Isolation of Salmonella from environmental samples collected in the reptile department of Antwerp Zoo using different selective methods

AIMS: To evaluate the environmental spread of Salmonella strains in the reptile department of Antwerp Zoo and to compare different isolation methods for Salmonella. METHODS AND RESULTS: One hundred environmental samples were collected in the service sections and public spaces of the reptile department. After pre-enrichment in buffered peptone water (BPW), selective enrichment was performed in Rappaport Vassiliadis Single Component Enrichment Broth (RVS), Selenite Cystine Broth (SEL) and Mueller Kauffman Tetrathionate Broth (MKTTn). Subculturing on Modified Semisolid Rappaport-Vassiliadis (MSRV) Medium, and the combined use of immunomagnetic separation (IMS) and RVS was evaluated. The isolation media used were Hektoen Enteric Agar (HE), Phenol Red Brilliant Green Agar (BG) and Xylose Lysine Decarboxylase Agar (XLD). Salmonella strains were found in 47 samples (47.0%). Most isolations were made on HE after combined IMS/RVS enrichment. Sixty-six Salmonella strains were serotyped, 29 belonged to Salmonella enterica ssp. enterica (I), 3 to ssp. salamae (II), 29 to ssp. arizonae or diarizonae (IIIa/b), 4 to ssp. houtenae (IV) and 1 strain showed autoagglutination. In addition, a 10-year survey (1995-2004) of Salmonella serovars isolated from reptiles at Antwerp Zoo is presented. CONCLUSIONS: A high prevalence of Salmonella strains was noted in the service sections of the reptile department. Only a few isolations were made in the public spaces. Selective enrichment in RVS was the most efficient. In combination with IMS, this method gave an even higher isolation rate than the International Standard method (ISO 6579:2002). SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms the importance of reptiles as spreaders of Salmonella in their surroundings. The possible infectious risks for zoo personnel and visitors are evaluated. Improved laboratory protocols for the isolation of Salmonella from the environment are suggested.     

Undescribed Serotype of Salmonella: Salmonella enteritidis ser. Lovelace

A new serotype of Salmonella is described. Culture 1505-68, serotype 13,22,36:l, v:1,5, was recovered from the stool of a female patient and designated Salmonella enteritidis ser. Lovelace.     

Additional Salmonella Serotype: Salmonella enteritidis ser. Madigan (44:c:1,5)

The antigenic composition of a new Salmonella type (44:c:1,5) isolated from the stool of an infant is described.     

Two Additional Salmonella Serotypes: Salmonella enteritidis Ser 58:a:- and Salmonella enteritidis Ser 44Z36, Z38:-

The antigenic compositions of two additional Salmonella serotypes isolated from the feces of man were determined to be 58:a:- and 44:Z(36), Z(38)-.     

Widespread occurrence of specific restriction endonucleases in Salmonella infantis, Salmonella thompson, and Salmonella blockley isolated from humans in Japan

Specific restriction endonucleases were detected in three serotypes of Salmonella spp. isolated from humans in Japan from 1970 to 1987: an isoschizomer of AvaII endonuclease at a frequency of 0.91 in Salmonella infantis, an isoschizomer of KpnI at a frequency of 0.34 in Salmonella thompson, and an isoschizomer of StyI at a frequency of 0.30 in Salmonella blockley. Of interest is that restriction endonuclease-producing S. thompson was detected at high frequencies in the 1970s but at low frequencies in the 1980s.     

New Salmonella Serotype: Salmonella enteritidis ser. Ordonez

The antigenic formula of a new serotype, Salmonella enteritidis serotype Ordonez, was characterized as (1),13,23,37:y:l,w.     

Growth inhibitory factors in bovine faeces impairs detection of Salmonella Dublin by conventional culture procedure

AIMS: To analyse the relative importance of different biological and technical factors on the analytical sensitivity of conventional culture methods for detection of Salmonella Dublin in cattle faeces. METHODS AND RESULTS: Faeces samples collected from six adult bovines from different salmonella-negative herds were split into subpools and spiked with three strains of S. Dublin at a concentration level of c. 10 CFU g(-1) faeces. Each of the 18 strain-pools was divided into two sets of triplicates of four volumes of faecal matter (1, 5, 10 and 25 g). The two sets were pre-enriched with and without novobiocin, followed by combinations of culture media (three types) and selective media (two types). The sensitivity of each combination and sources of variation in detection were determined by a generalized linear mixed model using a split-plot design. CONCLUSIONS: Biological factors, such as faecal origin and S. Dublin strain influenced the sensitivity more than technical factors. Overall, the modified semi-solid Rappaport Vassiliadis (MSRV)-culture medium had the most reliable detection capability, whereas detection with selenite cystine broth and Mueller Kauffman tetrathionate broth combinations varied more in sensitivity and rarely reached the same level of detection as MSRV in this experiment. SIGNIFICANCE AND IMPACT OF THE STUDY: The study showed that for MSRV-culture medium and xylose lysine decarboxylase agar as the indicative medium, the sensitivity of the faecal culture method may be improved by focusing on the strain variations and the ecology of the faecal sample. Detailed investigation of the faecal flora (pathogens and normal flora) and the interaction with chemical factors may result in developing an improved method for detection of S. Dublin.     

Widespread occurrence of specific restriction endonucleases in Salmonella infantis, Salmonella thompson, and Salmonella blockley isolated from humans in Japan.

Specific restriction endonucleases were detected in three serotypes of Salmonella spp. isolated from humans in Japan from 1970 to 1987: an isoschizomer of AvaII endonuclease at a frequency of 0.91 in Salmonella infantis, an isoschizomer of KpnI at a frequency of 0.34 in Salmonella thompson, and an isoschizomer of StyI at a frequency of 0.30 in Salmonella blockley. Of interest is that restriction endonuclease-producing S. thompson was detected at high frequencies in the 1970s but at low frequencies in the 1980s.     

Characterisation of the Salmonella O:8 antigen in the O-chain of the lipopolysaccharide produced by Salmonella virginia O:8.

The antigenic O-polysaccharide of the lipopolysaccharide produced by Salmonella virginia (O:8), analyzed by methylation, partial acid hydrolysis, and one- and two-dimensional nuclear magnetic resonance methods, was found to be a polymer of a repeating pentasaccharide unit composed of D-mannose, D-galactose, L-rhamnose, D-abequose, and O-acetyl (2:1:1:1:1.3) and having the following structure: [formula; see text] The disaccharide structure alpha-D-Abep-(1----3)-L-Rhap was identified as the Salmonella O:8 antigenic factor epitope, since the removal of alpha-D-Abep residues from the O-polysaccharide left a residual tetrasaccharide repeating unit backbone that did not show reaction with Salmonella type O:8 factor antiserum.     

Variation in the faecal shedding of Salmonella and E. coli O157:H7 in lactating dairy cattle and examination of Salmonella genotypes using pulsed-field gel electrophoresis

AIMS: To examine the variability in faecal shedding of Salmonella and Escherichia coli O157:H7 in healthy lactating dairy cattle and to evaluate the genetic relatedness of Salmonella isolates. METHODS: Faecal samples were obtained from lactating Holstein dairy cattle on four commercial farms in the southwestern US. All farms were within an 8-km radius and were sampled in August 2001, January 2002 and August 2002 (60 cows per farm per sampling; n = 720 total samples). Samples were cultured for E. coli O157:H7 and Salmonella and a portion of the recovered Salmonella isolates were examined for genetic relatedness using pulsed-field gel electrophoresis (PFGE). RESULTS: Faecal shedding of E. coli O157:H7 and Salmonella varied considerably between farms and at the different sampling times. Large fluctuations in the percentage of positive animals were observed from summer to summer for both of these pathogens. Similarly, Salmonella serotype and serotype prevalence varied from farm to farm and within farm from one sampling time to another. Multiple Salmonella genotypes were detected for a number of serotypes and identical genotypes were found on different farms with one genotype of Salmonella Senftenberg identified on three of the four farms. Significance and Impact of the STUDY: This study demonstrated the wide variability in pathogen shedding within and among dairy farms all located in a small geographical region and highlights the complexity of pathogen control at the farm level.     

Salmonella enteritidis temperature-sensitive mutants protect mice against challenge with virulent Salmonella strains of different serotypes

The protection conferred by temperature-sensitive mutants of Salmonella enteritidis against different wild-type Salmonella serotypes was investigated. Oral immunization with the single temperature-sensitive mutant E/1/3 or with a temperature-sensitive thymine-requiring double mutant (E/1/3T) conferred: (i) significant protection against the homologous wild-type Salmonella strains; (ii) significant cross-protection toward high challenge doses of S. typhimurium. Significant antibody levels against homologous lipopolysaccharide and against homologous and heterologous protein antigens were detected in sera from immunized mice. Moreover, a wide range of protein antigens from different Salmonella O serotypes were recognized by sera from immunized animals. Besides, primed lymphocytes from E/1/3 immunized mice recognized Salmonella antigens from different serotypes. Taken together, these results indicate that temperature-sensitive mutants of S. enteritidis are good candidates for the construction of live vaccines against Salmonella.     

沙门氏菌Ⅱ的三个新血清型

Eight cultures isolated from intestinal contents of reptiles were belonged to 3 new serotypes of Salmonella. They were all ducitol fermented, malonate utilized, but not attack lactose and salicin, no growth in KCN broth, ONPG negative. Therefore, they would be included in Salmonella II. They were all attacked by Felix phage O-I. Three represented strains were selected for antigen analysis. Their antigenic formula were identified as follows: S3194 Salmonella II 6,7:1,v:e,n,z15 S3196 Salmonella II 6, 7:y: e, n, z(1)5 S3195 Salmonella II 6, 8: e, h: 1,2 Among them, S3196 was indole positive belonging to a rare biotype. In addition, there were two other cultures as well as the formula of S3194, and three other cultures as well as the formula of S3196 (one of indole positive, two of indole negative).     

Immunochemical studies of Salmonella Dakar and Salmonella Telaviv O-antigens (serogroup O:28)

Salmonella Dakar and Salmonella Telaviv bacteria belong to serogroup O:28, which represents 107 serovars and possesses only the epitope O28. Salmonella Telaviv has the subfactors O28(1) and O28(2) , whereas S. Dakar has O28(1) and O28(3) . So far, only limited serological and immunological information for this serogroup is available in the literature. Knowledge of the structures of their O-polysaccharides and the immunochemical investigations performed in this work allowed to reveal the nature of subfactor O28(1) as attributed to the presence of 3-linked (or 3,4-disubstituted) α-d-GalpNAc in the main chains of S. Dakar and S. Telaviv O-polysaccharides. An explanation for the cross-reactions between Salmonella enterica O28 O-antigens and other Salmonella O-polysaccharides and their structural similarity to Escherichia coli O-serogroups is also given.     

Enzyme-linked immunosorbent assay for Salmonella typhimurium in food: feasibility of 1-day Salmonella detection

A microtitration plate, antibody-capture, enzyme-linked immunosorbent assay was developed for detection of Salmonella typhimurium. The assay utilizes a monoclonal detector antibody which shows no cross-reactions with non-Salmonella species and only a slight cross-reaction with one other Salmonella serotype. By using only one cultural stage (in a nonselective, chemically defined medium) prior to the enzyme-linked immunosorbent assay, low numbers of cells in food (10 cells 25 g-1) were detected in 19 h. Non-Salmonella competing organisms did not interfere with detection of S. typhimurium even when present in the ratio of 10(6):1 (non-Salmonella/Salmonella spp.). The assay shows the feasibility of rapid, 1-day testing for Salmonella spp. with antibody technology.     

Salmonella serotypes isolated from pet chews in New Zealand

AIMS: To survey the prevalence of Salmonella in imported and domestic pet chews for assessing their potential in introducing novel pathogenic and antimicrobial resistant Salmonella serotype clones into New Zealand, and as vehicles of salmonellosis in the domestic home environment. METHODS AND RESULTS: Three hundred samples, each of imported and domestic pet chews, were examined bacteriologically for the presence of Salmonella. Salmonella cells in the pre-enrichment culture were concentrated by using Dynabeads, and then selective enrichment and plating were performed by a method described in the Bacteriological and Analytical Manual, USFDA. Salmonella was isolated from 16 (5.3%) of the imported and 20 (6.7%) of the domestic pet chews, but the prevalences of Salmonella in imported and domestic products were not significantly different. All Salmonella isolates were serotyped and genotyped by pulsed-field gel electrophoresis and antimicrobial susceptibility determined by the Clinical and Laboratory Standards Institute disc diffusion methods. Salmonella Borreze has never been recorded earlier in New Zealand and was detected from Australian raw hide. Three isolates of Salmonella London were resistant to ampicillin and gentamicin, and two isolates of Salmonella Infantis were resistant to nalidixic acid, one of which was also resistant to streptomycin. CONCLUSIONS: Novel pathogenic and antimicrobial-resistant Salmonella are being introduced into New Zealand through the import of pet chews. This indicates that pet chews are a potential source of exposure to Salmonella in the domestic home environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Contaminated pet chews are potential sources of Salmonella infection for domestic pets, and humans are at risk of exposure either directly by contact through handling or inadvertently by cross-contamination of food or food-contact surfaces in home environments.     

Enzyme-linked immunosorbent assay for Salmonella typhimurium in food: feasibility of 1-day Salmonella detection.

A microtitration plate, antibody-capture, enzyme-linked immunosorbent assay was developed for detection of Salmonella typhimurium. The assay utilizes a monoclonal detector antibody which shows no cross-reactions with non-Salmonella species and only a slight cross-reaction with one other Salmonella serotype. By using only one cultural stage (in a nonselective, chemically defined medium) prior to the enzyme-linked immunosorbent assay, low numbers of cells in food (10 cells 25 g-1) were detected in 19 h. Non-Salmonella competing organisms did not interfere with detection of S. typhimurium even when present in the ratio of 10(6):1 (non-Salmonella/Salmonella spp.). The assay shows the feasibility of rapid, 1-day testing for Salmonella spp. with antibody technology.     

Organic acid and formaldehyde treatment of animal feeds to control Salmonella: efficacy and masking during culture

AIMS: To investigate whether treatment of animals feeds with organic acids/formaldehyde may mask the presence of Salmonella, when assessed by standard cultural methods. METHODS AND RESULTS: Four commercial treatments were applied at the manufacturers' recommended rates on feeds artificially inoculated with Salmonella. The recovery of Salmonella from these treated feeds was assessed after specific antagonists were added to the treatments during culture. A control group of treated feed received no antagonist. Masking of Salmonella was demonstrated when the addition of antagonists resulted in recovery of Salmonella from the treated feed, compared with a negative recovery when no antagonists were added. There were large variations in the efficacy of treatments, and masking was demonstrated with all four tested treatments. One formaldehyde-based product showed greater efficacy and less masking. Masking was greater when high levels of Salmonella were present in the feed. CONCLUSIONS: Some organic acid or formaldehyde-based feed treatments may mask the presence of Salmonella. SIGNIFICANCE AND IMPACT OF THE STUDY: Feeds may be deemed safe despite being contaminated with Salmonella. The use of antagonists during culture may help assess the level of Salmonella contamination when organic acid or formaldehyde treatments have been applied to feed ingredients.     

Microbial population profiles of the microflora associated with pre- and postharvest tomatoes contaminated with Salmonella typhimurium or Salmonella montevideo

Aims:  To determine the microflora profiles of pre- and postharvest tomatoes contaminated with Salmonella montevideo or S. typhimurium DT104.
Methods and Results:  Salmonella montevideo or S. typhimurium was inoculated onto the flowers of tomato plants with the microflora of the subsequent fruit examined using a combination of Source Carbon Utilization and 16S rDNA-PCR profiling. From 16S rDNA profiles it was evident that tomatoes derived from Salmonella inoculated plants harboured a different microbial population compared to nontreated controls. The same result was observed for tomatoes inoculated at postharvest and subsequently stored for 14 days at 15°C. From sequencing analysis it was found that tomatoes derived from Salmonella inoculated plants but testing negative for the enteric pathogen, frequently harboured Enterobacter and Bacillus spp. In contrast, both bacterial types were not found associated with tomatoes testing positive for Salmonella.
Conclusions:  Salmonella introduced onto tomatoes at pre- or postharvest alters the composition of the microbial community. The presence of Enterobacter and Bacillus spp negatively affects the persistence of Salmonella on preharvest tomatoes.
Significance and Impact of the Study:  Salmonella appears to modify rather than become integrated into the microbial communities associated with tomatoes. Yet, the presence of antagonistic bacteria appears to reduce the persistence of the enteric pathogen.     

Improvement in Salmonella detection in milk and dairy products: comparison between the ISO method and the Oxoid SPRINT Salmonella test

The Oxoid SPRINT Salmonella test was compared with the ISO method (ISO 6579: 1993) for the detection of Salmonella in milk and dairy products. Samples were artificially contaminated, in some cases with sublethally injured salmonellas. Experiments with raw milk, soft cheese made from heat-treated milk (mould-ripened and with smear) and soft cheese with smear made from raw milk showed no significant differences between the SPRINT and ISO methods. With dried milk products and mould-ripened soft cheese made from raw milk the reference method gave significantly more positive results. The addition of ferrioxamine E to pre-enrichment (ISO) or pre-enrichment/enrichment broth (SPRINT test) did not improve Salmonella detection.     

Salmonella isolated in sewage sludge traced back to human cases of salmonellosis

AIM: The main aim of this study was to investigate a possible connection between the Salmonella content in sewage sludge and human cases of salmonellosis. An additional aim was to survey the antimicrobial resistance situation in Salmonella isolated from Swedish sewage sludge. METHODS AND RESULTS: The Salmonella strains were compared by restriction enzyme analysis combined with pulsed field gel electrophoresis and antimicrobial susceptibility testing. This study suggests a link between Salmonella isolated from sewage sludge and human Salmonella isolates. CONCLUSIONS: This study demonstrates that Salmonella spp. isolated in sewage treatment plants (STP) originate from infected humans and survive treatment at STP. It also highlights the risk of spreading resistant Salmonella strains from sewage sludge to the environment. SIGNIFICANCE AND IMPACT OF THE STUDY: As Salmonella spp. originating from infected humans can survive the treatment at STP, the risk of Salmonella spp. being spread with sewage sludge to the environment and then to people and animals is enhanced. The threat to society is even worse if the bacteria are resistant to antimicrobial agents.