Role of antibody to extracellular proteins of Rhodococcus equi in protection against R. equi pneumonia in foals

Rhodococcus equi produces two exoenzymes (REE), a cholesterol oxidase in large amounts and a phospholipase C, which cause lysis of sheep red blood cells (SRBC) sensitized with Staphylococcus aureus beta toxin. Two immunization studies were done in foals to determine the role of antibody to REE in protection against R. equi pneumonia. In the first study, three foals (mean age 10 days) were vaccinated four times at 2-week intervals with over 1 million units of partially purified exoenzymes (PREE). In the second study, three foals (mean age 19 days) were administered plasma from an adult horse vaccinated with PREE. Relatively low titres (16-32) of neutralizing antibody were detected in the foals of the former group, and passive transfer of neutralizing antibody (titres 32-64) occurred in the latter. Following immunization, principal foals and an equal number of similarly aged nonimmunized foals were challenged by aerosol with 1 x 10(10) live R. equi per day for 5 consecutive days. No severe clinical pneumonia developed in either group and, with one exception, only minor and resolving lung abscesses developed in these foals. These studies showed that antibody response of foals to immunization with PREE was poor, antibody to PREE did not prevent foals from developing lung abscesses following experimental infection, and that foals even as young as 3 weeks of age may be largely refractory to aerosol challenge with virulent R. equi.     

Purification of the phospholipase D of Corynebacterium pseudotuberculosis by recycling isoelectric focusing

Recycling isoelectric focusing was investigated as a method for purification of phospholipase D (PLD) from cultures of Corynebacterium pseudotuberculosis. Supernatant fluids from cultures of equine isolate 155 in brain-heart infusion broth were dialyzed against distilled water, concentrated by lyophilization, and fractionated by preparative isoelectric focusing in free solution in a pH 3 to 13 gradient with 6M urea. Protein concentration, pH, and PLD activity of the 10 resulting fractions were determined. Two PLD activity assays were used: release of 14C choline from labeled sphingomyelin and synergistic hemolytic activity with Rhodococcus equi factors. Enzyme activity focused in 2 fractions at pH 8.5 to 9.8. The synergistic hemolytic assay was simple and rapid for detecting PLD in partially purified fractions. Electrophoretic examination of the fraction containing the highest concentration of PLD activity revealed protein bands at 14, 21, and 31.7 kD Mr, suggesting purification to near-homogeneity. Proteins from the 31.7-kD band were labeled by antibodies in serum from a goat with chronic C pseudotuberculosis infection.     

Two techniques for detection of antibodies against Corynebacterium (Rhodococcus) equi in horse sera

Two techniques were developed to detect antibodies against the exosubstance of C. equi called equi-factor. In the first technique serum samples are tested against native equi-factor produced by the growth of C. equi on agar medium. A positive result is manifested by the development of precipitation lines. The second test is based on neutralization of prepurified equi-factor by antibody, resulting in the inhibition of its hemolytic synergism with staphylococcal beta toxin. Sera (125 samples) from horses of different ages, kept in localities with a history of C. equi infections, were examined. The first technique detected 65.6%, and the second 40% of positive cases.     

Defences of the bovine mammary gland against infection and prospects for their enhancement

The equine alternative complement pathway has been partially characterized and compared to the equine classical activation pathway. A dose-dependent lysis of RbRBC was observed with peak lytic values noted within 10 minutes at 37°C when rabbit red blood cells (RbRBC) were used as an alternative pathway activator. Sheep red blood cells (SRBC) sensitized with rabbit hemolysin or partially purified equine IgM antibodies were equally sensitive to lysis. Dilution of the commercial hemolysin by $\text{1}\text{5}$ reduced lysis from 90% to 38% in the presence of constant cell numbers. Hemolysis of SRBC peaked at 10 minutes and the majority of lysis occurred within 10 minutes. Dilution of equine sera by as little as $\text{1}\text{5}$ decreased hemolytic activity for SRBC to 21.5% from greater than 90% with undiluted sera. The alternative pathway protein, equine factor B, was tested using RbRBC and monitored by its differential susceptibility to heat treatment at 50°C. This treatment led to almost complete inactivation after a 15-minute incubation. An apparent heat-dependent decay of certain classical pathway components was also observed after 50°C treatment. This sensitivity was indicated by a reduction in the lytic activity for sensitized SRBC. Treatment for 15 minutes at 56°C with either RbRBC or SRBC was sufficient to abolish hemolytic activity in all equine sera tested. Chelation of cations with 0.04 M EDTA blocked expression of alternative and classical pathway activation; however, chelation of Ca⁺⁺ ions with 10 mM EGTA containing 1 mM Mg⁺⁺ ions permitted lysis of the RbRBC but not the SRBC. A dose-related Mg⁺⁺-ion dependence for RbRBC hemolytic activity was observed as the concentration of Mg⁺⁺ was increased to 1.0 mM. In addition, our results obtained with pre-colostral foal serum strongly suggest that natural antibody to RbRBC was of little importance in the lysis observed with these cells. These results also show that the equine alternative pathway activation may require Ca⁺⁺ ions. If Ca⁺⁺ ions are required, the equine alternative pathway is quite different from any other mammalian complement system so far described. Our results suggest that the alternative pathway of activation is of major importance in the equine complement system. Confirmation of this hypothesis requires both purification of the components involved as well as further characterization.     

Hemolytic interactions of Dermatophilus congolensis.

The strains of Dermatophilus congolensis grew on blood agar with washed sheep erythrocytes with marked total hemolysis. In testing for hemolytic interactions they gave a significant synergistic effect of a characteristic shape with Rhodococcus equi and Streptococcus agalactiae, whereas with Staphylococcus aureus producing beta hemolysin and with Staphylococcus aureus producing delta hemolysin a simultaneous synergistic as well as antagonistic effect were observed. First of all a conspicuous inhibition of in the beta hemolysin zone began and then the hemolytic effect of D. congolensis was enhanced. A similar double reaction was also observed with Listeria ivanovii. With delta hemolysin there was an inhibition of the hemolytic effect of D. congolensis and at the same time a synergistic effect could be observed. Also D. congolensis gave a weak synergistic effect with Micrococcus lylae and Listeria monocytogenes, and a further weak antagonistic effect with alpha hemolysin of Staphylococcus aureus, Staphylococcus hyicus, Staphylococcus chromogenes and Micrococcus luteus. No interaction of D. congolensis was established with Corynebacterium pseudotuberculosis.     

A serologic method for the detection of Corynebacterium pseudotuberculosis infections in horses

A serologic technique useful for detecting antibodies formed in horses in response to infection with Corynebacterium pseudotuberculosis is described. The test relies on the ability of C. pseudotuberculosis toxin to produce a wide zone of hemolysis when applied to erythrocytes previously treated with a sterile filtrate of Corynebacterium equi broth culture. The synergistic hemolytic activity can be neutralized by anti-C. pseudotuberculosis serum. This test was used to analyze sera from 616 horses for the presence of C. pseudotuberculosis antitoxin. Of 177 animals (see Table 2) found positive, there were 34 horses with bacteriologically confirmed, active infections and 18 with active but unconfirmed infections. In addition, 13 animals had a history of having had the disease and 112 had no history or evidence of having had the infection. The other 439 horses had negative titers. Statistical treatments confirmed the value of the test as an epidemiological tool but precluded using only titers for the diagnosis of active clinical disease.     

Toxigenic characteristics of Clostridium perfringens type C in enterotoxemia of domestic animals.

Eleven Clostridium perfringens type C strains isolated from fatal cases of hemorrhagic enterotoxemia of Canadian calves, a piglet, and a foal were studied for the production of soluble antigens. All the isolates from calves and a foal failed to produce delta toxin, but were capable of producing large amounts of lethal beta toxin. A strain isolated from a piglet produced delta, but very little beta toxin. Other differences were relatively minor. The results indicated that young domestic animals may be susceptible to all subtypes of C. perfringens type C. A simple method of using blood agar plates coated with type A antiserum for demonstration of hemolytic patterns was found advantageous in differentiation of C. perfringens strains.     

Evaluation of heat-sensitive, neutrophil-toxic, and hemolytic activity of Haemophilus (Actinobacillus) pleuropneumoniae

Cytotoxic and hemolytic activity of Haemophilus (Actinobacillus) pleuropneumoniae serotype 1 strain CM5 was investigated because of the potential role as a virulence determinant. Viable bacteria were toxic for porcine and bovine neutrophils, whereas bacteria killed by heat treatment at 60 C for 1 hour were not. Similarly, bacteria-free culture supernatant was cytotoxic and hemolytic in assays that used porcine neutrophils and erythrocytes, whereas supernatant treated at 60 C for 1 hour had no activity. Erythrocytes from various species were susceptible to the hemolytic activity of bacteria-free culture supernatant, with ovine and bovine erythrocytes being most sensitive. The neutrophil-toxic and hemolytic activity of bacteria-free culture supernatant was inhibited by cholesterol and oxygen and abolished after trypsin digestion. The neutrophil-toxic and hemolytic activity was preserved during storage at or less than 4 C, but was lost rapidly at 56 C or 80 C. Neutralizing antibodies were demonstrated in serum of pigs and rabbits immunized with 10-fold concentrated culture supernatant of strain CM5 and in field pigs that had recovered from natural infection with H pleuropneumoniae serotype 1. Bacteria-free culture supernatants of 18 strains, including H pleuropneumoniae serotypes 1 through 10, Actinobacillus suis, and Haemophilus taxon minor group, were tested for heat-sensitive, neutrophil-toxic, and hemolytic activity. Fifteen strains were neutrophil toxic, but only 10 of these were hemolytic. Haemophilus pleuropneumoniae, serotype 1, strain VLS557; serotype 5, strain K17; and Haemophilus taxon minor group strain 33PN were neither cytotoxic nor hemolytic.     

Corynebacterium pseudotuberculosis exotoxin: fatal hemolytic anemia induced in gnotobiotic neonatal small ruminants by parenteral administration of preparations containing exotoxin

Inoculation of live Corynebacterium pseudotuberculosis, culture supernatant, ammonium sulfate-fractionated crude exotoxin, or chromatographically purified exotoxin preparations into gnotobiotic small ruminants (n = 13) caused death of the ruminants within 48 hours. Characteristic changes observed in animals living greater than or equal to 2 hours after inoculation included hemorrhage and edema at the site of injection, severe hemolytic anemia and hemoglobinuria, dark red fluid in body cavities, lung edema, and icterus. The crude exotoxin preparation caused a syndrome of acute shock in 2 lambs that died within 15 minutes after inoculation. Clinical and pathologic responses of animals inoculated with culture supernatant and purified toxin were similar. Histopathologic evidence indicated that the exotoxin caused necrotic changes in the proximal convoluted tubules of the kidneys. Inoculation with live organisms caused multiple foci of suppurative inflammation in skeletal muscle and adjacent adipose tissue, whereas such changes were not observed in animals administered exotoxin preparations. Although C pseudotuberculosis exotoxin induced a hemolytic anemia in the experimental animals, it did not lead to in vitro lysis of ovine, caprine, or bovine erythrocytes, unless they had been sensitized with Rhodococcus (Corynebacterium) equi filtrate. The toxic sphingomyelin-specific phospholipase D from C pseudotuberculosis had a molecular weight of 31,000 daltons and an isoelectric point of approximately 9.6. The elution profile of exotoxin on a carboxymethyl Sephadex column was studied and the majority of the enzymatic activity was eluted by a NaCl gradient (0.25M to 0.7M) with a maximum at 0.35M NaCl.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of the alpha toxin of Clostridium perfringens type A by ultrafiltration and gel chromatography

Clostridium perfringens type A toxin produced in Jayko & Lichstein medium was subjected to various concentration and purification procedures. The results obtained with 3 different ultrafiltration membranes followed by gel filtration showed that by using Millipore PSED OHV10 and Amicon XM-100 filter membranes in combination, a three-hundred-and-fivefold purification could be achieved as against a twelvefold increase obtained with ammonium sulphate/acetone precipitation. The lecitovitelin test was more sensitive than the haemolytic activity in determining the alpha toxin activity. The optical density, measured at 280 nm, did not reveal any alpha toxin activity in the relevant toxic fractions.     

Interaction of Rhodococcus equi with phagocytic cells from R. equi-exposed and non-exposed foals

The interaction of Rhodococcus equi with alveolar macrophages from adult horses, foals experimentally exposed to R. equi (sensitized foals) and non-exposed foals was studied using in vitro bactericidal assays, cytochemical staining and transmission electron microscopy. It was demonstrated that R. equi is a facultative intracellular parasite, able to survive and multiply within the alveolar macrophages of the host by interfering with phagosome-lysosome fusion. Opsonization of R. equi with antibody against capsular components was associated with increased phagosome-lysosome fusion and significantly enhanced (P less than 0.05) killing of the organism by alveolar macrophages from non-exposed foals. Macrophages from non-exposed foals were able to ingest the non-opsonized organism, but unable to kill greater than 65% of the infective dose by 6 h post-exposure. Alveolar macrophages from sensitized foals behaved as adult macrophages, able to kill greater than 95% of the infective dose by 6 h. Lymphocyte factors, derived by in vitro incubation of sensitized peripheral blood lymphocytes with R. equi surface antigens, enhanced macrophage bactericidal activity. Macrophages from non-exposed foals incubated in the presence of the lymphocyte factors had a 50% increase in killing of R. equi, while sensitized macrophages incubated with lymphocyte factors had a greater than 100% increase in killing capacity.     

Isolation and characterization of the eighth component of the bovine complement system

OBJECTIVE: To isolate and characterize the eighth component of the complement system (C8) in cattle. SAMPLE POPULATION: Fresh plasma obtained from beef cattle. PROCEDURES: Plasma samples were fractionated, using sequential precipitation and ion-exchange and gel-filtration chromatography, to yield C8. The protein was identified throughout the procedure on the basis of its hemolytic function. Electrophoresis in polyacrylamide gels was used to determine molecular weight and composition of polypeptide chains. Reconstitution of classical and alternative complement pathways was used to characterize the hemolytic function of bovine C8. RESULTS: The bovine C8 protein consisted of a disulfide-bonded alpha-gamma heterodimer that was noncovalently associated with a beta chain. Apparent molecular weight of the alpha, beta, and gamma chains under reducing conditions were 66, 61, and 23 kd, respectively. In the classical pathway of activation, bovine C8 and the ninth component of the complement system (C9) had species incompatibility with human C8 and C9 on sheep erythrocyte target cells. CONCLUSIONS: A simple 4-step fractionation procedure provided good yield of bovine C8 from plasma. The isolated protein was structurally comparable to C8 from other species. Purified bovine C8 may be useful in functional hemolytic assays to investigate the roles of complement-mediated lysis in the pathogenesis of inflammatory diseases and the killing of susceptible microorganisms.     

Stability, antigenicity, and aggregation of Moraxella bovis cytolysin after purification and storage

OBJECTIVES: To compare stability, antigenicity, and aggregation characteristics of Moraxella bovis cytolysins among isolates from geographically diverse areas. STUDY POPULATION: 8 isolates of M. bovis. PROCEDURE: Filter-sterilized broth culture supernatants of M. bovis were concentrated, diafiltered, and chromatographed. The endotoxin and cytolysin activities in samples were measured. Chromatographed cytolysins of M. bovis were examined by immunoblotting. Hemolytic and leukotoxic activities were measured from samples collected at each step of purification and before and after storage. Hemolysis was measured directly by use of washed bovine erythrocyte targets. Leukotoxicity was measured by use of a 51Cr release assay. RESULTS: Cytolysin was retained by a filter with 100-kd nominal molecular weight limit. Hemolytic activity, leukotoxic activity, and endotoxin were eluted together in void volume of a gel-filtration column (molecular mass exclusion limit = 4 X 10(7) d). Gel-column chromatographed diafiltered retentate had the greatest specific cytolytic activity and the highest endotoxin-to-protein ratio. Frozen diafiltered retentate(-80 degrees C, 4 months) was cytolytic after thawing. Immunoblots of gel-column chromatographed cytolysin contained 4 proteins with molecular masses between 90 and 68 kd. Fractions with high lytic activities also had additional protein bands with molecular masses of 98 and 63 kd. Immunoblots of gel-column chromatographed diafiltered retentate revealed proteins with molecular masses between 90 and 68 kd. CONCLUSIONS AND CLINICAL RELEVANCE: Diafiltered M. bovis cytolysin is aggregated with endotoxin. Antigenicity and cytolytic activities in diafiltered retentate are conserved among M. bovis isolates. Diafiltration could be useful for bulk semipurification of M. bovis cytolysin. Cytolysin-enriched vaccines of M. bovis could be contaminated by endotoxin.     

Cloning and characterization of a Moraxella bovis cytotoxin gene

OBJECTIVE: To identify the Moraxella bovis cytotoxin gene. PROCEDURE: Hemolytic and nonhemolytic strains of M. bovis were compared by use of western blotting to identify proteins unique to hemolytic strains. Oligonucleotide primers, designed on the basis of amino acid sequences of 2 tryptic peptides derived from 1 such protein and conserved regions of the C and B genes from members of the repeats in the structural toxin (RTX) family of bacterial toxins, were used to amplify cytotoxin-specific genes from M. bovis genomic DNA. Recombinant proteins were expressed, and antisera against these proteins were produced in rabbits. RESULTS: Several proteins ranging in molecular mass from 55 to 75 kd were unique to the hemolytic strain. An open reading frame encoding a 927-amino acid protein with a predicted molecular mass of 98.8 kd was amplified from M. bovis genomic DNA. The deduced amino acid sequence encoded by this open reading frame was homologous to RTX toxins. Antisera against the recombinant carboxy terminus encoded by this open reading frame neutralized hemolytic and cytolytic activities of native M. bovis cytotoxin. CONCLUSIONS AND CLINICAL RELEVANCE: A gene was identified in M bovis that encodes a protein with sequence homology to other RTX toxins. Results of cytotoxin neutralization assays support the hypothesis that M. bovis cytotoxin is encoded by this gene and belongs in the RTX family of bacterial exoproteins. Identification of this gene and expression of recombinant cytotoxin could facilitate the development of improved vaccines against infectious bovine keratoconjunctivitis.     

Beta-globin chain hemoglobin polymorphism and hemoglobin stability in black rhinoceroses (Diceros bicornis)

To evaluate the syndrome of acute intravascular hemolytic anemia in black rhinoceroses (Diceros bicornis), the hemoglobin of this species was evaluated by use of isopropanol- and heat-stability tests and was further characterized by electrophoretic studies. Samples were obtained from 22 apparently healthy captive North American black rhinoceroses, though 3 of the study animals had survived previous hemolytic events, and 3 others were parents of 3 offspring that had suffered hemolysis. The eastern African (Diceros bicornis michaeli) and the southern African subspecies (D b minor) were represented. Comparative samples were also obtained from 2 white (Ceratotherium simum) and 1 Indian (Rhinoceros unicornis) rhinoceroses. The hemoglobin of all 3 species appeared stable when tested by use of the heat and isopropanol methods. Thus, an unstable hemoglobin does not appear to be involved in the hemolytic crises of captive black rhinoceroses. Black rhinoceros hemoglobin had a striking polymorphism. Thirteen of the samples from black rhinoceroses had a single hemoglobin band, based on results of alkaline electrophoresis. Nine had, in addition to this major band, a slow (more cathodic) minor band that comprised about 10% of the total hemoglobin. Further studies indicated that the major band and the slower minor band may contain globin chains analogous to human beta- and delta-chains respectively; these bands have been tentatively designated B and C. Phenotypes B and BC are common, in a ratio of 4:3. A genetic mechanism is proposed that assumes beta b and beta c gene loci and that beta c-locus-expressed (beta c+) and beta c-locus-inhibited (beta c degrees) are common alleles for the beta c-locus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibody to equi factor(s) in the diagnosis of Corynebacterium equi pneumonia of foals.

Antibody to equi factor(s) in cases of Corynebacterium equi pneumonia in foals was detected using C. pseudotuberculosis exotoxin sensitized calf red blood cells. The test was standardized using antitoxin produced in rabbits by injection of equi factor(s). All sera from ten foals with culture-diagnosed C. equi pneumonia had antibodies to equi factor(s) (titre range 8-256, mean 74.0) and nine sera from 11 foals with suspected C. equi pneumonia also showed antibodies (titre range 4-512, mean 136.4). Two of five pneumonia foals with transtracheal aspirate cultures not yielding C. equi had such antibodies. Fifty-eight of 59 control horse sera had no antibodies; the one positive serum came from a foal on a farm where C. equi pneumonia was endemic. By contrast only five of 15 foals with experimentally-induced C. equi pneumonia had antibodies to equi factor(s), probably because the acute nature of the disease produced did not mimic the chronic course of the natural disease. Antibody to equi factor(s) can be used in the diagnosis of naturally-occurring corynebacterial pneumonia in foals.     

Cholesterol oxidase and resistance of Rhodococcus equi to peroxidative stress in vitro in the presence of cholesterol

Rhodococcus equi is a well-characterized bacterial pathogen which lyses cell membranes with the help of cholesterol oxidase (CO). Survival in macrophages is warranted by its ability to resist reactive radicals via catalase and superoxide dismutase (SOD). Therefore, CO production in the absence or presence of 0.1 % cholesterol and sensitivity to exogenous hydrogen peroxide (H2O2) and superoxide anion (SOA) were tested in seven strains of R. equi in vitro. When R. equi strains were grown on agar plates with cholesterol, the bacterial growth [colony-forming units (cfu)/plate] did not increase significantly in comparison with the growth on plates without cholesterol. The activity of CO increased, significantly for extracellular CO. In subsequent experiments, R. equi strains grown on cholesterol were stressed with H2O2 or SOA so that approximately 10 % of cfu/plate survived. During stress induced by SOA, membrane CO and SOD activity increased significantly. Catalase activity increased 2-fold with H2O2 and 3-fold with SOA exposure. These data suggest that the presence of cholesterol induces CO in bacteria grown on agar plates. Catalase, SOD and even membrane-bound CO respond to reactive oxygen species.     

Polyacrylamide gel electrophoresis of whole-cell preparations of Rhodococcus equi.

The whole-cell proteins of ten strains of Rhodococcus equi isolated from horses, pigs, or humans, including the type strain ATCC 6939, were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The protein profiles of seven different capsular serotypes and the type strain were very similar when bacteria were cultured under the same conditions. Protein profiles were largely unaffected by incubation at two temperatures (30 degrees C, 37 degrees C) or times (12 h, 48 h). There were generally minor differences in protein profiles between strains grown in different media (brain heart infusion, nutrient, minca broths, tryptic soy-blood agar) with the marked exception of a prominent diffuse 17.5 kd protein which was expressed in nutrient broth. This protein was not produced by the type strain and was lost on repeated passage in vitro (50th, 100th passage) in two of three other strains examined.     

Identification and characterization of serum complement activity in the American crocodile (Crocodylus acutus)

Incubation of unsensitized sheep red blood cells with serum from the American crocodile (Crocodylus acutus) resulted in a concentration-dependent hemolysis. The hemolytic activity was heat-sensitive, and inhibited by EDTA in a concentration-dependent manner. The EDTA-inhibited SRBC hemolysis could be restored by the addition of excess Ca²⁺ or Mg²⁺, but not Ba²⁺ or Cu²⁺, revealing the specificity of this activity for these two divalent cations. The hemolytic activity of crocodile serum was titer-dependent, with 329 μL producing 50% of maximal SRBC hemolysis. The complement activity was also temperature-dependent, with decreased activity at lower temperatures (5–15 °C) and maximal activity occurred at 30–40 °C. The hemolysis occurred relatively slowly, with near zero activity after 10 min, 40% of activity observed within 15 min of exposure to SRBCs, and maximal activity at 30 min.     

Assessment of IgE binding to native and hydrolyzed soy protein in serum obtained from dogs with experimentally induced soy protein hypersensitivity

OBJECTIVE: To assess binding of IgE to native, whole hydrolyzed, and separated hydrolyzed fractions of soy protein in serum obtained from dogs with experimentally induced soy protein hypersensitivity. ANIMALS: 8 na?ve Beagles (6 experimentally sensitized to native soy protein and 2 control dogs). PROCEDURES: 6 dogs were sensitized against soy protein by administration of allergens during a 90-day period. After the sensitization protocol was completed, serum concentrations of soy-specific IgE were measured and intradermal skin tests were performed in all 6 dogs to confirm that the dogs were sensitized against soy protein. Serum samples from each sensitized and control dog underwent western blot analysis to assess the molecular mass band pattern of the different allergenic soy fractions and evaluate reactivities to native and hydrolyzed soy protein. RESULTS: In sera from sensitized dogs, a characteristic band pattern with 2 major bands (approx 75 and 50 kd) and 2 minor bands (approx 31 and 20 kd) was detected, whereas only a diffuse band pattern associated with whole hydrolyzed soy protein was detected in the most reactive dog. Reactivity was evident only for the higher molecular mass peptide fraction. In control dogs, no IgE reaction to native or hydrolyzed soy protein was detected. CONCLUSIONS AND CLINICAL RELEVANCE: Data suggest that the binding of soy-specific IgE to the hydrolyzed soy protein used in the study was significantly reduced, compared with binding of soy-specific IgE to the native soy protein, in dogs with experimentally induced soy hypersensitivity.