Sep 18, 1995 - Lynette B. Corbeil, Ronald P. Gogolewski, Imre Kacskovics, Klaus H. Nielsen, Robert R. Corbeil,. James L. Morrill, Richard Greenwood, and ...
Bovine IgG2a Antibodies to Haemophilus somnus and Allotype Expression Lynette B. Corbeil, Ronald P. Gogolewski, Imre Kacskovics, Klaus H. Nielsen, Robert R. Corbeil, James L. Morrill, Richard Greenwood, and John E. Butler
ABSTRACT
gests IgG2a may be more protective than IgGl. To examine this further, the peptide specificity of these IgGl and IgG2a antibodies to p40 was examined. After limited proteolysis of p40, IgG2a antibodies reacted with 2 peptides not recognized by IgGl antibodies. Other peptides were recognized by both isotypes. Since these studies suggested that IgG2a may be important in protection against infection, we then investigated some aspects of the role of the 2 IgG2a allotypes, Al and A2. In retrospective studies of age differences in expression of IgG2a allotypes, no heterozygotes were detected in calves of 60 d old or less, and fewer heterozygotes were detected in calves 61-120 d old than in cattle older than 270 d (P < 0.01). In a subsequent prospective study of the time course of allotype expression, Holstein calves shown to be heterozygotes expressed the IgG2aAl allotype early but the IgG2aA2 allotype was not usually detected until 3 to 4 mo of age. Thus, both the retrospective and the prospective studies showed age related differences in expression of the IgG2aAl and A2 allotypes. This could have implication in protection.
Bovine IgG2a has been implicated in protection against pyogenic infections, including those caused by Haemophilus somnus. To further investigate the role of IgG2a in defense against H. somnus, IgGl and IgG2a antibodies were purified from antiserum against an immunodominant 40 kDa outer membrane protein (p40) of H. somnus, which was previously shown to passively protect calves against H. somnus pneumonia. The passive protective capacity of anti-p40 IgGl or IgG2a was evaluated in vivo in calves. Purified anti-p40 IgGl or IgG2a was incubated with H. somnus for 15 min before intrabronchial inoculation of calves. Bacteria incubated with anti-p40 IgGl or IgG2a were inoculated into one caudal lung lobe and bacteria incubated with IgGl or IgG2a from the respective preimmunization serum were inoculated into the contralateral lobe. The volumes of pneumonia in the right and left lungs were determined 24 h later. The difference in volume of pneumonia with H. somnus preincubated in IgGl pre- and postimmunization anti p40 was less (16 cm3, P = 0.298) than the difference in volume of pneumonia with H. somnus preincubated in IgG2a pre- and RESUME postimmunization anti p40 (30 cm3, Les immunoglobulines bovines P = 0.146). Although the differences in lesion size between pre- and pos- IgG2a ont ete impliquees dans la timmunization serum were not sta- protection contre les infections de tistically significant, the trend sug- nature pyogene, incluant celles
causees par Haemophilus somnus. Afin d'etudier un peu plus le role protecteur des IgG2a contre H. somnus, des anticorps IgGl et IgG2a furent purifies a partir de serums immuns, prealablement detmontre's comme protegeant passivement des veaux contre la pneumonie causee par H. somnus, dirige contre une proteine de 40 kDa (p40) de la membrane externe d'H. somnus. La capacite protectrice des IgGl et IgG2a anti-p40 fut evaluee in vivo chez des veaux. Des IgGl ou IgG2a anti-p40 purifies furent incubes durant 15 min avec H. somnus avant que ce dernier soit inocule par voie intrabronchique a des veaux. Les bacteries incubees avec les IgGl ou IgG2a anti-p40 furent inoculees dans un des lobes caudaux et les bacteries incubees avec des IgGl ou IgG2a provenant des serums respectifs mais preleves avant immunisation furent inocule'es dans le lobe contralateral. Le degre de pneumonie dans les poumons gauche et droit furent determines 24 h plus tard. La difference dans le volume de poumon atteint de pneumonie causee par H. somnus pre-incube avec les IgGl obtenues pre- et post-immunisation avec p40 etaient moindres (16 cm3, P = 0,298) que la difference de volume de pneumonie obtenue avec H. somnus pre-incube avec les IgG2a preet post-immunisation avec p40 (30 cm3, P = 0,146). Bien que les differences dans l'etendue des lesions observees soient statistiquement
Department of Pathology, University of California, 200 West Arbor Drive, San Diego, California 92103-8416 USA (Corbeil); P.O. Box 135, Ingleburn, NSW 2565, Australia (Gogolewski); Department of Microbiology, University of Iowa, Iowa City, Iowa 52242 USA (Kacskovics, Butler); Animal Disease Research Institute, Agriculture and Agri-Food Canada, 3851 Fallowfield Road, Nepean, Ontario K2H 8P9 (Nielsen); Mathematics and Computer Science Department, University of San Diego, Alcala Park, San Diego, California 92110 USA (Corbeil); Department of Animal Sciences and Industry, Kansas State University, Manhattan, Kansas 66506-1600 USA (Morrill, Greenwood). Address correspondence to Dr. L.B. Corbeil. This work was supported in part by USDA grant number 92-37204-8108 and by USDA Formula Funds for Animal Health Disease Research from the University of California, Davis. Received September 18, 1995.
Can J Vet Res 1997; 61: 207-213
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non-significatives, la tendance indique que les IgG2a protegeraient plus que les IgGl. Une etude subsequente fut entreprise dans le but d'examiner la specificite des IgGl et IgG2a anti-p40. Suite 'a une proteolyse limitee de p40, les anticorps IgG2a reagissaient avec deux peptides non reconnus par les anticorps IgGl. D'autres peptides etaient reconnus par les deux isotypes d'anticorps. Etant donne le fait que ces etudes suggerent un role protecteur important contre l'infection par H. somnus pour les IgG2a, quelques aspects du role des deux allotypes des IgG2a, Al et A2 furent e'tudies. Dans des etudes retrospectives sur les differences d'age quant a l'expression des allotypes d'IgG2a, aucun animal heterozygote ne fut detecte chez les veaux ages de 60 j ou moins, et moins d'heterozygotes furent detectes chez les veaux de 61-120 j que chez les animaux ages de plus de 270 j (P < 0,01). Lors d'une etude prospective subsequente sur la chronologie de l'expression allotypique, les veaux de race Holstein heterozygotes exprimaient tot l'allotype IgG2aAl alors que l'allotype IgG2aA2 n'etait pas detecte avant l'age de trois ou quatre mois. L'analyse retrospective, de meme que l'analyse prospective, montrent qu'il y a une difference dans l'expression des deux allotypes des IgG2a selon l'age de l'animal. Ceci pourrait avoir des implications au niveau de la protection chez les animaux. (Traduit par docteur Serge Messier)
INTRODUCTION The role of the IgG subisotypes in protection against infection is not generally well understood. Although immune defense against any infection is complex, it has been well known for many years that humoral immunity is most important for protection against extracellular pathogens. In cattle, IgG2a appears to be most important in defense against pyogenic extracellular microbes. Over 20 y ago, Nanssen (1) found that Red Danish milk cattle deficient in IgG2a were more susceptible than other cattle to pyogenic infections. Since then, sev208
eral laboratories have shown IgG2a antibodies to be most important in protection against bovine (2,3,4,5,6, 7,8,9,10) and ovine (11,12,13,14) pyogenic infections. Because of the importance of the IgG2a response in protection, we addressed mechanisms involving this response in Haemophilus somnus infection. This gram-negative bacterium causes pneumonia, septicemia, meningoencephalitis, abortion, arthritis and myocarditis in cattle (10,15,16,17,18,19). In our previous studies, the IgG2a response was implicated in protection against H. somnus disease for several reasons. Cattle with the lowest IgG2a titers to H. somnus before challenge were most susceptible to disease (10). Also, of all the immunoglobulin isotypes, IgG2a antibody titers specific for H. somnus increased the most after infection and persisted longest (9,10). In passive protection experiments, antiserum specific for a 40 kDa (p40) H. somnus surface antigen with high titers of both IgGI and IgG2a protected calves against pneumonia, whereas antiserum with predominantly IgGI antibodies against another surface antigen did not protect (3). The latter result could have been due to subclass differences, differences in epitope specificity or affinity, or a combination thereof. The importance of IgG2a in protection against this extracellular bacterial infection is consistent with work on other bovine and ovine extracellular infections reported within the last 2 to 3 decades (1,2,4,5,6,7,8,11,13,14). Protection by IgG2a could be the result of its antigen specificity and/or differences in Fc-mediated functions. The latter is complex for IgG2a since 2 allotypes are known which differ structurally (20,21). These differences are such that functional differences in the IgG2a between these allotypes could be expected. The purpose of this project was to study some roles of IgG2a in H. somnus infection. This was done by comparing passive protection with IgGI vs IgG2a antibodies to the H. somnus 40 kDa OMP (p40) and by analyzing the specificity of IgGI and IgG2a antibodies to p40. We further postulated that the 2 allotypes of IgG2a (Al and A2) that are encoded by 2 codominant alleles (22,23,24), could be
related to disease susceptibility if the function of the allotypes differed. This hypothesis was relevant because of the observation that some cattle in a feedlot or veal calf unit are more susceptible than others during outbreaks of pyogenic infection (such as pneumonia). The hypothesis was further supported by data indicating that certain human IgG2 allotypes are correlated with susceptibility to infection with extracellular bacteria such as Haemophilus influenzae, Streptococcus pneumoniae and Neisseria meningitidis (25). (The nomenclature of immunoglobulin is such that the IgG2a of cattle and IgG2a of humans are not necessarily homologous proteins. Nomenclature depends on order of discovery, relative concentration and electrophoretic mobility. Bovine IgG2a is structurally quite distinct from any human IgG subclass, including IgG2.) To begin to address this issue we examined allotype expression retrospectively in cattle of different ages and prospectively in young calves from birth to 8 mo of age. In this paper, we show that IgG2a antibodies to p40 tend to passively protect against H. somnus pneumonia better than IgG1 antibodies to p40 and that these IgG2a antibodies react with more peptides of p40 antigen than do the IgGI antibodies. We also show that allotype expression appears to be age related.
MATERIALS AND METHODS BACTERIAL CULTURE
Haemophilus somnus was grown on blood agar plates with 5% bovine blood in Brain Heart Infusion (BHI) Agar (Difco Laboratories, Detroit, Michigan, USA) or in BHI broth (Difco) with 1% Trizma base (Sigma Chemical Co, St. Louis, Missouri, USA) and 0.01% thiamine monophosphate (Sigma). Unless otherwise noted, H. somnus strain 2336 was used. This strain was obtained from a pneumonic lung of a veal calf and has been used to produce pneumonia in calves (3,16,26,27). ANTIBODIES TO H. somnus
The same monospecific antiserum to p40 that we used to show protection against H. somnus pneumonia (3)
was used for purification of monospecific IgGI and IgG2a antibodies to p40 in the studies reported here. This antiserum was from an animal of the Al/A2 IgG2 allotype immunized with gel purified p40 (3). Anion exchange fractions of the antiserum or preimmunization serum from the same calf were evaluated for immunoglobulin profile by dot blot using monoclonal antibodies to bovine IgG1, IgG2a and IgM (DAS16, DAS2 and DAS11 from Dr. Al Guidry, Beltsville, Maryland, USA) on separate blots. (The monoclonal used (DAS-2) recognizes only IgG2a but does not distinguish between IgG2a allotypes (31). The lack of bias for allotype was confirmed by ELISA of bovine anti H. somnus antibody of known allotype with H. somnus antigen on the solid phase [unpublished data].) Fractions containing only IgGI (no IgG2a or IgM) or only IgG2a (no IgGI or IgM) antibodies to p40 were pooled, dialyzed, lyophilized and the protein content of each pooled fraction determined. Based on our previous studies of protection with antiserum to p40 (3), we calculated that -34 mg of IgGI and -30 mg of IgG2a were used in the passive protection experiment using unfractionated antibodies. Since some denaturation of antibody may have occurred during isotype purification and concentration by lyophylization, we decided to use about twice that amount in these passive protection experiments (i.e. -65 mg per calf). Therefore 107 H. somnus bacteria were incubated in vitro for -15 min with 65 mg of IgGI or IgG2a from preimmunization serum or from serum collected after immunization with p40. This was a subagglutinating level. Fiberoptic bronchoscopy was used to infect 4 calves with 107 strain 2336 H. somnus plus preimmune IgGI on 1 side and 107 H. somnus plus postimmune IgG1 (antip40) on the other side of the lung. We had previously shown that the 2 mL inoculum size did not result in spill over to the other side of the lung (3,26). In another group of 4 calves, pre- and postimmune IgG2a was used. Calves were killed at 24 h, bronchial lavage fluid was collected for cultures and lesion size was determined by computer assisted image analysis as we previously reported (3,16,26,27).
POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE) AND WESTERN BLOTTING
Haemophilus somnus whole cells (at 108 cells per lane) or proteins were analyzed by SDS-PAGE as described (3) using 10% polyacrylamide in separating gels with 5% polyacrylamide in the stacking gels unless otherwise described. Separated proteins were lightly stained with Coomassie blue or electroblotted to nitrocellulose as previously described (3). The nitrocellulose strips were probed with the bovine antiserum to p40 described above. Antigen-antibody reactions were detected with peroxidase conjugated rabbit anti-bovine IgG (ICN Biomedicals, Lisle, Illinois, USA) and developed with 4-chloro-1-naphthol and hydrogen peroxide as previously described (3,15,26).
and Perry Laboratories Inc, Gaithersburg, Maryland, USA). A positive control 2nd antibody consisted of anti-bovine IgG conjugated to peroxidase as described above. IgG2a ALLOTYPING
Bovine sera from beef and dairy calves between 30 and 60 d of age, or 61 and 120 d of age and from beef and dairy cattle greater than 270 d of age were collected and stored at -20°C. One sample from each animal was available for retrospective studies. A 2nd study was done prospectively with serial plasma samples from 39 Holstein heifer calves in the dairy herd at Kansas State University. These calves were born in a maternity barn, remained there until 3 (+ 1) d of age and then were moved to individual hutches until they were 8 wk old. Calves received 1 to 2 quarts of their LIMITED PROTEOLYSIS dam's colostrum soon after birth, The H. somnus 40 kDa OMP anti- unless the dam was positive for gen was subjected to limited proteoly- bovine leucosis, in which case the sis as described by Josefsson and calves received stored colostrum from Randall (28) and modified for H. som- a leucosis-negative cow. Second nus surface proteins (29) in order to milking transition milk was fed on the determine which peptides reacted 2nd day after birth and whole milk with IgG1 or IgG2a antibodies raised was given on the 3rd day. While in to p40. In brief, the 40 kDa OMP was hutches, calves were given calf starter cut from SDS-PAGE gels of H. som- and water free choice. Whole milk nus proteins lightly stained with was fed twice a day until the calves Coomassie blue. The excised band were 3 wk or older. After 8 wk the was placed in the wells of a second calves were comingled and fed the SDS-PAGE gel along with 0.1 ,ug of same diet. Blood was collected Staphylococcus aureus V8 protease approximately 48 h after birth, then (Sigma) and electrophoresed into the weekly until 8 wk and then monthly stacking gel. Electrophoresis was until 8 mo of age. Plasma was stored stopped for 45 min to allow digestion at -20°C until all samples could be in the stacking gel and then restarted allotyped together. The IgG2a allotype of each serum to separate the peptide digestion products in the separating gel. Peptides sample in the 1st study was deterwere electroblotted to nitrocellulose, mined using a polyclonal rabbit antiprobed with antiserum to the 40 kDa IgG2a (84-y2) which distinguishes OMP and with second antibodies con- between Al/Al; A1/A2; and A2/A2 sisting of either murine monoclonal animals in a single immunodiffusion antibody to bovine IgGI (DAS 16, assay (30,3 1). Briefly 40 ,uL of 84b-y2 kindly provided by Dr. Al Guidry, was added to the central well of an Beltsville, Maryland, USA) or to Ouchterlony double diffusion plate bovine IgG2a (Sigma). Specificity of (25 mm Petri dish) and the surroundthe monoclonal antibodies was ing wells filled with 4 ,uL of test sera demonstrated since each reacted with diluted 1:20. Samples containing the target immunoglobulin (bovine IgG2a(A1) form a separate precipitin IgGI or IgG2a respectively) but not line than those containing IgG2a the untargeted Ig in immunodots. lacking the allotype. Heterozygotes Murine monoclonals were detected produce 2 precipitin lines. This with a 3rd layer consisting of goat method is described and illustrated in anti-mouse IgG and IgM conjugated more detail elsewhere (32). Plasma to horseradish peroxidase (Kirkegaard collected in the 2nd study were 209
TABLE I. Passive protection of calves with IgGl or IgG2a antibody to H. somnus p40 antigen Passive Mean lesion size (cm3) antibody Decreaseb Postimmunea n Preimmunea to p4O0 16 31 47 4 IgG 1 30 27 4 57 IgG2a Purified IgGI or IgG2a antibodies from serum collected before (preimmune) and after (postimmune) immunization with gel purified p40 from H. somnus were preincubated with H. somnus 15 min before intrabronchial inoculation. Lesion size was determined 24 h later bDecrease in lesion size on the side treated with postimmunization IgGl or IgG2a (i.e. difference between treated and control sides of the lung)
TABLE II. Distribution of IgG2a allotypes by age of animals Number of animals (Percentage) in each age group IgG2a > 270 d 61-120 d 38-60 d Allotype Detected 81 (50%) 13 (59%) 17 (74%) Al 61 (37%)5 (23%)0 (0%)a A1&A2 21 (13%) 4(18%) 6(26%) A2 163 22 23 Total aThe trend toward greater expression of heterozygosity with age was statistically significant
(P
