Contribution to the differentiation of cross-reacting antibodies in brucellosis serology--1. Reactions with various Yersinia serotypes and antibody avidity

Brucella and Yersinia enterocolitca O:9 (Y.e.O:9) have antigenic determinants in common causing cross-reactions which interfere strongly in Brucella serology. Hoffmann (1988) observed cross-reactivity between Y.e.O:9, Y.e.O:5, and Y.e.O:6 and used an ELISA for the differentiation between Brucella- and Yersinia antibodies since Y.e.O:5 and Y.e.O:6 produced only low-level cross-reactions with Brucella antigen. The observations of Hoffmann were confirmed and led to the construction of an ELISA with 4 antigens. Sera from natural outbreaks of brucellosis and from artificial infections with Brucella and Y.e.O:9 were properly identifiable by this system. Furthermore, the test material has been used to measure the antibody avidity by a specially designed ELISA. This test produced a further divergence between specific and non-specific reactions.     

Studies on the relationship between Yersinia enterocolitica IX and Brucella abortus agglutinins in naturally infected animals

Attempts were made to define the Brucella abortus and Yersinia enterocolitica IX infection status of animal populations by means of selected agglutination tests. The Brucella abortus O, the Yersinia enterocolitica IX OH and the Y enterocolitica IX H agglutinin titres were measured in a large number of cattle, goat and pig sera In the goats and, to a much lesser extent, the pigs, the relationships between these titres suggested that Yersinia infection was common. In contrast, the results from the cattle sera were complex and tended to indicate the presence of both Yersinia infection and brucellosis.     

Enzyme-linked immunosorbent assay for differentiation of the antibody response of cattle naturally infected with Brucella abortus or vaccinated with strain 19

Purified O chain of Brucella abortus was passively attached to polystyrene to differentiate antibody responses of cattle vaccinated with B abortus strain 19 from those of naturally infected cattle. In the indirect assay, using O polysaccharide as antigen, a single serum dilution was used and mouse monoclonal antibody to bovine L chain conjugated with horseradish peroxidase was the detection reagent. Measurable antibody was not found in sera of vaccinated cattle, except for 3 sera from cattle that were persistently infected with strain 19. Sera from 25 cattle infected with pathogenic strains contained antibody on the basis of results of indirect enzyme immunoassay, using smooth lipopolysaccharide or O chain as antigens, or results of competitive enzyme immunoassay, using the O-chain antigen. Results in sera from calves with experimentally induced Yersinia enterocolitica serotype 0:9 infection or inoculated with a low dose of B abortus strain 2308 were comparable with those in sera of cattle that were vaccinated with strain 19. The data correlated with those from competitive enzyme immunoassay, using one serum dilution and horseradish peroxidase-conjugated mouse monoclonal antibody to smooth lipopolysaccharide. On the basis of results of the indirect enzyme immunoassay, all sera (except those samples obtained before inoculation) contained antibody to smooth lipopolysaccharide.     

The serological response of cattle immunized with Yersinia enterocolitica O:9 or O:16 to Yersinia and Brucella abortus antigens in enzyme immunoassays

Sera from calves immunized with Yersinia enterocolitica serotypes O:9 or O:16 were tested by indirect enzyme linked immunosorbent assay (ELISA) using lipopolysaccharide (LPS) preparations from Brucella abortus or Y. enterocolitica O:9 or O:16 for their antibody content of the IgG1 or IgG2 subclasses. High IgG1 responses were present with the three antigens in both groups although some individual variations between animals were noted. The IgG2 responses were modest and in some cases not above background 'noise'. Thus IgG2 antibody was not measurable in sera from serotype O:9 injected calves when using serotype O:16 LPS or in serotype O:16 injected calves when using B. abortus or serotype O:9 LPSs. A competitive ELISA using B. abortus O-polysaccharide and a monoclonal antibody to B. abortus LPS (initially designed to differentiate the antibody responses of cattle naturally infected with B. abortus from those vaccinated with strain 19) was used on sera from both groups of calves. Using this test, no antibody was detected in the group immunized with serotype O:16 and except for one animal in the serotype O:9 immunized group, only low levels of antibody were transiently in evidence. One animal in this group responded with quite high levels of competing antibody which, however, declined towards the end of the test period. The competitive ELISA may prove a useful serological tool for differentiating vaccinal and field infection titers to B. abortus and also to eliminate cross-reactions observed with Y. enterocolitica serotypes.     

Studies on the use of Yersinia enterocolitica 09 antigens in rapid quantitative plate tests for the differentiation of Brucella and Yersinia infections in cattle and the diagnosis of bovine brucellosis

The use of the rapid quantitative plate agglutination test (QPAT) utilizing triphenyltetrazolium chloride (TTC) stained antigen for the differentiation of bovine brucellosis and yersiniosis is described. It was found with experimental laboratory animals and cattle, that titres against homologous antigen tended to exceed titres against heterologous antigen and that this reaction together with measurement of the Yersinia OH agglutinin titre could be used for purposes of differentiation. It was, however, not possible to differentiate these infections in serum from cattle with naturally-occurring antibodies. It was also shown that Brucella 0 agglutination in the QPAT was very weak in some sera with high titred anti-Brucella antibodies by serum agglutination, while Yersinia 0 agglutination was not. The QPAT, using Yersinia 0 antigen, therefore correlated well with the results obtained using Brucella 0 antigen in serum agglutination tests. It is suggested that Yersinia 0 antigen may be a suitable antigen for use in rapid agglutination tests because of this relative insusceptibility to false negative results.     

Passive immunity and serological immune response in dairy calves associated with natural Giardia duodenalis infections

In a previous study, Giardia infection patterns were studied in newborn dairy calves over a 4-month period. Chronic Giardia infections were observed in all calves with initial cyst excretion occurring at approximately 1 month of age. In the work presented here, the passive immunity and serological immune response associated with these Giardia infections were examined. Colostrum and milk samples were collected from the dams of these calves, and monthly serum samples were collected from each calf. The colostrum, milk and sera samples were analyzed by ELISA and Western blot for the presence of anti-Giardia IgG antibodies. In addition, the in vitro anti-Giardia activity of milk and colostrum was examined using a miniculture adherence assay. When examined by ELISA, mean anti-Giardia antibody titres were found to be significantly higher in colostrum compared to milk. The monthly mean serum antibody titres in the calves were not found to differ significantly at any time point during the study. Western blot analysis revealed that colostrum from the dams reacted strongly with many different Giardia antigens between 205 and 7.5kDa, while milk reacted with few antigens in the same size range. Sera collected from the calves when 30 and 60 days of age reacted with few Giardia antigens, but as the calves aged, IgG antibodies in their sera began to react with antigens of 21, 50, 65, 73 and 79kDa. The miniculture adherence assay demonstrated that colostrum had significantly more anti-Giardia activity in vitro compared to milk. These results suggest that the calves in this dairy did not mount a significant humoral immune response against Giardia following infection. However, colostrum contained a high level of anti-Giardia antibodies and exhibited anti-Giardia activity in vitro. Therefore, colostrum may have the potential to provide initial protection against Giardia infections in calves, but the lack of a strong, specific humoral immune response by these calves could account for the high prevalence and chronic duration of the infections.     

Serologic response to Brucella abortus of cattle and guinea pigs experimentally inoculated with a Yersinia enterocolitica serotype from milk

Ten strains of Yersinia enterocolitica belonging to ten various serogroups isolated from raw milk were inoculated into groups of five guinea pigs and five calves. Y. enterocolitica serotype 0:16 was the only serotype tested that induced an antibody response to Brucella abortus in calves. No anti-Brucella response could be demonstrated serologically in guinea pigs. Activity of the anti-Y. enterocolitica 0:16 calf sera against B. abortus antigen was shown by the tube agglutination test, and by the complement fixation test. The early agglutinating antibody response was partly sensitive to reduction by 2-mercaptoethanol. This sensitivity decreased later in the response. This is the first report of anti-Brucella responses induced by a serotype of Y. enterocolitica other than 0:9; sera from a group of five calves inoculated with 0:9 were tested by the same serological techniques for comparison.     

Antibodies in carrier wildebeest to the lymphoproliferative herpesvirus of malignant catarrhal fever

Six types of antibody to malignant catarrhal fever virus (MCFV) were measured in 132 sera collected from Wildebeest in Kenya Masailand. The titre of all types of antibody declined slowly with increasing age of the wildebeest. A significantly greater proportion of wildebeest calves had higher titres of antibodies to MCFV early antigens, IgM antibodies to MCFV late antigens and complement-fixing antibodies, than did older animals. One seronegative calf, reared in isolation without colostrum, became seropositive 4 1/2 weeks after birth but did not show any clinical signs indicative of MCFV infection. Similarities between MCFV infection of wildebeest calves and other inapparent infections with lymphoproliferative herpesviruses are discussed.     

Acetone-treated agglutinin against Yersinia pseudotuberculosis in free-living rats

Retrospective investigation was done on Yersinia pseudotuberculosis infection in barn rat populations living in an Yersinia endemic area and in zoo rat populations living in a nonendemic area. The OH agglutinins against Y pseudotuberculosis were normally distributed within both populations. Most of the OH agglutinin titers and many O agglutinin titers were reduced by treatment of sera with acetone. Sera with O and OH agglutinin titers were assigned to 2 groups, one with a titer less than 1:8 and the other with a titer of greater than or equal to 1:8. Rats harboring the organism did not possess O or OH agglutinin titers greater than 1:8 in acetone-treated sera. In most nontreated sera, OH agglutinin titers were higher than were the O agglutinin titers. However, in most acetone-treated sera, O agglutinin titers were equivalent to OH agglutinin titers. Differences by region and age were observed in acetone-treated O agglutinin titers of greater than or equal to 1:8 against Y pseudotuberculosis. Therefore, it is recommended that to detect antibody against Y pseudotuberculosis in rats, sera should be treated with acetone before the test and O antigen should be used.     

The bovine immune response to Brucella abortus IV. Studies with a double immunodiffusion test for antibody against A2.

A double immunodiffusion test for precipitins against Brucella antigen A2 was developed and applied to a variety of samples. The A2 precipitins were produced by a heifer infected with B. abortus strain 2308, cattle vaccinated with killed B. melitensis strain H38 or live B. abortus strain 19 and by a dog infected with B. canis. Precipitins were also detected in the second International Standard for anti-Brucella abortus serum, in several anti-B. canis sera and at low levels in one anti-B. ovis serum tested. Antisera produced in calves against Yersinia enterocolitica serotype 0:9 had no anti-A2 activity despite titers greater than or equal to 1/1024 and greater than or equal to 1/80 in standard Brucella agglutination and CF tests, respectively. The test for A2 precipitins lacked specificity as weak reactions were obtained with five of 295 sera from brucellosis-free herds. This test was relatively insensitive, detecting precipitins in only 16 of 24 sera from infected cattle and 27 of 54 sera positive by complement fixation and enzyme labelled antiglobulin tests performed with whole cell and smooth lipopolysaccharide antigens, respectively. The A2 precipitins were detected in nine sera from five cattle, in two infected herds, which were negative by agglutination and complement fixation tests.     

Serologic cross-reactivity of antibodies against Borrelia theileri, Borrelia burgdorferi, and Borrelia coriaceae in cattle.

OBJECTIVE: To evaluate the immune response induced by Borrelia theileri infection and to determine whether B theileri induces cross-reacting antibodies to other bovine borreliae. ANIMALS: Two 3-month-old calves, 1 of which was splenectomized. PROCEDURE: Calves were exposed to Boophilus microplus infected with B theileri. Rectal temperature, PCV, bacteremia, and clinical signs of infection were monitored. Serum was obtained weekly and used to evaluate the humoral response to homologous antigen and B burgdorferi and B coriaceae, using an indirect fluorescent antibody (IFA) test, and to B burgdorferi, using a commercially available ELISA. The identity of cross-reacting antigens was explored, using monoclonal antibodies to genus- and species-specific antigens in an IFA test. RESULTS: B theileri-infected calves produced antibodies that cross-reacted with B burgdorferi and B coriaceae whole-cell antigens. Borrelia theileri whole-cell antigen was recognized by genus-specific monoclonal antibody H9724 but not by species-specific antibody H5332. False-positive reactions were not observed when serum from B theileri-infected calves was tested by use of the ELISA for B burgdorferi. CONCLUSIONS: B theileri induces humoral responses in infected cattle that can be confused with those of other borrelial infections. Care must be taken to definitively distinguish between the various borreliae that may cause disease in cattle. CLINICAL RELEVANCE: Serologic cross-reactivity must be taken into account when making a serodiagnosis of Lyme borreliosis or epizootic abortion in epidemiologic studies involving cattle.     

Passive transfer of antibodies to Shiga toxin-producing Escherichia coli O26, O111 and O157 antigens in neonatal calves by feeding colostrum

To study whether or not passive immunity of neonatal calves against Shiga toxin-producing Escherichia coli (STEC) O26, O111, and O157 was obtained by colostrum administration, serum antibodies in calves after the feeding were determined by enzyme-linked immunosorbent assay (ELISA) in comparison with antibodies in colostrum and sera from donor dams. The highest antibody titers to STEC in colostrum from dams were detected soon after parturition. The antibody titers were found to be elevated in sera of neonatal calves (4-9 hr after birth) orally administered with colostrum with high antibody titers, suggesting that passive immunity of neonatal calves to STEC infection may be obtained by feeding colostrum. These results suggest that colostrum administration to neonatal calves may play an important role in elevating serum antibodies against STEC in neonatal calves.     

Humoral immune response of water buffalo monitored with three different antigens of Toxocara vitulorum

Humoral immune response of water buffalo naturally infected with Toxocara vitulorum was monitored using three different antigens of this parasite in serum and colostrum of buffalo cows and calves. Soluble extract (Ex) and excretory/secretory (ES) larval antigens and perienteric fluid antigen (Pe) of adult T. vitulorum were used to measure the antibody levels by an indirect ELISA. Serum of 7-12 buffalo cows for the first 365 days and colostrum of the same number of buffalo cows for the first 60 days of parturition, and serum of 8-10 buffalo calves for the first 365 days after birth were assayed. The ELISA detected antibodies against all three T. vitulorum antigens in the colostrum and serum of 100% of buffalo cows and calves examined. The highest antibody levels against Ex, ES and Pe antigens were detected in the buffalo cow sera during the perinatal period and were maintained at high levels through 300 days after parturition. On the other hand, colostrum antibody concentrations of all three antigens were highest on the first day post-parturition, but decreased sharply during the first 15 days. Concomitantly to the monitoring of immune response, the parasitic status of the calves was also evaluated. In calves, antibodies passively acquired were at the highest concentrations 24 h after birth and remained at high levels until 45 days coincidentally with the peak of T. vitulorum infection. The rejection of the worms by the calves occurred simultaneously with the decline of antibody levels, which reached their lowest levels between 76 and 150 days. Thereafter, probably because of the presence of adults/larvae stimulation, the calves acquired active immunity and the antibodies started to increase slightly in the serum and plateaued between the days 211 and 365. All three antigens were detected by the serum antibodies of buffalo calves; however, the concentration of anti-Pe antibody was higher than anti-EX and anti-ES, particularly after 90 days of age. By conclusion, the buffalo cows develop immunity and keep high levels of antibodies against T. vitulorum-Ex, ES and Pe antigens and these antibodies are transferred to their calves through the colostrum. This passively acquired immunity does not protect the calves against the acquisition of the infection, but these antibodies, passively or actively acquired, may have an important role during worm rejection by the calves and prevention of intestinal reinfection.     

Serological cross-reactions between different Brucella species and Yersinia enterocolitica. Immunodiffusion and immunoelectrophoresis

By immunodiffusion and immunoelectrophoresis tests in agarose serological cross-reactions were demonstrated between Yersinia enterocolitica type IX and Brucella strains from four species (Brucella abortus, Brucella melitensis, Brucella suis and Brucella neotomae). No qualitative differences between these strains in their tendencies to cross-react with Yersinia enterocolitica type IX were observed. Brucella canis and Brucella ovis, which have nonsmooth colonial morphology, gave no demonstrable cross-reaction with Yersinia enterocolitica type IX.The results of absorption tests and qualitative staining reaction of the obtained precipitation lines suggest that the antigenic determinants common to Brucella and Yersinia enterocolitica type IX seemed to be associated with the outer layer and in the lipopolysaccharide complex of the respective bacteria. By immunodiffusion and immunoelectrophoresis it was possible to identify in hyperimmune sera those antibodies that derive from Brucella and Yersinia enterocolitica type IX.Keyword: serological cross-reaction, Brucella species, Yersinia enterocolitica serotype IX, immunodiffusion, immunoelectrophoresis     

Cell-mediated immune responses differentiate infections with Brucella suis from Yersinia enterocolitica serotype O:9 in pigs

Due to almost identical lipopolysaccharide (LPS) O-antigens, infections with Yersinia enterocolitica serotype O:9 (YeO:9) cause false positive serological reactions (FPSR) in tests for Brucella and thus cause problems in National Brucella surveillance programs. As LPS are strong inducers of antibody responses it was hypothesized that cell-mediated immune responses to non-LPS antigens of the two bacteria can be used to separate immune responses to these two biologically very different infections. Following subclinical experimental infections with Brucella suis biovar 2, high interferon-gamma (IFN-gamma) assay responses with a commercial Brucella melitensis antigen preparation (Brucellergene OCB) preceded the development of antibodies. High IFN-gamma responses in the seven B. suis inoculated pigs with serological evidence of infection were consistent throughout a 20-week post-inoculation observation period. In contrast, IFN-gamma responses in two B. suis inoculated pigs without bacteriological or serological evidence of infection were below a cut-point of 25pg/ml at all samplings. IFN-gamma responses in repeated samplings from 5 uninfected control pigs and 18 pigs experimentally infected with YeO:9 were all negative, except for solitary false positives in 3.7% of the samples from both the experimentally YeO:9 infected pigs and control pigs. Skin tests using the same commercial Brucella antigen confirmed the ability of cell-mediated immune responses to differentiate between the two infections. In addition, a field evaluation of the diagnostic use of cell-mediated immune responses by IFN-gamma assay and skin test to resolve serological suspicions of Brucella was conducted in an YeO:9 infected pig herd. Following a screening of 200 pigs 39 pigs were identified with false positive serological Brucellosis reactions. While 36 of the 39 FPSR pigs were also FPSR in a second test, none of the pigs were test positive in whole blood IFN-gamma assay or Brucellergene OCB skin test. In conclusion, use of IFN-gamma assay and skin test as measurements of cell-mediated immune responses to non-LPS Brucella antigens were specific and sensitive in discriminating subclinical experimental infections with B. suis from both natural and experimental infections with YeO:9.     

Identification and isolation of a specific antigen with diagnostic potential from Dictyocaulus viviparus.

The purpose of the investigation was to isolate and identify a specific antigen of Dictyocaulus viviparus that can be used to diagnose lungworm infections in cattle. Somatic, excretion and secretion antigens of adult D. viviparus and somatic antigens of L3 larvae were examined in an indirect enzyme-linked immunosorbent assay (ELISA) to determine whether they cross-reacted with sera collected from calves with mono-infections of Fasciola hepatica. Ostertagia ostertagi, Ascaris suum, or Cooperia oncophora. Serum samples containing antibodies directed against F. hepatica, A. suum, and O. ostertagi cross-reacted with somatic antigens of adult D. viviparus; these sera cross-reacted less with excretion and secretion antigens. When somatic antigens of adult D. viviparus were analysed in a Western blot, a 17-kDa protein that did not react with the heterologous sera was detected. This protein was isolated by ultrafiltration and anion chromatography. Sera collected from calves infected with D. viviparus was tested in indirect ELISAs with either somatic antigens of adult D. viviparus or with a low molecular antigen fraction of this preparation containing the 17-kDa protein. The extinction values that were measured in both assays correlated well. We conclude that the 17-kDa protein isolated from somatic antigens of adult D. viviparus may be useful in developing an improved immunoassay to diagnose lungworm infections in cattle.     

Monoclonal antibodies reacting specifically with Francisella sp

Twenty two hybridoma strains producing monoclonal antibodies against Francisella tularensis ATCC 6223, var. tularensis, were characterized. In an enzyme-linked-immunosorbent-assay (ELISA) using formaldehyde fixed bacteria as antigens, neither cross-reactions with six different Brucella spp., with Yersinia enterocolitica 0:9 nor with two biotypes of Yersinia pseudotuberculosis could be detected. The antibodies gave comparable titres with the three strains of F.tularensis tested. ELISA binding studies indicated that fifteen of the antibodies bound with high affinities to their epitopes of the three Francisella strains, while the others each seemed to bind with low affinity to at least one of the antigens. Immunoblot analysis showed that six of the antibodies were directed to epitopes on the core moiety of the lipopolysaccharide molecule, while the other 16 antibodies bound to O side chain components.     

Demonstration of Pasteurella-specific immunoglobulin E in bovine serum

On the basis of recent observations that immunoglobulin (Ig) E antibodies specific for bacterial antigens occur in the serum of persons with chronic respiratory tract disease, we used bovine epsilon chain-specific antiserum to investigate the possibility that IgE antibodies are induced in cattle infected with Pasteurella. Using enzyme-linked immunosorbent assay and Western blotting techniques, we studied bovine sera to detect and quantitate the presence of IgE antibodies specific for antigens of Pasteurella. Immunoglobulin E antibodies reactive with whole formalinized P haemolytica, potassium thicyanate, and saline solution extracts were detected in serum of calves with bronchopneumonia, feedlot steers with interstitial pneumonia, as well as nonaffected penmates, and adult dairy cows. The role of parenteral vaccination in eliciting an IgE response was examined in healthy calves; vaccination with a Pasteurella bacterin failed to induce an IgE response. Adsorption studies were done to demonstrate the specificity of the antibodies for Pasteurella. Enzyme-linked immunosorbent assay absorbance values were significantly decreased by adsorption with P haemolytica, whereas adsorption with other gram-negative bacteria only moderately decreased serum absorbance values. To begin identification of the antigen(s) to which the IgE binds, Western blotting of P haemolytica extract with sera from calves with bronchopneumonia was done. A dense band of protein (approximately 60,000 daltons) reacted strongly with IgE in the highest titer sera. These results indicate that Pasteurella-specific IgE antibodies are not readily induced by parenteral vaccination, but can be found in serum of some cattle, possibly induced by existing or previous infection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Eimeria infections in cows in the periparturient phase and their calves: oocyst excretion and levels of specific serum and colostrum antibodies.

Faecal Eimeria oocyst excretion and levels of antibodies to first generation merozoite antigen of E. bovis in sera and colostra were followed in 86 and 70 cow-calf pairs in northern (group EF) and central Germany (group H), respectively, over periods of 3 weeks before to 3 weeks after calving in cows and from birth to an age of 63 days in calves. Oocysts were found in 30 and 7.7% of cows in groups EF and H, respectively. They belonged to 10 (group EF) and four Eimeria spp. (group H) with E. bovis, E. ellipsoidalis, E. auburnensis and E. zuerni as the most frequently occurring species. Prevalence and intensity of oocyst excretion varied with time resulting in peak values around the date of parturition, particularly in the case of E. bovis. Peak values at the time of parturition were also seen in case of strongyle egg excretion. Seven (group H) and nine Eimeria spp. (group EF) were found in the calves. The predominant species E. ellipsoidalis, E. zuerni, E. bovis and E. auburnensis were detected for the first time earlier after birth (3-5 weeks) than the others. The prevalence of Eimeria infections increased to 67.1% (group EF) and 50.1% (group H) 9 weeks after birth. Specific IgM and IgA antibody levels (the latter only determined in group EF) in cow sera remained almost constant throughout the observation period, whereas IgG(1) and IgG(2) levels were reduced at the time of parturition. Levels of specific antibodies in sera and colostra were significantly correlated. Except IgM antibodies, significant inverse correlations were found in cows between intensity of infection with E. bovis and specific serum IgG (group H) and IgG(2) (group EF) antibodies. Antibodies to E. bovis were detected in calves sera only after colostrum intake with significant correlations between levels in calves sera and colostra. Levels decreased, starting within the first week of life (most conspicuously in case of IgM and IgA) until the third week. Subsequently, but except IgG(1) antibody concentrations increased until the end of the observation period. Interrelations between antibody levels and the total amount of E. bovis oocysts excreted by the calves until the ninth week of life varied with the age of the animals. Inverse relationships in the first 3 weeks of life as suggested by negative correlation coefficients could not be proven statistically. Thus, there is no unambiguous proof for immunoprotection of calves against E. bovis via maternal immunity. Considering antibody levels in the 3-9 weeks old calves significant direct correlations with E. bovis oocyst excretion were found in case of IgM, IgG(2) and IgA, reflecting an active immune response of young calves to coccidial infection.     

Differentiation by western blotting of immune responses of cattle vaccinated with Brucella abortus strain 19 or infected experimentally or naturally with virulent Brucella abortus

Brucella abortus strain 19 salt-extractable proteins fractionated by differential ammonium sulfate precipitation were used in a western blotting method to detect bovine immunoglobulin G antibodies to B. abortus. Sera from infected cattle and from cattle vaccinated with strain 19 and subsequently exposed to virulent B. abortus bound to a common group of antigens ranging in molecular weights from 31,000 to 45,000 daltons. Immunoglobulin G antibodies in sera from the latter group in addition also bound to antigens with molecular weights of 66,000 to 71,000 daltons. Some sera from cattle vaccinated when sexually mature reacted similar to those from infected cattle, while immunoglobulin G antibodies in sera from Brucella-free cattle and vaccinated calves did not bind to either group of antigens. In general, fractionation of the proteins by ammonium sulfate precipitation offered no advantage for detecting differences between groups of sera. Ammonium sulfate fraction 0 to 35% reacted with a larger number of sera from a naturally infected group than fraction 0 to 70%. Both fractions reacted equally well with sera from the other groups of cattle, while fractions 35 to 70% and 70 to 100% reacted poorly in this technique. The attractive feature of the blot is that sera from calfhood-vaccinated cattle did not react.