Evaluation of the enzyme-linked immunosorbent assay in the detection of cattle infected with Brucella abortus

One group of 51 cattle was vaccinated with B. abortus S19 (S19) and a further 51 cattle were vaccinated with B. abortus S45/20 (S45/20). Forty-eight cattle (24 from each group) and a control group of 12 cattle were subsequently challenged with B. abortus S544. The enzyme-linked immunosorbent assay (ELISA) was used to detect specific IgG and IgM antibodies in these groups. All cattle vaccinated with S19 had high levels of IgG and IgM, but the S45/20 vaccine produced detectable antibody in only a few cattle. In those cattle where the challenge induced infection, the mean levels of IgG and IgM were much higher than those of the uninfected cattle in the same groups. When the isolation of B. abortus was compared at slaughter with the serological results, the ELISA, when used to detect specific IgG, was more sensitive but less specific than the serum agglutination test, complement fixation test and indirect haemolysis test, and more sensitive and more specific than the Rose Bengal test.     

Serological response of cattle after vaccination and challenge with Brucella abortus

New and currently used serological procedures were evaluated using sera from cattle that were challenged with B. abortus S544 (S544) after vaccination with either B. abortus S19 (S19) or B. abortus 45/20 (S45/20) as calves or adults. In animals vaccinated with S19, titres to the indirect haemolysis test (IHLT) rose more slowly, declined more rapidly and involved fewer animals than did titres to the complement fixation test (CFT). In animals vaccinated with S45/20 the rough antigen complement fixation test (RCFT) showed persistent titres. At slaughter the IHLT and CFT were found to be more specific and more sensitive than the Rose Bengal Plate Test (RBPT) and Serum Agglutination Test (SAT) in the detection of cattle infected with B. abortus.     

Serological cross-reactivity between Brucella abortus and Yersinia enterocolitica 0:9:: IV. Evaluation of the M- and C-epitope antibody response for the specific detection of B. abortus infections

Smooth lipopolysaccharides (SLPS) from Brucella abortus contain A-epitopes against which the majority of serum antibodies are directed during infections. SLPS from Yersinia enterocolitica 0:9 possesses identical epitopes, which are the cause for serological cross-reactivity. All Brucella spp. possess M- and C-epitopes which are not present in Y. enterocolitica 0:9. In order to examine the usefulness of these M- and C-epitopes for discrimatory serological testing, a panel of sera were used in this study, comprising sera from Y. enterocolitica 0:9-infected heifers, sera from B. abortus-infected cattle of comparable strength in the serological brucellosis tests to the sera from Y. enterocolitica 0:9-infected heifers, sera from B. abortus-infected bovines with strong serological reactions and sera from animals free from B. abortus or Y. enterocolitica infections. These sera were tested in blocking ELISAs with seven M- and one C-epitope-specific monoclonal antibodies in combination with SLPS from B. melitensis M16 high in M-epitopes as antigen. Strong B. abortus sera inhibited most strongly, while negative sera showed no or little inhibition. Sera with weak or intermediate titres blocked to a lower extent. Unexpectedly, the sera from Y. enterocolitica 0:9-infected heifers showed inhibition behaviour virtually identical to the comparable sera from B. abortus infected animals. Absorbing out of the A-epitope specific serum antibodies with either Y. enterocolitica 0:9 SLPS or with Y. enterocolitica 0:9 bacteria, indicated the presence of M- or C-epitope-specific serum antibodies in some sera from B. abortus-infected cattle but not in the sera from Y. enterocolitica 0:9-infected animals. These results demonstrate that the M- or C-epitope-specific antibody response in sera from B. abortus infected cattle is only of limited value for the serological discrimination between B. abortus and Y. enterocolitica 0:9 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.     

IMMUNOGLOBULIN CLASSES IN SERUM ANTIBODY REACTIONS IN CATTLE FOLLOWING VACCINATION WITH BRUCELLA ABORTUS STRAIN 19 AND KILLED 45/20 VACCINES

SUMMARY A group of 4 cows was vaccinated with Brucella abortus strain 19, followed 8 weeks later by a single dose of B. abortus 45/20 vaccine. A similar group received 2 doses of B. abortus 45/20 vaccine 8 weeks apart. The antibody responses of the groups were compared by testing whole serums and separated IgM and IgG fractions by the Rose Bengal Plate (RBP) agglutination and the complement fixation tests (CFT) using rough and smooth B. abortus antigens. Animals that had received B. abortus strain 19 responded to the 45/20 vaccine with increased titres to the smooth antigen. These relevant antibodies were predominantly of the IgG class. Standard CFT and RBP test antibodies could be detected in IgM and IgG fractions after the primary inoculation with B. abortus strain 19 vaccine.     

A study of cattle infected with Brucella abortus and which showed aberrant serological reactions

SUMMARY Sixty cows, 48 of which had been vaccinated with live Brucella abortus strain 19 (S19) or with killed B. abortus strain 45/20 (S45/20) and 12 of which were unvaccinated animals, were challenged with B. abortus strain 544. Ten of the 27 cattle found to be infected after challenge showed aberrant serological reactions to the Rose Bengal Plate test, serum agglutination test and/or complement fixation test. These 10 cattle were all previously vaccinated with S19 or S45/20. It was concluded that infection in cattle vaccinated with S19 or S45/20 may be more difficult to detect than infection in animals that have no history of vaccination.     

Differential antigen recognition by serum antibodies from three bovid hosts of Mycobacterium bovis infection

Cattle, bison and buffaloes are susceptible to Mycobacterium bovis, the causative agent for bovine tuberculosis. Accurate and timely identification of infected animals is critical for improved management and control of disease in these species. Bovids develop humoral immune responses to M. bovis infection making serological tests attractive for tuberculosis screening. However, optimization and validation of antibody assays designed for various animal species require understanding of antigen recognition patterns in each target host. The objective of this study was to characterize serological reactivity profiles generated by cattle, American bison, and African buffaloes in tuberculosis. Serum samples from M. bovis-infected animals were tested for the presence of IgM and IgG antibodies to MPB70/MPB83 and CFP10/ESAT6 chimeric proteins using Dual-Path Platform technology. All three host species showed IgG responses of higher magnitude and frequency than IgM responses; further, IgM seroreactivity was limited to MPB70/MPB83, whereas IgG antibodies recognized both test antigens. In cattle, the IgM and IgG responses were elicited mainly by MPB70/MPB83, whereas bison and buffaloes showed similar IgG seroreactivity rates for MPB70/MPB83 and CFP10/ESAT6 antigens. The findings demonstrate distinct patterns of predominant antigen recognition by different bovid species in M. bovis infection.     

Protective effects of recombinant Brucella abortus Omp28 against infection with a virulent strain of Brucella abortus 544 in mice

The outer membrane proteins (OMPs) of Brucella (B.) abortus have been extensively studied, but their immunogenicity and protective ability against B. abortus infection are still unclear. In the present study, B. abortus Omp28, a group 3 antigen, was amplified by PCR and cloned into a maltose fusion protein expression system. Recombinant Omp28 (rOmp28) was expressed in Escherichia coli and was then purified. Immunogenicity of rOmp28 was confirmed by Western blot analysis with Brucella-positive mouse serum. Furthermore, humoral- or cell-mediated immune responses measured by the production of IgG1 or IgG2a in rOmp28-immunized mice and the ability of rOmp28 immunization to protect against B. abortus infection were evaluated in a mouse model. In the immunogenicity analysis, the mean titers of IgG1 and IgG2a produced by rOmp28-immunized mice were 20-fold higher than those of PBS-treated mice throughout the entire experimental period. Furthermore, spleen proliferation and bacterial burden in the spleen of rOmp28-immunized mice were approximately 1.5-fold lower than those of PBS-treated mice when challenged with virulent B. abortus. These findings suggest that rOmp28 from B. abortus is a good candidate for manufacturing an effective subunit vaccine against B. abortus infection in animals.     

Circulating antibody response to Eimeria tenella oral and subcutaneous infections in chickens

Immune responses in chickens to Eimeria tenella using oral and subcutaneous routes of infection were investigated. The results obtained indicated that sporulated oocysts inoculated subcutaneously in doses up to 50 000 oocysts per bird were not fatal to 21-day-old chicks. Subcutaneous inoculation of oocysts was found to be less immunogenic than oral administration. The dynamics of the antibody responses were different for the two routes of infection. Orally administered oocysts stimulated a dramatic primary increase in the serum antibody titre with a tendency towards a decrease in the titre 14 days post infection irrespective of second infections at that time. However, a third oral dose of oocysts stimulated a slight increase in antibody titre. Two doses of oocysts injected subcutaneously induced only a slight increase in serum antibody titre. Such a low titre was dramatically increased following a subsequent oral dose of oocysts. Antibodies specific to E. tenella are IgM and IgG immunoglobulins. IgA immunoglobulin was not investigated.     

Infection of the chicken with a virulent or avirulent strain of Mycoplasma gallisepticum alone and together with Newcastle disease virus or E. coli or both

The influence of the virulence of M. gallisepticum (Mg) was studied in multiple infections of chickens involving Mg, Newcastle disease virus (NDV) and E. coli. Separate groups of 3-week-old chickens were inoculated supra-conjuctivally with a virulent and an avirulent strain of Mg alone and in combination with the La Sota strain of NDV, the 01 serotype of E. coli, and both NDV and E. coli. In addition, chickens were inoculated with NDV alone, E. coli alone, and with NDV and E. coli; one group was left uninfected.Clinical signs, lesions, recovery of the pathogens and the serological response were observed for all groups for 3 weeks after infection and for those involving Mg alone and Mg together with NDV for 18 weeks.No clinical signs were seen in any of the birds; in each infected group some showed mild lesions of the trachea and air sacs without any marked difference among the groups.Although the numbers of birds examined were small, the virulent strain of Mg was more readily recovered than the avirulent, from the respiratory tract, in the first 3 weeks following multiple infections. However, the virulence of Mg had no influence on the recovery of the other pathogens.For the first 6 weeks after infection there was a direct relationship between the virulence of the Mg and the proportion of birds with agglutinins to the Mg rapid serum agglutination (RSA) test in single or multiple infections; multiple infections enhanced the antibody response to both virulent and avirulent mycoplasma. For NDV, multiple infections, particularly involving E. coli, enhanced the peak titre of haemagglutination-inhibition antibodies and accelerated their appearance; the virulence of the Mg had no apparent effect on this.Neither the mycoplasma nor NDV were detected in the trachea by immunofluorescence.     

Bovine IgM and IgM response to Leptospira interrogans serovar hardjo as measured by enzyme immunoassay

The enzyme-linked immunosorbent assay (ELISA) was used to detect specific IgG and IgG antibodies in the sera of cattle infected or immunized with Leptospira interrogans serovar hardjo. IgM appeared first but was quickly followed by IgG which persisted longer than IgM. The levels of antibody detectable by ELISA and by the microscopic agglutination test (MAT) did not correlate, suggesting that the two techniques measured different antigen-antibody systems. The transient nature of the IgM response as measured by ELISA indicates potential usefulness as a serodiagnostic test for detecting current leptospiral infections.     

Q fever in pregnant goats: humoral and cellular immune responses

Q fever is a zoonosis caused by the intracellular bacterium Coxiella burnetii. Both humoral and cellular immunity are important in the host defence against intracellular bacteria. Little is known about the immune response to C. burnetii infections in domestic ruminants even though these species are the major source of Q fever in humans. To investigate the goat’s immune response we inoculated groups of pregnant goats via inhalation with a Dutch outbreak isolate of C. burnetii. All animals were successfully infected. Phase 1 and Phase 2 IgM- and IgG-specific antibodies were measured. Cellular immune responses were investigated by interferon-gamma, enzyme-linked immunosorbent spot test (IFN-γ Elispot), lymphocyte proliferation test (LPT) and systemic cytokines. After two weeks post inoculation (wpi), a strong anti-C. burnetii Phase 2 IgM and IgG antibody response was observed while the increase in IgM anti-Phase 1 antibodies was less pronounced. IgG anti-Phase 1 antibodies started to rise at 6 wpi. Cellular immune responses were observed after parturition. Our results demonstrated humoral and cellular immune responses to C. burnetii infection in pregnant goats. Cell-mediated immune responses did not differ enough to distinguish between Coxiella-infected and non-infected pregnant animals, whereas a strong-phase specific antibody response is detected after 2 wpi. This humoral immune response may be useful in the early detection of C. burnetii-infected pregnant goats.     

Use of enoyl coenzyme A hydratase of Mycobacterium avium subsp. paratuberculosis for the serological diagnosis of Johne's disease

Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP), remains difficult to control because of the lack of specific and sensitive diagnostic tests. In order to improve the specificity of sero-diagnosis for JD, the phage display library derived from genomic DNA of MAP was immunoscreened to identify novel antigenic targets. We selected a clone using antibodies from MAP experimentally infected cattle, and annotated its coding sequence as MAP1197 in the MAP genome, which encoded “echA12_2” in the MAP protein (Map-echA) belonging to Enoyl-CoA hydratase, known as a crotonase enzyme. The Map-echA was expressed in Esherichia coli and purified as a histidine-tag recombinant protein (rMap-echA), and the diagnostic potential of the protein was further evaluated by enzyme-linked immunosorbent assays (ELISA). Antibody responses to rMap-echA were higher in MAP-infected cattle than in uninfected cattle. The specificity of the Map-echA ELISA was also confirmed by evaluation with hyper-immune sera against various kinds of Mycobacterium species. Furthermore, in all experimentally infected cattle the antibody against rMap-echA was detected 2–7 months earlier than by a commercially available ELISA kit. These results suggested that Map-echA can be used as a specific and sensitive serological diagnostic antigen for the detection of MAP infection.     

Characterization of culture supernatant proteins from Brucella abortus and its protection effects against murine brucellosis

In this study, we characterized the secreted proteins of Brucella abortus into the enriched media under the bacterial laboratory growth condition and investigated the pathogenic importance of culture supernatant (CS) proteins to B. abortus infection. CS proteins from stationary phase were concentrated and analyzed using 2D electrophoresis. In MALDI TOF/TOF analysis, more than 27 proteins including CuZn SOD, Dps, Tat, OMPs, Adh, LivF, Tuf, SucC, GroEL and DnaK were identified. Cytotoxic effects of CS proteins were found to increase in a dose-dependent manner in RAW 264.7 cells. Upon B. abortus challenge into phagocytes, however, CS proteins pre-treated cells exhibited lower bacterial uptake and intracellular replication compared to untreated cells. Immunization with CS proteins induced a strong humoral and cell mediated immune responses and exhibited significant higher degree of protection against virulence of B. abortus infection compared to mice immunized with Brucella broth protein (BBP). Taken together, these results indicate that B. abortus secreted a number of soluble immunogenic proteins under laboratory culture condition, which can promote antibody production resulted in enhancing host defense against to subsequently bacterial infection. Moreover, further analysis of CS proteins may help to understand the pathogenic mechanism of B. abortus infection and host–pathogen interaction.     

Serum anti-trichostrongyle antibody responses of first and second season grazing calves

Serum anti-Ostertagia ostertagi and anti-Cooperia oncophora antibody responses were assessed in first season and second season calves grazing permanent paddocks. Calves without previous exposure to trichostrongyles were found to mount significant parasite-specific IgG1 antibody responses within two months of introduction to the pastures. A significant serum IgA response to O ostertagi and IgG2 responses to both O ostertagi and C oncophora antigens were also observed, but these responses were weaker. No consistent serum anti-trichostrongyle IgM responses were discernible in either age group. Second season grazing calves had significantly elevated IgG1, IgG2 and IgA antibody levels at turnout when compared to first season calves, but only IgA antibody levels against O ostertagi increased during the second grazing season. Comparison of serum antibody levels in first and second season calves grazed separately or together suggests that mixed grazing had no discernible effect on antigen priming.     

Application of indomethacin as a potentiator of lymphocyte blastogenesis in Brucella abortus exposed cattle

Lymphocytes from $\text{Brucella abortus}$ field strain infected, strain 19 vaccinated, non-exposed and field strain infected, but immunologically unresponsive cattle were incubated with $\text{B. abortus}$ antigen and indomethacin. There were significant increases (P < 0.005) in the blastogenic responses, as measured by [³H] thymidine uptake, in cultures with indomethacin as compared to cultures without indomethacin. Lymphocyte blastogenic responses to $\text{B. abortus}$ antigen were potentiated by indomethacin in both $\text{B. abortus}$ exposed and non-exposed cultures. However, potentiation of sensitized lymphocyte blastogenic responses by indomethacin was significantly greater (P < 0.005) than that in non-exposed lymphocytes. Additionally, indomethacin significantly potentiated Brucella-induced lymphocyte blastogenic responses in lymphocytes from anergic cattle.     

Efficacy of strain RB51 vaccine in protecting infection and vertical transmission against Brucella abortus in Sprague-Dawley rats

Immunizing animals in the wild against Brucella (B.) abortus is essential to control bovine brucellosis because cattle can get the disease through close contact with infected wildlife. The aim of this experiment was to evaluate the effectiveness of the B. abortus strain RB51 vaccine in protecting infection as well as vertical transmission in Sprague-Dawley (SD) rats against B. abortus biotype 1. Virgin female SD rats (n = 48) two months of age were divided into two groups: one group (n = 24) received RB51 vaccine intraperitoneally with 3 × 10¹⁰ colony forming units (CFU) and the other group (n = 24) was used as non-vaccinated control. Non-vaccinated and RB51-vaccinated rats were challenged with 1.5 × 10⁹ CFU of virulent B. abortus biotype 1 six weeks after vaccination. Three weeks after challenge, all rats were bred. Verification of RB51-vaccine induced protection in SD rats was determined by bacteriological, serological and molecular screening of maternal and fetal tissues at necropsy. The RB51 vaccine elicited 81.25% protection in SD rats against infection with B. abortus biotype 1. Offspring from rats vaccinated with RB51 had a decreased (p < 0.05) prevalence of vertical transmission of B. abortus biotype 1 compared to the offspring from non-vaccinated rats (20.23% and 87.50%, respectively). This is the first report of RB51 vaccination efficacy against the vertical transmission of B. abortus in the SD rat model.     

Immunization with a multisubunit vaccine considerably reduces establishment of infective larvae in a rodent model of Brugia malayi

Although recombinant vaccines have several advantages over conventional vaccines, protection induced by single antigen vaccines is often inadequate for a multicellular helminth parasite. Therefore, immunoprophylactic efficacy of cocktail antigen vaccines comprised of several combinations of three Brugia malayi recombinant proteins BmAF-Myo, Bm-iPGM and Bm-TPP were evaluated. Myosin + TPP and iPGM + TPP provided the best protection upon B. malayi infective larval challenge with ∼70% reduction in adult worm establishment over non-vaccinated animals that was significantly higher than the protection achieved by any single antigen vaccine. Myosin + iPGM, in contrast did not provide any enhance protection over the single recombinant protein vaccines. Specific IgG, IgM level, IgG antibody subclasses levels (IgG1, IgG2a, IgG2b, IgG3), lymphocyte proliferation, reactive oxygen species level and cytokines level were also determined to elucidate the characteristics of the protective immune responses. Thus the study undertaken provided more insight into the cocktail vaccination approach to combat LF.     

Immune response of calves following the inoculation of Mycoplasma dispar and Mycoplasma bovis

A small but significant reduction in the number of Mycoplasma dispar colonising the respiratory tract after intratracheal challenge was observed in gnotobiotic-calves previously inoculated subcutaneously three times with formalin-killed organisms and oil adjuvant. Injection of M. dispar by the intramuscular route, with oil adjuvant, and 2 weeks later by the intratracheal route, without adjuvant, failed to induce immunity to subsequent intratracheal challenge.Following the subcutaneous injection of killed M. dispar, the amount of antibody detected by single radial haemolysis (SRH) increased markedly with increasing age in groups of calves with average ages of 16 to 155 days when first injected. Most calves aged less than 40 days failed to produce an antibody response to a singel injection of M. dispar. With M. bovis a smaller difference was observed between antibody levels generated in calves of different ages; also, calves less than 40 days old produced a detectable SRH antibody response following a single injection of killed M. bovis.IgG1 and IgG2 antibody to M. dispar and M. bovis were measured by ELISA. IgG1 appeared before IgG2 antibody and this was particularly pronounced in younger calves. Also, for both mycoplasmas IgG2 antibody levels were lower in younger than older calves. The IgG1 response to M. dispar was compared in three groups of calves with average ages of 16, 55 and 155 days and was greatest in the oldest and least in the youngest animals. In contrast, the IgG1 response to M. bovis varied little in calves of different ages. It therefore appears that the immune response of young calves to M. dispar is impaired or defective.     

A comparative study of ELISA and other methods for the detection of Brucella antibodies in bovine sera

Enzyme-linked immunosorbent assay (ELISA), using β-galactosidase and a fluorigenic substrate, was used for the detection of antibodies to Brucella abortus in bovine sera.Among 677 animals from 9 brucellosis-free herds, none reacted in the ELISA. Among 785 animals from 23 brucellosis-infected herds, 336 were positive in ELISA, 229 in the slow agglutination test (SAT), 185 in the complement fixation test (CFT), and 165 in the Rose-Bengal test (RBT).Experimental infections were conducted with two B. abortus strains. At slaughter on day 101, after intraconjunctival infection of heifers with B. abortus strain 19 organisms, 3 animals were positive in the SAT, 3 in the CFT, 4 in the RBT and 11 in the ELISA, and Brucella organisms could be cultivated from 10 animals; among these, 2 scored positive in the SAT, 3 in the CFT, 3 in the RBT and 8 in the ELISA test. Seventeen heifers were infected with organisms of B. abortus strain 2308. On day 101, 11 heifers were found to be carriers, all of which yielded positive results in the CFT, RBT and ELISA tests, but not in the SAT.