IMMUNE RESPONSE OF SHEEP TO HAEMONCHUS CONTORTUS : SERUM ANTIBODIES AGAINST CROSS REACTING ANTIGENS BETWEEN PARASITES

Transcription

1 IMMUNE RESPONSE OF SHEEP TO HAEMONCHUS CONTORTUS : SERUM ANTIBODIES AGAINST CROSS REACTING ANTIGENS BETWEEN PARASITES Joëlle Charley, C. Bourdieu, G. Luffau, P. Pery To cite this version: Joëlle Charley, C. Bourdieu, G. Luffau, P. Pery. IMMUNE RESPONSE OF SHEEP TO HAEMONCHUS CONTORTUS : SERUM ANTIBODIES AGAINST CROSS REACTING ANTIGENS BETWEEN PARASITES. Annales de Recherches Vétérinaires, INRA Editions, 1981, 12 (2), pp <hal > HAL Id: hal Submitted on 1 Jan 1981 HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

2

3 IMMUNE RESPONSE OF SHEEP TO HAEMONCHUS CONTORTUS : SERUM ANTIBODIES AGAINST CROSS REACTING ANTIGENS BETWEEN PARASITES Joëlle CHARLEY C. BOURDIEU G. LUFFAU P. PERY Station de Recherches de Virologie, et d lmmunologie, lnra, Thiverval-Grignon, France Résumé RÉPONSE IMMUNOLOGIQUE DES OVINS À HAEMONCHUS CONTORTUS : ANTICORPS SÉRIQUES CONTRE DES ANTIGÈNES COMMUNS ENTRE PARASITES. - Lorsque des sérums normaux et des sérums de moutons infestés par H. contortus sont analysés par la technique ELISA contre des antigènes parasitaires à différents stades, une activité anticorps est détectée dans tous les cas. Les anticorps présents dans le sérum normal sont en partie éliminés après absorption par un extrait de vers adultes de Nippostrongylus brasiliensis. Les antigènes de N. brasiliensis et d H. contortus ont été comparés par cross immunoelectrophorèse en tandem à l aide d un sérum de lapin anti-n. brasiliensis : au moins sept antigènes communs ont pu être détectés. L extrait de vers adultes d H. contortus a été épuisé par le sérum de lapin anti-n. brasiliensis : après absorption tous les antigènes communs ont été éliminés, par contre il reste au moins un antigène qui réagit avec un sérum de lapin anti-h. contortus. Lorsque cet antigène est utilisé dans le test ELISA une faible activité anticorps est retrouvée dans le sérum normal. A multitude of cross reactions between parasites have already been related (Biguet et a/., 1962a, b ; Soulsby, 1963 ; Capron et al., 1968 ; Pery and Luffau, They have been detected by a variety of serological test using various antigen preparations (Soulsby, 1962). Studying immune response of sheep to H. contortus we observed that extracts of the parasite react with sera from both normal and infected animals. The following studies were designed to elucidate this reaction with the aim to abolish it. In this paper we demonstrate that activity in normal serum was due to antibodies directed, mostly, against cross-reacting antigens between parasites. Materials and Methods Sheep Lambs of the Prealpes du Sud breed (from the INRA farm at Brouessy) were reared indoors under N, contortus free conditions. Infective larvae The strain of H. contortus maintained in the Institute for several years was a gift from

4 G.M. Urquhart and E.W. Allonby of Glasgow Veterinary School. the Larval antigens We have used : * From H. contortus : L3 infective larvae, a mixture of L3 - QL obtained from larvae cultured in vitro, adult worms and eggs. * From N. brasiliensis : adult worms. Soluble antigens were prepared by grinding frozen parasites in phosphate buffered saline ph : 7.5. The suspension was cleared by centrifugation at g, 30 min. Each preparation was estimated for protein concentration (Bradford, 1976). Sera Blood samples were collected from the jugular vein, left to clot and the serum harvested after centrifugation. Anti-ovine immunoglobulin antiserum It was prepared by injecting a mixture of L and H y chains (Franek, 1961) in rabbits. Tested in an ELISA test, this antiserum reacts against the three immunoglobulin classes : IgG, IgA, IgM. Assay for anti-h. contortus antibodies Serum antibodies against the different stages of the parasites were studied using an enzyme linked immunosorbent assay ELISA (Engvall, 1972). Sequential incubations, for 3 h at 37 C or overnight at room temperature of 100 gl of the following reagents were carried out : 1) Antigen dissolved in 0.1 M sodium carbonate buffer ph 9.6, concentration selected after preliminary tests were 0.02 mg/ml. 2) Serum diluted in PBS 0.05 M ph 7.4 were added to duplicate of each antigen preparations. 3) Anti IgG diluted 1/100 in PBS. 4) Pig anti-rabbit immunoglobulin conjugated with alkaline phosphatase by the method of Avrameas and Ternynck (1971). Each incubation was followed by washing in saline solution. Each buffer contained 0.02% sodium azide and after antigen washing 0.05% Tween 20. Alkaline phosphatase was revealed by adding 200 pl of P-nitrophenyl phosphate (1 mg/ml) dissolved in 0.05 M Sodium carbonate buffer ph : 9.8 containing 10-3 M MgC6 and measurement of the optical density at 400 nm. Isolation of sheep lgg and Fab fragments Immunoglobulins from normal serum were prepared by adding ammonium sulfate at 40% saturation. The precipitate found was dialysed and applied on a DEAE cellulose column. A mixture of IgG, and IgG 2 was eluted at 0.03 M ph 8 (Lisowski, 1975). Fab and Fc fragments were obtained by the technique described by Levieux ( Anti-N. brasiliensis and anti-h. contortus antisera They were prepared by injecting into rabbits N. brasiliensis and H. contortus adult worms antigens. Rabbit anti-n. brasiliensis was insolubilized by the Avrameas (1969) technique. Tandem cross immunoelectrophoresis (T-CIEP) analyses were performed according to the procedure of KrØIl ( Results Presence of ELISA activity in normal serum against H. contortus antigens In an ELISA test, normal and infected serum were tested against different stages of parasites (fig. 1). In all cases a clear positive reaction was detected. The activity is due to antibodies Fab and Fc fragments of IgG were purified from normal serum and tested against adult worm antigens (fig. 2). Positive activity was found in normal serum and Fab fragments but not in Fc fragments and foetal calf serum. Absorption of sheep sera with N. brasiliensis antigens Normal and infected sera were absorbed with an immunoadsorbent prepared by insolubilizing N. brasiliensis adult worm extract and tested against H. contortus adult worm (fig. 31. After this absorption antibody activity was considerably reduced both in normal and infected serum. Cross reacting antigens between N. brasiliensis and H. contortus N. brasiliensis and H. contortus adult worm

5 antigens were at the same time analysed against an anti-n. brasiliensis rabbit serum in a tandem cross immunoelectrophoresis. A minimum of seven fused precipitins peaks, corresponding to common antigens between the two parasites were formed (fig. 4). Elimination of common antigens The insolubilized anti N.brasiliensis rabbit serum was used to absorb H. contortus adult worm extract : this absorption appears to eliminate all common antigens. However, at least one antigen remains when tested against an anti H. contortus serum (fig. 5). When absorbed H. contortus antigen is used in ELISA system. Antibody activity in normal and infected serum is considerably reduced when compared to the test realized with unabsorbed antigen (fig. 6). Discussion When studying humoral immune response anti-h. contortus in an ELISA system, positive activity was detected in infected sera as well as in normal sera. This activity was revealed with all stages of the parasites. These results are not consistent with Smith s data (1977)

6

7 who found no activity in uninfected serum using a mixture of third and fourth stage H. contortus larvae as antigen. With the aim to eliminate this high background in normal sera, we have looked for its nature. The activity of normal serum is found in Fab fragments but not Fc fragments, indicating that antibodies are responsible for this phenomenon and not unspecific immunoglobulin fixation on a parasitic Fc IgG receptor, like in the case of Schistosoma mansoni (Torpier, Absorption of normal and infected serum by N. brasiliensis adult worm antigen reduced very much antibody activity. This indicates that most antibodies are directed against common antigens between parasites. Antigenic relationships between N. brasiliensis and H. contortus were analysed with anti-n. brasiliensis rabbit serum by TCIEP. At least seven common antigens to these parasites have been detected. This cross reacting phenomenon has been used to prepare a more specific H. contortus antigen. After absorption of H. contortus antigen by anti-n. brasiliensis rabbit serum common antigens between H. contortus and N. brasiliensis were eliminated. On the contrary at least one antigen is detected by an anti H. contortus rabbit serum. In ELISA test, using the absorbed antigen, very little activity was detected in normal serum. Nevertheless a light background is maintained. This was probably due to cross reacting antigens with other parasites. Indeed it is not possible to ascertain that sheep have always been free of any other parasite than H. contortus. Accepted for publication, May l2th, Summary Normal and H. contortus infected sera were studied by ELISA technique against different stages of the parasites. In all cases antibody activity was detected. This activity in serum is partially eliminated after absorption with an adult worm extract of N. brasiliensis. N. brasiliensis and H. contortus antigens were analysed by TCIEP with a rabbit anti-n. brasiliensis serum to examine shared antigens of H. contortus. A minimum of seven cross reacting antigens were detected. H. contortus adult worm extract was absorbed by the rabbit anti-n. brasiliensis serum. After absorption all cross reacting antigens were removed but at least one antigen reacting with a rabbit serum anti-h. contortus is maintained. When this antigen is tested in ELISA technique only a weak antibody activity is found in normal serum. References AVRAMEAS S., TERNYNCK T., The cross-linking of protein with glutaraldehyde and its use for the preparation of immunosorbents. lmmunochemistry, 6, 57. AVRAMEAS S., TERNYNCK T., Peroxidase labelled antibody and Fab conjugates with enhanced intracellular penetration. lmmunochemistry, 8, BIGUET J., ROSE F., CAPRON A., TRAN VAN K.Y.P., Contribution de I analyse immunoelectrophoretique a la connaissance des antig6nes vermineux. Incidence pratique sur leur standardisation, leur purification et diagnostic des Helminthiases par immunoelectrophorese. Rev. lmmunol. Paris, 29, BRADFORD M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, CAPRON A., BIGUET J., VERNES A., AFCHAIN D., Structure antig6nique des helminthes. Aspects immunologiques des relations h6te-parasite. Pathol. Biol., 16, ENGVALL E., PERLMANN P., Enzyme-linked immunosorbent assay, ELISA. III Quantitation of specific antibodies by enzyme labelled anti-immunoglobulin in antigen coated tubes. J. Immunol., 109,

8 FRANEK F., Dissociation of animal 7Sy globulins by cleavage of disulphide bonds. Biochem. Biophys. Res. Commun., 4, KRØLL J., A manual of quantative immunoelectrophoresis. Scand. J. Immunol., 2, LEVIEUX D., Immunoglobulines bovines et brucellose. I. Purification des immunoglobulines et pr6paration de leurs antis6rums spécifiques. Ann. Rech. VEt., 5, LISOWSKI J., JANUSZ M., BRONISLAW T., MORAWIECKI A., GOLAB S., BIALKOWSKA H., Immunoglobulins of colostrum. III Comparative studies of ovine serum and colostral IgG, and IgG 2. lmmunochemistry, 12, 159. PERY P., LUFFAU G., Antigens of helminths. /n : SELA M., The antigens, 5, 83. Academic Press, New York. SMITH W.D., Antilarval antibodies in the serum and abomasal mucus of sheep hyperinfected with Haemonchus contortus. Res. Vet. Sci. 22, 334. SOULSBY E.J.L., The nature and origin of the functional antigens in helminth infections. Ann. N. Y. Acad. Sci., 113, 492.