Feedstuffs as a vehicle of cattle exposure to Escherichia coli O157:H7 and Salmonella enterica

Feed has been reported as a vehicle for transmission of Salmonella enterica in cattle and several lines of evidence suggest that feed can be a vehicle for transmitting Escherichia coli O157:H7 as well. To show whether microbial contamination of feeds could contribute to the populations of S. enterica and E. coli O157:H7 on a farm, we compared isolates from feed samples to bovine fecal isolates from the same farm using pulsed-field gel electrophoresis (PFGE). Four of 2365 component feed samples (0.2%) and 1 of 226 feed mill samples (0.4%) were positive for E. coli O157:H7. Twenty of 2405 (0.8%) component feed samples and none of 226 feed mill samples were positive for Salmonella. PFGE profiles from E. coli O157:H7 isolated from a component feed sample closely resembled that from a fecal isolate collected later from the same farm, and a similar observation was made of a Salmonella Tyhpimurium isolate from component feed on another farm. There were indistinguishable PFGE profiles from component feed Salmonella Tyhpimurium DT104 isolates and fecal isolates from the same farm. These results provide evidence for a role of cattle feed in transmission of E. coli O157:H7; S. enterica; cattle-bacteria.     

Comparison of methods for differentiation of Salmonella enterica serovar Enteritidis phage type 4 isolates

OBJECTIVE: To compare molecular typing methods for the differentiation of Salmonella enterica serovar Enteritidis phage type (PT) 4 isolates that allowed for the determination of their genetic relatedness. SAMPLE POPULATION: 27 Salmonella Enteritidis PT 4 strains isolated in the United States and Europe. PROCEDURE: Several molecular typing methods were performed to assess their ability to genetically differentiate among Salmonella Enteritidis PT 4 isolates. Results of pulse-field gel electrophoresis (PFGE), repetitive polymerase chain reaction (PCR) assay, 16S rRNA gene sequencing, random amplification of polymorphic DNA (RAPD), PCR-restriction fragment length polymorphism of 16S rRNA, and antimicrobial susceptibility were evaluated. RESULTS: Compared with results for other techniques, results for the RAPD typing method with the RAPD1 primer reveal that it was the most discriminatory fingerprinting technique, and it allowed us to cluster Salmonella Enteritidis PT 4 isolates on the basis of their genetic similarity. CONCLUSIONS AND CLINICAL RELEVANCE: This study revealed the value of RAPD with the RAPD1 primer as a tool for epidemiologic investigations of Salmonella Enteritidis PT 4. It can be used in conjunction with PFGE and phage typing to determine the genetic relatedness of Salmonella Enteritidis isolates involved in outbreaks of disease. A reliable and highly discriminatory method for epidemiologic investigations is critical to allow investigators to identify the source of infections and consequently prevent the spread of Salmonella Enteritidis PT 4.     

Comparison of pulsed-field gel electrophoresis with random amplified polymorphic DNA for typing of Mycoplasma synoviae

Pulsed-field gel electrophoresis (PFGE) analysis was developed and compared with random amplified polymorphic DNA (RAPD) method to type 18 Mycoplasma synoviae (MS) strains. All analysed strains were typeable by RAPD but only 89% of MS strains were typeable by PFGE because of DNA degradation. The discriminatory power of RAPD was greater than that of PFGE but the two techniques had a discriminatory index superior to 0.95, the threshold value for interpreting typing results with confidence. The in vitro, in ovo and in vivo reproducibility of both typing techniques was 100%. However, the interpretation of RAPD patterns was complicated because of inconsistent band intensity. Thus, these molecular typing techniques should be helpful for epidemiological studies of avian mycoplasma infections.     

Multilocus variable-number of tandem-repeats analysis of Salmonella enterica serotype Gallinarum and comparison with pulsed-field gel electrophoresis genotyping

Salmonella enterica serovar Gallinarum is the causative agent of fowl typhoid, a severe disease of poultry, responsible for heavy economic losses. Epidemiologic investigation of fowl typhoid significantly benefits from molecular typing tools, RAPD and PFGE have been proposed for this purpose. PFGE, a well established technique, is still the gold standard among typing methods for most bacteria, including salmonella. Nevertheless, it has some limitations regarding execution and reproducibility, in particular it is labour intensive and requires good technical expertise. Furthermore, it needs accurate standardization and results can be ambiguous to interpret. Such limitations can hamper reproducibility and transfer of results. As a possible alternative to PFGE, multilocus variable-number of tandem-repeats analysis (MLVA) has recently emerged as an effective genotyping method for many bacterial pathogens showing high discriminatory power associated to robustness. We developed a six-loci MLVA protocol for Salmonella Gallinarum and compared it to PFGE performed with SpeI, XbaI and NotI on fifty isolates. The proposed MLVA has a high discriminatory power, equivalent to that of the three-enzyme PFGE (Simpson's index 0.94 for MLVA, 0.93 for three-enzyme PFGE) but it is simpler to perform and straightforward in genotype identification, allowing unambiguous exchange of results. Stability of selected VNTR loci, assessed in vitro and in vivo, is good but not absolute, reflecting the sensitivity of MLVA to detect evolutionary changes of bacteria. Clustering of the isolates as determined by MLVA typing is substantially confirmed by PFGE.     

Pulsed-field gel electrophoresis-based subtyping of DNA degradation-sensitive Salmonella enterica subsp. enterica serovar Livingstone and serovar Cerro isolates obtained from a chicken layer farm

Salmonella enterica serovar subsp. enterica Livingstone and serovar Cerro isolates from a commercial egg-producing farm, which had previously been untypeable by pulsed-field gel electrophoresis (PFGE) because of DNA degradation during the PFGE process, successfully gave banding patterns using electrophoresis buffer supplemented with 50 microM thiourea. By PFGE in the presence of thiourea, DNA degradation-sensitive S. enterica serovar Cerro isolates from the commercial egg-producing farm were found to be genetically unrelated to S. enterica serovar Cerro isolates that gave the patterns in the absence of thiourea. Forty-five of 50 (90%) S. enterica serovar Livingstone isolates from the farm showed arbitrarily designated XbaI-digested patterns X1 and X2 that were distinguished by one-band difference and had an identical BlnI-digested pattern. In one of the two layer houses in the farm, the numbers of isolates having the pattern X2 increased from 57% in 1997 to 89% in 1998, whereas virtually all the isolates obtained from the other house in the same period showed the profile X1. This suggests that strains having the pattern X2 might have an advantage to preferentially colonize in the former house.     

The prevalence of Salmonella enterica in Spanish feed mills and potential feed-related risk factors for contamination

A cross-sectional study was conducted in Spain to estimate the prevalence of Salmonella enterica in feed mills and to identify and evaluate potential risk factors associated with feed contamination. A total of 3844 samples were collected from 523 different feed mills using a stratified sampling method. Samples were tested for the presence of Salmonella using conventional culture methods. When the presence of Salmonella was detected, samples were further characterised using serotyping at the National Reference Laboratory (NRL) for animal feed. Additional data about the biosecurity and hygiene measures, feed material used and compound feed produced, were collected by official veterinarians using a questionnaire in situ. In 144 of the feed mills visited (28%), Salmonella were present. However, it was only isolated from 4.8% of samples taken from all of the feed mills (3.5% from feed materials, 3.2% from compound feed and 12.5% from dust of the feed mill facilities). Salmonella serovars of public health importance (Enteritidis, Typhimurium, Infantis, Virchow and Hadar), were detected in only 2.7% of feed mills and in 0.3% of the samples studied. Logistic regression was used to investigate potential feed-mill risk factors for the isolation of Salmonella. Feed mill intake pits were demonstrated to have an increased risk of culture-positive dust samples (OR=6.4; 95% CI: 2.7-15.1). The feed material used in the production of compound feed was associated with recovery of Salmonella. Of the feed material used, cotton seeds were identified as having the highest odds of contamination (OR=3.8; 95% CI: 1.7-8.3). Pelleting appears to reduce the chance of contamination because non-pelleted compound feed is 8 times more likely to be contaminated than pelleted compound feed (OR=8.2; 95% CI: 2.5-26.6). The role of the feed itself in the epidemiology of Salmonella seems to be of limited importance as compound feed is not frequently contaminated at the feed mill level. This should not preclude Salmonella control measures from including all stages of feed production and they should have a risk-based approach according to the findings of this study.     

Molecular Differentiation of Mycoplasma gallisepticum and Mycoplasma imitans Strains by Pulsed‐Field Gel Electrophoresis and Random Amplified Polymorphic DNA

Pulsed‐field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) analysis were used to compare 21 Mycoplasma gallisepticum strains and five M. imitans strains. Each strain of M. gallisepticum typed by PFGE and RAPD methods was genetically quite unique and RAPD and PFGE fingerprinting enabled strain characterization. Relationships between the M. gallisepticum and M. imitans strains were established and dendrograms were drawn from PFGE and RAPD patterns. PFGE group A and RAPD group D were significantly associated with M. imitans strains (P < 0.05). Three M. imitans strains shared the same PFGE and RAPD patterns. The two M. gallisepticum vaccine strains had singular PFGE and RAPD patterns. Thus, PFGE and RAPD can be used to investigate disease outbreaks in vaccinated flocks or for epidemiological tracking. For M. gallisepticum, the RAPD and PFGE discriminatory powers were superior to 0.95 and the in vitro, in ovo and in vivo reproducibility of RAPD and PFGE was 100%. The RAPD drawback was the inconsistent band intensity complicating the interpretation of patterns, while the PFGE limit was its low typeability (86%). Thus, these two molecular typing methods seemed complementary for M. gallisepticum epidemiological studies.     

Molecular typing by random amplification of polymorphic DNA (RAPD) and detection of virulence genes of Salmonella enterica subspecies enterica serovar Gallinarum biovar gallinarum

Salmonella enterica subspecies enterica serovar Gallinarum biovar Gallinarum is the causative agent of fowl typhoid in chickens, outbreaks of which have devastated poultry populations in Korea since 1992. In order to identify genetic differences among S. Gallinarum isolates, bacteria were examined using the random amplified polymorphic DNA (RAPD) method. Of 13 arbitrary primers screened initially, the primer designated as universal rice primer-6 (URP-6) was selected for subsequent typing assays because it produced a distinctive and reproducible DNA fingerprint for a S. Gallinarum reference strain. URP-6-based RAPD analysis assigned 30 S. Gallinarum isolates into 6 types, with 26 isolates (86.6%) belonging to 2 major RAPD types. The distribution of virulence genes in S. Gallinarum isolates was examined by Southern hybridization. All tested isolates had the invasion gene, invA, the virulence plasmid gene, spvB, and the S. Enteritidis fimbrial gene, sefC. The distribution of virulence genes among S. Gallinarum isolates did not correlate with any specific RAPD type.     

Genomic relatedness among Actinobacillus pleuropneumoniae field strains of sterotypes 1 and 5 isolated from healthy and diseased pigs.

Forty-four Actinobacillus pleuropneumoniae isolates recovered from both healthy and diseased pigs were characterized by random amplified polymorphic DNA analysis (RAPD), pulsed field gel electrophoresis (PFGE) and apx toxin gene typing. Nine RAPD types and 14 PFGE patterns were identified. No common RAPD or PFGE patterns were found between strains of serotype 1 and those of serotype 5. The RAPD analysis indicated that the 15 serotype 1 strains isolated from diseased pigs were assigned to 4 RAPD types, with 66% of strains characterized by the same RAPD type. By contrast, the 5 strains of serotype 1 isolated from healthy carriers were dispersed in 4 RAPD types. These data suggest that the diversity of strains isolated from healthy pigs could be higher than that of strains recovered from diseased pigs. In addition, all serotype 5 strains exhibited a unique RAPD type. Unlike RAPD, PFGE analysis allowed discrimination among isolates of serotype 1 and among those of serotype 5. All but 3 isolates showed the same apx genotype as their respective serotype reference strain. These data indicate that RAPD analysis is a valuable rapid tool for routine subtyping of strains of serotype 1. For strains of serotype 5, a combination of several typing methods, such as PFGE and apx gene typing, is needed to provide useful information on the molecular epidemiology of swine pleuropneumonia.     

Antimicrobial resistance, virulence-associated genes, and pulsed-field gel electrophoresis profiles of Salmonella enterica subsp. enterica serovar Typhimurium isolated from piglets with diarrhea in Korea

Salmonella enterica subsp. enterica serovar Typhimurium was isolated from diarrheic piglets in 2 periods, 2000-2001 (n = 25) and 2005-2006 (n = 17). To compare the characteristics of the isolates collected during the 2 periods, all isolates were tested for antimicrobial resistance, the presence of virulence genes, and pulsed-field gel electrophoresis (PFGE) patterns. All 42 isolates were resistant to at least 1 of the 20 antimicrobials tested, and 39 (93%) were resistant to 2 or more antimicrobials. One isolate was resistant to 12 antimicrobials. Profiles of antimicrobial resistance revealed 20 resistance types. Several isolates were also resistant to quinolones and expanded-spectrum cephalosporins. Ten isolates (24%) were resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (ACSSuT); only one isolate had been isolated in 2000-2001, indicating that this type of resistance has rapidly disseminated. Polymerase chain reaction (PCR) assays revealed that all the isolates carried invA. Among the 25 strains isolated in 2000-2001, all carried the sipA, sopA, sopD, sopE2, and ssaR genes, and 96% carried sopB and sifA. Among the 17 strains isolated in 2005-2006, all carried sifA, and approximately 90% carried sipA, sopA, sopB, sopD, sopE2, and ssaR. However, only 6 (14%) of the 42 isolates carried spvC. By PFGE analysis, all 42 strains were classified into 4 major clusters, basically by collection period. The genetic similarity according to PFGE suggests that the strains isolated from diarrheic piglets of this region within the same period may be closely related.     

Prevalence of Escherichia coli O157:H7 prophage-like sequences among German Salmonella enterica serotype Typhimurium phage types and their use in detection of phage type DT104 by the polymerase chain reaction

A 1.6kb DNA fragment identified by random amplifiable polymorphic DNA differentiation (RAPD) from a Salmonella enterica serotype Typhimurium phage type DT104 isolate was used to investigate the prevalence of the region in 160 DT104 isolates, 83 other epidemiological important S. Typhimurium phage types and 20 strains selected from 17 other Salmonella serotypes. PCR screening tests using two different primer-sets derived from the RAPD fragment's nucleotide sequence showed that 76% of the 160 DT104 isolates investigated, including subtypes DT104A, DT104B, DT104B low, DT104H and DT104L, reacted positively. High sensitivity was shown for DT104 strains expressing at least the penta-resistance pattern ACSSuT (97% of 104 strains tested). DT104 susceptible strains showed only a sensitivity of 35% (17 strains tested). In contrast, 83% of the 83 strains from the other S. Typhimurium phage types reacted negatively. Strains from five out of the 17 other serotypes showed a positive signal with one primer-set. The other primer-set exhibited only a positive reaction with one S. Dublin isolate. The analysis of a 2415bp extended sequence revealed homologies to genes encoded by Escherichia coli O157:H7 prophages, suggesting that the described region contains genes of a prophage specific for DT104 and related phage types.     

Typing of Salmonella enterica subsp. enterica serovar Mbandaka isolates

Recently, Salmonella enterica subsp. enterica serovar Mbandaka (S. Mbandaka) has gained some importance in the epidemiology of salmonellosis in Poland. Since biotyping, resistance typing, and plasmid profiling were insufficient for strain differentiation, genome macrorestriction by means of pulse-field gel electrophoresis (PFGE) was applied and proved to be the method of choice in S. Mbandaka epidemiological studies. XbaI and BcuI macrorestriction produced 15 and 14 pulse-field profiles (PFP), respectively, but in the case of each enzyme one profile was prevalent. When macrorestriction profiles were combined, a total 24 patterns were found. Based on the similarity of the profiles, four clonal lineages were identified. One clonal lineage contained the majority of poultry, feed and human isolates. Poultry was concluded to be an important source of S. Mbandaka for humans in Poland. Complementary use of various typing techniques improved efficacy of epidemiological studies giving possibility to subdivide S. Mbandaka into 35 types and the index of discrimination reached 0.947.     

Molecular characterization reveals Salmonella enterica serovar 4,[5],12:i:- from poultry is a variant Typhimurium serovar

Although Salmonella remains one of the leading causes of foodborne illnesses in the United States, the Salmonella enterica serovars and genetic types associated with most infections appear to fluctuate over time. Recently, the Center for Disease Control and Prevention (CDC) has reported an increase in cases of salmonellosis caused by Salmonella 4,[5],12:i:-. Similarly, this unusual Salmonella serovar has been isolated from cattle and poultry in the state of Georgia. We examined the genetic relatedness of Salmonella 4,[5],12:i:-, isolated from several different poultry companies and dairy farms in Georgia, by pulsed-field gel electrophoresis (PFGE). Several Salmonella 4,[5],12:i:- isolates had PFGE patterns identical or similar to PFGE patterns of Salmonella Typhimurium isolated from numerous animal sources. We identified distinct PFGE patterns for Salmonella 4,[5],12:i:- and matching Salmonella Typhimurium PFGE patterns, identifying four "distinct" strains. We focused a more specific analysis on the poultry Salmonella 4,[5],12:i:- and Salmonella Typhimurium isolates and found that of these Salmonella 4,[5],12:i:- isolates, 32% lacked the entire phase 2 antigen gene, fljB; 61% contained partial deletion(s); and 4% had partial deletion(s) in fljB and an adjacent gene hin, 5' to fljB. Thirteen percent contained smaller deletions or point mutations not identified by our DNA probes. The Salmonella 4,[5],12:i:- isolates were positive for several genes present in the Salmonella Typhimurium, including lpfE (100%), sseI(96%), and spvC (93%). Genetic analysis indicates independent, spontaneous mutations in fljB in at least four distinct Salmonella Typhimurium strains of animal origin circulating in nature.     

Phenotypic and genetic characterization of Salmonella enterica subsp. enterica serovar Typhimurium isolated from pigs in Rio Grande do Sul, Brazil

This study aimed to investigate the relatedness of porcine Salmonella enterica subsp. enterica (S.) serovar Typhimurium strains isolated in Southern Brazil. Sixty-six isolates from pigs belonging to three commercial companies were submitted to phage typing, XbaI-macrorestriction (PFGE), IS200 hybridization, rep-PCR, antimicrobial susceptibility testing, and PCR assay targeting the spvR region. All strains presented a unique rep-PCR pattern and 63 strains had a common IS200 profile. One pulse-type (XA) was the most prevalent (39/66 strains) and included strains of phage types DT177, DT192, DT194 and RDNC. The spvR region was detected in three strains, which harboured plasmids of 90 kb. High rates of tetracycline, sulfonamide and streptomycin resistance were found. Isolates from farms located in different geographic regions but associated to the same commercial companies clustered together and presented a common resistance profile. Results suggested that clonal groups of S. Typhimurium are present in pig commercial companies in Southern Brazil.     

Comparison of Salmonella enterica serovar Abortusequi isolates of equine origin by pulsed-field gel electrophoresis and fluorescent amplified-fragment length polymorphism fingerprinting

Equine paratyphoid is caused by Salmonella enterica serovar Abortusequi, and manifests mainly as abortion in the mare. We compared S. Abortusequi strains isolated in Japan and other countries using pulsed-field gel electrophoresis (PFGE) and fluorescent amplified-fragment length polymorphism (FAFLP) analysis. PFGE analysis of S. Abortusequi strains gave 21-27 fragments ranging in size from 33 to 602kb. Although two PFGE profiles were observed among the 20 S. Abortusequi isolates in Japan, the restriction fragments originating from the chromosome were common between the two profiles. The similarity index of the two profiles was 90.9%, while those between Japanese and five other S. Abortusequi strains were 29.8-37.5%. On the other hand, FAFLP analysis of S. Abortusequi strains generated 64-67 amplified fragments ranging in size from 100 to 400bp. One polymorphic fragment was observed among the 20 S. Abortusequi isolates in Japan. These data indicate the close relation of this agent in Japan. S. Abortusequi strains sharing a common ancestry might have been conserved in Japan.     

Pulsed field gel electrophoresis for animal Salmonella enterica serovar Typhimurium isolates in Taiwan

Salmonella enterica subspecies enterica serovar Typhimurium is a common pathogen for humans and animals. In order to trace the clonal relationship and to find the circulating strains between human and animal isolates, chromosomal DNAs from 87 serovar Typhimurium strains isolated from animals (pigs were the majority) were subjected to XbaI and SpeI digestion and pulsed field gel electrophoresis (PFGE). For the 87 animal isolates, 38 PFGE pattern combinations were obtained. As the subtyping results from animal isolates were compared with those from the 45 human isolates, it was found that 14 of the animal isolates and 13 of the human isolates shared a common PFGE pattern combination, i.e., pattern XgSf (or called X5S4). When these human and animal isolates were subjected to antibiotic susceptibility test using 11 antibiotics, it was found that strains of pattern XgSf (X5S4) belong to a common antibiogram pattern which is tetracycline, gentamicin, ampicillin, streptomycin and chloramphenicol resistant. Since most of the animal and human strains in pattern XgSf were originally isolated from various areas over different years, strains of this PFGE pattern may be the most epidemic strains which circulating between human and animal sources.     

Molecular genotyping of Salmonella enterica Abortusovis by pulsed field gel electrophoresis

Genotyping of Salmonella strains is an important tool to discriminate among isolates and to improve epidemiological studies when an outbreak occurs. No phagetyping scheme is available for Salmonella enterica subsp. enterica serovar Abortusovis (SAO) and molecular methods previously used were not standardized and were time consuming. Among the DNA-based methods of genotyping, pulsed field gel electrophoresis (PFGE) is currently in use to subtype Salmonella isolates. In this study we evaluated the feasibility of genotyping of SAO by XbaI and BlnI restrictions. Separation of restricted fragments was performed by PFGE. To test the possibility to apply this methodology to epidemiological investigation, a collection of 38 SAO strains isolated in different regions of Italy were analyzed. Eighteen and 29 different PFGE profiles were defined for XbaI and BlnI digestions, respectively. The method demonstrated an adequate typing ability and an excellent discriminatory power. Results from this study show that PFGE may represent a powerful tool to discriminate within the SAO serovar, and provide useful information in support of traditional epidemiological investigations. In particular, this method could be used to identify the origin of infection during outbreaks within a single flock or in different herds.     

Diversity of swine Bordetella bronchiseptica isolates evaluated by RAPD analysis and PFGE

The degree of genetic diversity in 45 Bordetella (B.) bronchiseptica strains comprised of a vaccine strain (N = 1), reference strains (N = 3) and field isolates (N = 41) was evaluated using random amplified polymorphic DNA (RAPD) fingerprinting and pulsed-field gel electrophoresis (PFGE). Three candidate primers were selected for RAPD analysis after screening 20 random decamer oligonucleotides for their discriminatory abilities. The OPA-07, OPA-08 and OPA-18 primers yielded 10, 10, and 6 distinct fingerprint patterns, respectively. The most common identical RAPD pattern was produced by OPA-07 which was shared by 32 isolates (71.1%), the pattern produced by OPA-08 was shared by 26 isolates (57.8%), and the pattern produced by OPA-18 was shared by 40 isolates (88.9%). The RAPD patterns of the vaccine strain and the 3 reference strains did not match any of the patterns produced by the field isolates when primers OPA-07 and OPA-08 were used. PFGE using the restriction endonuclease XbaI produced a total of 15 patterns consisting of 4 PFGE types (A, B, B1 and C, differing by ≥ 4 bands) and 11 A subtypes (differing by ≤ 3 bands). Most of the field isolates exhibited identical type A and B patterns, suggesting that they were related. The vaccine strain and the three reference strains showed different PFGE patterns as compared to the identical type A strains.     

Evaluation of the association between feeding raw meat and Salmonella enterica infections at a Greyhound breeding facility

OBJECTIVE: To investigate Salmonella enterica infections at a Greyhound breeding facility. DESIGN: Cross-sectional study. ANIMAL AND SAMPLE POPULATIONS: 138 adult and juvenile dogs and S. enterica isolates recovered from the dogs and their environment. PROCEDURES: The investigation was conducted at the request of a Greyhound breeder. Observations regarding the environment and population of dogs were recorded. Fecal, food, and environmental specimens were collected and submitted for Salmonella culture. Isolates were serotyped and tested for susceptibility to 16 antimicrobials. Isolates underwent genetic analyses by use of pulsed-field gel electrophoresis and ribotyping. RESULTS: S. enterica was recovered from 88 of 133 (66%) samples of all types and from 57 of 61 (93%) fecal samples. Eighty-three (94.3%) of the isolates were serotype Newport, 77 (87.5%) of which had identical resistance phenotypes. Genetic evaluations suggested that several strains of S. enterica existed at the facility, but there was a high degree of relatedness among many of the Newport isolates. Multiple strains of Salmonella enterica serotype Newport were recovered from raw meat fed on 1 day. CONCLUSIONS AND CLINICAL RELEVANCE: S. enterica infections and environmental contamination were common at this facility. A portion of the Salmonella strains detected on the premises was likely introduced via raw meat that was the primary dietary constituent. Some strains appeared to be widely disseminated in the population. Feeding meat that had not been cooked properly, particularly meat classified as unfit for human consumption, likely contributed to the infections in these dogs.     

Evaluation of molecular typing methods for Salmonella enterica serovar Typhimurium DT104 isolated in Germany from healthy pigs

The discriminatory power of four different DNA based typing methods was tested for the molecular subtyping of Salmonella Typhimurium phage type DT104 isolates. German DT104 strains (n = 133) originating from slaughter pigs were analysed by plasmid profiling, and 32 of them by pulsed-field gel electrophoresis (PFGE) using the restriction enzymes XbaI, SpeI or BlnI, random amplification of polymorphic DNA (RAPD) using 13 different primers and IS200 typing. A resulting subtyping scheme was obtained which is based on the most discriminatory power of the individual methods i.e. plasmid profiling and PFGE with all three enzymes. The index of discrimination obtained by the subtyping scheme was 0.909 closely approaching the maximum value of one. Although minor differences occurred in the molecular DNA pattern of single DT104 strains, a dominating subtyping pattern was observed confirming other studies which showed, that S. Typhimurium DT104 isolates are highly clonal.