The efficacy, biodistribution and safety of an inhibin DNA vaccine delivered by attenuated Salmonella choleraesuis.

DNA vaccines, the third-generation vaccines, were extensively studied. The attenuated Salmonella choleraesuis (S. choleraesuis) was widely focused as a carrier to deliver DNA vaccines in the chromosome-plasmid balanced-lethal system. The efficacy of inhibin DNA vaccine delivered by attenuated S. choleraesuis was proved in mice and cows in our previous studies. In this study, the efficacy of inhibin DNA vaccine was confirmed in rhesus monkeys. To further study the biodistribution and safety, the mice were immunized under laboratory conditions. The results of the rhesus monkeys showed the plasma IgA and IgG titres against inhibin were elevated, and the oestradiol (E2 ) and progesterone (P4 ) levels were increased with immunizing inhibin DNA vaccine. The biodistribution and safety assessment displayed the body weight, pathological change and haematology indexes where there is no significant difference between vaccinated mice and control. And the genomics analysis showed there was no integration of the inhibin gene into the mouse genome 2 months after immunization. This study indicated the inhibin DNA vaccine delivered by attenuated S. choleraesuis was safe. And this vaccine was a potential means to improve their reproductive traits in primates and other animals.

Antimicrobial profile of Salmonella enterica serotype Choleraesuis from free-range swine in Kakamega fish market, western Kenya.

INTRODUCTION: Salmonella enterica subspecies enterica serovar Choleraesuis is a host-adapted, facultative, intracellular pathogen that causes swine paratyphoid. Its antimicrobial resistance presents a challenge to feed manufacturing industries. However, stopping antibiotics in animal feed would have economic implications for the industry. METHODOLOGY: Conventional microbial methods for isolation and identification of S. Choleraesuis were employed. The isolates were subjected to screening against 17 antimicrobial agents and genotyping of resistance markers by PCR. The data were then analyzed and presented in percentages. RESULTS: Phenotypically, 43 out of 95 isolates showed multidrug resistance. Among the 17 antibiotics tested, resistance was observed as follows: sulphonamides (45.2%), nalidixic acid (44.25%), tetracycline (42%), ampicillin (36.8%), erythromycin (34.7%), carbenicillin (31.5%), chrolamphenical (28.4%), gentamicin (27.3%), kanamycin (24.2%), spectinomycin (21%), sulfamethoxazole-trimethoprim (16.8%), streptomycin (12.6%), cephalothion (8.4%), ofloxacin (5.2%), ciprofloxacin (4.2%), and norfloxacin (4.2%). Fifty-two isolates were susceptible to the antimicrobial agents tested. A total of 3.1% of the isolates had the integron gene pattern combination of dfrA2-aadA2 (2100 bp), dfrA12 (2100 bp); 4.2% had dfrA12-aadA2-sulI (2100 bp); 2.1% had dfrA12-aadA2 (2100 bp); and 1% had dfrA2-aadA2-sulI (2100 bp), oxa1-aadA2 (1500 bp), dfrA12-aadA2-sulI, and blaPSE (2100 bp). CONCLUSIONS: The isolated S. Choleraesuis were resistant to more than 10% of the antimicrobial agents used in this study. Appropriate surveillance is warranted to gain more information about the epidemiology, as stopping antibiotics in animal feed would have economic implications for the industry.

Efficacy and safety of an oral somatostatin DNA vaccine without antibiotic resistance gene in promoting growth of piglets.

This study aimed to evaluate the efficacy and safety of an oral DNA vaccine against somatostatin (SS) (pGS/2SS-asd, encoding two copies of somatostatin genes) mediated by attenuated Salmonella choleraesuis C500 without antibiotic resistance gene on piglets growth. A total of 50 piglets were uniformly divided into five groups. The animals in the first three groups were orally given vaccine in dose of either 5 9 1010, 5 9 109 or 5 9 108 colony-forming units (CFU).The remaining two groups were orally administered with either bacteria C500(containing pVAX-asd plasmid without somatostatin gene) or phosphate buffered saline (PBS) as controls. The results indicated that the vaccine induced SS-specific antibodies in a dose-dependent pattern. Compared with the PBS control, animals in the high-dose group showed lower SS levels and higher growth hormone (GH) levels in sera. Average daily gain of animals in the high dose group was increased by 32.88% and 26.46% during 4 and 8 weeks,respectively. Anti-SS antibodies were positively correlated with either GH levels or average daily gain at week 8 after primary immunization (P < 0.05). Faecal,soil and water samples originating from immunized piglets and surrounding environment were collected. The target gene (the fusion gene GS/2SS) of C500(pGS/2SS-asd) was not detected by PCR amplification in these samples,indicating that the surrounding environment was not contaminated by residual recombinant bacteria. In conclusion, the vaccine without antibiotic resistance gene is attributable to improve growth performance of piglets through an influence on GH secretion. Moreover, the immunization did not contaminate the surrounding environment of animals.

[Development of modified molecular beacon based real-time PCR assay for the rapid detection of Salmonella choleraesuis and Salmonella paratyphi C].

OBJECTIVE: To develop real-time PCR assay based on modified molecular beacon for simultaneous detection of S. choleraesuis and S. paratyphi C. The established method was applied to the rapid detection of S. choleraesuis in food and stool samples of food poisoning, and then was applied to the identification of Salmonella C. METHODS: Based on the sequences (CP000857.1) published in GenBank, Two sets of primers and modified molecular beacon were designed. The Real-time PCR assay for the simultaneous detection of S. paratyphi C and S. choleraesuis was developed with optimized PCR procedures and PCR components, while other 11 different bacterial species were as the control. Then the sensitivity and specificity of the assay were tested using 77 Samonella strains. The assay was applied to the detection of 70 food samples. RESULTS: The limit of detection achieved was 10 fg/reaction or 20 CUF/reaction, Only Salmonella paratyphi C and Salmonella choleraesuis strains generated fluorescent signals. No cross-reaction was observed with other 11 bacterium, the sensitivity and specificity were both 100%. No samples among 70 food samples were found Salmonella positive by both real-time PCR assay and traditional culture method. It could be finished within 2 hours from template preparation to detection and the overall test would be finished within one day. CONCLUSION: The real-time PCR assay was rapid, sensitive and specific. It could be applied to the rapid diagnosis of S. paratyphi C and S. choleraesuis in food and stool samples of food poisoning and the identification of Salmonella C to guarantee food safety.

Engineering Escherichia coli for efficient production of 5-aminolevulinic acid from glucose.

5-Aminolevulinic acid (ALA) recently received much attention due to its potential applications in many fields. In this study, we developed a metabolic strategy to produce ALA directly from glucose in recombinant Escherichia coli via the C5 pathway. The expression of a mutated hemA gene, encoding a glutamyl-tRNA reductase from Salmonella arizona, significantly improved ALA production from 31.1 to 176mg/L. Glutamate-1-semialdehyde aminotransferase from E. coli was found to have a synergistic effect with HemA(M) from S. arizona on ALA production (2052mg/L). In addition, we identified a threonine/homoserine exporter in E. coli, encoded by rhtA gene, which exported ALA due to its broad substrate specificity. The constructed E. coli DALA produced 4.13g/L ALA in modified minimal medium from glucose without adding any other co-substrate or inhibitor. This strategy offered an attractive potential to metabolic production of ALA in E. coli.

Molecular characterization of extended-spectrum cephalosporinase-producing Salmonella enterica serovar Choleraesuis isolates from patients in Thailand and Denmark.

The objective of this study was to characterize extended-spectrum cephalosporinase (ESC)-producing isolates of Salmonella enterica serovar Choleraesuis recovered from patients in Thailand and Denmark. Twenty-four blood culture isolates from 22 patients were included in the study, of which 23 isolates were recovered from 21 Thai patients during 2003, 2007, or 2008 and one isolate was recovered from a Danish traveler to Thailand. ESC production was confirmed in 13 out of the 24 isolates by MIC testing. Microarray and plasmid profiling (replicon typing and restriction fragment length polymorphism [RFLP]) were used to characterize the genetic mechanisms of antimicrobial resistance in the 13 ESC-producing isolates. Pulsed-field gel electrophoresis (PFGE) and MIC testing were used to compare the clonality between the 13 ESC-producing isolates and the 11 non-ESC-producing isolates. Based on susceptibility patterns, the ESC-producing isolates were more closely related than non-ESC-producing isolates. Microarray, PCR, plasmid profiling, and replicon typing revealed that the 13 ESC-producing isolates harbored either bla(CMY-2) containing incA/C or bla(CTX-M-14) containing incFIIA, incFrepB, and an unknown replicon located on plasmids ranging in size from 75 to 200 kb. The RFLP and replicon typing clustered the isolates into four distinct groups. PFGE revealed 16 unique patterns and five clusters; each cluster contained two or three of the 24 isolates. The isolate from the Danish patient was indistinguishable from two Thai clinical isolates by PFGE. This study revealed the emergence of the bla(CTX-M-14) gene among several clones of Salmonella serovar Choleraesuis. Numerous plasmids were identified containing up to two different ESC genes and four distinct replicons. A "travel-associated" spread was confirmed. Overall, a high degree of clonal diversity between isolates resistant and susceptible to cephalosporins was observed. The findings represent a serious threat to public health for the Thai people and tourists.

[Construction of a dual-promoter expression plasmid delivered by Salmonella choleraesuis C500].

Salmonella choleraesuis C500 strain is an attenuated vaccine preventing piglet from paratyphoid and can also be used as a live vector of other DNA vaccines. Through mucosal immunization, immune response to specific antigens carried by it can be induced. To enhance the immune efficiency of DNA vaccine it carried, promoter Ptrc was inserted into the down stream of the human cytomegalovirus (CMV) immediate early promoter of eukaryotic expression plasmid pEGFP-C1. Then transcription terminator rrnbT1T2 was inserted into down stream of the multiple clone sites of pEGFP-C1, and the dual-promoter expression vector pEGFPPtrcR was constructed. Using 1xTSS method, we transformed the recombinant plasmid into C500, and obtained C500/pEGFPPtrcR. We used SDS-PAGE and Western blotting to detect the expression of report gene EGFP. Strong green fluorescence was observed under fluorescent microscope. The stable passages of this recombinant bacterium were at least 20 generations in vitro. Using liposome we transfected plasmid pEGFPPtrcR into Vero cell. After 24 h, green fluorescent was observed, showing the expression of EGFP in nuclei and endochylema. The construction of dual-promoter expression vector pEGFPPtrcR was successful. The foreign gene was expressed in Salmonella strain C500 and somatocytes, resulting in increased antigen expression. This research provides a foundation for the research of new DNA vaccines which use Salmonella C500 as carrier.

Salmonella paratyphi C: genetic divergence from Salmonella choleraesuis and pathogenic convergence with Salmonella typhi.

BACKGROUND: Although over 1400 Salmonella serovars cause usually self-limited gastroenteritis in humans, a few, e.g., Salmonella typhi and S. paratyphi C, cause typhoid, a potentially fatal systemic infection. It is not known whether the typhoid agents have evolved from a common ancestor (by divergent processes) or acquired similar pathogenic traits independently (by convergent processes). Comparison of different typhoid agents with non-typhoidal Salmonella lineages will provide excellent models for studies on how similar pathogens might have evolved. METHODOLOGIES/PRINCIPAL FINDINGS: We sequenced a strain of S. paratyphi C, RKS4594, and compared it with previously sequenced Salmonella strains. RKS4594 contains a chromosome of 4,833,080 bp and a plasmid of 55,414 bp. We predicted 4,640 intact coding sequences (4,578 in the chromosome and 62 in the plasmid) and 152 pseudogenes (149 in the chromosome and 3 in the plasmid). RKS4594 shares as many as 4346 of the 4,640 genes with a strain of S. choleraesuis, which is primarily a swine pathogen, but only 4008 genes with another human-adapted typhoid agent, S. typhi. Comparison of 3691 genes shared by all six sequenced Salmonella strains placed S. paratyphi C and S. choleraesuis together at one end, and S. typhi at the opposite end, of the phylogenetic tree, demonstrating separate ancestries of the human-adapted typhoid agents. S. paratyphi C seemed to have suffered enormous selection pressures during its adaptation to man as suggested by the differential nucleotide substitutions and different sets of pseudogenes, between S. paratyphi C and S. choleraesuis. CONCLUSIONS: S. paratyphi C does not share a common ancestor with other human-adapted typhoid agents, supporting the convergent evolution model of the typhoid agents. S. paratyphi C has diverged from a common ancestor with S. choleraesuis by accumulating genomic novelty during adaptation to man.

Rapid multiplex PCR and real-time TaqMan PCR assays for detection of Salmonella enterica and the highly virulent serovars Choleraesuis and Paratyphi C.

Salmonella enterica is a human pathogen with over 2,500 serovars characterized. S. enterica serovars Choleraesuis and Paratyphi C are two globally distributed serovars. We have developed a rapid molecular-typing method to detect serovars Choleraesuis and Paratyphi C in food samples by using a comparative-genomics approach to identify regions unique to each serovar from the sequenced genomes. A Salmonella-specific primer pair based on oriC was designed as an internal control to establish accuracy, sensitivity, and reproducibility. Serovar-specific primer sets based on regions of difference between serovars Choleraesuis and Paratyphi C were designed for real-time PCR assays. Three primer sets were used to screen a collection of over 100 Salmonella strains, and both serovars Choleraesuis and Paratyphi C gave unique amplification patterns. To develop the technique for practical use, its sensitivity for detection of Salmonella spp. in a food matrix was determined by spiking experiments. The technique was also adapted for a real-time PCR rapid-detection assay for both serovars Choleraesuis and Paratyphi C that complements the current procedures for Salmonella sp. isolation and serotyping.

[Construction and characterization of a novel somatostatin prokaryotic expression].

In the current work, the fusion gene including somatostatin (SS) and the hepatitis B surface antigen gene was cloned into a balanced lethal system plasmid (pYA3493), and then transformed into asd- attenuated Salmonella choleraesuis C500 strain, the positive transformant without antibiotic resistance gene was confirmed by restriction analysis and DNA sequencing, designated as pYA-SS. The expression and immunogenicity of fusion protein were detected by SDS-PAGE and Western blot analysis. These results show that the recombinant prokaryotic expression plasmid pYA-SS could express the SS fusion protein with good immunogenicity in C500 strain. In above all, this study could provide reliable materials to develop novel, good and safe vaccine in enhancing the growth of animals.

[Salmonella choleraesuis C500 delivering DNA immunization against classical swine fever virus].

Classical Swine Fever Virus (CSFV) E2 protein eukaryotic expression plasmid pVAXE2 was constructed. The plasmid pVAXE2 was transformed into Salmonella choleraesuis C500 (S. C500) attenuated vaccine strain by electroporation to generate Salmonella choleraesuis engineering strain S. C500/pVAXE2. The characterization of S. C500/pVAXE2 in morphology, growth, biochemistry and serology indicated that it retained the same properties as its original strain S. C500 with exception of kanamycin resistance originated from the plasmid pVAXE2. The plasmid stable in the bacteria after 15 passages. Kunming mice and rabbits were vaccinated three times at two weeks interval with S. C500/pVAXE2 in oral and intramuscular routes at the dosage of 1 x 10(8) CFU for mice and 2 x 10(9) CFU for rabbits each time. The specific antibody response against CSFV and Salmonella choleraesuis was detected by ELISA. Two weeks after the third boost the immunized rabbits were challenged with 20 ID50 of hog cholera lapinized virus (HCLV), followed by a virulent strain of Salmonella choleraesuis two week later than HCLV challenge. The results showed that all immunized mice and rabbits produced significant antibodies against CSFV and Salmonella choleraesuis, and the immunized rabbits demonstrated the effective protection against the challenge of HCLV and virulent Salmonella choleraesuis. These results indicated the potential of developing multiplex swine DNA vaccine by using this bacteria as the vector.

Phenotypic and genotypic characterization of Salmonella arizonae from an integrated turkey operation.

Fifty cases submitted between 2000 and 2002 were selected for retrospective analysis to evaluate possible relationships between Salmonella arizonae isolated from breeder flocks, hatching eggs, and meat bird flocks belonging to a single turkey integrator. In all the meat bird cases selected for this study, arizonosis was the primary diagnosis. In birds under 1 month of age, clinical signs and pathologic changes were observed in older birds. The Salmonella arizonae isolates were analyzed by antibiotic resistance pattern and serotype and genotyped by pulsed-field gel electrophoresis (PFGE). Serotyping and PFGE yielded similar results, but the antibiotic resistance patterns did not correspond to either serotyping or PFGE typing. The presence of common pulsed-field patterns in breeder flocks, eggs, and meat bird flocks suggested that S. arizonae was being transmitted vertically from the breeder flock.

Molecular diversity of the genetic loci responsible for lipopolysaccharide core oligosaccharide assembly within the genus Salmonella.

The waa locus on the chromosome of Salmonella enterica encodes enzymes involved in the assembly of the core oligosaccharide region of the lipopolysaccharide (LPS) molecule. To date, there are two known core structures in Salmonella, represented by serovars Typhimurium (subspecies I) and Arizonae (subspecies IIIA). The waa locus for serovar Typhimurium has been characterized. Here, the corresponding locus from serovar Arizonae is described, and the molecular basis for the distinctive structures is established. Eleven of the 13 open reading frames (ORFs) are shared by the two loci and encode conserved proteins of known function. Two polymorphic regions distinguish the waa loci. One involves the waaK gene, the product of which adds a terminal alpha-1,2-linked N-acetylglucosamine residue that characterizes the serovar Typhimurium core oligosaccharide. There is an extensive internal deletion within waaK of serovar Arizonae. The serovar Arizonae locus contains a novel ORF (waaH) between the waaB and waaP genes. Structural analyses and in vitro glycosyltransferase assays identified WaaH as the UDP-glucose:(glucosyl) LPS alpha-1,2-glucosyltransferase responsible for the addition of the characteristic terminal glucose residue found in serovar Arizonae. Isolates comprising the Salmonella Reference Collections, SARC (representing the eight subspecies of S. enterica) and SARB (representing subspecies I), were examined to assess the distribution of the waa locus polymorphic regions in natural populations. These comparative studies identified additional waa locus polymorphisms, shedding light on the genetic basis for diversity in the LPS core oligosaccharides of Salmonella isolates and identifying potential sources of further novel LPS structures.

Characterization of the spv locus in Salmonella enterica serovar Arizona.

Salmonella enterica serovar Arizona (S. enterica subspecies IIIa) is a common Salmonella isolate from reptiles and can cause serious systemic disease in humans. The spv virulence locus, found on large plasmids in Salmonella subspecies I serovars associated with severe infections, was confirmed to be located on the chromosome of serovar Arizona. Sequence analysis revealed that the serovar Arizona spv locus contains homologues of spvRABC but lacks the spvD gene and contains a frameshift in spvA, resulting in a different C terminus. The SpvR protein functions as a transcriptional activator for the spvA promoter, and SpvB and SpvC are highly conserved. The analysis supports the proposal that the chromosomal spv sequence more closely corresponds to the ancestral locus acquired during evolution of S. enterica, with plasmid acquisition of spv genes in the subspecies I strains involving addition of spvD and polymorphisms in spvA.

The excision of intervening sequences from Salmonella 23S ribosomal RNA.

Novel, approximately 90 bp intervening sequences (IVs) were discovered within the 23S rRNA genes of S. typhimurium and S. arizonae. These non-rRNA sequences are transcribed and then excised during rRNA maturation. The rRNA fragments that result from the excision of the extra sequences are not religated. This results in fragmented 23S rRNAs. The excision of one IVS was shown to be catalyzed in vivo and in vitro by ribonuclease III. These IVSs are highly volatile evolutionarily, sometimes occurring in only some of the multiple rRNA operons of a particular cell. The sporadic nature of the occurrence of fragmented rRNAs among closely related organisms argues that such fragmentation is a derived state, not a primitive one. Possible sources of these IVSs, their parallels with internal transcribed spacers and introns in eukaryotes, and their possible roles in the evolutionary process are discussed.

In vivo transfer of antibiotic resistance to a strain of Salmonella arizonae.

Two trials were conducted to determine the transferability of antibiotic resistance in vivo between two strains of enteric bacteria. Newly hatched turkey poults were inoculated per os with a strain of Escherichia coli resistant to kanamycin, tetracycline, and ampicillin, a strain of Salmonella arizonae resistant to nalidixic acid and streptomycin, or both strains. Kanamycin was added to the drinking water of some poults. To test for S. arizonae cells which had received transferable resistance determinants in vivo from the E. coli strain, samples were collected by swabbing the recta of the poults and by removing segments of the intestines and livers after the birds were sacrificed. Nalidixic acid was added to the isolation media to prevent in vitro transfer from occurring after the samples were collected. Salmonella arizonae resistant to nalidixic acid, streptomycin, kanamycin, tetracycline, and ampicillin were isolated from 20% of the rectal samples taken from poults that had received both bacterial strains. S. arizonae cells which had received resistance determinants in vivo were also isolated from 73% of the intestinal samples and 8% of the liver samples taken from birds inoculated with both donor E. coli and recipient S. arizonae. Salmonella arizonae demonstrating resistance to all five antibiotics were recovered from all intestinal samples taken from birds given kanamycin in their drinking water immediately after the last S. arizonae inoculation but from only 43% of such samples taken from birds given no kanamycin.

Incidence and in vitro acquisition of transferable drug resistance in Salmonella arizonae.

Fifty-eight cultures of Salmonella arizonae isolated in 1983 from poultry sources and 21 cultures isolated in 1972 to 1974 were screened for resistance to nalidixic acid, chloramphenicol, triple sulfa, ampicillin, tetracycline, streptomycin, and kanamycin by the standardized disc susceptibility method. Sensitivity to all seven drugs was observed in 51% of the cultures tested (76% of the older cultures and 41% of the recent isolates). Resistance to two or more drugs was observed in 36% of the cultures tested (14% of the older cultures and 43% of the newer ones). The highest incidence of resistance was to streptomycin. Salmonella arizonae cultures sensitive to all seven drugs were tested for the ability conjugatively to acquire kanamycin, streptomycin, tetracycline, and ampicillin resistance determinants from a multiply resistant strain of Escherichia coli K12NA. Complete or intermediate resistance to all antibiotics was transferred to 93% of the sensitive cultures at 41 C and 85% at 28 C. Kanamycin resistance was transferred most frequently, and ampicillin resistance least frequently.

Plasmid- and chromosome-coded aerobactin synthesis in enteric bacteria: insertion sequences flank operon in plasmid-mediated systems.

Large plasmids were detected in two aerobactin-producing enteric bacterial species (Aerobacter aerogenes 62-I, Salmonella arizona SA1, and S. arizona SL5301) and designated pSMN1, pSMN2, and pSMN3, respectively. Other Salmonella spp., namely, S. arizona SL5302, S. arizona SLS, Salmonella austin, and Salmonella memphis, formed aerobactin but contained no detectable large plasmids. S. arizona SL5283 made no aerobactin. A probe consisting of the aerobactin biosynthetic genes cloned on plasmid pABN5 hybridized to a HindIII digest of pSMN1 but not to digests of pSMN2 or pSMN3. A larger probe, the insert of pABN1 containing the complete aerobactin operon, hybridized to four fragments in HindIII digests of the parent plasmid, pColV-K30. A 2.0-kilobase PvuII fragment responsible for this multiple-hybridization pattern was cloned into vector pUC9 to form pSMN30. The latter was mapped and shown to correspond to either IS1 or to a closely related insertion sequence.

R plasmid-mediated gentamicin resistance in salmonellae isolated from turkeys and their environment.

Gentamicin-resistant salmonellae were isolated from turkey poults, hatching eggs, and litter on three unrelated farms and from a scrub jay (Aphelocoma coerulescens) on a fourth farm unrelated to the other three. The isolates were Salmonella arizonae from three of the farms (poults, eggs, scrub jay) and S. thompson (litter) from the fourth farm. The genes responsible for gentamicin resistance were located on the same conjugal plasmid. This plasmid also encoded resistance to kanamycin, streptomycin, ampicillin, and sulfadiazine.

Plasmid-specified sucrose fermentation in Salmonella arizonae.

Thirty cultures of Salmonella arizonae 47:r:253 (Ar 23:24-25) were isolated over 7 months from the faeces of a captive reptile. All were unusual in their inability to produce a positive o-nitrophenyl-beta-D-galactosidase reaction, and in their ability to ferment sucrose. These S. arizonae carried a plasmid having a molecular mass of 72 megadaltons which specified tetracycline resistance and a plasmid of 5 megadaltons which coded for the ability to ferment sucrose. The small size of this sucrose plasmid clearly distinguishes it from others which have been reported.