A Comparative Study of the Nutritional Requirements of Salmonella typhosa, Salmonella pullorum and Salmonella gallinarum.
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A total of 197 Salmonella strains were isolated from 1717 stool samples on salmonella-shigella agar and modified semisolid Rappaport-Vassiliadis medium before and after enrichment in selenite broth. Better sensitivity was obtained with salmonella-shigella agar than in direct plating (89.2% vs. 64.4%, P<0.0001) and after broth enrichment (96.4% vs. 88.1%, P<0.01). The incidence of false-positive results using modified semisolid Rappaport-Vassiliadis medium was higher than that obtained using salmonella-shigella agar combined with the oxidase and C8 esterase tests in direct plating (33 vs. 2 strains) and after enrichment (43 vs. 0 strains). Thus, based on its performance modified semisolid Rappaport-Vassiliadis medium could be a suitable option for isolation of salmonellae from stool samples in clinical microbiology laboratories.
Cloning and sequence analysis of rfbS gene identified two polymorphic nucleotides, one at position 598 (Salmonella gallinarum-specific) and other at position 237 (Salmonella pullorum-specific). Based on S. gallinarum-specific nucleotide found at position 598, an allele-specific PCR method was developed for serotype-specific detection of S. gallinarum. This PCR method was able to discriminate pure cultures of S. gallinarum from S. pullorum and other Salmonella serotypes from serogroup D in less than 3 h. Serotype-specific detection of S. gallinarum was possible in less than 24 h when the PCR was applied on the presumptive Salmonella colonies obtained after overnight incubation of selective media plates streaked with the clinical material from diseased chickens. As little as 100 pg of genomic DNA could be detected with S. gallinarum-specific primers; no PCR product was detected in non-S. gallinarum serotypes of serogroup D and other closely related non-salmonella organisms. This rfbS allele-specific amplification assay is specific, reproducible and less time consuming than the standard bacteriological methods used to detect S. gallinarum and could be an effective molecular tool for rapid definitive diagnosis of fowl typhoid in the areas of endemicity where fowl typhoid infection exists.
BACKGROUND: CKS257 (Salmonella typhimurium SL1344 DeltaphoP/phoQDelta aroA Deltaasd DeltastrA/strB pSB2131) is a live oral vaccine vector expressing HIV Gag. METHODS: HIV Gag was expressed as a fusion protein of a Salmonella Type III secretion system protein SopE, from a balanced lethal asd-based plasmid. Eighteen healthy adults were given single escalating oral doses of 5 x 10(6) to 1 x 10(10)CFU of CKS257 and were monitored for clinical events, shedding and immune responses. RESULTS: Adverse events were mild except at the highest dose. Volunteers shed the organism an average of 5.1 days (range 0-13 days). Eighty-three percent (15/18) of subjects had a mucosal immune response to Salmonella LPS and flagella by IgA ELISPOT assay. Seventy-two percent (13/18) of subjects seroconverted to Salmonella antigens. No volunteer had a response to recombinant Gag as measured by serology, IgA ELISPOT, or immediate ex vivo gamma-interferon ELISPOT response to Gag peptide pools. Two volunteers responded to Gag peptides by IL-2 ELISPOT, and 4 of 10 volunteers receiving >or=5 x 10(8)CFU had a response to HIV peptides in a cultured gamma-interferon ELISPOT assay. CONCLUSIONS: Although immunogenicity of the HIV antigen needs augmentation, the attenuated Salmonella strain proved to be an excellent platform for vaccine development.
In Denmark, a serological Salmonella surveillance programme in finishing pig herds has been in place since 1995. The programme was founded on data from experimental studies, which demonstrated a strong association between Salmonella serology and the prevalence of these bacteria. The current study was carried out in three Danish abattoirs to evaluate the correlation under field conditions. A total of 160 Danish finishing pig herds were included. Seven out of these were examined twice, yielding a total of 167 observations. The herds were selected according to their herd serology based on data from the national surveillance. From each herd, samples were taken from 10 finishers at slaughter. The prevalence of Salmonella bacteria was measured at four sites: (1) caecal-content; (2) carcass surface; (3) pharynx; and (4) caecal lymph nodes. A logistic regression model was constructed for each sampling site. Abattoir, sanitary slaughter and herd seroprevalence were used as explanatory variables. The results demonstrated that there was a strong association between herd serology and the prevalence of Salmonella bacteria measured at three of the sampling sites: caecal-content, pharynx, and carcass surface. For these sites, the odds for being culture-positive for Salmonella varied from 1.3 to 1.5 for each increase of 10% in herd serology (P < 0.0001). For caecal lymph nodes, however, no linear association was found.
The production and secretion of Salmonella enteritidis whole cell antigen-specific antibodies in the oviducts and in the serum of laying hens experimentally infected with Salmonella enteritidis, was analyzed by ELISA. The dynamics of the antibody levels in the oviducts were identical to that in the serum. Subclasses of antibodies (IgA, IgG, and IgM) in the infected hens were found to increase significantly (p < 0.01) compared to those in the control uninfected hens throughout the experiment. IgG and IgM levels in both oviducts and in sera reached to a peak by 14 days post-inoculation, and remained elevated throughout. The secretion of IgA seemed to be transient since the IgA levels increased to a peak 7 days after both primary and secondary inoculations, and declined rapidly. The elevated levels of antibodies were followed by partial clearance of Salmonella organisms from the oviducts. The present results indicate a significant local immune reaction against the Salmonella infection and suggest an association of the local antibodies with the clearance of Salmonella from the oviducts at least partially.
Monoclonal antibody (MAb) 8aC10 against Salmonella O:5 antigen was obtained after immunization of BALB/c mice with live attenuated mutant of Salmonella typhimurium. Antigen specificity of the MAb was characterized by ELISA, immunoblotting, passive hemagglutination (PHA), passive hemolysis and agglutination tests. In ELISA, PHA and immunoblotting the MAb reacted only with lipopolysaccharides (LPS) of Salmonella strains from group O:4 (B), expressing O:5 antigen. The MAb agglutinated in addition Salmonella strains with O:8 antigen from group C(2)-C(3) but did not react with purified LPS. These results demonstrate O:5 specificity of MAb 8aC10. Cross-agglutination with group C(2)-C(3) suggests the presence of similar but not identical epitope in O:8 expressing strains, which is possibly localized onto O-acetyl-abequose and abequose residues bound with a alpha-1-->3 linkage to the basic polysaccharide backbone of Salmonella LPS with O:5 and O:8 antigen respectively.
The purpose of this investigation was to study the host specific infection of Salmonella Gallinarum in chickens and to determine the contribution of intestinal invasion and macrophage survival in relation to systemic infection in the host. This was carried out by comparing the kinetics of infection of S. Gallinarum to that of other Salmonella host-adapted (S. Cholerae-suis, S. Dublin and S. Typhimurium) and host-specific (S. Pullorum and S. Abortus-ovis) serovars. Establishment of the rate of colonisation in intestinal tissue, bursa and systemic sites was carried out by oral infection in day-old and week-old birds. Salmonella Gallinarum was the only serovar capable of causing systemic infection in chickens, however, general colonising ability in the intestine and bursa demonstrated no apparent selective advantage for S. Gallinarum. Further quantification of gastrointestinal invasion was carried out using ligated loops in the small intestine. Invasion in the jejunum of the chicken intestine over 3h demonstrated that Salmonella Typhimurium invasion was statistically higher (P<0.01) when compared with S. Gallinarum. Specific sites of high lymphoid tissue concentration in the chicken, including the bursa of Fabricius and caecal tonsils, were also targeted in invasion assays to investigate possible areas of tissue tropism. S. Typhimurium demonstrated significantly higher (P<0.01) invasion at these sites when compared with S. Gallinarum. Infection of chicken macrophages with S. Gallinarum did not demonstrate increased multiplication and survival intracellularly when compared with other Salmonella serotypes. The only difference seen was with S. Abortus-ovis, which demonstrated a significantly lower (P<0.05 to 0.001) intracellular survival. Together these data suggest that although S. Gallinarum host specificity in the chicken correlates with systemic infection, intestinal and lymphoid tissue invasion in the bursa and caeca, and macrophage survival does not influence this outcome.
The Salmonella plasmid virulence genes (spv) are commonly found on plasmids contained in a small number of serotypes of Salmonella belonging to subspecies I, where they are important for survival within macrophages and the establishment of successful systemic infection. However, in this study, spv genes were detected by the polymerase chain reaction in the chromosome of a plasmid-free strain of S. IV 16:z4, z32:- (Salmonella subspecies IV). The full range of spv genes (spvR, spvA, spvB, spvC and spvD) was demonstrated, but a 216-bp deletion, accompanied by an insertion of 59-bp cryptic DNA, was present in spvA. S. IV 16:z4, z32:- was avirulent in mice and did not become virulent with the introduction of a fully functionally serotype-associated virulence plasmid (SAP) from S. typhimurium. By use of an spvRAB'-chloramphenicol acetyl transferase fusion gene, it was demonstrated that S. IV 16:z4, z32:- did not express the spv genes. Salmonella subspecies IV is monophasic, and in phylogenetic analyses it clusters distantly to Salmonella subspecies I, where all the serotypes that normally carry SAPs are found. The mechanisms by which spv genes have been transferred to this serotype remain unknown.
Bacteriologically proved cases of salmonellosis presenting at the University Hospital have increased nearly threefold since 1957. The most striking change has been a considerable increase in the incidence of Salmonella heidelberg and Salmonella derby in the last 5 years, probably resulting from hospital acquired infection. About 80 cases of typhoid fever are reported each year in Jamaica. There has been little change in the prevalence of different phage types of Salmonella typhi since 1961. Paratyphoid fever is rare. Standardized antimicrobial disk-sensitivity testing was performed on selected surviving salmonella strains since 1964. S. typhi has remained fully sensitive to all the agents tested except streptomycin. Other salmonellas, however have shown a significant increase in antimicrobial resistance since 1970. Most of this increase was due to multiple resistance in S. heidelberg and S. derby, and the survival and dissemination of these strains in the hospital environment may be related to antibiotic usage.
Salmonella was the most frequent bacterial pathogen isolated from patients with acute diarrhoea in Hong Kong. In Queen Mary Hospital, the major hospital on Hong Kong Island, 94.7% of salmonellae isolated from faecal specimens from patients during the period 1973-82 belonged to the gastroenteric group, while 5.3% belonged to the enteric fever group. Amongst the gastroenteric group, 68 salmonella serotypes were identified, with Salmonella derby, S. typhimurium and S. anatum being the predominant ones. Three outbreaks caused by S. johannesburg, S. worthington and S. wandsworth were detected. Of S. typhimurium, 61.6% were resistant to multiple antibiotics and belonged to four major phage types: 193, 22, 138 and U288. The majority (96.8%) of S. johannesburg strains which caused a widespread epidemic were multiply-resistant. Multiple antibiotic resistance was rarely observed in most other gastroenteric salmonellae. S. typhi was the commonest of the enteric fever group isolated from the blood of patients. Nineteen phage types were identified; E1 being the commonest (18.5%) while 21% were nontypable. Many of these isolated were resistant to streptomycin or sulphadiazine, but none were resistant to ampicillin, chloramphenicol or trimethoprim.
A multiplex-PCR-based assay (m-PCR) was developed for the detection of Salmonella and for the identification of the two serotypes Enteritidis and Typhimurium. Three sets of primers selected from different genomic sequences amplified a 429 bp fragment specific for the genus Salmonella within a randomly cloned sequence, a 559 bp target specific for Salmonella Typhimurium within the fliC gene and a 312 bp fragment specific for Salmonella Enteritidis within the sefA gene. The m-PCR-based assay was used for detecting Salmonella from 1078 environmental swabs of poultry houses. Prior to PCR, these swabs were pre-enriched in phosphate-buffered peptone water for 18-20 h and then sub-cultured on a Modified Semi-solid Rappaport Vassiliadis medium (MSRV) for 18-20 h. The m-PCR combined with MSRV had a better sensitivity (95%) than the bacteriological method (92.5%). The MSRV-m-PCR assay and the bacteriological method had an agreement rate of 95.6%.
An indirect immunofluorescence (IFL) method was used for diagnosis of infections caused by Salmonella enteritdis during an epidemic. Antiserum prepared against the synthetic disaccharide tyvelose 1 leads to alpha 3 D-mannose, representative of salmonella O antigen 9, covalently linked to bovine serum albumin, was used. Of 43 decal samples examined, 28 were positive for S. enteritidis by culture. IFL was applied (1) directly on fecal smears, (2) on enrichment broth cultures after incubation for 18-24 hr, and (3) on agar-grown colonies after incubation for 18-48 hr. The percentage of correctly identified Salmonella and the approximate gain of time for IFL as compared with conventional culture were 75% and 72 hr for (1), 89% and 48 hr for (2), and 100% and 24 hr for (3). Serum samples from 26 of the Salmonella-infected patients were examined by an enzyme-linked immunosorbent assay (ELISA). Twenty-four (92%) of the 26 patients acquired elevated ELISA titers of antibody to lipopolysaccharide, representative of Salmonella serogroup D.
We analyzed 18 Salmonella Paratyphi A and 12 Salmonella Typhi isolates from domestic and imported cases in Aichi, Japan, using pulsed-field gel electrophoresis. Paratyphoid fever cases have increased and outbreaks of Salmonella Paratyphi A occasionally occur in Japan, but S. Paratyphi A has not been extensively analyzed. Our study suggests significant genetic homogeneity among Salmonella Paratyphi A belonging to different phage types, which is in contrast to the genetic heterogeneity of Salmonella Typhi. These results suggest that a limited number of clones are responsible for paratyphoid fever.
Mice immunized intravenously 10 days earlier (but not those immunized 2 months earlier) with an attenuated Salmonella typhimurium SL3261 aroA live vaccine and tested for delayed-type hypersensitivity by injection of crude Salmonella extracts in the footpad can die within 24 to 48 h of an unexplained allergic reaction. The lethal reaction could be prevented by prior administration of anti-tumor necrosis factor alpha serum. Injection of lipopolysaccharide (LPS) (either purified phenol-water-extracted [Westphal] LPS or protein-rich trichloracetic acid-extracted [Boivin] LPS) was also lethal for mice immunized 10 days before. An LPS-rich crude Salmonella extract was more toxic than one which contained less LPS, suggesting that LPS may have been involved in the lethal reactions to crude antigens. Mild alkaline hydrolysis removes O-linked acyl groups from lipid A and eliminates many toxic effects of LPS; however, both Boivin LPS and Westphal LPS remained toxic for immunized mice after alkaline hydrolysis. In contrast, alkaline hydrolysis of crude whole Salmonella extracts (which caused marked protein degradation) reduced the lethal toxicity of the extracts, especially for an LPS-rich preparation. Mice immunized orally with the live vaccine did not show hypersensitivity to either LPS or crude extracts. The results suggest that the lethal reaction to crude Salmonella antigens in mice immunized 10 days earlier is complex, that tumor necrosis factor alpha is involved, and that allergic reactions to crude antigens (but not to LPS alone) can be reduced by mild alkaline hydrolysis.
Our laboratory has previously shown that after immunization with a strain of Salmonella typhimurium, SL3235, made avirulent by a blockage in the pathway of aromatic synthesis, murine splenocytes were profoundly suppressed in their capacity to mount an in vitro antibody plaque-forming cell (PFC) response to sheep erythrocytes. Evidence indicated that suppression was mediated by nitric oxide (NO), since the in vitro addition of NG-monomethyl-L-arginine blocked suppression. The present studies examined the effect of blocking NO production on Salmonella-induced immunosuppression by in vivo administration of aminoguanidine hemisulfate (AG). AG was administered to C3HeB/FeJ mice in their drinking water (2.5% solution) for 7 days prior to intraperitoneal inoculation with SL3235. AG treatment inhibited the increase in nitrate and nitrite levels in plasma and nitrite levels in the spleen seen in immunized mice. Importantly, AG treatment completely blocked suppression of the splenic PFC response and markedly attenuated the suppression of the response to concanavalin A in immunized mice, providing further evidence that Salmonella-induced immunosuppression is mediated by NO. AG treatment also alleviated the majority of the splenomegaly associated with SL3235 inoculation, which correlated with a blockage of influx of neutrophils and macrophages into spleens, as assessed by flow cytometry. AG treatment unexpectedly resulted in 90% mortality in mice injected with the highly attenuated vaccine strain of Salmonella, SL3235. Increased mortality in AG-treated mice correlated with inability to clear organisms from the spleen by day 15 postinoculation and with persistent bacteremia, compared with control mice. Collectively, these in vivo results underscore the dual biological consequences of NO production following Salmonella infection, with NO being necessary for host defense, but also having the potentially adverse effect of immunosuppression. A unifying hypothesis to explain how these seemingly paradoxical effects could both result from NO production is presented.
Recombinant Salmonella strains expressing foreign heterologous genes have been extensively studied as live oral vaccine delivery vectors. We have investigated the mucosal and systemic immune responses following oral immunization with a recombinant Salmonella enterica serovar Typhimurium expressing the hemagglutinin HagB from Porphyromonas gingivalis, a suspected etiological agent of adult periodontal disease. We have previously shown a primary mucosal and systemic response following oral immunization with chi4072/pDMD1 and recall responses following boosting at 14 weeks after primary immunization. In this study, we examined the effects of earlier boosting as well as the effects of deliberately induced immunity to the Salmonella carrier strain on subsequent immune responses. Mice boosted at week 7 following immunization, a point which corresponded to the peak of the primary response, generally showed lower responses than those boosted at week 14. When mice were preimmunized with the Salmonella carrier alone and then immunized with the recombinant strain 7 or 14 weeks later, significant reductions were seen for serum immunoglobulin G (IgG) antibodies at week 14 and for salivary IgA at week 7. No reductions were seen in serum IgA or vaginal wash IgA antibodies. Mice appear to be refractory to boosting with orally administered salmonellae at 7 weeks. Deliberate immunization with the carrier strain did not appreciably affect recall responses at 14 weeks, with the exception of the serum IgG responses, nor did it affect colonization of the Peyer's patches.