Engineered deltaguaB-A deltavirG Shigella flexneri 2a strain CVD 1205: construction, safety, immunogenicity, and potential efficacy as a mucosal vaccine

Shigella flexneri 2a strain CVD 1204, which was constructed by introducing a specific, in-frame deletion mutation in the guaB-A operon, was compared with deltaaroA strain CVD 1201. CVD 1204 was less invasive for HeLa cells than CVD 1201, whereas following invasion, the abilities of the two mutants to proliferate intracellularly were similarly impaired. The reduction in invasiveness was independent of the guanine auxotrophic phenotype and fully recovered when the chromosomal deletion mutation in CVD 1204 was repaired. Following inoculation of the conjunctival sac of guinea pigs (Serény test) at high doses (10(9) CFU per eye), both strains evoked minimal, short- lived conjunctival inflammation, which was significantly milder with strain CVD 1204. Double mutant deltaguaB-A deltavirG (also called icsA) strain CVD 1205 induced, after a single intranasal dose, high mucosal immunoglobulin A antilipopolysaccharide titers, which were significantly boosted further following a second dose of vaccine given 14 days later. Upon Serény test challenge with wild-type S. flexneri 2a, CVD 1205-vaccinated animals were significantly protected against keratoconjunctivitis (zero of eight vaccinees versus five of seven controls, P = 0.03; vaccine efficacy, 100%). CVD 1205 is an attractive candidate for human clinical trials.     

Effect on viraemia of an American and a European serotype PRRSV vaccine after challenge with European wild-type strains of the virus

Three groups of 10 pigs were vaccinated with an American serotype porcine reproductive and respiratory syndrome virus (PRRSV) vaccine and three groups of 10 pigs were vaccinated with a European serotype PRRSV vaccine. A control group of 12 pigs was left unvaccinated. Four weeks after vaccination the PRRSV-specific antibody titres were determined and each group was challenged with either a Spanish, German or Dutch PRRSV wild-type strain. The serological responses four weeks after vaccination confirmed that the two vaccines were of different serotypes. Vaccination with the American serotype vaccine hardly reduced the level of viraemia after challenge with the European PRRSV wild-type strains, and only after challenge with the Spanish PRRSV strain was a moderate, statistically significant reduction in viraemia observed. In contrast, after vaccination with the European serotype vaccine, viraemia was completely suppressed after challenge with the German PRRSV isolate and almost completely suppressed after challenge with the Spanish and Dutch PRRSV isolates.     

Safety, immunogenicity, and transmissibility in humans of CVD 1203, a live oral Shigella flexneri 2a vaccine candidate attenuated by deletions in aroA and virG

We evaluated the safety and immunogenicity of attenuated Shigella flexneri 2a vaccine candidate CVD 1203, which harbors precise deletions in the plasmid gene virG and in the chromosomal gene aroA. CVD 1203 invades epithelial cells but undergoes minimal intracellular proliferation and cell-to-cell spread. Fasting healthy volunteers, aged 18 to 40 years, were randomly allocated (double-blind design) to receive either CVD 1203 vaccine or placebo, along with sodium bicarbonate buffer, on days 0 and 14, as follows. At the time of the first inoculation, 10 subjects received placebo (group 1) and 22 subjects received either 1.5 x 10(8) (group 2; 11 subjects) or 1.5 x 10(9) (group 3; 11 subjects) CFU of CVD 1203. Fourteen days later, subjects from group 1 received 1.2 x 10(6) CFU of CVD 1203 and subjects from groups 2 and 3 received 1.2 x 10(8) vaccine organisms. Clinical tolerance was dose dependent. After a single dose of CVD 1203 at 10(6), 10(8), or 10(9) CFU, self-limited (<48-h duration) objective reactogenicity (fever, diarrhea, or dysentery) developed in 0, 18, and 72% of subjects, respectively, and in no placebo recipients. CVD 1203 induced immunoglobulin G seroconversion to S. flexneri 2a lipopolysaccharide (LPS) in 30, 45, and 36% of subjects from groups 1, 2, and 3, respectively, and stimulated immunoglobulin A-producing anti-LPS antibody-secreting cells in 60, 91, and 100% of subjects, respectively. After vaccination, significant rises in tumor necrosis factor alpha concentration in serum (groups 1, 2, and 3) and stool (group 2) samples were observed. We conclude that engineered deletions in virG and aroA markedly attenuate wild-type S. flexneri but preserve immunogenicity; however, less reactogenic vaccines are needed.     

Identification of Shigella flexneri subserotype 1c in rural Egypt

In a population-based study of diarrhea in rural, northern Egypt, 60 Shigella flexneri strains were identified, of which 10 could not be definitively serotyped. Serological analysis with commercial reagents suggested that they were serotype 1, but the strains failed to react with subserotype 1a- or 1b-specific antibodies. All 10 strains reacted with MASF 1c, a monoclonal antibody specific for a provisional S. flexneri subserotype, 1c, first identified in Bangladesh and not previously detected outside of that region. Our results show that S. flexneri subserotype 1c is not unique to Bangladesh and that the inability to detect it may reflect both the limited use of suitable screening methods and the rarity of this subserotype.     

Mucosal DNA vaccine immunization against measles with a highly attenuated Shigella flexneri vector

An intranasal vaccine vector would elicit protective immunity at the respiratory mucosa, the portal of entry and the primary site for replication for measles virus (MV) and other respiratory viruses. In a murine model of pulmonary Shigella, we demonstrate here that a candidate-attenuated Shigella vaccine vector is safely tolerated in IFN-gamma deficient mice at an inoculum that is 1 million-fold higher than the inoculum of the wild-type parent strain that would be lethal for greater than 90% of these mice. Also, following intranasal inoculation, the Deltaasd Shigella harboring a DNA MV vaccine plasmid induces a vigorous MV-specific Th1-type (both CD8+ CTL and IFN-gamma) and, to a lesser degree, Th2-type responses among splenocytes in addition to low levels of IgG and IgA in the serum. Priming for MV-specific CTL responses was possible in mice that had prior infection with a wild-type Shigella of the same serotype. Remarkably, mice immunized by the intranasal route with attenuated Shigella harboring the DNA MV vaccine plasmid had a level of MV-specific CTL activity among splenocytes that was comparable with levels observed in mice immunized by the i.p. route with attenuated Salmonella typhi harboring the same DNA vaccine plasmid, despite the fact that Shigella remained localized to the lungs, yet Salmonella disseminated to the spleen following inoculation. Thus, Deltaasd Shigella represents a very useful vector for delivery of DNA vaccines to mucosal lymphoid tissues.     

Mechanism of bacteriophage SfII-mediated serotype conversion in Shigella flexneri

We have isolated the lysogenic bacteriophage SfII, which mediates glucosylation of Shigella flexneri O-antigen, resulting in expression of the type II antigen. SfII belongs to group A of the Bradley classification and has a genome size of 42.3kb. DNA sequencing of a 4 kb BamHI subclone identified four open reading frames (ORFs), of which only two were found to be necessary for serotype conversion. These genes were named bgt, which encodes a putative bactoprenol glucosyl transferase, and gtrII, encoding the putative type II antigen determining glucosyl transferase. These genes are adjacent to the integrase gene (int) and attachment site (attP), which are highly homologous to those of Salmonella bacteriophage P22. Another ORF encoded a highly hydrophobic protein of 120 amino acids with homologues in Escherichia coli, Salmonella bacteriophage P22 and S. flexneri. Previous studies identified gtrX, the glucosyl transferase gene, of bacteriophage SfX, which also glucosylates the O-antigen specifically. We determined that gtrX-mediated expression of the group 7,8 antigen also requires bgt. This allowed us to identify gtrII as being the serotype antigen II determining glucosyl transferase. Southern hybridization and polymerase chain reaction (PCR) analyses indicated that bgt homologues exist in the genomes of all S. flexneri serotypes and in E. coli K-12, whereas gtrII was only detected in strains of serotype 2. Transposon TnphoA-derived chromosomal mutations of bgt and gtrII in S. flexneri serotype 2a were isolated and characterized. [35S]-methionine labelling and the use of a T7 RNA polymerase expression system identified a protein of 34kDa corresponding to Bgt. However, GtrII, which has a predicted molecular weight of 55 kDa, was not detected. We propose that the function of Bgt is to transfer the glucose residues from the UDP-glucose onto bactoprenol and GtrII then transfers the glucose onto the O-antigen repeat unit at the rhamnose III position. The chromosomal organization of these serotype-converting genes, when compared with their homologues in E. coli K-12 chromosome and the P22 bacteriophage genome, were very similar. This suggests that the regions encode similar functions in these organisms and have a similar evolutionary origin.     

Differential features of transglottic carcinoma

Actinobacillus actinomycetemcomitans is considered to be an aetiological agent in various forms of periodontitis, with serotype b-specific carbohydrate being the immunodominant antigen of A. actinomycetemcomitans Y4 in high-responder patients. Lipopolysaccharide (LPS) of the organism may also be an important antigen. The purpose of the present study was to clarify the importance of LPS as an antigen of A. actinomycetemcomitans. Twenty patients who had high antibody titres to strain Y4 were selected, and the reactivity of their sera with LPS was determined by ELISA and Western blotting. Two groups of patients were observed: group 1 had high IgG titres only to serotype b strain, whereas group 2 had high IgG titres to serotypes a, b and c strains. The results of adsorption tests showed that anti-A. actinomycetemcomitans Y4 antibody in group 1 patients mostly consisted of antibody reactive with the serotype b-specific carbohydrate, whereas the antibody in group 2 patients mostly consisted of antibody reactive with the LPS of all serotypes. These data show that anti-LPS antibody is present and predominant in anti-A. actinomycetemcomitans Y4 antibody from some high-responder patients, and indicate an important role for LPS as an antigen in the humoral immune response to the organism.     

The screening detection of the antigens of pathogenic enterobacteria in the biological fluids of patients with chronic intestinal diseases

Screening detection of the antigens of some pathogenic enterobacteria in the coagglutination test with the use of Shigella (S. sonnei, S. flexneri 2a, 1b, 6), Salmonella (groups A, B, C, D, E) and Yersinia (Y. pseudotuberculosis, serovars I and III, Y. enterocolitica 03 and 09) diagnostica in patients with chronic enteric diseases, hospitalized in a noninfectious ward, has demonstrated a high incidence of the above antigens. S. flexneri antigens have shown the highest detection rate. The results thus obtained indicate that these infective agents probably take part in the development of exacerbations of chronic enteric diseases and, therefore, a comprehensive examination of this group of patients is necessary with a view to the detection of bacterial enteric infection.     

Inhibition of Salmonella intracellular proliferation by non-phagocytic eucaryotic cells

Listeria monocytogenes is a Gram-positive, facultative intracellular bacterium that causes invasive, often fatal, disease in susceptible hosts. As a foodborne pathogen, the bacterium has emerged as a significant public health problem and has caused several epidemics in the United States and Europe. Three serotypes (1/2a, 1/2b, 4b) of L. monocytogenes are responsible for nearly 95% of all reported cases of human listeriosis. L. monocytogenes serotype 4b has caused all well-characterized foodborne epidemic outbreaks in North America and Europe between 1981 and 1993. However, most of the genetic studies to characterize virulence factors of L. monocytogenes have been done by using serotypes 1/2a and 1/2c. In this investigation, we examined three virulence-associated genes (hly encoding listeriolysin, plcA encoding phosphotidylinositol-specific phospholipase C, and inlA encoding internalin) of two serotype 4b and two serotype 1/2b strains. We chose these virulence-associated genes on the basis of published sequence differences among strains from Listeria subgroups containing serotypes 1/2a and 1/2c versus 4b, respectively. They correspond to sequence homologies that include very highly conserved (hlyA), highly conserved (plcA) and mostly conserved (inlA). We found by using nucleotide sequence analysis of the hly, plcA, and inlA genes, the two L. monocytogenes strains (including a strain associated with a foodborne disease outbreak in California in 1985) in this study, two serotype 1/2b strains from a study that we recently reported, and other similar published data for serotypes 1/2a, 1/2c, and 4b, had a high degree of sequence conservation at the gene and protein levels for all three genes. However, the sequences for the hly gene of L. monocytogenes strains of serotypes 1/2b and 4b were more closely related to each other and showed significant divergence from serotypes 1/2a and 1/2c. A unique nonsynonymous mutation was found in the hly gene of L. monocytogenes isolates that were associated with the 1985 California outbreak and were the epidemic phage type. When 158 L. monocytogenes isolates from the collection at the Centers for Disease Control and Prevention were screened, the mutation was found only in one other strain that had been isolated in California 3 years before the epidemic. Although the California epidemic clone was lactose negative, other L. monocytogenes serotype 4b isolates that were lactose negative did not possess the unique mutation observed in that epidemic clone.     

Development of a fluorescent focus identification assay using serotype-specific monoclonal antibodies for detection and quantitation of rotaviruses in a tetravalent rotavirus vaccine

A fluorescent focus identification assay (FFIDA) was developed for use in experimental studies and for quantitation of the components in a tetravalent live oral rotavirus vaccine. The assay utilizes four serotype-specific neutralizing monoclonal antibodies (MAb) to detect and quantify individual rotaviruses by immunofluorescence staining of fixed virus-infected monkey kidney cells. In mixed virus infections, all four MAb, W1 (serotype 1), 1C10 (serotype 2), R1 (serotype 3), and S4 (serotype 4), specifically stain the relevant homologous serotype without exhibiting any cross-reactivity against the other serotypes. Furthermore, the test is sensitive enough to differentiate at least twofold (0.3 log) differences in virus titer. The results of testing four individual experimental vaccine lots three or more consecutive times showed that all four lots contained similar proportions of the four vaccine strains as detected by the classical plaque neutralization identification test. The rapidity and efficiency of the FFIDA are desirable attributes that make it suitable for use in studies requiring identification and quantitation of one or more of the four major rotavirus serotypes.     

Direct detection of Streptococcus mutans in human dental plaque by polymerase chain reaction

Streptococcus mutans is an etiological agent in human dental caries. A method for the detection of S. mutans directly from human dental plaque by polymerase chain reaction has been developed. Oligonucleotide primers specific for a portion of the dextranase gene (dexA) of S. mutans Ingbritt (serotype c) were designed to amplify a 1272-bp DNA fragment by polymerase chain reaction. The present method specifically detected S. mutans (serotypes c, e and f), but none of the other mutans streptococci: S. cricetus (serotype a), S. rattus (serotype b), S. sobrinus (serotypes d and g), and S. downei (serotype h), other gram-positive bacteria (16 strains of 12 species of cocci and 18 strains of 12 species of bacilli) nor gram-negative bacteria (1 strain of 1 species of cocci and 20 strains of 18 species of bacilli). The method was capable of detecting 1 pg of the chromosomal DNA purified from S. mutans Ingbritt and as few as 12 colony-forming units of S. mutans cells. The S. mutans cells in human dental plaque were also directly detected. Seventy clinical isolates of S. mutans isolated from the dental plaque of 8 patients were all positive by the polymerase chain reaction. These results suggest that the dexA polymerase chain reaction is suitable for the specific detection and identification of S. mutans.     

Antimicrobial susceptibility of Shigella from a rural community in Bangladesh

Of the 63 Shigella strains isolated from stool cultures from 200 patients who attended a district hospital in Bangladesh with bloody diarrhoea, 37 (59%) were S. dysenteriae type 1, 25 (39%) were S. flexneri and only one (2%) was S. sonnei. Over half (54%) of the Shigella isolates came from children aged < 10 years. Most (89%) of the isolates of S. dysenteriae type 1 were resistant to ampicillin, cotrimoxazole, nalidixic acid, tetracycline and chloramphenicol. Although many (60%) of the isolates of S. flexneri were resistant to ampicillin and cotrimoxazole, only 4% of them were resistant to nalidixic acid. However, all of the S. dysenteriae and S. flexneri were sensitive to ciprofloxacin. The need for periodic monitoring to determine the resistance pattern in remote areas is emphasised.     

Virulent strains of Streptococcus suis serotype 2 and highly virulent strains of Streptococcus suis serotype 1 can be recognized by a unique ribotype profile

The ribotype profiles of 42 different Streptococcus suis strains were studied. These strains belonged to five serotypes and differed in their virulence for pigs as well as in the expression of the muramidase-released protein and the extracellular protein factor. For the ribotyping, chromosomal DNAs were digested with EcoRI and were hybridized with a 1,066-bp ribosomal DNA probe. The hybridization patterns showed genetic heterogeneity within and between the serotypes. Pathogenic strains of serotype 2 and highly pathogenic strains of serotype 1 could be recognized by their unique ribotype profiles. Nonpathogenic strains showed a high degree of genetic heterogeneity. Moreover, by comparing the 16S ribosomal DNA sequences of a number of S. suis strains, we were able to design two DNA probes which specifically hybridized with S. suis strains.     

Shigella isolates in stool

A total of 447 Shigella strains were isolated from stool samples during 1989-1991. Of these 270 (60%) were from children. Among the different species and serotypes Sh. flexneri 60 (13.4%) and Sh. sonnei Phage 139 (65%) were the most frequently isolated strains. 154 (34.4%) strains were resistant to three and 179 (40%) to more than three antibiotics. Some strains of Shigella were found to be resistant to furazolidine and neomycin.     

Structural determination of the O-antigenic polysaccharide from the enterotoxigenic Escherichia coli O147

The structure of the O-antigenic polysaccharide from enterotoxigenic Escherichia coli O147 has been determined by NMR spectroscopy, and component and methylation analyses. The sequence of the sugar residues could be determined by NOESY and heteronuclear-multiple-bond-connectivity NMR experiments. It is concluded that the polysaccharide is composed of tetrasaccharide repeating units with the following structure: -->4)-beta-D-GalpA-(1-->3)-beta-D-GalpNAc-(1-->2)-alpha-L-Rhap+ ++-(1-->2)-alpha-L-Rhap-(1-->, where Rha represents 6-deoxymannose. The O-antigen of E. coli O147 is identical to the repeating unit of Shigella flexneri serotype 6 lipopolysaccharide, except that the latter contains an O-acetyl group at C3 of the rhamnosyl residue substituted by the N-acetylgalactosamine residue. Immunochemical analyses using a monoclonal antibody specific for the S. flexneri serotype 6 O-antigen showed an identical reactivity with both lipopolysaccharides.     

Is undifferentiated seronegative spondyloarthropathy a forme fruste of reactive arthritis?

Undifferentiated spondyloarthropathy (USpa) may either represent a forme fruste of other spondyloarthropathies like reactive arthritis or be a different disease entity. To study the link between USpa and reactive arthritis, we studied the presence of IgA antibodies to Yersinia enterocolitica, Salmonella typhimurium, Shigella flexneri, Campylobacter jejuni and Chlamydia trachomatis in sera from 14 patients with USpa (European Spondyloarthropathy Study Group criteria) using ELISA. Escherichia coli was used as a control antigen. An OD value of more than the mean +/- 2 S.D. of 51 blood donors was considered positive. Five patients had elevated IgA antibodies to S. flexneri, while two patients each had elevated antibody levels to S. typhimurium and Chlamydia. No patient had elevated antibodies to Y. enterocolitica, C. jejuni and E. coli. Among 51 normals, 1, 4, 3, 2 and 3 had elevated antibodies to S. flexneri, S. typhimurium, Y. enterocolitica, C. jejuni and E. coli, respectively. Nine of 14 patients with USpa had antibodies to one of the bacteria implicated in reactive arthritis: of these, antibodies to Shigella were the most frequent. Thus, a proportion of patients with USpa may in fact have reactive arthritis.     

Cloning of the gene encoding the Actinobacillus actinomycetemcomitans serotype b OmpA-like outer membrane protein

The gene encoding an outer membrane protein A (OmpA)-like, heat-modifiable Omp of Actinobacillus actinomycetemcomitans ATCC 43718 (strain Y4, serotype b) was cloned by a PCR cloning procedure. DNA sequence analysis revealed that the gene encodes a protein of 346 amino acid residues with a molecular mass of 36.9 kDa. The protein expressed by the cloned gene reacted with a monoclonal antibody to the previously described 29-kDa Omp (Omp29) of strain Y4. This monoclonal antibody reacted specifically with Omp29 of A. actinomycetemcomitans (serotype b), but not with any Omp of Escherichia coli, including OmpA. This protein exhibited characteristic heat modifiability on sodium dodecyl sulfate-polyacrylamide gels, showing an apparent molecular mass of 29 kDa when unheated and a mass of 34 kDa when heated. The N-terminal amino acid sequence of the protein expressed in E. coli perfectly matched those deduced from the purified Omp29 of strain Y4. The deduced amino acid sequence of the gene coding for Omp29 from serotype b matched completely (except for valine at position 321) that of a recently reported omp34 gene described for A. actinomycetemcomitans serotype c (NCTC 9710). Because of the conserved nature of the gene within these serotypes, we designated the gene described herein from serotype b as omp34.     

Nonsteroidal anti-inflammatory drug use and Alzheimer-type pathology in aging

Infant rats were passively immunized to determine the protective capacity of pneumococcal anticapsular antibodies. Animal-passaged strains of Streptococcus pneumoniae serotypes 1, 4, 5, 6b, 7f, 9v, 14, 18c, 19f, and 23f were used as challenge inocula (1-1500 cfu) in a model of pulmonary infection that resulted in bacteremia, meningitis, and death. From untreated control animals, histologic sections of lung demonstrated infiltrative pneumonia and lung homogenate cultures grew S. pneumoniae at concentrations of 10(3)-10(8) cfu per gram of lung tissue. A type-specific anti-capsular antibody serum concentration of 0.1-1.15 microg/mL resulted in a statistically significant reduction in mortality compared with the reduction in untreated controls, except for serotype 14, which required 2.32 microg/mL for a significant reduction in mortality. The serum antibody level that provided 50% reduction in mortality ranged from 0.1-3.5 microg/mL for all serotypes.     

Serogrouping of Bacteroides nodosus isolates from 62 sources in the United States

Bacteroides nodosus isolates from 62 sources in the United States were obtained from sheep with infectious foot diseases. Serotypic analysis of these isolates revealed 21 serotypes (designated I-XXI). These serotypes were compared with British and Australian/New Zealand B nodosus strains by use of reciprocal tube agglutination tests. These tests, as well as the cross-matching tube agglutination tests of the US serotypes, resulted in arranging the US serotypes into 11 serogroups, and comparing these serogroups with their Australian/New Zealand serogroup and British serotype counterparts. Three US serogroups and 1 additional British serotype had little or no relationship to any of the Australian/New Zealand serogroups A-H (the vaccine strains). One or more of these unrelated serogroups were found in 29% of the sources studied. The most frequently found US serotype was serotype XV at 29%. The most frequently found US serogroups were the serogroups analogous to serogroup B (43.5%) and serogroup H (37%); the other serogroups were found in 22.6% or less of the sources studied. Evaluation of 3 sources revealed that multiple serotypes in a single flock are common, multiple serotypes from a single lesion are possible, B nodosus isolates obtained from goats (unlike those from cattle) appear identical to the isolates obtained from sheep, and disease can appear in vaccinated animals, even in a flock that appears to be harboring only a single serogroup-B serotype (the serogroup for which there are 3 strains in the current vaccine).     

Vocim analysis of laryngeal images: is breathiness related to the glottic area?

289 Shigella strains were isolated from children at the paediatrics department of Ankara University. 75% of the isolates were S. sonnei and 24.8% were S. flexneri. Each strain was tested for resistance to 9 antimicrobial agents. 79% of the isolates were resistant to streptomycin (S), 56% to tetracycline (T), 55.7% to trimethoprim-sulfamethoxazole (SXT), 27.7% to ampicillin (Am) and 19.7% to chloramphenicol (C). None of the isolates was resistant to ciprofloxacin, nalidixic acid, cephalothin, ampicillin-sulbactam and ceftriaxone. 56% of the isolates were resistant to 3 or more antimicrobial agents. The most frequent pattern of resistance of S. sonnei and S. flexneri strains was SXT, T, S (39.6%) and Am, SXT, T, S, C (48.6%), respectively (p < 0.0001). These results demonstrate that trimethoprim-sulfamethoxazole should not be used in the treatment of shigellosis.