In search of a combined brucellosis and tuberculosis vaccine for cattle

Bovine brucellosis is caused by Brucella abortus. The bacterial pathogen causes economic losses because it induces abortion in cattle. Vaccination of calves with live B. abortus strain 19 induces a certain level of protection but induces persistent antibodies against cell envelope lipopolysaccharide that make it difficult to Distinguish Infected from Vaccinated Animals (DIVA). Live vaccine B. abortus strain RB51 was developed to eliminate such interfering antibodies and therefore, facilitate the differentiation of infected from vaccinated animals and help in the eradication of the disease. Vaccination with strain RB51 induces levels of protection similar to strain 19 but neither of the two vaccines give complete protection. We have been working to enhance protection induced by strain RB51 vaccine. Protective Brucella antigens can be over-expressed in strain RB51 by introducing a plasmid containing the leuB gene and the genes encoding such antigens. To avoid the expression of antibiotic resistance genes, we produced a leuB deficient strain RB51 and introduced a plasmid containing the leuB gene and the genes to be over-expressed. This new strain maintains the plasmid and has induced significantly high protection levels in mice. In addition, it allowed the construction of an RB51 vaccine strain able to express Mycobacterium bovis protective antigens so that the vaccine could protect against brucellosis and tuberculosis simultaneously

Resposta sorológica de bovinos adultos após vacinação com as amostras 19 e RB51 de Brucella abortus

Adult cattle vaccinated once or twice with 2x10(9) viable B. abortus strain RB51 bacteria did not seroconvert in the rose bengal, serum agglutination and mercaptoethanol tests. Animals vaccinated while pregnant did not abort and no B. abortus was isolated from their vaginal mucus and milk.Bovinos adultos vacinados uma ou duas vezes com 2x10(9) bactérias viáveis da amostra RB51 de Brucella abortus não reagiram sorologicamente nas provas de rosa de bengala, soro-aglutinação em tubos e mercaptoetanol. Animais vacinados durante a prenhez não abortaram e não foi isolada B. abortus das secreções vaginais ou do leite

Brucella suis urease encoded by ure1 but not ure2 is necessary for intestinal infection of BALB/c mice

BACKGROUND: In prokaryotes, the ureases are multi-subunit, nickel-containing enzymes that catalyze the hydrolysis of urea to carbon dioxide and ammonia. The Brucella genomes contain two urease operons designated as ure1 and ure2. We investigated the role of the two Brucella suis urease operons on the infection, intracellular persistence, growth, and resistance to low-pH killing. RESULTS: The deduced amino acid sequence of urease-α subunits of operons-1 and -2 exhibited substantial identity with the structural ureases of alpha- and beta-proteobacteria, Gram-positive and Gram-negative bacteria, fungi, and higher plants. Four ure deficient strains were generated by deleting one or more of the genes encoding urease subunits of B. suis strain 1330 by allelic exchange: strain 1330Δure1K (generated by deleting ureD and ureA in ure1 operon), strain 1330Δure2K (ureB and ureC in ure2 operon), strain 1330Δure2C (ureA, ureB, and ureC in ure2 operon), and strain 1330Δure1KΔure2C (ureD and ureA in ure1 operon and ureA, ureB, and ureC in ure2 operon). When grown in urease test broth, strains 1330, 1330Δure2K and 1330Δure2C displayed maximal urease enzyme activity within 24 hours, whereas, strains 1330Δure1K and 1330Δure1KΔure2C exhibited zero urease activity even 96 h after inoculation. Strains 1330Δure1K and 1330Δure1KΔure2C exhibited slower growth rates in tryptic soy broth relative to the wild type strain 1330. When the BALB/c mice were infected intraperitoneally with the strains, six weeks after inoculation, the splenic recovery of the ure deficient strains did not differ from the wild type. In contrast, when the mice were inoculated by gavage, one week after inoculation, strain 1330Δure1KΔure2C was cleared from livers and spleens while the wild type strain 1330 was still present. All B. suis strains were killed when they were incubated in-vitro at pH 2.0. When the strains were incubated at pH 2.0 supplemented with 10 mM urea, strain 1330Δure1K was completely killed, strain 1330Δure2C was partially killed, but strains 1330 and 1330Δure2K were not killed. CONCLUSION: These findings suggest that the ure1 operon is necessary for optimal growth in culture, urease activity, resistance against low-pH killing, and in vivo persistence of B. suis when inoculated by gavage. The ure2 operon apparently enhances the resistance to low-pH killing in-vitro

Characterization of recombinant B. abortus strain RB51SOD toward understanding the uncorrelated innate and adaptive immune responses induced by RB51SOD compared to its parent vaccine strain RB51

Brucella abortus is a Gram-negative, facultative intracellular pathogen for several mammals, including humans. Live attenuated B. abortus strain RB51 is currently the official vaccine used against bovine brucellosis in the United States and several other countries. Overexpression of protective B. abortus antigen Cu/Zn superoxide dismutase (SOD) in a recombinant strain of RB51 (strain RB51SOD) significantly increases its vaccine efficacy against virulent B. abortus challenge in a mouse model. An attempt has been made to better understand the mechanism of the enhanced protective immunity of RB51SOD compared to its parent strain RB51. We previously reported that RB51SOD stimulated enhanced Th1 immune response. In this study, we further found that T effector cells derived from RB51SOD-immunized mice exhibited significantly higher cytotoxic T lymphocyte activity than T effector cells derived from RB51-immunized mice against virulent B. abortus-infected target cells. Meanwhile, the macrophage responses to these two strains were also studied. Compared to RB51, RB51SOD cells had a lower survival rate in macrophages and induced lower levels of macrophage apoptosis and necrosis. The decreased survival of RB51SOD cells correlates with the higher sensitivity of RB51SOD, compared to RB51, to the bactericidal action of either Polymyxin B or sodium dodecyl sulfate (SDS). Furthermore, a physical damage to the outer membrane of RB51SOD was observed by electron microscopy. Possibly due to the physical damage, overexpressed Cu/Zn SOD in RB51SOD was found to be released into the bacterial cell culture medium. Therefore, the stronger adaptive immunity induced by RB51SOD did not correlate with the low level of innate immunity induced by RB51SOD compared to RB51. This unique and apparently contradictory profile is likely associated with the differences in outer membrane integrity and Cu/Zn SOD release.Peer reviewedVeterinary Health Science

Recombinant Ochrobactrum anthropi Expressing Brucella abortus Cu,Zn Superoxide Dismutase Protects Mice against B. abortus Infection Only after Switching of Immune Responses to Th1 Type

The members of the genus Brucella are gram-negative, facultatively intracellular bacterial pathogens that cause brucellosis in many animal species and humans. Although live, attenuated vaccines are available to protect several animal species from the disease, there is no safe and effective vaccine for human use. Here we report that a bacterium that is closely related to Brucella species, Ochrobactrum anthropi, can be used as a vaccine vector for the delivery of Brucella antigens to mice, leading to the elicitation of protective immunity against brucellosis. Brucella abortus Cu,Zn superoxide dismutase (SOD), a protective Brucella antigen, was expressed in large amounts in O. anthropi strain 49237 by use of the broad-host-range plasmid pBBR1MCS. Neither O. anthropi strain 49237 nor the recombinant O. anthropi strain 49237SOD, expressing B. abortus Cu,Zn SOD, provided protection against virulent Brucella infection in mice. Analysis of immune responses indicated that strains 49237 and 49237SOD stimulated a mix of Th1 and Th2 type responses in the mice. After the immune response was switched to a Th1-biased response by addition of oligonucleotides containing unmethylated CpG motifs, both O. anthropi strain 49237 and the recombinant O. anthropi strain 49237SOD induced protection in mice. However, the protection conferred by strain 49237SOD was significantly better than that induced by the parental strain, 49237

An indirect ELISA to detect the serologic response of elk (Cervus elaphus nelsoni) inoculated with Brucella abortus strain RB51

An indirect enzyme-linked immunosorbent assay (ELISA) was developed to identify elk (Cervus elaphus nelsoni) with Brucella abortus strain RB51 (RB51)-specific antibodies using a mouse monoclonal antibody specific for bovine IgG1. This test was relatively easy to perform, accurate, and easily reproducible; therefore it could be standardized for use between laboratories. In addition, we attempted to compensate for inherent variabilities encountered when comparing ELISA readings from multiple samples taken from many animals over time. Optical density (OD) readings for each sample were converted into a percent positivity value for analysis. A negative cutoff value was determined above which a sample was considered to have a significantly elevated anti-RB51 antibody level. Pre- and postvaccination sera from 64 6-8 mo old elk, divided into four groups (females subcutaneously inoculated with saline (control animals), females ballistically inoculated with RB51, females subcutaneously inoculated with RB51, and males subcutaneously inoculated with RB51) were used. All serum samples were collected between 27 April and 15 November 1995. Values for all saline controls were appropriately below the negative cutoff value. All subcutaneously and ballistically inoculated elk were serologically positive to RB51 for at least two sampling periods during the study. The difference in percent positivity values for the ballistically compared to the subcutaneously inoculated groups was not statistically significant at 8, 10, 14, or 18 wk postvaccination. This suggests that processing RB51 into lactose based pellets and ballistically inoculating elk with these pellets does not alter the detectable elk antibody response. Also, inoculated and control animals can be accurately identified with ELISA at 4-8 weeks postvaccination