BOVINE IMMUNOGLOBULINS AND BRUCELLOSIS IV

BOVINE IMMUNOGLOBULINS AND BRUCELLOSIS IV. STUDY BY MICROCHROMATOGRAPHIC TECHNIQUE OF CLASS TYPES OF PERSISTENT ANTIBODIES FOUND IN HEIFERS VACCINATED 12 TO 14 MONTHS PREVIOUSLY WITH BRUCELLA ABORTUS STRAIN 19 D. Levieux To cite this version: D. Levieux. BOVINE IMMUNOGLOBULINS AND BRUCELLOSIS IV. STUDY BY MICROCHRO MATOGRAPHIC TECHNIQUE OF CLASS TYPES OF PERSISTENT ANTIBODIES FOUND IN HEIFERS VACCINATED 12 TO 14 MONTHS PREVIOUSLY WITH BRUCELLA ABORTUS STRAIN 19. Annales de Recherches Vétérinaires, INRA Editions, 1981, 12 (2), pp.193199. <hal 00901324> HAL Id: hal00901324 https://hal.archivesouvertes.fr/hal00901324 Submitted on 1 Jan 1981 HAL is a multidisciplinary 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.

BOVINE IMMUNOGLOBULINS AND BRUCELLOSIS IV. STUDY BY MICROCHROMATOGRAPHIC TECHNIQUE OF CLASS TYPES OF PERSISTENT ANTIBODIES FOUND IN HEIFERS VACCINATED 12 TO 14 MONTHS PREVIOUSLY WITH BRUCELLA ABORTUS STRAIN 19 D. LEVIEUX 1. N. R. A., Station de Pathologie de la Reproduction, 37380 Nouzilly, France Résumé IMMUNOGLOBULINES BOVINES ET BRUCELLOSE. IV. ÉTUDE DES CLASSES DES ANTI CORPS PERSISTANTS EXISTANT CHEZ DES GÉNISSES VACCINÉES PAR BRUCELLA ABORTUS 19, 12 À 14 MOIS AUPARAVANT. &horbar; La microchromatographie avec Biogel A 1.5 est proposée pour différencier les anticorps IgM ou IgG présents dans les liquides biologiques. Cette technique permet, avec seulement un matériel simple, une séparation complète des deux classes en deux heures avec moins de 1 ml de prélèvement. La technique appliquée à l étude des sérums des génisses vaccinées par B. abortus 19, 12 à 14 mois auparavant a montré que les anticorps persistants sont presque exclusivement du type IgM chez des animaux présentant un titre agglutinant de plus de 30 unités. Comme les sérums des animaux infectés chroniques sont généralement du type IgG, cette analyse pourrait permettre de mettre en évidence l origine infectieuse ou vaccinale des agglutinines résiduelles. Differentiation of low levels of brucella agglutinins arising from vaccination and those arising from infection is an unresolved problem. Agglutinins can persist several months after vaccination of calves or adult cows by B. abortus strain 19 and in general the older the animal at the moment of vaccination the more the agglutinins persist (Lambert et al., 1961 ; Cunningham and O Reilly, 1968). The study of classes of immunoglobulins responsible for agglutination could furnish the means by which this problem could be resolved if it were possible to demonstrate that 1 : present address Laboratoire des Maladies Nutritionnelles, CRVZ de Theix, 63110 Beaumont, France postvaccination and postinfection antibodies belonged to different classes. Numerous studies have followed the appearance of IgM and IgG class antibodies after vaccination of heifers by B. abortus strain 19. While most authors generally agree that IgM type antibodies appear first in the immune response, some believe that antibodies which persist in the following months are also IgM (Rose and Roepke, 1964; Renoux et al., 1971 a), or IgG (Rice et al., 1966 ; Rice et al., 1967 ; Amerault et al., 1961, 1962 ; Pilet and Toma, 1969 ; Beh, 1974). Since the wide range of techniques used by these authors could be responsible for most of the contradictory results observed it seemed opportune to reexamine this question by microchromatography since this permits a

Separation Sensitization IgG more rapid and reliable separation of IgG and IgM than techniques used previously. Materials and Methods 1. Cattle Nineteen brucellosis free Black and White Friesian heifers (Frisonne Pie Noire), imported from the Netherlands, were housed in a closed yard protected from any subsequent contamination by brucella ( Plommet et al., 1970). They received, at between 7 and 12 months old, a subcutaneous injection of 8 x 10 10 B. abortus strain 19. 2. Sera Sterile blood was collected from the jugular vein and allowed to clot for 24 h at room temperature. The exudate was centrifuged at 3 000 g for 20 min and stored at 20 C. 3. Serology Seroagglutination was carried out by the microtechnique of Renoux et al. (1971 bl. The antigen utilized (Roger Bellon) was standardised with respect to International Serum and results expressed in international units. 19 during gestation and having then aborted. The anti brucella antibodies are essentially IgG type (Levieux, 1974 b). Control «IgM» serum (365) : serum obtained from a cow vaccinated by B. abortus strain 19 two weeks previously. Antibrucella antibodies are quite exclusively IgM type. 5. and lgm titre in chromatography fractions measured by inhibition of hemagglutination of hen red cells 5 mg of a purified preparation of either bovine IgGl or of IgM were mixed with 10 ml of 20% suspension (in NaCl 0.15 M) of hen red cells prewashed three times in NaCl (0.15 M). One ml of glutaraldehyde (2.5%) was added drop by drop with stirring and the mixture left at room temperature for an hour. The red cells were then washed three times in NaCI (0.15 M), incubated for 1 hour with a 0.1 M solution of lysine and rewashed with NaCl (0.15 M). They were then resuspended in phosphate 4. of IgM and IgG by microchromatography Chromatography columns (30 x 1 cm) were made from bacteriological pipettes (25 ml) whose tips were blocked by a small plug of glass wool. They were filled with 25 ml of Biogel A1. 5, 200400 mesh (Biorad Laboratories), packed under 80 cm of hydrostatic pressure and washed with phosphate buffer (0.01 M, ph 6.9) in NaCI (0.15M). From 0.3 to 1 ml of serum was deposited at the buffergel interface by means of a syringe fitted with a needle whose point had been bent back 90, this prevents any disturbance of the gelbed when loading the sample. Separation was achieved by eluting the column with phosphate buffer (0.01 M, ph 6.9) in NaCl (0.15 M) at a rate of about 12 ml per hour with a peristaltic pump or Mariotte Flask (hydrostatic pressure of about 18 cm). Fractions of 0.6 ml were collected and their optical density at 280 nm measured spectrophotometrically. Control «IgG» serum (B1) : this serum is a mixture of twenty sera from cows vaccinated then infected by B. abortus strain

Separation Study buffer (200 ml, 0.01 M, ph 6.9) in NaCI, 0.15 M containing human serum albumin (2 g per litre) and sodium azide (1 g per litre). Titre From 0.025 ml, twofold dilutions of chromatography fractions or of control IgM or IgG1 preparations were carried out in the wells of microtiter plates ; 0.025 ml of a specific anti IgM or anti IgGl antiserum (Levieux, 1974 a) containing 5 to 10 hemagglutinating units were then added. The mixture was incubated for 10 min at room temperature and sensitized red cells (0.025 ml) were then added to each well. After allowing 20 to 30 min for red cells sedimentation, the last dilution to show a 50% inhibition was recorded. Results 1. of lggl and lgm by microchromatography Figure 1 shows the absorption at 280 nm of the eluate of bovine serum passed through a microcolumn. The positions of IgM and IgG as determined by inhibition of hemagglutination are also shown. It should be noted that crosscontamination of IgG by IgM and vice versa was less than 1 %. Figure 2 shows the pattern obtained with control IgG and [gm sera. The distribution of agglutinating activity in different fractions here again demonstrates the quality of separation. Chromatograms were run with sample quantities varying between 0.3 and 1 ml and with elution rates varying from 5 to 25 ml per hour. These higher values should not be exceeded if the quality of separation is to be maintained ; with a reasonable rate of between 12 to 15 ml per hour, separation can be completed in two hours. The same column can be used to separate twenty samples successively without any appreciable loss of efficiency. 2. of postvaccination agglutinins illustrates the agglutinating activity Table 1 of sera from 19 heifers vaccinated with B. abortus strain 19, 12 and 14 months before was carried out with hand. Seroagglutination the sera at the time of sampling (S.A. 1) and redone after chromatography (S.A. 2) ; that is after storage at 20 C for 8 months and thawing. While the titres in S.A. 2 are fre

Gradient Sephadex quently lower than those in S.A. 1, the difference never exceeds one dilution. The sera containing more than 30 international agglutinating units in the S.A. 1 series (9 out of 19, 12 months after vaccination ; 7 out of 14 at 14 months) were chromatographied. Serological activity in IgM and IgG fractions from analysis of 0.75 ml of serum was calculated using dilution factors (arising from the chromatography) of four for IgM and five for [gg. (The dilution factor was determined by hemagglutination inhibition and by the titre of serological activity of sera «IgM» and «IgG»). For all sera, agglutinating activity was present essentially in the IgM fraction. Only serum 305 shows a detectable activity in the IgG fraction, inferior nevertheless to its IgM fraction ; this serum is also the only one to show a slight complement fixing activity ( + + at 1/5) due no doubt to the activity of this IgG fraction. Discussion The microchromatography technique described permits a gentle, sensitive and rapid separation of serum IgM and IgG, with simple material and small sample volumes. This contrasts with classical techniques used to distinguish IgM and IgG antibody classes responsible for serological activity of serum : density ultrafiltration is a gentle and reliable technique, but is long, delicate and needs complex equipment. G200 chromatography in large columns of 100 x 2.5 cm is also a gentle and reliable technique, but complete separation requires more than 24 hours and the amount of sample required (at least 2 ml) is not always available. Titre of antibody activity before and after inactivation of IgM (heat inactivation, Rivanol

The Enzymelinked precipitation, reduction with mercaptoethanol) has the major inconvenience that it can only be used for sera in which the titre of IgM class antibodies is higher than those of the ]gg class (it is necessary to achieve a diminution of serological titre of at least one dilution for the technique to be significant). In addition, inactivation is not always specific for IgM and particularly in the case of mercaptoethanol different modes of treatment have been described for the serology of bovine brucellosis which give completely contradictory results (Levieux, personal results). technique of Nash et al. (1969), which is based on measurement by radial immunodiffusion of the immunoglobulin classes before and after absorption by the antigen has two major disadvantages : it can only be used for high titre sera : the accuracy of measurement being 5 to 10 percent, the minimum measurable in brucellosis affected cattle for example is 1 to 2 mg of IgG per ml and 0.1 to 0.2 mg of IgM per ml which corresponds to an agglutinating activity of more than 250500 units. Since serological activities of IgM and IgG at equal concentrations are very different one cannot therefore extrapolate from the titre by immunodiffusion to the contribution to serological activity. immunosorbent assay (ELISA) is a potential tool for serodiagnosis in bacteriology, virology and parasitology (reviewed by Piroird and Lombard, 1980) and has been evaluated for brucella antibodies by Byrd et al. (1979). As immunofluorescent assays, this technique is still very dependent upon the specificity of the antisera available and subject to competition for the antigen when there is too great an inbalance between the quantities of antibodies in each one of the different immunoglobulin classes. Most workers therefore prefer to separate IgM from IgG before testing. Protein A affinity chromagraphy has been investigated recently (Field et al., 1980) as a means of removing IgG from sera before testing for rubellaspecific IgM. Only 98% of IgG but also 5060% of IgM were removed. In the ruminant protein A reacts weakly with IgG (Lind et a/., 1970). Moreover, while approximatively 95% of the cow and sheep IgG2 bound to protein ASepharose, 75% of IgGl did not (Goudswaard et al., 1978). Consequently this technique cannot be applied to bovine sera. Results of analysis of the postvaccination residual antibody type in this report can really only be compared with those obtained by ultracentrifugation or G200 chromatography. They are hence in perfect agreement with the results of Rose and Roepke (1964) and Patterson et al. (19761. These authors demonstrated that the antibodies persisting 3 months after the vaccination of 8 month old heifers, with B19 vaccine were almost exclusively IgM. While it seems clear that the persistant antibodies postvaccination are IgM type it is difficult on the other hand to determine the nature of the antibodies present at low titre in chronically infected animals since it would be necessary to keep these animals on experiment for several years. Nevertheless judging from results from field observations one generally discerns that agglutinating antibodies tend to disappear in infected animals whereas complement fixing antibodies remain (O Reilly and Cunnigham, 1971) indicating the persistance of the subclass IgGI. Analysis of distribution of IgM or IgG antibodies in sera of animals with low serological titre can thus be a strong indication of the vaccination or infective origin of persistant agglutinins. Another promising approach has been investigated by Diaz et al. (1979). A brucella antigen containing polysaccharide but lacking smooth lipopolysaccharide was used in a radial immunodiffusion test. With this test, it would be possible to identify cattle infected with B. abortus in recently vaccinated herds which have high numbers of reactions to standard diagnostic tests. Accepted for publication, June 22nd, 1981 Summary Microchromatography with Biogel A 1.5 is proposed to distinguish IgM or IgG antibodies present in biological fluid. The technique allows, with only simple material, a complete separation of the two classes in two hours, with a sample volume of less than 1 ml.

The technique, applied to the study of sera from heifers vaccinated 12 to 14 months beforehand with B. abortus strain 19 demonstrated that the persistant antibodies were nearly exclusively of IgM type in animals retaining an agglutinating titre of more than 30 units. Since the sera of chronically infected animals are classically IgG type, this type of analysis could provide good evidence of vaccination or infection origin of residual agglutinins. References AMERAULT T.E., MANTHEI C.A., GOOD E.R., LAMBERT G., 1961. A heat inactivation test for differentiating specific and nonspecific agglutination reactions for bovine Brucellosis. Am. J. Vet. Res., 22, 564 569. AMERAULT T.E., LAMBERT G., MANTHEI C.A., 1962. The heat stability of Brucella agglutinins in bovine serum. Am. J. Vet. Res., 23, 10231026. BEH K.J., 1974. Quantitative distribution of Brucella antibody amongst immunoglobulin classes in vaccinated and infected cattle. Res. Vet. Sa:, 17, 14. BYRD J.W., HECK F.C., HIDALGO R.G., 1979. Evaluation of the enzymelinked immunosorbent assay for detecting Brucella abortus antibodies. Am. J. Vet. Res., 40, 896898. CUNNINGHAM B., 0 REILLY D.J., 1968. Agglutinin responses in blood serum and milk, following vaccination of cattle of various age with living S19 and killed 45/20 adjuvant Brucella abortus vaccines. Vet. Rec., 82, 678680. DIAZ R., GARATEA P., JONES L.M., MORIYON 1., 1979. Radial immunodiffusion test with a Brucella polysaccharide antigen for differentiating infected from vaccinated cattle. J. Clin. Microbiol., 10, 3741. FIELD P.R., SHANKER S., MURPHY A.M., 1980. The use of protein ASepharose affinity chromatography for separation and detection of specific IgM antibody in acquired rubella infection : a comparison with absorption by staphylococci containing protein A and density gradient ultracentrifugation. J. lmmunol. Meth., 32, 5970. GOUDSWAARD J., VAN DER DONK J.A., NOORDZIJ A., VANDAM R.H., VAERMAN J.P., 1978. Protein A reactivity of various mammalian immunoglobulins. Scand. J. Immunol., 8, 2128. LAMBERT G., AMERAULT T.E., MANTHEI C.A., GOOD E.R., 1961. Immunogenic response of calves vaccinated at different ages with Brucella abortus strain 19. Proc. 65th Annual Meeting United States Livestock Sanitary Association, 9399. LEVIEUX D., 1974a. Immunoglobulines bovines et Brucellose. I. Purification des immunoglobulines et pr6paration de leurs antis6rums spécifiques. Ann. Rech. V!t., 5, 329342. LEVIEUX D., 1974b. Immunoglobulines bovines et Brucellose. II. Activite des IgGI, IgG2 et IgM du serum dans les reactions d agglutination, de Coombs, de fixation du compi6ment et dans le test au Rose Bengale. Ann. Rech. V6t., 5, 343353. LEVIEUX D., 1978. Bovine immunoglobulins and Brucellosis. 3. Activity of IgGI, IgG2 and IgM versus different commercial batches of Rose Bengal antigen. Ann. Rech. Vet., 9, 489493. LIND I., LIVE I., MANSA B., 1970. Variation in Staphylococcal protein A reactivity with y Gglobulins of different species. Acta Path. Microbiol. Scand., 78, 673682. NASH D.R., HEREMANS J.F., 1969. A quantitative antibodybinding method for the determination of specific antibody within different immunoglobulin classes. Application to four Ig classes in the mouse. lmmuno%gy, 17, 685694. 0 RE!LLY D.J., CUNNINGHAM B., 1971. An assessment of Brucellosis card test. Vet. Rec., 88, 590594. PATTERSON J.M., DEYOE B.L., STONE S.S., 1976. Identification of immunoglobulins associated with complement fixation, agglutination, and low ph buffered antigen tests for Brucellosis. Am. J. Vet. Res., 37, 319324. PILET C., TOMA B., 1969. Sur I emploi du test au mercaptoethanol pour 1 6tude des agglutinines brucelliques. Rec. Méd. V!t., 145, 11551172. PIROIRD R., LOMBARD M., 1980. Les méthodes immunoenzymatiques et leurs applications s6rologiques. Rev. Méd. Vét., 131, 2542. PLOMMET M., RENOUX G., PHILIPPON A., LORENTZ C., GESTIN J., 1970. Brucellose bovine exp6rimentale. I. Comparaison de 1 efficacitb des vaccins B19 et H38. Ann. Rech. V!t., 1, 189201.

RENOUX G., PHILIPPON A., PLOMMET M., 1971a. Brucellose bovine experimentale. IX. Evolution des anticorps IgM et IgG chez des génisses primogestantes, vaccinées par vaccin vivant B19, vaccin inactiv6 H38 ou aprbs leur infection par Brucella abortus. Ann. Rech. V6t., 2, 173183. RENOUX G., PLOMMET M., PHILIPPON A., 1971b. Microréactions d agglutination et de fixation du compi6ment pour le diagnostic des Brucelloses. Ann. Rech. Vet., 2, 263269. RICE C.E., TAILYOUR J., COCHRANE D., 1966. Ultracentrifugal studies of sera from cattle vaccinated or sera from cattle vaccinated or naturally infected with Brucella abortus. Can. J. Comp. Med. Vet. Sci., 30, 270278. RICE C.E., ALEXANDER D.C., BARRETT B.B., 1967. Chromatographic studies of sera from calves vaccinated with Brucella abortus strain 19. Can. J. Comp. Med. Vet. Sci., 31, 114121. ROSE J.E., ROEPKE M.H., 1964. Physicochemical studies on postvaccinal Brucella agglutinins in bovine serum. Am. J. Vet. Res., 25, 325328.