Estimating plume reach and trapping radii for male and female Cydia pomonella (Lepidoptera: Tortricidae) captured in pheromone-kairomone baited traps in Washington State apple orchards under mating disruption.

Male Cydia pomonella (L.) (Lepidoptera: Tortricidae) dispersion has largely been studied in nonmating disrupted orchards due to synthetic pheromone interference with capture in monitoring traps. Little is known about female dispersion. This study aimed to characterize male and female dispersion in mating disrupted commercial apple orchards. Sterile C. pomonella recapture data from single-trap multiple-release experiments using PHEROCON CM-DA COMBO + AA Lure-baited orange Pherocon VI delta traps was interpreted to determine pheromone-kairomone lure-baited trap effective area, trap deployment density for effective monitoring, and absolute male and female C. pomonella density in mating disrupted Washington commercial apple orchards. The maximum plume reach of the pheromone-kairomone lure in mating disrupted orchards was <5 m from the baited trap for both sexes. Maximum dispersive distances for 95% of the released C. pomonella in mating disrupted orchards were 106 and 135 m for males and females, yielding trapping areas of 3.87 and 6.16 ha, respectively. Estimates were consistent across 3 growing seasons and represent the first records of male and female dispersal distance and monitoring trap efficacy from commercial C. pomonella mating disrupted apple orchards. With relevance to commercial monitoring programs and economic thresholds in mating disrupted orchards, traps should be deployed at a density of 1 per 3-6 ha. Capture of a single male or female C. pomonella corresponds to at least 82-104 C. pomonella within the 3-6 ha trapping area. This refined C. pomonella capture interpretation in pheromone-kairomone baited traps in mating disrupted commercial apple orchards yields more precise damage estimates and assists in insecticide-use decision making.

Escherichia coli isolates from commercial chicken meat and eggs cause sepsis, meningitis and urinary tract infection in rodent models of human infections.

The zoonotic potential of Escherichia coli from chicken-source food products is important to define for public health purposes. Previously, genotypic and phenotypic screening of E. coli isolates from commercial chicken meat and shell eggs identified some E. coli strains that by molecular criteria resembled human-source extraintestinal pathogenic E. coli (ExPEC). Here, to clarify the zoonotic risk of such chicken-source E. coli, we compared selected E. coli isolates from chicken meat and eggs, stratified by molecularly defined ExPEC status, to human-source ExPEC and to laboratory E. coli for virulence in rodent models of sepsis, meningitis and UTI, and evaluated whether specific bacterial characteristics predict experimental virulence. Multiple chicken-source E. coli resembled human-source ExPEC in their ability to cause one or multiple different ExPEC-associated infections. Swimming ability corresponded with urovirulence, K1 capsule corresponded with ability to cause neonatal meningitis, and biofilm formation in urine corresponded with ability to cause sepsis. In contrast, molecularly defined ExPEC status and individual genotypic traits were uncorrelated with ability to cause sepsis, and neither complement sensitivity nor growth in human urine corresponded with virulence in any infection model. These findings establish that chicken-derived food products contain E. coli strains that, in rodent models of multiple human-associated ExPEC infections, are able to cause disease comparably to human-source E. coli clinical isolates, which suggests that they may pose a significant food safety threat. Further study is needed to define the level of risk they pose to human health, which if appreciable would justify efforts to monitor for and reduce or eliminate them.

Evaluating the efficacy of antimicrobial cycling programmes and patient isolation on dual resistance in hospitals.

Antibiotic-resistant bacteria cause a number of infections in hospitals and are considered a threat to public health. A strategy suggested to curb the development of resistant hospital-acquired infections is antimicrobial cycling, in which antibiotic classes are alternated over time. This can be compared with a mixing programme in which, when given two drugs, half of the physicians prescribe one drug over the other. A mathematical model of antimicrobial cycling in a hospital population setting is developed to evaluate the efficacy of a cycling programme with an emphasis on reducing the emergence and significance of dual resistance. The model also considers the effects of physician compliance and isolating patients harbouring dual-resistant bacteria. Simulation results show that the optimal antimicrobial drug usage programme in hospital populations depends upon the type of resistance being targeted for treatment; a cycling programme is more effective against dual resistance compared with mixing. Patient isolation and high compliance to a cycling programme is also shown to dramatically decrease dual resistance in hospitalized populations. Ultimately, the exclusive use of antimicrobials in fighting nosocomial infection does not solve the problem but just slows down what appears to be a losing battle against drug resistance. We hope that this paper serves to instigate discussion on the many dimensions of the complex problem of drug resistance in hospital settings.

Allelic loss of the active X chromosome during bladder carcinogenesis.

CONTEXT: Previous studies have shown that loss of the X chromosome is involved in the carcinogenesis of certain human malignancies. OBJECTIVE: To determine whether X-linked allelic losses occur during bladder tumorigenesis and whether such losses involve the active or the inactive X chromosome. DESIGN: We analyzed the deletion status of the X-linked human androgen receptor gene locus in 6 female patients who underwent radical cystectomies for muscle-invasive urothelial carcinoma of the urinary bladder. Four patients had coexisting urothelial carcinoma in situ. Analysis for inactivation of the X chromosome was carried out in parallel. RESULTS: Three cases were informative. Invasive tumor samples showed loss of heterozygosity involving the active allele at the androgen receptor locus in all 3 positive cases, whereas carcinoma in situ showed nonrandom X chromosome inactivation but not allelic deletion. CONCLUSIONS: Our data suggest that allelic loss of the activated X chromosome is involved in bladder carcinogenesis and cancer progression.

Molecular genetic alterations in the laser-capture-microdissected stroma adjacent to bladder carcinoma.

BACKGROUND: Urothelial carcinoma commonly manifested loss of heterozygosity (LOH) at different regions of chromosomes 17p, 3p, and 9q. Recent studies suggested that bladder stromal cells may be implicated in the growth and progression of urothelial carcinoma. To better understand the genetic alterations in the stromal cells in patients with bladder carcinoma, the authors evaluated the prevalence of allelic loss at three microsatellite polymorphic markers on chromosomes 17p13 (TP53), 3p25-26 (D3S3050), and 9q32-33 (D9S177). In addition, the pattern of X-chromosome inactivation of the stromal cells was evaluated by analyzing the DNA methylation pattern at a polymorphic site on the androgen receptor gene. METHODS: The authors studied 18 female patients who underwent either transurethral resection (n = 2) or radical cystectomy (n = 16) for high-grade muscle-invasive urothelial carcinoma of the urinary bladder. Genomic DNA samples from the stromal cells immediately adjacent to the tumor and the tumor itself were prepared from formalin-fixed, paraffin-processed tissues using laser-assisted microdissection and LOH was determined. RESULTS: The stromal cells showed a high frequency of LOH on chromosomes 17p13 (29%), 3p25-26 (61%), and 9q32-33 (47%) with no clear concordance with the adjacent tumor cells. Fourteen specimens (78%) showed LOH in the stroma in at least 1 of 3 markers examined. Nonrandom X-chromosome inactivation was frequent in the stromal cells (50% of informative specimens). CONCLUSIONS: The current study revealed that some of the genetic changes that commonly occur with invasive urothelial carcinoma were frequently found in the adjacent stroma and suggested that the stroma of urothelial carcinoma may play an important role in bladder carcinogenesis.

Bacteremia associated with live attenuated chi8110 Salmonella enterica serovar Typhi ISP1820 in healthy adult volunteers.

Live attenuated chi8110 Salmonella enterica serovar Typhi ISP1820 vaccine was given in a dose-escalation trial to healthy, adult volunteers. Positive stool and blood cultures were noted, but limited, as were immune responses measured by ELISA and ELISPOT. Only volunteers with bacteremia developed immune responses; however, no symptoms were associated with bacteremia. The vaccine was insufficiently immunogenic for use as a vaccine. It is possible that reduced survival in the gut and reduced immunogenicity may have been due to the thawing of frozen inocula immediately prior to use.

Identification of Salmonella typhi genes expressed within macrophages by selective capture of transcribed sequences (SCOTS).

Salmonella enterica serovar Typhi (S. typhi) is a human-restricted pathogen which causes typhoid fever. Relatively little is known about S. typhi host interaction as animal models of this disease are severely limited by the lack of virulence of S. typhi in other hosts. The virulence of other Salmonella serovars in animal models is dependent on the abilities of these bacteria to survive within host macrophages. We have used selective capture of transcribed sequences (SCOTS) to identify S. typhi genes expressed during growth in human macrophages. This positive cDNA selection technique identified 28 distinct clones representing previously identified as well as novel, uncharacterized and hypothetical gene sequences that are expressed intracellularly. Transcripts for the Vi capsular antigen and genes whose products are involved in stress responses and nutrient acquisition were obtained from intracellular bacteria using SCOTS. Most of these clones are present in the S. typhimurium genome and are also expressed in murine macrophages. Nineteen of these gene sequences were disrupted insertionally in S. typhi, and most of the resulting mutants exhibited a lower level of survival within macrophages compared with the wild-type parent strain. Mutant strains, transformed with a copy of a wild-type gene, exhibited a macrophage survival level similar to that of the parental strain.

MlrA, a novel regulator of curli (AgF) and extracellular matrix synthesis by Escherichia coli and Salmonella enterica serovar Typhimurium.

Production of curli (AgF) adhesins by Escherichia coli and Salmonella enterica serovar Typhimurium (S. typhimurium) is associated with extracellular matrix production and is optimal at low temperature during stationary phase. Curli and extracellular matrix synthesis involves a complex regulatory network that is dependent on the CsgD (AgfD) regulator. We have identified a novel regulator, termed MlrA, that is required for curli production and extracellular matrix formation. Two cosmids from a genomic library of avian pathogenic E. coli chi7122 conferred mannose-resistant haemagglutination (HA) and curli production to E. coli HB101, which is unable to produce curli owing to a defective regulatory pathway. The rpoS gene, encoding a known positive regulator of curli synthesis, and the E. coli open reading frame (ORF) of unknown function, yehV, identified on each of these cosmids, respectively, conferred curli production and HA to E. coli HB101. We have designated yehV as the mlrA gene for MerR-like regulator A because its product shares similarities with regulatory proteins of the MerR family. HA and curli production by strain chi7122 were abolished by disruption of rpoS, mlrA or csgA, the curli subunit gene. Both csgD and csgBA transcription, required for expression of curli, were inactive in an mlrA mutant grown under conditions that promote curli production. An mlrA homologue was identified in S. typhimurium. Analysis of mlrA-lac operon fusions demonstrated that mlrA was positively regulated by rpoS. mlrA mutants of wild-type S. typhimurium SL1344 or SR-11 no longer produced curli or rugose colony morphology, and exhibited enhanced aggregation and extracellular matrix formation when complemented with the mlrA gene from either S. typhimurium or E. coli present on a low-copy-number plasmid. However, inactivation of mlrA did not affect curli production and aggregative morphology in an upregulated curli producing S. typhimurium derivative containing a temperature- and RpoS-independent agfD promoter region. These results indicate that MlrA is a newly defined transcriptional regulator of csgD/agfD that acts as a positive regulator of RpoS-dependent curli and extracellular matrix production by E. coli and S. typhimurium.

Intranasal immunogenicity of a Delta cya Delta crp-pabA mutant of Salmonella enterica serotype Typhimurium for the horse.

The aim of this study was to investigate the intranasal immunogenicity for the horse of a Deltacya Deltacrp-pabA mutant (MGN-707) of Salmonella enterica serotype Typhimurium (S. typhimurium). MGN-707 caused no sign of disease, was not detected in feces and a single administration induced strong Salmonella-specific serum and nasal mucosal antibody responses. All ponies had made strong salmonella specific serum IgGa, IgGb, IgA and IgM antibody responses by day 25 after the first immunization. IgM responses to salmonella lipopolysaccharide (LPS) were short lived whereas salmonella specific serum IgGa and IgGb persisted at high levels in all ponies until 83 and 140 days, respectively. Specific nasal mucosal antibody responses dominated by IgA and IgM were evident by day 25 in all ponies except one in which only specific IgGa and IgGb were evident. Specific nasal mucosal IgA persisted in most ponies until day 69. A second immunization on day 140 boosted antibody responses, and stimulated a strong nasal mucosal IgA response in the pony that failed to make an IgA response after primary immunization. At the termination of the experiment, IgA and IgGb dominated jejunal antibody responses whereas vaginal responses were mainly IgA. The latter response unequivocally confirms the existence of a common mucosal immune system in equids. The results indicate that a S. typhimurium Deltacya Deltacrp-pabA mutant has potential as an intranasal vaccine against salmonellosis in the horse.

Intranasal immunogenicity of a Deltacya Deltacrp-pabA mutant of Salmonella enterica serotype Typhimurium for the horse.

The aim of this study was to investigate the intranasal immunogenicity for the horse of a Deltacya Deltacrp-pabA mutant (MGN-707) of Salmonella enterica serotype Typhimurium (S. typhimurium). MGN-707 caused no sign of disease, was not detected in feces and a single administration induced strong Salmonella-specific serum and nasal mucosal antibody responses. All ponies had made strong salmonella specific serum IgGa, IgGb, IgA and IgM antibody responses by day 25 after the first immunization. IgM responses to salmonella lipopolysaccharide (LPS) were short lived whereas salmonella specific serum IgGa and IgGb persisted at high levels in all ponies until 83 and 140 days, respectively. Specific nasal mucosal antibody responses dominated by IgA and IgM were evident by day 25 in all ponies except one in which only specific IgGa and IgGb were evident. Specific nasal mucosal IgA persisted in most ponies until day 69. A second immunization on day 140 boosted antibody responses, and stimulated a strong nasal mucosal IgA response in the pony that failed to make an IgA response after primary immunization. At the termination of the experiment, IgA and IgGb dominated jejunal antibody responses whereas vaginal responses were mainly IgA. The latter response unequivocally confirms the existence of a common mucosal immune system in equids. The results indicate that a S. typhimurium Deltacya Deltacrp-pabA mutant has potential as an intranasal vaccine against salmonellosis in the horse.

Secretion of virulence determinants by the general secretory pathway in gram-negative pathogens: an evolving story.

Secretion of proteins by the general secretory pathway (GSP) is a two-step process requiring the Sec translocase in the inner membrane and a separate substrate-specific secretion apparatus for translocation across the outer membrane. Gram-negative bacteria with pathogenic potential use the GSP to deliver virulence factors into the extracellular environment for interaction with the host. Well-studied examples of virulence determinants using the GSP for secretion include extracellular toxins, pili, curli, autotransporters, and crystaline S-layers. This article reviews our current understanding of the GSP and discusses examples of terminal branches of the GSP which are utilized by factors implicated in bacterial virulence.

Expression of the rfa, LPS biosynthesis promoter in Salmonella typhimurium during invasion of intestinal epithelial cells.

Salmonella regulates transcription of many of its genes in response to environmental conditions encountered inside or outside the eukaryotic cells it infects. In this paper, we examined Salmonella typhimurium gene expression within epithelial cells, by using bacterial luciferase as a reporter. We focused on gene expression controlled by Salmonella rfa promoter, using lac promoter as a control. We observed down regulation for both promoters during the initial 2 h of invasion. The decreased levels of luciferase activity appeared to be due to metabolic changes, since we observed similar results with tissue culture medium alone. Gene expression stabilized to a new steady state for the Salmonella rfa promoter, while a lac promoter activity steadily decreased. Bacterial luciferase activity was a good indicator of intracellular numbers and allowed us to detect as few as 1000 bacterial cells/infected monolayer. Both promoters were not dependent on host protein synthesis for expression.

Relationship between the Tsh autotransporter and pathogenicity of avian Escherichia coli and localization and analysis of the Tsh genetic region.

The temperature-sensitive hemagglutinin Tsh is a member of the autotransporter group of proteins and was first identified in avian-pathogenic Escherichia coli (APEC) strain chi7122. The prevalence of tsh was investigated in 300 E. coli isolates of avian origin and characterized for virulence in a 1-day-old chick lethality test. Results indicate that among the tsh-positive APEC isolates, 90.6% belonged to the highest virulence class. Experimental inoculation of chickens with chi7122 and an isogenic tsh mutant demonstrated that Tsh may contribute to the development of lesions within the air sacs of birds but is not required for subsequent generalized infection manifesting as perihepatitis, pericarditis, and septicemia. Conjugation and hybridization experiments revealed that the tsh gene is located on a ColV-type plasmid in many of the APEC strains studied, including strain chi7122, near the colicin V genes in most of these strains. DNA sequences flanking the tsh gene of strain chi7122 include complete and partial insertion sequences and phage-related DNA sequences, some of which were also found on virulence plasmids and pathogenicity islands present in various E. coli pathotypes and other pathogenic members of the Enterobacteriaceae. These results demonstrate that the tsh gene is frequently located on the ColV virulence plasmid in APEC and suggest a possible role of Tsh in the pathogenicity of E. coli for chickens in the early stages of infection.

Effects of DksA and ClpP protease on sigma S production and virulence in Salmonella typhimurium.

Salmonella typhimurium responds to a variety of environmental stresses by accumulating the alternative sigma factor sigmaS. The repertoire of sigmaS -dependent genes that are subsequently expressed confers tolerance to a variety of potentially lethal conditions including low pH and stationary phase. The mechanism(s) responsible for triggering sigmaS accumulation are of considerable interest, because they help to ensure survival of the organism during encounters with suboptimal environments. Two genes associated with regulating sigmaS levels in S. typhimurium have been identified. The first is clpP, encoding the protease known to be responsible for degrading sigmaS in Escherichia coli. The second is dksA, encoding a protein of unknown function not previously associated with regulating sigmaS levels. As predicted, clpP mutants accumulated large amounts of sigmaS even in log phase. However, dksA mutants failed to accumulate sigmaS in stationary phase and exhibited lower accumulation during acid shock in log phase. DksA appears to be required for the optimal translation of rpoS based upon dksA mutant effects on rpoS transcriptional and translational lacZ fusions. The region of rpoS mRNA between codons 8 and 73 is required to see the effects of dksA mutations. This distinguishes the role of DksA from that of HF-I (hfq ) in rpoS translation, as the HF-I target area occurs well upstream of the rpoS start codon. DksA appears to be involved in the expression of several genes in addition to rpoS based on two-dimensional SDS-PAGE analysis of whole-cell proteins. As a result of their effects on gene expression, mutations in clpP and dksA decreased the virulence of S. typhimurium in mice, consistent with a role for sigmaS in pathogenesis.

Genomic subtractive hybridization and selective capture of transcribed sequences identify a novel Salmonella typhimurium fimbrial operon and putative transcriptional regulator that are absent from the Salmonella typhi genome.

Salmonella typhi, the etiologic agent of typhoid fever, is adapted to the human host and unable to infect nonprimate species. The genetic basis for host specificity in S. typhi is unknown. The avirulence of S. typhi in animal hosts may result from a lack of genes present in the broad-host-range pathogen Salmonella typhimurium. Genomic subtractive hybridization was successfully employed to isolate S. typhimurium genomic sequences which are absent from the S. typhi genome. These genomic subtracted sequences mapped to 17 regions distributed throughout the S. typhimurium chromosome. A positive cDNA selection method was then used to identify subtracted sequences which were transcribed by S. typhimurium following macrophage phagocytosis. A novel putative transcriptional regulator of the LysR family was identified as transcribed by intramacrophage S. typhimurium. This putative transcriptional regulator was absent from the genomes of the human-adapted serovars S. typhi and Salmonella paratyphi A. Mutations within this gene did not alter the level of S. typhimurium survival within macrophages or virulence within mice. A subtracted genomic fragment derived from the ferrichrome operon also hybridized to the intramacrophage cDNA. Nucleotide sequence analysis of S. typhimurium and S. typhi chromosomal sequences flanking the ferrichrome operon identified a novel S. typhimurium fimbrial operon with a high level of similarity to sequences encoding Proteus mirabilis mannose-resistant fimbriae. The novel fimbrial operon was absent from the S. typhi genome. The absence of specific genes may have allowed S. typhi to evolve as a highly invasive, systemic human pathogen.

Construction and evaluation of a delta cya delta crp Salmonella typhimurium strain expressing avian pathogenic Escherichia coli O78 LPS as a vaccine to prevent airsacculitis in chickens.

Avian pathogenic strains of Escherichia coli cause a number of extraintestinal diseases in poultry, including airsacculitis and colisepticemia. Expression of O78 lipopolysaccharide (LPS) is frequently associated with pathogenic isolates. Salmonella, a common poultry contaminant, is a major public health concern. The purpose of this work was to develop an E. coli vaccine for poultry with the use of an attenuated Salmonella typhimurium carrier that would benefit both the bird and the consumer. Orally administered attenuated S. typhimurium delta cya delta crp strains have been shown to provide excellent protection against wild-type Salmonella challenge in chickens. This work describes the construction of a delta cya delta crp derivative of an avian pathogenic S. typhimurium that expresses both the homologous group B determinants (O1,4,5,12) and the heterologous E. coli O78 LPS O antigens. This was accomplished by inserting the E. coli rfb region, which encodes the genes required for O78 expression, into the chromosomal cya gene of S. typhimurium, creating a defined deletion/insertion mutation. A delta crp mutation was introduced in a subsequent step. Expression of both O antigens was stable in vitro and in vivo. Vaccination of white leghorn chicks at day of hatch and 14 days with the recombinant vaccine strain induced serum immune responses against both S. typhimurium and E. coli LPS and protected the birds against subsequent challenge with an avian pathogenic E. coli O78 strain. Introduction of a mutation in rfc, which encodes the O antigen polymerase, reduced the chain length of the S. typhimurium LPS without affecting the expression of O78. The rfc mutation further enhanced the ability of the vaccine strain to protect chickens against E. coli challenge.

Attenuation and immunogenicity of Deltacya Deltacrp derivatives of Salmonella choleraesuis in pigs.

Six different isogenic Deltacya Deltacrp derivatives of a strain of Salmonella choleraesuis var. kunzendorf-chi3246 virulent for pigs were constructed by transposon-mediated deletion mutagenesis. These strains were evaluated for virulence and ability to elicit a protective immune response in young weaned pigs after oral administration and were compared to a commercially available vaccine which lacks the 50-kb virulence plasmid (vpl(-)). These derivatives were Deltacya Deltacrp vpl(+), Deltacya Deltacrp vpl(-), Deltacya Delta(crp-cdt) vpl(+), Deltacya Delta(crp-cdt) vpl(-), Deltacya Deltacrp pmi-3834 vpl(+), and Deltacya Delta(crp-cdt) pmi-3834. In experiments to evaluate safety, no significant adverse effects of any of the vaccine constructs were observed, except that two of the strains which carried the virulence plasmid (vpl(+)) caused a small, short-term elevation in maximum temperature compared to pretreatment temperature values. Orally immunized animals, except for those vaccinated with the Deltacya Deltacrp pmi-3834 vpl(+) strain or SC-54, developed significant serum antibody responses 21 days postvaccination as measured by enzyme-linked immunosorbent assay. No cell-mediated immune responses to heat-killed S. choleraesuis were noted at the same time point as measured with heat-killed bacteria as antigen in a lymphocyte proliferation assay. In an oral challenge exposure model with a highly virulent heterologous strain of S. choleraesuis, the Deltacya Deltacrp strains with deletions in pmi were not protective. As measured by morbidity scores, the responses to challenge of the pigs vaccinated with the other four Deltacya Deltacrp derivatives were significantly better than those of the nonvaccinated, challenged group. With the exception of temperature elevation and slight differences in diarrhea scores postchallenge, none of these strains differed significantly from each other in the other clinical parameters analyzed. While the commercial vaccine was protective by most of the parameters measured, it was not fully protective against challenge with virulent S. choleraesuis as judged by diarrhea scores and temperature elevation. Collectively, these data demonstrate that Deltacya Deltacrp derivatives, with or without the virulence plasmid but not with deletions in the pmi gene, are candidates for vaccines for protection against salmonellosis in pigs.