Multiplex PCR-based method for identification of common clinical serotypes of Salmonella enterica subsp. enterica

A multiplex PCR method has been developed to differentiate between the most common clinical serotypes of Salmonella enterica subsp. enterica encountered in Washington State and the United States in general. Six genetic loci from S. enterica serovar Typhimurium and four from S. enterica serovar Typhi were used to create an assay consisting of two five-plex PCRs. The assays gave reproducible results with 30 different serotypes that represent the most common clinical isolates of S. enterica subsp. enterica. Of these, 22 serotypes gave unique amplification patterns compared with each other and the other 8 serotypes were grouped into four pairs. These were further resolved by two additional PCRs. We compared the data from PCR serotyping with conventional serotyping and found that PCR serotyping was nearly as discriminatory as conventional serotyping was. The results from a blind test screening 111 clinical isolates revealed that 97% were correctly identified using the multiplex PCR assay. The assay can be easily performed on multiple samples with final results in less than 5 h and, in conjunction with pulsed-field gel electrophoresis, forms a very robust test method for the molecular subtyping of Salmonella enterica subsp. enterica.     

Fingerprinting of Salmonella enterica subsp. enterica serovar Enteritidis by ribotyping

Objective: To carry out an epidemiologic evaluation of Salmonella enterica subsp. enterica serovar Enteritidis outbreaks in households and small communities by means of rRNA gene restriction pattern analysis (ribotyping).
Method: One hundred Enteritidis isolates dating from 1989 to 1994 which could be allocated epidemiologically to different sources or to small community outbreaks were investigated with ribotyping, a fingerprinting method in which bacterial DNA is hybridized with the biotin-labeled plasmid pKK 3535 containing a ribosomal RNA operon of Escherichia coli to determine the ribosomal RNA gene restriction patterns.
Results: Four different ribotyping patterns were found with the restriction endonuclease Sma I and nine with Sph I. Ribotypes of isolates which could be allocated epidemiologically to a common source usually corresponded. Almost 60% of the Enteritidis infections had the ribotyping pattern Sph I-A. In contrast, this pattern was not found in any of the five Enteritidis strains isolated in 1989. The suspicion that Enteritidis phage type 4 infections are caused by consumption of insufficiently heated eggs is supported by the fact that the ribotyping pattern Sph 1-A was found in isolates from eggs and from human specimens.
Conclusions: As patterns Sph I-A and Sma I-J appeared in 58% and 75% of the isolates, respectively, ribotyping cannot be used for the differentiation between various outbreaks with these two patterns. In cases where the Enteritidis strains showed less frequent patterns, ribotyping seems to be a practical tool for the identification of infection chains. In addition newly appearing ribotyping patterns can give information about the epidemiologic development of Enteritidis infection.     

Salmonella enterica subsp. enterica serovar Agona infections in commercial pheasant flocks.

Infections in pheasant flocks due to Salmonella enterica subsp. enterica serovar Agona occurred in a commercial pheasant farm in 1995 and 2000. S. enterica serovar Agona isolates obtained from the affected birds in both years and an environmental sample from 1998 showed an identical pulsed-field gel electrophoresis pattern, indicating that the farm was continually contaminated with the strain during this period. Nine hundred and seventy-three of 1850 birds (56.2%) died at 4 to 5 days of age in 1995, whereas 80 of 2004 birds (4%) died at 15 to 25 days of age in 2000. Pericarditis were found in the birds in both years although infiltration of heterophils in the lesion was more remarkable in the birds in 2000 than those in 1995, indicating that the S. enterica serovar Agona infection in pheasants in 1995 may have led to the rapid death. These observations suggest that susceptibility of pheasants to S. enterica serovar Agona is age dependent.     

Profile of Salmonella enterica subsp. enterica (subspecies I) serotype 4,5,12:i:- strains causing food-borne infections in New York City

Strains of newly emerging Salmonella enterica subsp. enterica (subspecies I) serotype 4,5,12:i:- causing food-borne infections, including a large food poisoning outbreak (n = 86) characterized by persistent diarrhea (14% bloody), abdominal pain, fever, and headache, were examined. The organisms were found in the stool samples from the patients. The biochemical profile of the organisms is consistent with that of S. enterica subsp. I serotypes, except for decreased dulcitol (13%) and increased inositol (96%) utilization. Twenty-eight percent of the strains showed resistance to streptomycin, sulfonamides, or tetracycline only; all three antimicrobial agents; or these agents either alone or in combination with ampicillin, trimethoprim, and trimethoprim-sulfamethoxazole. None of the serotype 4,5,12:i:- strains showed resistance or decreased susceptibility to chloramphenicol or ciprofloxacin. On pulsed-field gel electrophoresis (PFGE), the strains showed 11 or 12 resolvable genomic fragments with 18 banding patterns and three PFGE profile (PFP) clusters (i.e., PFP/A, PFP/B, and PFP/C). Seventy-five percent of the isolates fingerprinted were closely related (zero to three band differences; similarity [Dice] coefficient, 86 to 100%); 63% of these were indistinguishable from each other (PFP/A(1)). PFP/A(1) was common to all strains from the outbreak and 11 hospital sources. Strains from six other hospitals shared clusters PFP/B and PFP/C. PFP/C(4), of the environmental isolate, was unrelated to PFP/A and PFP/B. Nine band differences (similarity coefficient, 61%) were noted between PFP/A(1) and PFP/E of the multidrug-resistant S. enterica subsp. enterica serotype Typhimurium definitive type 104 strains. Whether these emerging Salmonella strains represent a monophasic, Dul(-) variant of serotype Typhimurium or S. enterica subsp. enterica serotype Lagos or a distinct serotype of S. enterica subsp. I is not yet known. Some of the phenotypic and genotypic properties of the serotype 4,5,12:i:- strains are described here.     

Variable number of tandem repeats in Salmonella enterica subsp. enterica for typing purposes

The genomic sequences of Salmonella enterica subsp. enterica strains CT18, Ty2 (serovar Typhi), and LT2 (serovar Typhimurium) were analyzed for potential variable number tandem repeats (VNTRs). A multiple-locus VNTR analysis (MLVA) of 99 strains of S. enterica supsp. enterica based on 10 VNTRs distinguished 52 genotypes and placed them into four groups. All strains tested were independent human isolates from France and did not reflect isolates from outbreak episodes. Of these 10 VNTRs, 7 showed variability within serovar Typhi, whereas 1 showed variability within serovar Typhimurium. Four VNTRs showed high Nei's diversity indices (DIs) of 0.81 to 0.87 within serovar Typhi (n = 27). Additionally, three of these more variable VNTRs showed DIs of 0.18 to 0.58 within serovar Paratyphi A (n = 10). The VNTR polymorphic site within multidrug-resistant (MDR) serovar Typhimurium isolates (n = 39; resistance to ampicillin, chloramphenicol, spectinomycin, sulfonamides, and tetracycline) showed a DI of 0.81. Cluster analysis not only identified three genetically distinct groups consistent with the present serovar classification of salmonellae (serovars Typhi, Paratyphi A, and Typhimurium) but also discriminated 25 subtypes (93%) within serovar Typhi isolates. The analysis discriminated only eight subtypes within serovar Typhimurium isolates resistant to ampicillin, chloramphenicol, spectinomycin, sulfonamides, and tetracycline, possibly reflecting the emergence in the mid-1990s of the DT104 phage type, which often displays such an MDR spectrum. Coupled with the ongoing improvements in automated procedures offered by capillary electrophoresis, use of these markers is proposed in further investigations of the potential of MLVA in outbreaks of salmonellosis, especially outbreaks of typhoid fever.     

Molecular characterization of multidrug-resistant Salmonella enterica subsp. enterica serovar Typhimurium isolates from swine

As part of a longitudinal study of antimicrobial resistance among salmonellae isolated from swine, we studied 484 Salmonella enterica subsp. enterica serovar Typhimurium (including serovar Typhimurium var. Copenhagen) isolates. We found two common pentaresistant phenotypes. The first was resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline (the AmCmStSuTe phenotype; 36.2% of all isolates), mainly of the definitive type 104 (DT104) phage type (180 of 187 isolates). The second was resistance to ampicillin, kanamycin, streptomycin, sulfamethoxazole, and tetracycline (the AmKmStSuTe phenotype; 44.6% of all isolates), most commonly of the DT193 phage type (77 of 165 isolates), which represents an unusual resistance pattern for DT193 isolates. We analyzed 64 representative isolates by amplified fragment length polymorphism (AFLP) analysis, which revealed DNA fingerprint similarities that correlated with both resistance patterns and phage types. To investigate the genetic basis for resistance among DT193 isolates, we characterized three AmKmStSuTe pentaresistant strains and one hexaresistant strain, which also expressed resistance to gentamicin (Gm phenotype), all of which had similar DNA fingerprints and all of which were collected during the same sampling. We found that the genes encoding the pentaresistance pattern were different from those from isolates of the DT104 phage type. We also found that all strains encoded all of their resistance genes on plasmids, unlike the chromosomally encoded genes of DT104 isolates, which could be transferred to Escherichia coli via conjugation, but that the plasmid compositions varied among the isolates. Two strains (strains UT08 and UT12) had a single, identical plasmid carrying bla(TEM) (which encodes ampicillin resistance), aphA1-Iab (which encodes kanamycin resistance), strA and strB (which encode streptomycin resistance), class B tetA (which encodes tetracycline resistance), and an unidentified sulfamethoxazole resistance allele. The third pentaresistant strain (strain UT20) was capable of transferring by conjugation two distinct resistance patterns, AmKmStSuTe and KmStSuTe, but the genes were carried on plasmids with slightly different restriction patterns (differing by a single band of 15 kb). The hexaresistant strain (strain UT30) had the same plasmid as strains UT08 and UT12, but it also carried a second plasmid that conferred the AmKmStSuGm phenotype. The second plasmid harbored the gentamicin resistance methylase (grm), which has not previously been reported in food-borne pathogenic bacteria. It also carried the sul1 gene for sulfamethoxazole resistance and a 1-kb class I integron bearing aadA for streptomycin resistance. We also characterized isolates of the DT104 phage type. We found a number of isolates that expressed resistance only to streptomycin and sulfamethoxazole (the StSu phenotype; 8.3% of serovar Typhimurium var. Copenhagen strains) but that had AFLP DNA fingerprints similar or identical to those of strains with genes encoding the typical AmCmStSuTe pentaresistance phenotype of DT104. These atypical StSu DT104 isolates were predominantly cultured from environmental samples and were found to carry only one class I integron of 1.0 kb, in contrast to the typical two integrons (InC and InD) of 1.0 and 1.2 kb, respectively, of the pentaresistant DT104 isolates. Our findings show the widespread existence of multidrug-resistant Salmonella strains and the diversity of multidrug resistance among epidemiologically related strains. The presence of resistance genes on conjugative plasmids and duplicate genes on multiple plasmids could have implications for the spread of resistance factors and for the stability of multidrug resistance among Salmonella serovar Typhimurium isolates.     

Molecular follow-up of Salmonella enterica subsp. enterica serovar Agona infection in cattle and humans

Salmonella enterica subsp. enterica serovar Agona was not frequently encountered in Finland until an increase in rates of isolation among animal and feed was seen in 1994. A small outbreak among cattle farms in the regions of Oulu and Vaasa in northwestern Finland in 1994-1995 included eight farms. After the outbreak, an increase in the number of serovar Agona infections in humans was seen in 1999: the number of annual microbiologically confirmed cases in humans increased from about 10 from 1990 to 1998 to 84 in 1999, including an outbreak in which more than 50 people were infected. To gather epidemiological data on serovar Agona and to trace the origin of the human infections, 110 serovar Agona isolates isolated from animal, feed, and other sources as well as from humans with cases of salmonellosis of domestic and foreign origin, which were recovered from 1984 to 1999, were analyzed for their pulsed-field gel electrophoresis (PFGE), plasmid, and IS200 profiles and antibiograms. Of these typing methods, PFGE with restriction endonucleases XbaI, BlnI, NotI, and SpeI was the most useful. The PFGE profile of the strain causing an outbreak among cattle in Finland in 1994-1995 was not seen previously. The strain with this profile was later only sporadically found in human infections. The profile of the strain causing the human outbreak in 1999 was not found among isolates from cattle or any other sources. Molecular typing was valuable in showing that although the outbreaks in cattle and humans seemed to be related regionally, they were not related otherwise.     

Molecular typing of Salmonella enterica subsp. enterica serovar Wien by rRNA gene restriction patterns.

Analysis of digested DNA from 40 Salmonella enterica subsp. enterica serovar Wien isolates revealed five different rRNA gene restriction patterns for HindIII (H1 to H5) and five for PstI (P1 to P5). The isolates had been selected on the basis of the year of isolation, the geographic origin and the antibiotic resistance pattern and were distinguished as follows: a) 13 pre-epidemic isolates from different French towns in 1958-69 and from Senegal in 1968; b) 7 epidemic isolates from Algiers in 1969; c) 20 post-epidemic isolates from different French and Italian towns in 1970-90. Three different rRNA patterns (H1P1, H3P3 and H4P4) were observed among the pre-epidemic isolates. Conversely, the epidemic isolates, which were characterized by the previously described large antibiotic resistance patterns and by the presence of 1.3 and 80-109 MDa plasmids, belonged to the same H1P1 ribotype. All but two post-epidemic isolates were of the H1P1 ribotype. The determination of rRNA gene restriction patterns together with the plasmid content proved to be useful for a better characterization of the serovar Wien endemic and epidemic isolates.     

Characterization of extended-spectrum beta-lactamase (TEM-52)-producing strains of Salmonella enterica serovar typhimurium with diverse resistance phenotypes

Two Salmonella enterica serovar Typhimurium strains from different clonal origins, both producing an extended-spectrum beta-lactamase (TEM-52), were isolated from a patient. This enzyme was encoded on a single plasmid and was found at very low levels in one strain, while being encoded on multiple plasmids and in multiple different EcoRI fragments in the other strain.     

Multiple-locus variable-number tandem repeat analysis for subtyping of Salmonella enterica subsp. enterica serovar Enteritidis

Salmonella enterica subsp. enterica serovar Enteritidis is a major food-borne pathogen that caused most of Salmonella infections worldwide. S. Enteritidis phage type 4 (PT4) especially presents a real challenge for the classical typing methods. We developed a simple multiple-locus variable-number tandem repeat analysis (MLVA) assay based on three hypervariable variable-number tandem repeat (VNTR) loci for subtyping of Salmonella Enteritidis. Testing an arbitrary chosen strain collection of 110 S. Enteritidis isolates, comprising PTs 4, 8, and 21, the MLVA assay yielded a higher discriminatory power, corresponding to a Simpson's index of diversity (ID) of 0.91, when compared to pulsed-field gel electrophoresis (PFGE) which had a Simpson's ID of 0.41. To simplify interpretation of results, we developed a VNTR allele code based on the repeat unit number. This code can easily be exchanged. In conclusion, MLVA is a promising new tool to investigate outbreaks of S. Enteritidis and constitutes a useful addition to the current phage typing scheme.     

Characterization of beta-lactamases responsible for resistance to extended-spectrum cephalosporins in Escherichia coli and Salmonella enterica strains from food-producing animals in the United Kingdom

Nine epidemiologically unrelated isolates [1 Salmonella Bredeney from turkeys, and 8 Escherichia coli [3 environmental isolates (2 from chickens, 1 from pigs), and 5 isolates from cattle with neonatal diarrhea]] were examined both pheno- and genotypically for extended-spectrum beta-lactam (ESBL) resistance. Resistance phenotypes (ampicillin, aztreonam, cefotaxime, cefpodoxime, ceftazidime, and ceftriaxone) suggested the presence of an ESBL enzyme, but cefoxitin MICs (>/= 32 mg/L) suggested the presence of an AmpC-like enzyme. Synergism experiments with benzo(b)thiophene-2-boronic acid (BZBTH2B) and isoelectric focusing (IEF) revealed the presence of an AmpC beta-lactamase with a pI >/= 9. amp C multiplex PCR, sequence, and Southern analyses indicated that only the Salmonella isolate had a plasmid-encoded AmpC beta-lactamase CMY-2 on a nonconjugative 60-MDa plasmid. PCR and sequence analysis of the E. coli ampC promoter identified mutations at positions -88(T), -82(G), -42(T), -18(A), -1(T) and +58(T) in all the isolates. In addition one strain had two extra-mutations at positions +23(A) and +49(G), and another strain had one extra-mutation at position +32(A). DNA fingerprinting revealed that all the E. coli isolates were different clones. It also showed that the U.K. Salmonella isolate was indistinguisable from a Canadian Salmonella isolate from turkeys; both had identical resistance phenotypes and produced CMY-2. This is the first report of a CMY-2 Salmonella isolate in the United Kingdom. These data imply that beta-lactam resistance in animal isolates can be generated de novo as evidenced by the E. coli strains, or in the case of the Salmonella strains be the result of intercontinental transmission due to an acquired resistance mechanism.     

Fecal excretion of Salmonella enterica serovar typhimurium following a food-borne outbreak

Fecal excretion of Salmonella enterica serovar Typhimurium organisms was observed in patients and in people not showing symptoms who were involved in an outbreak of food-borne infection with this organism. Excretion of organisms was prolonged in the patients who were given antimicrobial drugs compared with those who were not. The isolates were indistinguishable by their pulsed-field gel electrophoresis patterns and biotyping from the strain recovered from the roast pork that had been consumed by all of the people. This indicates that these isolates obtained from the infected people had originated in the contaminated pork.     

Use of biochemical kinetic data to determine strain relatedness among Salmonella enterica subsp. enterica isolates

Classical biotyping characterizes strains by creating biotype profiles that consider only positive and negative results for a predefined set of biochemical tests. This method allows Salmonella subspecies to be distinguished but does not allow serotypes and phage types to be distinguished. The objective of this study was to determine the relatedness of isolates belonging to distinct Salmonella enterica subsp. enterica serotypes by using a refined biotyping process that considers the kinetics at which biochemical reactions take place. Using a Vitek GNI+ card for the identification of gram-negative organisms, we determined the biochemical kinetic reactions (28 biochemical tests) of 135 Salmonella enterica subsp. enterica strains of pig origin collected in Spain from 1997 to 2002 (59 Salmonella serotype Typhimurium strains, 25 Salmonella serotype Typhimurium monophasic variant strains, 25 Salmonella serotype Anatum strains, 12 Salmonella serotype Tilburg strains, 7 Salmonella serotype Virchow strains, 6 Salmonella serotype Choleraesuis strains, and 1 Salmonella enterica serotype 4,5,12:-:- strain). The results were expressed as the colorimetric and turbidimetric changes (in percent) and were used to enhance the classical biotype profile by adding kinetic categories. A hierarchical cluster analysis was performed by using the enhanced profiles and resulted in 14 clusters. Six major clusters grouped 94% of all isolates with a similarity of > or =95% within any given cluster, and eight clusters contained a single isolate. The six major clusters grouped not only serotypes of the same type but also phenotypic serotype variations into individual clusters. This suggests that metabolic kinetic reaction data from the biochemical tests commonly used for classic Salmonella enterica subsp. enterica biotyping can possibly be used to determine the relatedness between isolates in an easy and timely manner.     

Discrimination of d-tartrate-fermenting and -nonfermenting Salmonella enterica subsp. enterica isolates by genotypic and phenotypic methods

A multiplex PCR and an improved lead acetate test were developed to discriminate d-tartrate-fermenting and -nonfermenting Salmonella enterica subsp. enterica strains. Both methods showed an accuracy of 100% when 125 Salmonella strains belonging to 15 serovars were tested. Special emphasis was given to S. enterica subsp. enterica serovar Paratyphi B isolates because of the clinical importance of its d-tartrate-nonfermenting variant and the recently increasing numbers of cases of human outbreaks caused by its fermenting variant (formerly Salmonella serovar Java). The lead acetate test described previously (G. A. Alfredsson, R. M. Barker, D. C. Old, and J. P. Duguid, J. Hyg. 70:651-666, 1972) was modified in the inoculation and incubation procedure. The PCR assay was based on the genotypic difference of the presence (d-tartrate-fermenting strains) or absence (d-tartrate-nonfermenting strains) of the ATG start codon for the gene STM 3356, which encodes a putative cation transporter. Sequence data revealed a nucleotide exchange from G to A within the ATG start codon of gene STM 3356 in the d-tartrate-nonfermenting strains. In order to increase the reliability of the PCR assay, a positive control based on a Salmonella genus-specific primer set for the detection of Salmonella DNA was included. The PCR-based discrimination needs only several hours compared to 6 days needed by the improved lead acetate test to obtain reliable results. Consequently, the PCR d-tartrate assay should be the method of choice for the discrimination of d-tartrate-fermenting and -nonfermenting Salmonella strains in the future.     

Evaluation and comparison of molecular techniques for epidemiological typing of Salmonella enterica subsp. enterica serovar dublin.

A total of 28 unrelated isolates of the Salmonella enterica subsp. enterica serovar dublin (S. dublin) collected during a 6-year period, as well as four samples of the S. dublin live vaccine strain Bovisaloral and its prototype strain S. dublin 442/039, were investigated by different molecular typing methods for the following reasons: (i) to find the most discriminatory method for the epidemiological typing of isolates belonging to this Salmonella serovar and (ii) to evaluate these methods for their capacity to discriminate among the live vaccine strain Bovisaloral, its prototype strain S. dublin 442/039, and field isolates of the serovar dublin. Five different plasmid profiles were observed; a virulence plasmid of 76 kbp as identified by hybridization with an spvB-spvC gene probe was present in all isolates. The detection of 16S rRNA genes and that of IS200 elements proved to be unsuitable for the epidemiological typing of S. dublin; only one hybridization pattern could be observed with each of these methods. The results obtained from macrorestriction analysis strongly depended on the choice of restriction enzyme. While the enzyme NotI yielded the lowest discriminatory index among all enzymes tested, it was the only enzyme that allowed discrimination between the Bovisaloral vaccine strain and its prototype strain. In contrast to the enzymes XbaI and SpeI, which only differentiated among the S. dublin field isolates, XhoI as well as AvrII also produced restriction fragment patterns of the Bovisaloral strain and of its prototype strain that were not shared by any of the S. dublin field isolates. Macrorestriction analysis proved to be the most discriminatory method not only for the epidemiological typing of S. dublin field isolates but also for the identification of the S. dublin live vaccine strain Bovisaloral.     

ERIC-PCR genotyping of field isolates of Salmonella enterica subsp. enterica serovar Gallinarum biovars Gallinarum and Pullorum

Salmonella Gallinarum (SG) and Salmonella Pullorum (SP) have been classified as biovars belonging to Salmonella enterica subsp. enterica serovar Gallinarum. Genetic diversity among isolates of the same biovar can be detected by DNA fingerprinting techniques which are useful in epidemiological investigations. In this study, we applied the PCR amplification of Enterobacterial Repetitive Intergenic Consensus sequences (ERIC-PCR) to analyse 45 strains of SG and SP, most of which were isolated from diseased poultry of different Brazilian regions over a period of 27 years until 2014. The ERIC-genotypes obtained were used to describe the epidemiological relationship amongst the strains. Our findings showed that there were six ERIC-patterns for SG strains at 80% similarity. In addition, some of the SG isolates recovered from different regions and years clustered with 100% similarity, suggesting that transfer of genotypes between these regions has taken place. The commercial rough vaccine strain 9R showed a unique profile. Meanwhile, more genetic diversity was observed among SP strains where ten ERIC-patterns were also formed at 80% similarity.     

DNA sequence-based subtyping and evolutionary analysis of selected Salmonella enterica serotypes

While serotyping and phage typing have been used widely to characterize Salmonella isolates, sensitive subtyping methods that allow for evolutionary analyses are essential for examining Salmonella transmission, ecology, and evolution. A set of 25 Salmonella enterica isolates, representing five clinically relevant serotypes (serotypes Agona, Heidelberg, Schwarzengrund, Typhimurium, and Typhimurium var. Copenhagen) was initially used to develop a multilocus sequence typing (MLST) scheme for Salmonella targeting seven housekeeping and virulence genes (panB, fimA, aceK, mdh, icdA, manB, and spaN). A total of eight MLST types were found among the 25 isolates sequenced. A good correlation between MLST types and Salmonella serotypes was observed; only one serotype Typhimurium var. Copenhagen isolate displayed an MLST type otherwise typical for serotype Typhimurium isolates. Since manB, fimA, and mdh allowed for the highest subtype discrimination among the initial 25 isolates, we chose these three genes to perform DNA sequencing of an additional 41 Salmonella isolates representing a larger diversity of serotypes. This "three-gene sequence typing scheme" allowed discrimination of 25 sequence types (STs) among a total of 66 isolates; STs correlated well with serotypes and allowed within-serotype differentiation for 9 of the 12 serotypes characterized. Phylogenetic analyses showed that serotypes Kentucky and Newport could each be separated into two distinct, statistically well supported evolutionary lineages. Our results show that a three-gene sequence typing scheme allows for accurate serotype prediction and for limited subtype discrimination among clinically relevant serotypes of Salmonella. Three-gene sequence typing also supports the notion that Salmonella serotypes represent both monophyletic and polyphyletic lineages.     

Dissemination of Salmonella enterica subsp. enterica Serovar Typhimurium var. Copenhagen clonal types through a contract heifer-raising operation

Salmonella enterica subsp. enterica serovar Typhimurium var. Copenhagen isolates from a heifer-raising operation and from 11 dairy herds that had their calves contracted to the heifer-raising operation were examined for their phenotypic and genotypic characteristics. Results of the study showed that the heifer-raising operation could serve as a clearinghouse for Salmonella serovar Typhimurium var. Copenhagen and perhaps other Salmonella serotypes.     

Evaluation of two enzyme-linked immunosorbent assays for detecting Salmonella enterica subsp. enterica Serovar Dublin antibodies in bulk milk.

Two enzyme-linked immunosorbent assays (ELISAs) for the detecting Salmonella enterica subsp. enterica serovar Dublin antibodies in bulk milk were developed and evaluated for potential use in control programs. The ELISAs were based on either lipopolysacharide (LPS ELISA) or flagellar antigen (GP ELISA). Sensitivity was determined with 79 case herds with a wide range of clinical signs. Specificity was determined with 125 Dutch and 200 Swedish control herds. The relation between antibodies in bulk milk, antibodies in serum, and the level of milk production of individual cows was studied with 61 case herds. The optimal optical density (OD) values of the LPS ELISA and the GP ELISA were determined to be 0.2 and 0.5, respectively. The sensitivities of the LPS ELISA and the GP ELISA were 54 and 63%, respectively, with a specificity of 98% for both ELISAs with samples from the Dutch control herds. The specificities for samples from the Swedish herds were 100% for the LPS ELISA and 95% for the GP ELISA. The sensitivity of the combination of tests was 65% when samples were run in parallel, and the specificity was 100% when samples were run in series, irrespective of whether the samples came from Dutch or Swedish control herds. The variance (R(2)) in the OD value for bulk milk samples could be explained by the percentage of seropositive lactating cows in a herd with the LPS ELISA for 51% of the samples and with the GP ELISA for 72%. The variance in the OD value was best explained by the combination of the percentage of seropositive lactating cows in the herd and the mean log(10) serum antibody titer for that herd (R(2) = 62% for the LPS ELISA and R(2) = 75% for the GP ELISA). Case herds more often tested negative by the ELISA with bulk milk when the percentage of seropositive lactating cows was less than 5%. It is concluded that both ELISAs with bulk milk can be used in control programs to distinguish between infected and noninfected herds. Specificity can be increased by using the two tests in combination. Sensitivity was relatively low for both single tests and both tests combined.     

Fatal case of Salmonella enterica subsp. arizonae gastroenteritis in an infant with microcephaly

Salmonella enterica subsp. arizonae is a common gut inhabitant of reptiles, with snakes as the most common reservoir. Though human cases due to this organism are exceedingly rare, it may infect young infants and immunocompromised individuals with a history of intimate associations with reptiles. Gastroenteritis is the most common presentation; others include peritonitis, pleuritis, osteomyelitis, meningitis, and bacteremia. We report a fatal case of S. enterica subsp. arizonae gastroenteritis in a 3-month-old child with microcephaly, with a review of earlier cases and problems encountered in identification of this rare human pathogen.