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Entry into intestinal epithelial cells is an essential feature in the pathogenicity of Salmonella typhi, which causes typhoid fever in humans. This process requires intact motility and secretion of the invasion-promoting Sip proteins, which are targets of the type III secretion machinery encoded by the inv, spa and prg loci. During our investigations into the entry of S. typhi into cultured epithelial cells, we observed that the secretion of Sip proteins and flagellin was impaired in Vi-expressing strains. We report here that the production of Sip proteins, flagellin and Vi antigen is differentially modulated by the RcsB-RcsC regulatory system and osmolarity. This regulation occurs at both transcriptional and post-translational levels. Under low-osmolarity conditions, the transcription of iagA, invF and sipB genes is negatively controlled by the RcsB regulator, which probably acts in association with the viaB locus-encoded TviA protein. The cell surface-associated Vi polysaccharide, which was maximally produced under these growth conditions, prevented the secretion of Sip proteins and flagellin. As the NaCl concentration in the growth medium was increased, transcription of iagA, invF and sipB was found to be markedly increased, whereas transcription of genes involved in Vi antigen biosynthesis was greatly reduced. The expression of iagA, whose product is involved in invF and sipB transcription, occurred selectively during the exponential growth phase and was maximal in the presence of 300mM NaCl. At this osmolarity, large amounts of Sips and flagellin were secreted in culture supernatants. As expected from these results, and given the essential role of Sip proteins and motility in entry, RcsB and osmolarity modulated the invasive capacity of S. typhi. Together, these findings might reflect the adaptive response of S. typhi to the environments encountered during the different stages of pathogenesis.

Polyphasic taxonomic methods were employed to characterize a new species of slowly growing, nonpigmented mycobacteria. We propose the name Mycobacterium triplex sp. nov. for this new taxon. Conventional identification testing demonstrated a group of similar organisms that were geographically widespread in the United States. Commercially available nucleic-acid probes specific for the Mycobacterium avium complex were unreactive for these strains. High-performance liquid chromatography analysis of the mycolic acids revealed mycolate profiles that closely resembled Mycobacterium simiae. Comparative 16S rRNA sequence data confirmed the phylogenetic relationship of the strains with the slowly growing mycobacteria. Representative-type strains have been deposited in the American Type Culture Collection as strain ATCC 700071 [corrected].

Identification of mycobacteria from BACTEC 12B cultures is achieved in 7 to 21 days by reverse-phase high-performance liquid chromatography (HPLC) using a UV spectrophotometer to detect nonpolar p-bromophenylacyl mycolic acid derivatives. However, cultures grown in BACTEC and other liquid media seldom contain sufficient mycolic acids to permit reliable identification under usual HPLC assay conditions, so the sample size must be increased. Unfortunately, samples prepared from cultures in liquid media such as BACTEC cultures also contain large amounts of extraneous polar and strongly nonpolar contaminants that interfere with the analysis and hasten deterioration of the HPLC column. The contaminants were removed from 10 samples simultaneously by solid-phase extraction (SPE), i.e., by passing the crude suspension containing the mycolic acid derivatives into disposable 500-mg tC18 SPE columns in place of the usual final filtration step used to prepare specimens for HPLC. Fifteen milliliters of 20% (vol/vol) dichloromethane in methanol was passed through the columns (< 3 ml/min) to wash through the undesired contaminants and bind the mycolic acid derivatives. The mycolates were quantitatively eluted in 3 ml of dichloromethane for analysis by HPLC. Treating a panel of 31 strains of frequently isolated mycobacteria by SPE reduced the content of contaminants by 89.3 to 99.9% without altering the chromatographic patterns compared with the same strains grown on conventional solid media and processed without SPE. Peak heights of mycolates prepared from BACTEC cultures were increased from < or = 6 to > or = 25 absorbance milliunits with SPE, sufficient for reliable interpretation by visual inspection of chromatograms obtained with a UV detector. Also, removal of the contaminants improved column longevity.

Over a 1-year period, 502 mycobacterial cultures submitted to the Microbial Diseases Laboratory were identified by high-performance liquid chromatography (HPLC) in parallel with standard biochemical methods. Identification by HPLC using a curvilinear gradient was achieved by comparing the chromatograms of the unknown cultures to chromatograms for known reference strains, together with calculation of peak height or peak area ratios, as necessary. The overall agreement between HPLC and biochemical identification was 97.2%. In addition, 7 of 12 cultures of Mycobacterium bovis were identified by HPLC as the BCG strain. Of 111 cultures biochemically identified as members of the M. avium complex (MAC), 108 were confirmed as MAC by DNA probe and 106 were confirmed by HPLC. Of the latter 106, 58 probe-positive strains were identified as M. avium, 38 were identified as M. intracellulare, and 10 were identified as Mycobacterium sp. strain "X" by HPLC. Of the remaining five nonchromogenic cultures, four had MAC-like chromatograms that did not match any in our library sufficiently to permit definitive identification. Of the latter four, two were confirmed as MAC strains by DNA probe and two were not. The last of the cultures biochemically identified as MAC (1 of 111) was a mixture of MAC and non-MAC strains. Overall, only 2 of 502 cultures yielded results by HPLC that differed from those obtained by standard biochemical methods. The HPLC result was confirmed in both cases by an independent national reference laboratory. In the 12 instances in which HPLC did not provide identification, the chromatograms were either uninterpretable or did not match available reference chromatograms. These findings show that the identification obtained by HPLC concurs well with that obtained by both the standard biochemical methods and the DNA probes. Thus, identification by HPLC provides mycobacteriology laboratories with a reproducible and specific method for accurate and timely identification of most medically important mycobacteria.

An immunoglobulin G3(kappa) monoclonal antibody (MAb), MAb 32-1-E3, which was prepared in BALB/c mice by using a heated, alcohol-acetone-extracted Salmonella newport CDC 50 antigen, reacted with protein-free lipopolysaccharides from Salmonella groups C2 (O:6,8) and C3 (O:-,8) but not with those from any other serogroup tested. Sodium periodate did not inhibit antigen reactivity, which was consistent with its identity as the abequose-containing disaccharide O:8 antigen. Reactivity was inhibited by competition with serogroup C2 (O:6,8) and C3 (O:-,8) antigens but not with non-O:8 antigens. Reactivity was also inhibited by preincubation of the antigen with polyclonal rabbit antiserogroup C2 or C3 antibodies but not with antisera to serogroup C1 or other Salmonella serogroups. The MAb agglutinated with all strains of Salmonella serogroups C2 and C3 tested but not with other bacteria. Agglutination was inhibited by preabsorbing the MAb with either of two serogroup C3 Salmonella strains, S. virginia CDC 189 or S. haardt MDL 83A4545, which contain only O:8, but not by preabsortion with O:8-negative S. cholerasuis MDL 81A7623 (group C1; O:6,7), S. paratyphi type B CDC 157 (group B; O:1,[4],5,12), or Escherichia coli (O:157) (which contains no Salmonella serogroup antigens). The MAb reacted strongly (4+ agglutination) with all 140 wild-type strains of group C2 and C3 Salmonella spp. tested and showed no reaction with any of 1,324 wild-type strains of non-C2 or non-C3 Salmonella spp. tested. The MAb is useful as a replacement for absorbed, polyclonal, single-factor O:8 antiserum to discriminate Salmonella serogroups C2 and C3 from serogroup C1.

Seventy-nine strains of Mycobacterium avium complex bacteria (MAC), previously characterized by genetic probe analysis, were assayed using two methods of reverse phase high-performance liquid chromatography (HPLC) that employed curvilinear gradients. Although different in column length and cycle time, the methods produced equivalent results, yielding seven distinct chromatographic patterns (chromatotypes) of M. avium and M. intracellulare based on the ratio of mycolate concentrations in the late vs. the middle of three peak clusters (L:M ratio). The M. avium strains (n = 36) were assigned to chromatotypes 1 through 4 (L:M ratios less than 3), and the M. intracellulare strains (n = 25) to chromatotypes 5 through 7 (L:M ratios greater than 4). Of 18 Mycobacterium 'X' strains, seven resembled M. avium, seven others resembled M. intracellulare, and four were intermediate between M. avium and M. intracellulare.

A commercial system (Biolog, Hayward, CA) that uses reduction of an indicator dye to determine the oxidation of 95 different carbon substrates contained in a defined minimal medium was tested with 35 isolates of Brucella spp. to determine if the system could be used in place of respirometric methods to identify species. Of 95 substrates contained in this system, three were oxidized by all the Brucella strains tested, 48 were oxidized by none of the strains tested, and 44 were oxidized differentially. Brucella melitensis, B. abortus, and B. suis could be distinguished from each other on the basis of their oxidation reactions in seven in these substrates; epidemiologically related strains could not be unambiguously differentiated. This carbon substrate utilization method may prove to be a useful alternative to respirometry as a means to identify strains of Brucella spp. to species level, provided that personnel are protected from exposure to this highly infectious agent.

Inoculation of neonatal CD-1 mice by multiple routes with an amyocarditic temperature-sensitive (ts) mutant (ts 1) derived from a myocarditic parent variant of coxsackievirus B3 (CVB3(m)) resulted in approximately half of the neonates surviving to adolescence. Challenge of the ts 1 survivors with CVB3(m) did not induce myocarditis, as assessed by histological examination of heart tissues. Virus was not detected in heart tissues of adolescent ts 1 survivors, but inoculation of these mice with CVB3(m) resulted in virus concentrations similar in titers to those found in CVB3(m)-inoculated normal adolescent mice. The ts 1 survivors did not contain detectable levels of anti-CVB3(m) neutralizing antibody, but upon challenge with CVB3(m) they produced antibody more rapidly and to higher titers than did normal CD-1 adolescents after primary inoculation with CVB3(m). Cell-mediated immunity in ts 1 survivors was compared with that of normal mice after challenge with CVB3(m). The capacity for production of migration inhibitory factor was assessed by the agarose droplet cell migration inhibition assay, using peritoneal exudate cells and a CVB3(m) cell lysate or KCl-extracted antigens from heart tissues of CVB3(m)-inoculated mice. Migration inhibitory factor activity was not detected in cultures of splenic leukocytes from ts 1 survivors of CVB3(m)-inoculated ts 1 survivors, but it was readily detected in cultures of splenic leukocytes from CVB3(m)-inoculated normal adolescent mice. The [(3)H]thymidine stimulation assay, performed with splenic lymphoid cells and purified CVB3(m) particles, revealed that lymphocytes from normal mice, whether inoculated with CVB3(m) or not, were not stimulated by CVB3(m) particle antigens, whereas lymphoid cells from a significantly higher proportion of ts 1 survivors, whether inoculated with CVB3(m) or not, responded with a stimulation index >/=2.0. The cells responding with positive stimulation were T lymphocytes. A higher proportion of normal mice and ts 1 survivors, both inoculated with CVB3(m), contained splenic cytotoxic T lymphocytes with higher reactivity against CVB3(m)-infected neonatal skin fibroblasts than against normal skin fibroblasts, as assessed by a (51)Cr release assay. The group of uninoculated ts 1 survivors present as a high proportion of individuals with cytotoxic T-lymphocyte reactivity against both uninoculated and CVB3(m)-inoculated skin fibroblasts. However, ts 1 survivors and normal mice possessed the same proportions of splenic lymphocytes carrying either allele for Lyt 1 and Lyt 2 surface markers. The results suggest two mechanisms by which ts 1 survivors exhibit resistance to CVB3(m) induction of myocarditis, namely, the rapid production of high-titered anti-CVB3(m) neutralizing antibody in response to CVB3(m) inoculation and altered cell-mediated immune responses against CVB3(m)-induced viral or novel cellular antigens. The data are compatible with the notion that an immune deviation mechanism, thought to be controlled through a mechanism requiring suppressor cell activity which inhibits macrophage activation in ts 1 survivors, protects these mice from induction of myocarditis.

The importance of the genus Aeromonas in human disease has recently become better appreciated through the use of improved methodologies for the recovery and identification of aeromonads from biologic specimens and as a result of medical studies aimed at determining the clinical significance of these bacteria when isolated from localized and invasive infections of humans. Taxonomic schemes currently allow for the identification of six Aeromonas species (five mesophilic and one psychrophilic), four of the five mesophilic species having been recovered from infectious processes or isolated from clinical material of human origin. Two major categories of human illness attributed to Aeromonas species have been observed: acute gastroenteritis in both pediatric and adult populations and disseminated disease (e.g., bacteremia) in persons with underlying hematologic malignancies or hepatic dysfunctions. Although the role of mesophilic aeromonads as important agents of bacterial gastroenteritis remains unconfirmed because of the inability to fulfill Koch's postulates (no animal model), several lines of compelling clinical and laboratory evidence indicate that these microorganisms are significant enteric pathogens.

Autoagglutination (AA phenotype) of mesophilic aeromonads in broth was found to be a virulence-associated marker. There were two kinds of AA+ strains: those that spontaneously pelleted (SP+), and those that pelleted only after boiling (PAB+). Of 79 strains tested, 24 (30%) were AA+, and 18 of these were recovered from clinical specimens. Most of the AA+ strains (n = 21) were identified as either Aeromonas sobria or Aeromonas hydrophila. Of the well-documented clinical isolates of A. sobria and A. hydrophila available, 5 (46%) of 11 from invasive disease and 4 (14%) of 29 from noninvasive disease were SP- PAB+. The SP- PAB+ phenotype was significantly associated with invasive infections (e.g., bacteremia and peritonitis [chi 2, P less than 0.05]). All seven of the SP- PAB+ A. sobria and A. hydrophila strains tested killed mice within 48 h after intraperitoneal infection with 1 x 10(7) to 3 x 10(7) CFU, whereas only two of four SP+ PAB+ strains tested were lethal. All of the SP- PAB+ A. sobria and A. hydrophila isolates examined shared common O somatic antigens and possessed an external layer peripheral to the cell wall as determined by thin-section electron micrography. The LL1 strain of A. hydrophila used by Dooley et al. (J. S. G. Dooley, R. Lallier, and T. J. Trust, Vet. Immunol. Immunopathol. 12:339-344, 1986) to demonstrate an S membrane protein component in aeromonads virulent for fish also was SP- PAB+ and possessed the peripheral membrane, suggesting an association between these two components. Seven AA- and three SP+ strains tested lacked this layer; furthermore, 22 (71%) of 31 such isolates did not kill mice. The AA phenotype was a stable characteristic upon long-term passage of isolates in vitro. Study of SP+ and PAB+ aeromonads by surface charge and hydrophobicity analyses indicated that neither property correlated with either virulence or the presence of an external layer.

The capacity of immune spleen, lymph node, peritoneal, bone marrow, and thymic cells to protect C58/wm mice from syngeneic transplanted line Ib leukemia was quantified. Cells harvested 14 to 15 days after primary immunization were used for adoptive protection tests. Regression curves were computer analyzed and log10, PD50 values compared. For immune spleen, lymph node, peritoneal, bone marrow, and thymic cells the PD50 values were 4.53, 5.92, 4.88, 5.51, and 5.59, respectively. When immune spleen cells were treated with anti-Thy 1.2 serum the PD50 value was increased from 4.73 to 6.09, i.e., protection was reduced greater than or equal to 95%. Similar treatment of immune thymic cells reduced protection below measureable values. Anti-B cell sera (anti-IgM and anti-Ly 4.2) did not reduce the protective effect of immune spleen or marrow cells. These results indicate that a major protective cell population in each of these compartments was theta-positive. Experiments were carried out to characterize the cortisone (CS) and x-ray sensitivity of immune spleen, thymic, and marrow cells . When donor mice were treated with 12.5 mg of cortisone acetate/day for 2 days before lymphoid cells were harvested, the orotective effects of immune spleen cells, but not immune thymic or marrow cells, was reduced. When immune spleen cells were x-irrated in vitro, their protective effect was reduced by 350 R and abolished by 1000 R. When mice received whole boyd x-irradiation 24 hr before immune spleen cells were transferred their protective effect was reduced by 1000 R but only slightly lowered by 350 R. The possible significance of the multicompartmental nature of immunity to leukemia was discussed.

Immune polioencephalomyelitis (IPE) was induced by the i.p. injection of x-irradiated (10, 000 R) syngeneic line Ib malignant lymphocytes into C58 mice that were 7 or more months old and in young mice immunosuppressed by x-ray or drugs. The occurrence of IPE in young immunosuppressed C58 mice was systematically analyzed. When mice less than 2 weeks old were x-irradiated with 600 R, IPE could not be induced. The incidence in 1-month-old mice was approximately 50% and increased progressively with the age except for a drop in incidence at 3 months. An analysis of the dose effects of x-irradiation on the occurrence of IPE in mice of different ages revealed a marked increase in the incidence in 3- and 5-month-old mice beginning at dose levels of 450 R and 300 R, respectively. Considered together, these data indicated that two subpopulations of immunocytes differing in x-ray sensitivity interacted to protect mice from IPE. It appears that under natural conditions an x-ray sensitive cell population, possibly having suppressor function, decreased with age and made mice susceptible ot induction of IPE. Five-month-old mice were immunosuppressed with an LD10 of cyclophosphamide, prednisolone, or methotrexate to determine whether mice immunosuppressed with drugs also were susceptible to the induction of IPE. The incidence was 89%, 13%, and 5%, respectively. The mouse strain specificity of IPE induction also was studied. In 6- to 8-month-old mice suppressed with 600 R, IPE could not be induced in non-H-2k strains: BALB, C57BL/6, NZB. Of the H-2K strains tested (CBA/J, C3H/He, AKR/J, C58), the disease could be induced only in the C58 and AKR/J strains. Histopathologic studies showed that CNS lesions in immunosuppressed C58 and AKR/J mice did not differ significantly from those in old C58 mice with IPE. Taken together, the results of these studies indicate that IPE can be used as a model for analyzing age-dependent diseases of suspected immunopathologic etiology.

Levamisole administration to several strains of adolescent mice at the time of or up to 4 days post-inoculation (p.i.) with a myocarditic variant of coxsackievirus B3 (CVB3m) increased the number of myocarditic lesions above that found in CVB3m-inoculated mice. Virus replication in heart tissues in vivo was not affected by levamisole administration to the mice, nor was production of neutralizing antibody to CVB3m. Lymphocytes from nodes of virus-inoculated mice treated with levamisole at 2 days p.i. exhibited an increased reactivity to phytohemagglutinin on days 6 and 8 p.i., compared with respective responses by nodal T lymphocytes from CVB3m-inoculated mice. Levamisole treatment of CVB3m-inoculated mice also increased the reactivity of splenic and peripheral blood T lymphocytes to phytohemagglutinin on day 8 p.i., but not day 6 p.i., compared with the respective responses by lymphocytes from CVB3m-inoculated mice. The proportion of theta antigen-bearing lymphocytes in the total lymphocyte population in peripheral blood of CVB3m-inoculated mice was not altered by levamisole treatment. However, CVB3m-induced reduction in this subpopulation of lymphocytes in the nodes was restored to control levels by levamisole treatment. Reactivities of cytotoxic T lymphocytes from CVB3m-inoculated mice were increased against both normal and CVB3m-inoculated target cells after levamisole treatment of these mice. The results suggest that levamisole may contribute to CVB3m induction of myocarditis by several mechanisms, such as increasing the blastogenic activity of the phytohemagglutinin-responding subset of T lymphocytes, by possibly altering T-lymphocyte distribution in the body and by nonspecifically increasing reactivities of cytotoxic T lymphocytes.

Terminal dilution, adoptive cell transfer techniques were developed to quantify the protective effect of lymphoid cells in the pathogenesis of immune polioencephalomyelitis (IPE). The pathogenic effects of lymphoid cell populations were quantified by deleting the step of antigenic challenge. Regression curves were computer analyzed and PD50 values were compared. Immune spleen cells (ISC) from 4- to 6-week-old donors were more protective (PD50 = 4.9 +/- 1.3) than ISC from 12-month-old animals (PD50 greater than 7.0). The slopes of the regression curves also differed markedly (young mice, -0.24; old mice, -0.09). ISC were less protective in 12-month-indicator mice than in 5-month-old recipients (PD50 values of 5.2 +/- 0.8 and 3.7 +/- 0.8, respectively). When adoptive cell transfer tests were used to quantify the pathogenetic effects of donor cells it was found that ISC were pathogenetic at doses of 10(5) or less, but protective at higher doses. IPEC were pathogenetic at all test doses. When ISC were x-irradiated or sonicated the were only pathogenetic. Normal spleen or peritoneal exudate cells were neither protective nor pathogenetic. A model was developed in which mice were either thymectomized at birth (Tx), or Tx at birth and x-irradiated (500 R) 8 weeks later (Tx-XR). Sham Tx or Tx-XR mice served as controls. All of the mice were challenged with antigen (10(4) x-irradiated Ib cells). Only a portion (8/24) of the Tx mice developed IPE, indicating that resistance was T cell dependent but also involved a significant T cell independent component. The data also indicated that T cells were not pathogenetic effector cells in this model. Tx mice were not reconstituted by ISC (7/18 developed IPE), Tx-XR mice were partially reconstituted (3/12 developed IPE), but sham Tx-XR were fully restored (0/20 had IPE). Normal spleen cells did not reconstitute any of the mice.

Modification of Sepharose 6MB affinity beads by conjugation with CIBA blue dextran and albumination together with siliconization and modification of the bed supports of glass chromatography columns permitted the construction of affinity columns with low non-specific binding characteristics. When used with staphylococcal protein A as the affinity ligand to fractionate antibody-coated lymphocytes, non-specific adherence was reduced to 2--3% of the viable input cells and essentially the entire input cell number could be recovered in viable, functional form. The capacity of the columns permitted quantitative separation of T and B lymphocytes in 250 microliter amounts at cell densities up to 5 X 10(8)/ml on 1.2 ml columns. Once used, the columns may be regenerated and reused numerous times without alteration of their characteristics. The method thus appears useful for preparative separation without loss of large numbers of highly purified cells having normal in vitro reactivity.

Two variants of coxsackievirus B3 (CVB3) were compared with the original myocarditic parent variant (CVB3m) for myocarditic properties in several strains of mice. The ts1R variant produced little to no myocarditis in any of the nine mouse strains examined. The ts10R variant and CVB3m could be differentiated on the basis of the extent of myocarditis induced in mice of selected H-2b and H-2k haplotypes and in the female versus the male responses of two other inbred strains. Virus quantities recovered from the hearts of myocarditic mice did not correlate with the extent of disease. The three variants could not be differentiated on the basis of: (i) rate and extent of adsorption to heart tissue homogenates, (ii) kinetic neutralization rates with antiserum directed against CVB3m, (iii) 125I labeling of surface regions of polypeptides on purified particles, or (iv) rates of heat inactivation of infectivity at 50 degrees C. These data suggest that differences in pathogenicity cannot be attributed to major alterations in capsid polypeptides. Oligonucleotide fingerprint maps of T1 RNase digests of the genomes of purified particles of the three CVB3 variants showed distinct differences. Thus, the extent of myocarditis induced by CVB3 variants in a mouse model is affected by some subtle expression of the genome, presumably not involving capsid polypeptides, as well as by the haplotype and sex of a given mouse host species.

The surface characteristics of 24 autoagglutinating (AA+) mesophilic aeromonads were investigated. One group of 16 was found to be highly related serologically by their reactive pattern against O antisera generated against three reference strains. Subsequent characterization of 11 of these isolates (group 1) indicated that they had the following properties in common: precipitation after boiling (PAB+), membership of serogroup O:11 (typing scheme of Sakazaki and Shimada), resistance to lysis by bacteriophage Aeh1, and possession of a surface layer (S layer) as determined by transmission electron microscopy. Strains not exhibiting the same serologic reactivity pattern belonged to diverse serogroups (other than O:11), were generally susceptible to lysis by Aeh1, and were S layer negative by transmission electron microscopy (group 2). Analysis of selected isolates representing both groups indicated that group 2 strains were usually more hydrophobic than group 1 isolates in several different assays; both groups, however, possessed high surface charge as determined by binding to DEAE-cellulose. Group 1 isolates were more virulent than group 2 strains tested as determined by lower 50% lethal doses for mice. On the basis of the results of the kinetics of autoagglutination in broth, relative surface hydrophobicity, uptake of Congo red, agglutination of yeast cells, and electrophoretic protein profiles of whole-cell extracts, the surface layer associated with O:11 mesophilic aeromonads appears to be distinct from that of Aeromonas salmonicida. The results suggest that a new pathogenic group of mesophilic aeromonads linked through a common AA phenotype, serogroup, and S layer cause serious infections in both humans and animals (fish).