Bacterial persistence and immunity in goats vaccinated with a purE deletion mutant or the parental 16M strain of Brucella melitensis.

To evaluate host responses, young goats were inoculated subcutaneously with a genetic deletion mutant (deltapurE201) of Brucella melitensis (n = 6), its virulent parental strain 16M (n = 6), or saline (n = 6). No clinical evidence of brucellosis was seen in any goat. Serum antibody titers peaked at postinoculation day (PID) 14. Bacteria in lymph nodes that drained sites of vaccination reached peak numbers of >10(6) CFU/g in both infected groups at PID 7 and progressively declined to PID 84. At necropsy, bacteria were present in mammary lymph nodes or spleen of 33% of goats given virulent 16M but in none of goats given the purE mutant. Lymphadenitis, most severe in goats given 16M, involved depletion of lymphocytes and germinal centers, proliferation of lymphoblasts, and vasculitis. By PID 28, lymph node architecture was restored; there was marked germinal center formation and medullary plasmacytosis. Brucellar antigens, detected with immunoperoxidase techniques, were prominent in capsular granulomas but not in lymph node cortices. Ultrastructurally, bacteria were found in macrophages (>97%) and small lymphocytes (<3%) but not in large lymphocytes. Bacteria were intact in small lymphocytes but in macrophages were in various stages of degradation. The deltapurE phenotype of deltapurE201 was preserved during infection of goat lymph nodes. Unlike Salmonella spp. purE mutants, strain deltapurE201 may be a candidate for efficacy testing; it produced immune responses, was cleared from visceral tissues, and produced less severe pathologic changes than its wild-type parent.     

Bovine NK cells can produce gamma interferon in response to the secreted mycobacterial proteins ESAT-6 and MPP14 but not in response to MPB70

Bovine NK cells have recently been characterized and the present study describes the interaction between NK cells, antigen-presenting cells, and secreted mycobacterial proteins. Gamma interferon (IFN-gamma) production by NK cells was seen in approximately 30% of noninfected calves in response to the Mycobacterium tuberculosis complex-specific protein ESAT-6, MPP14 from Mycobacterium avium subsp. paratuberculosis, and purified protein derivative (PPD) from M. tuberculosis. In contrast, no response was induced by MPB70, which is another M. tuberculosis complex-specific secreted antigen. The production of IFN-gamma by NK cells in whole blood in response to ESAT-6 and MPP14 was demonstrated using intracellular staining together with surface labeling for the NK cell-specific receptor, NKp46, or CD3. Furthermore, the depletion of NK cells from peripheral blood mononuclear cells completely abolished the IFN-gamma production. The response was mediated through stimulation of adherent cells and was largely independent of contact between adherent cells and the NK cells. Neutralization of interleukin-12 only partly inhibited IFN-gamma production, showing that other cytokines were also involved. The demonstration of NK cell-mediated IFN-gamma production in young cattle provides an explanation for the nonspecific IFN-gamma response frequently encountered in young cattle when using the IFN-gamma test in diagnosis of mycobacterial infections.     

Genetic structure of population of Bacillus cereus and B. thuringiensis isolates associated with periodontitis and other human infections

The genetic diversity and relationships among 35 Bacillus cereus and Bacillus thuringiensis isolates recovered from marginal and apical periodontitis in humans and from various other human infections were investigated using multilocus enzyme electrophoresis. The strains were isolated in Norway, except for three strains isolated from periodontitis patients in Brazil. The genetic diversity of these strains was compared to that of 30 isolates from dairies in Norway and Finland. Allelic variation in 13 structural gene loci encoding metabolic enzymes was analyzed. Twelve of the 13 loci were polymorphic, and 48 unique electrophoretic types (ETs) were identified, representing multilocus genotypes. The mean genetic diversity among the 48 genotypes was 0.508. The genetic diversity of each source group of isolates varied from 0.241 (periodontal infection) to 0.534 (dairy). Cluster analysis revealed two major groups separated at a genetic distance of greater than 0.6. One cluster, ETs 1 to 13, included solely isolates from dairies, while the other cluster, ETs 14 to 49, included all of the human isolates as well as isolates from dairies in Norway and Finland. The isolates were serotyped using antiflagellar antiserum. A total of 14 distinct serotypes were observed. However, little association between serotyping and genotyping was seen. Most of the strains were also analyzed with pulsed-field gel electrophoresis, showing the presence of extrachromosomal DNA in the size range of 15 to 600 kb. Our results indicate a high degree of heterogeneity among dairy strains. In contrast, strains isolated from humans had their genotypes in one cluster. Most strains from patients with periodontitis belonged to a single lineage, suggesting that specific clones of B. cereus and B. thuringiensis are associated with oral infections.     

Distinct differences in repertoires of low-molecular-mass secreted antigens of Mycobacterium avium complex and Mycobacterium tuberculosis

Antigens in a 4-week-old culture filtrate (CF) of Mycobacterium avium subsp. avium were separated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by Western blotting. The culture had minimal lysis of bacilli, giving a CF preparation consisting mainly of secreted proteins. Comparison with a similar CF of Mycobacterium tuberculosis with almost no contamination with intracellular proteins showed the presence of cross-reactive antigens homologous to the four components of the antigen 85 complex, as well as MPT32. These were major constituents of the M. avium subsp. avium CF. In addition, there were several low-molecular-mass bands (<15 kDa) in both species that did not cross-react with polyclonal and polyvalent rabbit antibodies in Western blotting. Furthermore, these bands were not detected in corresponding sonicate preparations, indicating high localization indexes, which is typical of soluble secreted proteins. A 14-kDa protein was selected for purification and more detailed characterization. The N-terminal amino acid sequence was determined, and a matching gene was found within the genomic sequence of M. avium subsp. avium which was highly homologous to Rv0455c of M. tuberculosis. The gene encoded a signal peptide typical of secreted mycobacterial proteins. A rabbit antiserum was raised against the purified protein, and the antigen was demonstrated by Western blotting in CFs of M. avium subsp. avium, Mycobacterium avium subsp. paratuberculosis, Mycobacterium intracellulare, and Mycobacterium scrofulaceum but was not detected in M. tuberculosis. This is a new example of a highly homologous gene being differentially expressed by different mycobacterial species.     

Immune and pathologic responses in mice infected with Brucella abortus 19, RB51, or 2308.

Immune and pathologic responses were measured for 20 weeks after infection of mice with Brucella abortus 19, RB51, or 2308. Live bacteria and bacterial antigens of 19 and RB51 persisted in spleens for 10 and 4 weeks after infection, respectively, whereas 2308 bacteria and bacterial antigens persisted for at least 20 weeks. Small germinal centers and profound lymphoid depletion occurred in spleens of mice during the first 4 weeks of infection with strain 19 or 2308; however, mice infected with strain RB51 had much larger germinal centers but no lymphoid depletion. At 4 weeks, only spleen cells from RB51-infected mice proliferated when incubated with 2308 bacteria. Large germinal centers in the spleen and spleen cell proliferative responses to 2308 did not appear in strain 19-infected mice until 6 weeks or in strain 2308-infected mice until 10 weeks. Similar proliferative responses to 2308 occurred in mice infected with strain 19 or RB51 at 6 weeks and in mice infected with strain 19, RB51, or 2308 at 10 weeks. However, at 20 weeks, spleen cell proliferative responses to 2308 occurred in mice infected with strain 19 or 2308 but not in mice infected with strain RB51. Mice infected with strain RB51 had lower and less persistent antibody titers to 2308 than did mice infected with strain 19 or 2308. Collectively, these results indicate that RB51-infected mice have less persistent immune responses to 2308 than do mice infected with 19 or 2308. The shorter duration of the responses probably resulted because RB51 is considerably less pathogenic and is cleared more rapidly from mice than are 19 and 2308.     

Lymphocyte proliferation in response to Brucella abortus RB51 and 2308 proteins in RB51-vaccinated or 2308-infected cattle.

Cattle vaccinated with Brucella abortus strain RB51 (SRB51) or infected with strain 2308 (S2308) had lymph node lymphocytes which proliferated most when incubated with 32-, 27-, 18-, or <18-kDa proteins of either SRB51 or S2308. Some S2308-infected cattle but no SRB51-vaccinated cattle had lymphocytes which proliferated in response to 80- and 49-kDa proteins of SRB51 and S2308. These results suggest that cattle vaccinated with SRB51 or infected with S2308 have lymphocytes which proliferate in response to most of the same S2308 proteins and that the immunodominant protein antigens of SRB51 and S2308 have similar molecular masses of 32, 27, 18, and <18 kDa.     

Near-infrared spectrophotometry determined brain oxygenation during fainting

During orthostatic hypotension we evaluated whether presyncopal symptoms relate to a reduced brain oxygenation. Nine subjects performed 50° head-up tilt for 1 h and eight subjects were followed during 2 h of supine rest and during 1 h of 10° head-down tilt. Cerebral perfusion was assessed by transcranial Doppler determined middle cerebral artery blood velocity (MCA v_mean), while brain blood oxygenation was assessed by near-infrared spectrophotometry determined concentration changes for oxygenated (ΔHbO₂) and deoxygenated haemoglobin and brain cell oxygenation by the oxidized cytochrome c concentration (ΔCytO₂). During head-up tilt, six volunteers developed presyncopal symptoms and mean arterial pressure (88 (78–103) to 68 (57–79) mmHg; median and range), heart rate (96 (72–111) to 65 (50–107) beats min^?1), MCA v_mean (59 (51–82) to 41 (29–56) cm s^?1), ΔHbO₂ (by ?5.3 (?3.0 to ?14.8) μmol l^?1) and ΔCytO₂ were reduced (by ?0.2 (?0.1 to ?0.4) μmol l^?1; P < 0.05). During tilt down the cardiovascular variables recovered immediately and ΔHbO₂ increased to 2.2 (?0.9–12.0) mmol L^?1 above the resting value and also ΔCytO₂ recovered. In the nonsyncopal head-up tilted subjects as in the controls, blood pressure, heart rate, MCA v_mean and brain oxygenation indices remained stable. The results suggest that during orthostasis, presyncopal symptoms relate not only to cerebral hypoperfusion but also to reduced brain oxygenation.     

Studies of immunological function in mice with defective androgen action. Distinction between alterations in immune function due to hormonal insensitivity and alterations due to other genetic factors.

The presence of androgen receptors in thymocytes and the well-described effects of exogenous androgens on thymus size suggest a role for androgenic hormones in thymocyte growth and maturation. Testicular feminization (Tfm/Y) mice which bear a heritable defect in the androgen receptor protein were studied to investigate how androgens might influence immune phenotype and function. These mice were compared to two types of controls; their Tabby/Y normal male littermates and male mice of the C57 Bl/6 strain from which the Tabby and Tfm mice were derived. Thymuses and spleens from Tfm/Y mice were larger than both types of controls. Phenotypic differences in thymocyte and splenocyte subpopulations identified by the T-cell markers CD3, CD4 and CD8 suggested that T-cell maturation was altered in the androgen-resistant animal. However, both Ta/Y and Tfm/Y were found to be high producers of interleukin-4 (IL-4) by both spleen and thymus cells, while cells from the C57 mice produced predominantly IL-2. These findings suggest that some immunological features of the Tfm/Y mouse may be related to its defect in androgen action, but that high levels of IL-4 production are probably related to other genetic changes in the C57 background.