Cynthia L. Baldwin

Interferon-gamma is crucial for surviving a Brucella abortus infection in both resistant C57BL/6 and susceptible BALB/c mice.

Brucella abortus is an intracellular bacterial pathogen that causes chronic infections in humans and a number of agriculturally important species of animals. It has been shown that BALB/c mice are more susceptible to infections with virulent strains of Brucella abortus than C57BL/6 or C57BL/10 strains. In experiments described here, gene knock‐out mice were utilized to elucidate some of the salient components of resistance. Resistant C57BL/6 mice with gene deletions or disruptions in the interferon‐γ (IFN‐γ), perforin or β2‐microglobulin genes had decreased abilities to control intracellular infections with B. abortus strain 2308 during the first week after infection. However, only the IFN‐γ knock‐out mice had a sustained inability to control infections and this resulted in death of the mice at approximately 6 weeks post‐infection. These mice had a continual increase in the number of bacterial colony‐forming units (CFU) in their spleens until death. When BALB/c mice with the disrupted IFN‐γ gene were infected they had more splenic CFU at one week post‐infection than control mice but the increase was not statistically significant and by 3 weeks they did not have more CFU than control mice. Moreover, the number of splenic bacteria did not increase in the BALB/c IFN‐γ knock‐out mice between 6 and 10·5 weeks, although they died at 10·5 weeks, the time by which normal BALB/c mice were clearing the infection. Death in both strains of IFN‐γ gene disrupted mice coincided with symptoms of cachexia and macrophages comprised ≥ 75% of the splenic leucocytes.

Protective Killed Leptospira borgpetersenii Vaccine Induces Potent Th1 Immunity Comprising Responses by CD4 and γδ T Lymphocytes

ABSTRACT Leptospira borgpetersenii serovar hardjo is the most common cause of bovine leptospirosis and also causes zoonotic infections of humans. A protective killed vaccine against serovar hardjo was shown to induce strong antigen-specific proliferative responses by peripheral blood mononuclear cells (PBMC) from vaccinated cattle by 2 months after the first dose of vaccine. This response was absent from nonvaccinated control cattle. The mean response peaked by 2 months after completion of the two-dose vaccination regimen, and substantial proliferation was measured in in vitro cultures throughout the 7 months of the study period. Variations in magnitude of the response occurred among the vaccinated animals, but by 7 months postvaccination there was a substantial antigen-specific response with PBMC from all vaccinated animals. Up to one-third of the PBMC from vaccinated animals produced gamma interferon (IFN-γ) after 7 days in culture with antigen, as ascertained by flow cytometric analysis, and significant levels of IFN-γ were measured in culture supernatants by enzyme-linked immunosorbent assay. Two-color immunofluorescence revealed that one-third of the IFN-γ-producing cells were γδ T cells, with the remaining cells being CD4+ T cells. The significance of this study is the very potent Th1-type immune response induced and sustained following vaccination with a killed bacterial vaccine adjuvanted with aluminum hydroxide and the involvement of γδ T cells in the response. Moreover, induction of this Th1-type cellular immune response is associated with the protection afforded by the bovine leptospiral vaccine against L. borgpeterseniiserovar hardjo.

Fundamentals of host immune response against Brucella abortus: what the mouse model has revealed about control of infection

The studies reviewed here evaluated the role cellular immune system components play in control of brucellosis by conducting comparative studies with brucella-resistant C57BL/10 or C57BL/6 mice and susceptible BALB/c mice. We have shown by both in vitro and in vivo studies that activation of macrophages with interferon-gamma (IFN-gamma) is an important factor for control of infection with B. abortus in the mouse model and that the mechanism of anti-brucella activity largely involved reactive oxygen intermediates. Differences in control of the organism by resistant and susceptible mice was not related to inherent differences in the ability of their macrophages to control infection either with or without IFN-gamma activation nor was it attributable to NK cells since we found no role for them in control of brucellosis in either mouse strain. However, relative resistance to brucellosis did correlate with increased production of IFN-gamma by CD4 T cells during the first weeks after infection while IL-10 contributed to susceptibility in BALB/c mice. Moreover, by 3 weeks post-infection splenocytes from the susceptible BALB/c mice failed to produce IFN-gamma and relied on TNF-alpha as well as CD8 T cells to control infection until the end of the plateau phase around 6 weeks post-infection when IFN-gamma production resumed and clearance began. In contrast, IFN-gamma was crucial for control throughout the infection in the more resistant C57BL/6 mice and the mice died in its absence by 6 weeks post-infection compared to 12 weeks for the more susceptible mice that relied on additional mechanisms of control. In contrast to the IFN-gamma knock-out mice, both beta2 microglobulin knock-out C57BL/6 mice, which do not express conventional MHC class I molecules and thus cannot present antigen to CD8 T cells, or perforin knock-out C57BL/6 mice, which have no T cell cytotoxic activity, controlled and cleared the infection as well as normal C57BL/6 mice. The hiatus of IFN-gamma production in BALB/c mice correlated with very high levels of total IL-12 and it was postulated that the lack of IFN-gamma was a consequence of p40 homodimer blocking activity. However, reduction of p40 IL-12 in vivo through administration of indomethacin reduced the infection without a concomitant measurable increase in IFN-gamma. Current studies are aimed at elucidating the mechanism of the IFN-gamma hiatus.

γδ T Cell Function Varies with the Expressed WC1 Coreceptor

WC1 molecules are transmembrane glycoproteins belonging to the scavenger receptor cysteine-rich family and uniquely expressed on γδ T cells. Although participation of WC1+ γδ T cells in immune responses is well established, very little is understood regarding the significance of expressing different forms of the WC1 molecule. Two forms previously identified by mAbs, i.e., WC1.1 and WC1.2, are expressed by largely nonoverlapping subpopulations of γδ T cells. In this study it was shown that expression of the WC1.1 coreceptor was the main indicator of proliferation and IFN-γ production in response to autologous and bacterial Ags as well as for IFN-γ production without proliferation in Th1-polarizing, IL-12-containing cultures. Nevertheless, after culture in either Th1-polarizing or neutral conditions, mRNA was present for both T-bet and GATA-3 as well as for IL-12Rβ2 in WC1.1+ and WC1.2+ subpopulations, and neither produced IL-4 under any conditions. Although the steady decrease in the proportion of WC1.1+ cells, but not WC1.2+ cells, within PBMC with animal aging suggested that the two subpopulations may have different roles in immune regulation, cells bearing either WC1.1 or WC1.2 expressed mRNA for regulatory cytokines IL-10 and TGF-β, with TGF-β being constitutively expressed by ex vivo cells. Overall, the results demonstrate that the form of the WC1 coreceptor expressed on γδ T cells divides them into functional subsets according to IFN-γ production and proliferative capacity to specific stimuli as well as with regard to representation within PBMC. Finally, evidence is provided for minor differences in the intracytoplasmic tail sequences of WC1.1 and WC1.2 that may affect signaling.

Improved methods for purification and depletion of monocytes from bovine peripheral blood mononuclear cells: Functional evaluation of monocytes in responses to lectins

We have compared different techniques for the enrichment and depletion of monocytes from bovine peripheral blood mononuclear cells. Adherence to plasma-coated gelatin was the most efficient and reproducible method for enrichment of monocytes (80% monocytes), whereas depletion of peripheral blood mononuclear cells of monocytes (0.3% monocytes and less) was best achieved by defibrination of the blood from which the PBM were separated. In both instances, purity of the cell population could be improved further by an additional step, namely, FACS sorting with a monocyte-specific monoclonal antibody to purify monocytes (97% monocytes and more), and adherence to polystyrene to remove residual monocytes from defibrinated PBM (0.1% monocytes and less). Depletion of monocytes abolished the response of PBM to concanavalin A and phytohaemagglutinin. The lectin-induced response could be restored by adding gelatin/plasma purified monocytes. This activity of monocytes could be replaced by 2-mercaptoethanol.

Evaluation of Type 1 Immune Response in Naïve and Vaccinated Animals following Challenge with Leptospira borgpetersenii Serovar Hardjo: Involvement of WC1+ γδ and CD4 T Cells

ABSTRACT Organisms within the Hardjo serovar of Leptospira species are harbored in cattle throughout the world, causing abortion in pregnant animals as well as being shed in the urine, thereby providing sources of zoonotic infection for humans. We recently showed that sterile immunity in vaccinated cattle is associated with induction of a type 1 (Th1) cell-mediated immune response. Here naïve and previously vaccinated pregnant cattle were challenged with a virulent strain of serovar Hardjo and subsequently evaluated for expression of a type 1 immune response. Lymphocytes that responded in a recall response to antigen by undergoing blast transformation were evident in cultures of peripheral blood mononuclear cells (PBMC) from vaccinated cattle throughout the postchallenge test period while those from naïve cattle were evident at one time point only. Nevertheless, beginning at 2 weeks after challenge, gamma interferon (IFN-γ) was measured in supernatants of antigen-stimulated PBMC cultures from nonvaccinated animals although the amount produced was always less than that in cultures of PBMC from vaccinated animals. IFN-γ+ cells were also evident in antigen-stimulated cultures of PBMC from vaccinated but not from nonvaccinated animals throughout the postchallenge period. The IFN-γ+ cells included CD4+ and WC1+ γδ T cells, and a similar proportion of these two subpopulations were found among the dividing cells in antigen-stimulated cultures as ascertained by carboxyfluorescein succinimidyl ester loading. Finally, while naïve and vaccinated animals had similar levels of antigen-specific immunoglobulin G1 (IgG1) following challenge, vaccinated animals had twofold-more IgG2. In conclusion, while infection may induce a type 1 response we suggest that it is too weak to prevent establishment of chronic infection.

Identification of expressed bovine class I MHC genes at two loci and demonstration of physical linkage

A cDNA library prepared from lymphocytes of a cow (E98), homozygous at major histocompatibility complex (MHC) loci (BoLA phenotype w10, KN104), was screened with a bovine MHC class I probe. Of the cDNA clones isolated, two, (2.1 and 5.1) were selected and showed divergence at both 5′ and 3′ termini. E98 DNA was digested with rare-cutter enzymes (Sfi I, Mlu I, Not I, and Cla I) and fragments were size-separated by field inversion gel electrophoresis (FIGE). Hybridization with an entire class I cDNA probe revealed multiple fragments generated by each enzyme. When the 3′ untranslated regions (UT) of 2.1 and 5.1 were used as probes, only one fragment was revealed in each digested sample, showing locus specificity of these probes in cattle. Further, DNA of transfected mouse fibroblasts L4 (expressing KN104) and L10 (expressing w10) hybridized to the 3UT regions of clones 2.1 and 5.1, respectively, Northern blot analysis of the mRNA of the L4 and L10 transfected cells provided further evidence that the cDNA clones 2.1 and 5.1 code for the BoLA-KN104 and BoLA-w10 class I molecules respectively, and thus these represent the products of two different genes. A long range physical mapping of the BoLA-w10 and KN104 genes was performed using FIGE analysis of DNA of and homozygous and an heterozygous animal. This analysis revealed that the BoLA-w10 and KN104 genes are separated by not more than 210 kilobases (kb) and that they are components of a multigene family spanning 1550 kb. As the] w10 gene is at the BoLA-A locus we assign the KN104 gene to a B locus.

Comparison of three different leptospiral vaccines for induction of a type 1 immune response to Leptospira borgpetersenii serovar Hardjo

Leptospira serovar Hardjo are bacterial pathogens of cattle that cause zoonotic infections of humans. Monovalent serovar Hardjo vaccines protect cattle from serovar Hardjo while pentavalent vaccines do not even though they contain serovar Hardjo organisms. Here, cattle vaccinated with either of two monovalent vaccines had lymphocytes that made interferon-gamma (IFN-gamma) and IgG(1) and IgG(2) antibodies to Hardjo antigen while those from cattle vaccinated with a pentavalent Leptospira vaccine did not. IFN-gamma-producing cells were mainly CD4(+), but included CD8(+) and gamma delta TCR(+) cells. Despite their monovalent composition, those vaccines also induced IFN-gamma responses to serovar Grippotyphosa antigens. Thus, induction of a type 1 immune response is consistent with protective immunity to serovar Hardjo infections.

Bovine helper T‐cell clones specific for lymphocytes infected with Theileria parva (Muguga)

Summary T‐cell clones specific for lymphocytes infected with Theileria parva were derived from animals immunized by infection with T. parva (Muguga). These clones were non‐cytolytic and had the BoT4+ BoT8‐ surface phenotype, BoT4 and BoT8 being the bovine analogues of human CD4 and CD8 molecules. The clones proliferated in response to irradiated autologous lymphoblasts infected with T. parva (Muguga) but not to autologous uninfected lymphoblasts or monocytes. They were parasite strain‐specific, in that they did not respond to autologous lymphoblasts infected with another parasite stock, T. parva (Marikebuni). The clones proliferated in the absence of exogenous T‐cell growth factor (TCGF) and produced TCGF when stimulated with concanavalin A. Induction of proliferation of the cloned T‐cells was genetically restricted, and evidence was obtained which indicated that they were restricted by determinants on class II major histocompatibility complex (MHC) molecules. These findings demonstrate that infections with T. parva stimulate antigen‐specific MHC‐restricted T‐cells with the properties of T‐helper cells. The results also provide further evidence for the expression of a parasite strain‐specific antigen on the surface of T. parva‐infected lymphocytes.

Genetic Organization and Iron-Responsive Regulation of the Brucella abortus 2,3-Dihydroxybenzoic Acid Biosynthesis Operon, a Cluster of Genes Required for Wild-Type Virulence in Pregnant Cattle

ABSTRACT Brucella abortus reportedly produces the monocatechol siderophore 2,3-dihydroxybenzoic acid (2,3-DHBA) in response to iron limitation. Nucleotide sequence analysis of the cloned DHBA biosynthesis locus from virulent B. abortus 2308 and genetic complementation of defined Escherichia coli mutants were used to identify the B. abortus genes (designated dhbC, -B, and -A) responsible for synthesis of this siderophore. Reverse transcriptase PCR analysis of total RNA with dhb-specific primers demonstrated that dhbC, -B, and -A are transcribed as components of an operon, together with dhbE, a functional homolog of the Escherichia coli entE gene. Homologs of the E. coli entD and Vibrio cholerae vibH genes were also detected in the flanking regions immediately adjacent to the B. abortus dhbCEBA operon, suggesting that B. abortus has the genetic capacity to produce a more complex 2,3-DHBA-based siderophore. Slot blot hybridization experiments and primer extension analysis showed that transcription of the B. abortus dhbCEBA operon originates from two iron-regulated promoters located upstream of dhbC. Consistent with their iron-dependent regulation, both of the dhbCEBA promoter sequences contain typical consensus Fur-binding motifs. Although previously published studies have shown that 2,3-DHBA production is not required for the establishment and maintenance of chronic spleen infection by B. abortus in mice, experimental infection of pregnant cattle with the B. abortus dhbC mutant BHB1 clearly showed that production of this siderophore is essential for wild-type virulence in the natural ruminant host.