemonchus L 3. The serum IgG anti-Haemonchus response was estimated by enzyme-linked immunosorbent assay (ELISA) using soluble proteins from adults and L~. Previously infected Manchego lambs failed to mount a protective immune response against challenge, at least as assessed by faecal egg counts and pre-patency periods. Primary infection did not provoke any rise in specific anti-parasite serum antibodies, whereas a weak but significant rise was observed in challenged 6.5-month-old lambs which was very similar in both infected and non-infected lambs. The serum IgG anti-parasite response was higher against larval antigens than adult soluble proteins. Preliminary characterization of adult and larval soluble proteins by electrophoresis under reducing and denaturing conditions and Western blotting showed high cross-reactivity of both extracts. Immunoblots of adult H. contortus probed with infected and challenged lambs' sera did not yield conclusive results, although some low molecular weight peptides were recognized.

contortus and challenged 2 months later with 5000

INTRODUCTION

Ovine haemonchosis constitutes an important cause of economic losses in all parts of the world where there are significant concentrations of sheep (Blood and Radostits, 1989 ). Under semi-arid conditions, such as those prevailing in most sheep-rearing areas in Spain, chronic haemonchosis is the rule. However, more intensive management systems using irrigated pastures put the lambs at risk of acute disease after weaning. Apparently, there is an age-resistance phenomenon associated with this *Author to w h o m correspondence should be addressed.

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© 1991 - - Elsevier Science Publishers B.V.

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parasitic disease. Most sheep breeds seem to reach their immunological maturity against Haemonchus contortus when they are 6-12 months old or later (Manton et al., 1962; L6pez and Urquhart, 1967 ), whereas other breeds are able to m o u n t a protective i m m u n e response against H. contortus challenges very early (Berezkho et al., 1987; Neilson and Van de Walle, 1987). Immunoprophylactic control of lamb haemonchosis is far from being a reality in spite of the partial protection obtained with irradiated larvae (Urquhart et al., 1966), metabolic products of the parasite (Boisvenue et al., 1987; Neilson and Van de Walle, 1987 ) or 'contortin'-enriched adult somatic extracts of the parasite ( M u n n et al., 1987). The control of this disease relies on anthelmintic treatment following accurate diagnosis. Present-day laboratory diagnosis is restricted to patent infections, is time consuming and requires a high level of expertise. Unfortunately, the studies on the specific antiHaemonchus i m m u n e response in sheep have produced inconsistent results (Smith, 1977a, b; Duncan et al., 1978; Adams and Beh, 1981; Charley-Poulain et al., 1984) and the characterization of H. contortus antigens (Ags), from both larval and adult sources, has been relatively rare, bearing in mind the economic importance of sheep haemonchosis. Notable exceptions are the preliminary studies by Neilson (1969) and Ozerol and Silverman (1969, 1970) on somatic and metabolic Ags, and the more recent contribution by Cox et al. (1989) on surface Ags. Since there is no information concerning the i m m u n e response of Manchego breed lambs to H. contortus infection, 16-18-week-old lambs were infected and subsequently challenged 2 months later, estimating the faecal output of parasite eggs and the serum specific IgG anti-parasite (somatic Ags of infective larvae and adults) response by enzyme-linked immunosorbent assay (ELISA). In addition, a preliminary characterization ofH. contortus Ags by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDSPAGE) and Western blotting using sera from infected and reinfected lambs was carried out. MATERIAL AND METHODS

Infective material Haemonchus contortus infective larvae (L3) were kindly supplied by Merck, Sharp and Dohme, Spain. This strain was maintained at our laboratories by serial passage in lambs. The L3 used for the experimental infections were obtained through faecal culture at 26 ° C for 10 days, baermanization of the faeces, purification by partial desiccation on filter paper and recovery of the clean larvae (Ministry of Agriculture, Fisheries and Food, 1971 ). The larvae obtained were stored in tapwater at 4-10 °C until use.

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Lambs and experimental design Four-six-week-old Manchego lambs (three males and six females) were obtained from a local producer. At their arrival, a slight infection with Strongyloides and coccidia was detected, and the animals were treated with Amprolium and Thiabendazole. Lambs were housed under conditions precluding helminth infections and fed with commercial pelleted food, alfalfa and tapwater ad libitum. When the lambs were 16-18 weeks old, they were randomly allocated to two groups: Group A (two males and three females) and Group B (one male and three females). Group A was infected with 2500 L 3 per animal and Group B lambs were kept as controls. The experimental design included the removal of primary infections before challenge, given the low reproducibility of self-cure reactions (Adams, 1983 ). Seven weeks after previous infection, both groups (infected and uninfected) received an anthelmintic treatment (Thiabendazole, 88 mg kg-~ live weight) and 12 days post-treatment all animals were challenged with an infective dose of 5000 H. contortus L 3. During the experimental period, individual blood samples were taken twice a week by jugular venipuncture using evacuated tubes. Blood samples were allowed to clot at room temperature and the sera were labelled and stored at - 2 0 ° C until use. Coproscopical analyses (McMaster technique) were carried out daily up to the 41 st day post-previous infection and 2-3 times a week afterwards.

Haemonchus contortus antigens Soluble Ags from adult and third larval stage H. contortus were used. Adult H. contortus, largely females, were obtained from the abomasa of experimentally infected lambs. Worms were collected, extensively washed in cold phosphate-buffered saline (PBS) and stored at - 2 0 ° C . L 3 w e r e obtained after faecal culture as above. Frozen samples were subjected to freezing and thawing cycles (eight for the adults and three for the larvae), homogenized in a glass-in-glass Potter-Elvehjem-type homogenizer at 4 °C and the extracts centrifuged (30 000 × g at 4 ° C for 30 min) (Klesius et al., 1986). Supernatants from both sources were aliquoted and cryopreserved at - 7 0 to - 8 0 ° C . Protein content determinations were performed by the Bradford method (Bradford, 1976).

ELISA conditions O p t i m u m ELISA conditions were determined in a checkerboard manner. Soluble adult Ag was used at a concentration of 10/~g m l - ~and larval extracts at 5/~g ml -I in 0.05 M carbonate buffer (pH 9.6) as coating solution for 16 h at 4°C. Bovine serum albumin (BSA) 5% was used as blocking solution

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(1 h, 37°C). Sera were used at a 1/50 dilution in PBS-Tween 20 (0.05%) for 1 h at 37 °C and the anti-species conjugate, alkaline phosphatase-labelled rabbit anti-sheep IgG (Bio Rad, U.S.A.), was used at 1/800 for 1 h at 37°C. Colourwas developed with 1,4 p-nitrophenol phosphate (PNPP), 1 mg ml l for 30 rain at 37°C. The reaction was stopped with 50 #1 3 N NaOH and the optical density (OD) was read at 405 nm in a EAR 400 ELISA reader. Final volumes were 100 #1 in all steps except the blocking solution (200/d) and flat-bottomed 96-well microtitre plates (Nunc) were used. Electrophoresis and Western blotting

Soluble protein extracts ( ~ 1 mg ml -~ ) were fractionated by 12.5% SDSPAGE in 10 × 10 × 0.1-cm gel plates at 150 V. Low molecular weight (LMW) markers were from Pharmacia (Sweden). Gels were electroblotted (overnight at 0.1 A + 1.5 h at 0.56 A) on to Immobilon (Millipore) membranes. Western blotting was carried out by a modification of previously described methods (Jenkins and Dame, 1987; Shepherd and McManus, 1987; Cross et al., 1988 ). Briefly, membranes were washed three times ( 10, 5 and 5 min) with 10 mM Tris-HC1 with 0.15 M NaC1 (TBS) and 0.05% Tween 20, blocked in TBS-Tween and 5% powdered skimmed milk (Molico, Nestld) at room temperature (RT) for 30 min. After washing, membrane strips were incubated with 1/ 10 diluted sera in blocking solution for 3 h at 37 °C. Anti-species antibody used was affinity-purified rabbit anti-sheep IgG horseradish peroxidase labelled (HRPE) (Bio Rad) at a 1/100 dilution and incubated for 1 h at 37 ° C. Strips were washed twice ( 10 and 5 rain) with TBS-Tween and twice ( 5 and 5 min) in TBS to avoid precipitates, and developed with 0.5 mg m l 4-chloronaphthol, 20% methanol and 0.015% H202 in TBS at RT for ~ 10 min under shaking. The reaction was stopped by rinsing the strips in water. Hyperimmune anti-larval serum was obtained in rabbits by intramuscular injection of 500 /tg soluble protein in Freund's complete coadjuvant and weekly boosters. Western blots involving hyperimmune serum were developed with goat anti-rabbit IgG HRPL (Bio Rad) as above. RESULTS

Faecal H. contortus eggs output

Figure 1 shows the results found when studying the faecal output of//. contortus eggs. The results did not show any significant difference between both groups (infected and control lambs) when they were challenged with 5000 H. contortus L3. Both animal groups exhibited similar egg counts and actually, on average, the Group B (uninfected) lambs reached lower counts than the previously infected Group A lambs. Moreover, pre-patency periods were very

135

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close in all cases and sex differences were not noticeable within each group. These results suggest that Manchego lambs were not able to mount a protective i m m u n e response against subsequent challenge with 5000 L3. The nonlinear relationship found between egg counts and infective doses used in 1618-week-old lambs (Group A) and 6-month-old lambs (Groups A and B) with similar faecal egg output, (despite the different (2500 vs. 5000 L3) infective doses) could possibly be the result of the age-related increase in faecal deposition and, therefore, to the dilution of parasites in older animals. ELISA of serum anti-Haemonchus IgG response

Sixteen-eighteen-week-old infected lambs (Group A) did not show any specific serum IgG anti-Haemonchus response, at least as assessed by ELISA. Optical density (OD) values remained at extremely low levels during the experimental infection, without significant differences to the uninfected control lambs (Group B) and with OD readings close to those found at pre-infection (Figs. 2 and 3 ). When both groups were challenged 2 months later with 5000 L3, a weak but significant rise in IgG was observed. This increase began at about patency and reached m a x i m u m values 24-25 days after patency using both antigenic extracts (L 3 and adult soluble extracts). Anti-larval IgG levels exhibited a more clear and rapid rise than anti-adult IgG. These results, agreeing well with the coproscopical findings, could be evidence for the unresponsiveness of young Manchego lambs to H. contortus infections and a potentially serious shortcoming for any specific diagnosis system for ovine haemonchosis in recently weaned animals. However, and according to our

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6 months old which could be used, if purified, for the specific diagnosis of sheep haemonchosis. In addition, infected and challenged lambs (Group A) exhibited higher reactivity at the same Mw and a greater antigenic heterogeneity than uninfected and challenged animals (Group B ). Whether or not these higher reactivity levels, not clear in ELISA readings, are factual or represent a consequence of the more than likely inversion of the albumin/globulin ratio in twice-infected lamb (owing to albumin losses through the abomasal mucosa) deserves more research but, if confirmed, could be evidence of some immunological memory not previously demonstrated (Smith, 1977b) and which could be exploited for diagnosis. In particular the time course studies during the primary infections (by Western blotting) and the certain cross-reactivity with other commonly found gastrointestinal nematodes of sheep should be explored since there is evidence of shared antigens with some related (Anderson et al., 1989) and unrelated nematodes (Charley-Poulain et al., 1981; Shamansky et al., 1989). ACKNOWLEDGEMENTS

Excellent technical assistance by Susana M6ndez is acknowledged. M. Cuquerella was supported by a fellowship from the Plan Nacional de Investiga-

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ci6n and the research project received financial support from CICYT GAN89/ 324.

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