Characterization of Salmonella occurring at high prevalence in a population of the land iguana Conolophus subcristatus in Galápagos Islands, Ecuador

The aim of the study was to elucidate the association between the zoonotic pathogen Salmonella and a population of land iguana, Colonophus subcristatus, endemic to Galápagos Islands in Ecuador. We assessed the presence of Salmonella subspecies and serovars and estimated the prevalence of the pathogen in that population. Additionally, we investigated the genetic relatedness among isolates and serovars utilising pulsed field gel electrophoresis (PFGE) on XbaI-digested DNA and determined the antimicrobial susceptibility to a panel of antimicrobials. The study was carried out by sampling cloacal swabs from animals (n = 63) in their natural environment on in the island of Santa Cruz. A high prevalence (62/63, 98.4%) was observed with heterogeneity of Salmonella subspecies and serovars, all known to be associated with reptiles and with reptile-associated salomonellosis in humans. Serotyping revealed 14 different serovars among four Salmonella enterica subspecies: S. enterica subsp. enterica (n = 48), S. enterica subsp. salamae (n = 2), S. enterica subsp. diarizonae (n = 1), and S. enterica subsp. houtenae (n = 7). Four serovars were predominant: S. Poona (n = 18), S. Pomona (n = 10), S. Abaetetuba (n = 8), and S.Newport (n = 5). The S. Poona isolates revealed nine unique XbaI PFGE patterns, with 15 isolates showing a similarity of 70%. Nine S. Pomona isolates had a similarity of 84%. One main cluster with seven (88%) indistinguishable isolates of S. Abaetetuba was observed. All the Salmonella isolates were pan-susceptible to antimicrobials representative of the most relevant therapeutic classes. The high prevalence and absence of clinical signs suggest a natural interaction of the different Salmonella serovars with the host species. The interaction may have been established before any possible exposure of the iguanas and the biocenosis to direct or indirect environmental factors influenced by the use of antimicrobials in agriculture, in human medicine or in veterinary medicine.     

Comparison of Salmonella enterica Serovar Typhimurium LT2 and Non-LT2 Salmonella Genomic Sequences, and Genotyping of Salmonellae by Using PCR

Genes of Salmonella enterica serovar Typhimurium LT2 expected to be specifically present in Salmonella were selected using the Basic Local Alignment Search Tool (BLAST) program. The 152 selected genes were compared with 11 genomic sequences of Salmonella serovars, including Salmonella enterica subsp. I and IIIb and Salmonella bongori (V), and were clustered into 17 groups by their comparison patterns. A total of 38 primer pairs were constructed to represent each of the 17 groups, and PCR was performed with various Salmonella subspecies including Salmonella enterica subsp. I, II, IIIa, IIIb, IV, VI, and V to evaluate a comprehensive DNA-based scheme for identification of Salmonella subspecies and the major disease-causing Salmonella serovars. Analysis of PCR results showed that Salmonella enterica subsp. I was critically divided from other subspecies, and Salmonella strains belonging to S. enterica subsp. I were clustered based on their serovars. In addition, genotypic relationships within S. enterica subsp. I by PCR results were investigated. Also, Salmonella signature genes, Salmonella enterica serovar Typhimurium signature genes, and Salmonella enterica subsp. I signature genes were demonstrated based on their PCR results. The described PCR method suggests a rapid and convenient method for identification of Salmonella serovars that can be used by nonspecialized laboratories. Genome sequence comparison can be a useful tool in epidemiologic and taxonomic studies of Salmonella.     

Pet Snakes as a Reservoir for Salmonella enterica subsp. diarizonae (Serogroup IIIb): a Prospective Study

Reptile-associated Salmonella infections are an increasing problem for humans. We have prospectively screened two breeding groups of 16 pet snakes for colonization with Salmonella species. Various serovars of S. enterica subsp. diarizonae were found in 81% of the snakes. To avoid transmission, strict hygienic precautions should be applied when reptiles are handled.     

Detection of Salmonella spp. Using a Generic and Differential FRET-PCR

To facilitate the detection of Salmonella and to be able to rapidly and conveniently determine the species/subspecies present, we developed and tested a generic and differential FRET-PCR targeting their tetrathionate reductase response regulator gene. The differential pan-Salmonella FRET-PCR we developed successfully detected seven plasmids that contained partial sequences of S. bongori and the six S. enterica subspecies. The detection limit varied from ∼5 copies of target gene/per PCR reaction for S. enterica enterica to ∼200 for S. bongori. Melting curve analysis demonstrated a T _m of ∼68°C for S. enterica enterica, ∼62.5°C for S. enterica houtenae and S. enterica diarizonae, ∼57°C for S. enterica indica, and ∼54°C for S. bongori, S. enterica salamae and S. enterica arizonae. The differential pan-Salmonella FRET-PCR also detected and determined the subspecies of 4 reference strains and 47 Salmonella isolated from clinically ill birds or pigs. Finally, we found it could directly detect and differentiate Salmonella in feline (5/50 positive; 10%; one S. enterica salamae and 4 S. enterica enterica) and canine feces (15/114 positive; 13.2%; all S. enterica enterica). The differential pan-Salmonella FRET-PCR failed to react with 96 non-Salmonella bacterial strains. Our experiments show the differential pan-Salmonella FRET-PCR we developed is a rapid, sensitive and specific method to detect and differentiate Salmonella.     

Phylogenomic Analysis Identifies Gene Gains That Define Salmonella enterica Subspecies I

Comparative methods for analyzing whole genome sequence (WGS) data enable us to assess the genetic information available for reconstructing the evolutionary history of pathogens. We used the comparative approach to determine diagnostic genes for Salmonella enterica subspecies I. S. enterica subsp. I strains are known to infect warm-blooded organisms regularly while its close relatives tend to infect only cold-blooded organisms. We found 71 genes gained by the common ancestor of Salmonella enterica subspecies I and not subsequently lost by any member of this subspecies sequenced to date. These genes included many putative functional phenotypes. Twenty-seven of these genes are found only in Salmonella enterica subspecies I; we designed primers to test these genes for use as diagnostic sequence targets and data mined the NCBI Sequence Read Archive (SRA) database for draft genomes which carried these genes. We found that the sequence specificity and variability of these amplicons can be used to detect and discriminate among 317 different serovars and strains of Salmonella enterica subspecies I.     

An evaluation of the species and subspecies of the genus Salmonella with whole genome sequence data: Proposal of type strains and epithets for novel S. enterica subspecies VII,VIII, IX,X and XI

Species and subspecies within the Salmonella genus have been defined for public health purposes by biochemical properties; however, reference laboratories have increasingly adopted sequence-based, and especially whole genome sequence (WGS), methods for surveillance and routine identification. This leads to potential disparities in subspecies definitions, routine typing, and the ability to detect novel subspecies. A large-scale analysis of WGS data from the routine sequencing of clinical isolates was employed to define and characterise Salmonella subspecies population structure, demonstrating that the Salmonella species and subspecies were genetically distinct, including those previously identified through phylogenetic approaches, namely: S. enterica subspecies londinensis (VII), subspecies brasiliensis (VIII), subspecies hibernicus (IX) and subspecies essexiensis (X). The analysis also identified an additional novel subspecies, reptilium (XI). Further, these analyses indicated that S. enterica subspecies arizonae (IIIa) isolates were divergent from the other S. enterica subspecies, which clustered together and, on the basis of ANI analysis, subspecies IIIa was sufficiently distinct to be classified as a separate species, S. arizonae. Multiple phylogenetic and statistical approaches generated congruent results, suggesting that the proposed species and subspecies structure was sufficiently biologically robust for routine application. Biochemical analyses demonstrated that not all subspecies were distinguishable by these means and that biochemical approaches did not capture the genomic diversity of the genus. We recommend the adoption of standardised genomic definitions of species and subspecies and a genome sequence-based approach to routine typing for the identification and definition of novel subspecies.     

Salmonella enterica Diversity in Central Californian Coastal Waterways

Salmonella enterica is one of the most important bacterial enteric pathogens worldwide. However, little is known about its distribution and diversity in the environment. The present study explored the diversity of 104 strains of Salmonella enterica isolated over 2 years from 12 coastal waterways in central California. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing were used to probe species diversity. Seventy-four PFGE patterns and 38 sequence types (STs) were found, including 18 newly described STs. Nineteen of 25 PFGE patterns were indistinguishable from those of clinical isolates in PulseNet. The most common ST was consistent with S. enterica serovar Typhimurium, and other frequently detected STs were associated with the serovars Heidelberg and Enteritidis; all of these serovars are important etiologies of salmonellosis. An investigation into S. enterica biogeography was conducted at the level of ST and subspecies. At the ST and subspecies level, we found a taxon-time relationship but no taxon-area or taxon-environmental distance relationships. STs collected during wet versus dry conditions tended to be more similar; however, STs collected from waterways adjacent to watersheds with similar land covers did not tend to be similar. The results suggest that the lack of dispersal limitation may be an important factor affecting the diversity of S. enterica in the region.     

Distributions of Salmonella Subtypes Differ between Two U.S. Produce-Growing Regions

Salmonella accounts for approximately 50% of produce-associated outbreaks in the United States, several of which have been traced back to contamination in the produce production environment. To quantify Salmonella diversity and aid in identification of Salmonella contamination sources, we characterized Salmonella isolates from two geographically diverse produce-growing regions in the United States. Initially, we characterized the Salmonella serotype and subtype diversity associated with 1,677 samples collected from 33 produce farms in New York State (NYS). Among these 1,677 samples, 74 were Salmonella positive, yielding 80 unique isolates (from 147 total isolates), which represented 14 serovars and 23 different pulsed-field gel electrophoresis (PFGE) types. To explore regional Salmonella diversity associated with production environments, we collected a smaller set of samples (n = 65) from South Florida (SFL) production environments and compared the Salmonella diversity associated with these samples with the diversity found among NYS production environments. Among these 65 samples, 23 were Salmonella positive, yielding 32 unique isolates (from 81 total isolates), which represented 11 serovars and 17 different PFGE types. The most common serovars isolated in NYS were Salmonella enterica serovars Newport, Cerro, and Thompson, while common serovars isolated in SFL were Salmonella serovars Saphra and Newport and S. enterica subsp. diarizonae serovar 50:r:z. High PFGE type diversity (Simpson''s diversity index, 0.90 ± 0.02) was observed among Salmonella isolates across both regions; only three PFGE types were shared between the two regions. The probability of three or fewer shared PFGE types was <0.000001; therefore, Salmonella isolates were considerably different between the two sampled regions. These findings suggest the potential for PFGE-based source tracking of Salmonella in production environments.     

Salmonella Pathogenicity and Host Adaptation in Chicken-Associated Serovars

SUMMARY

Enteric pathogens such as Salmonella enterica cause significant morbidity and mortality. S. enterica serovars are a diverse group of pathogens that have evolved to survive in a wide range of environments and across multiple hosts. S. enterica serovars such as S. Typhi, S. Dublin, and S. Gallinarum have a restricted host range, in which they are typically associated with one or a few host species, while S. Enteritidis and S. Typhimurium have broad host ranges. This review examines how S. enterica has evolved through adaptation to different host environments, especially as related to the chicken host, and continues to be an important human pathogen. Several factors impact host range, and these include the acquisition of genes via horizontal gene transfer with plasmids, transposons, and phages, which can potentially expand host range, and the loss of genes or their function, which would reduce the range of hosts that the organism can infect. S. Gallinarum, with a limited host range, has a large number of pseudogenes in its genome compared to broader-host-range serovars. S. enterica serovars such as S. Kentucky and S. Heidelberg also often have plasmids that may help them colonize poultry more efficiently. The ability to colonize different hosts also involves interactions with the host''s immune system and commensal organisms that are present. Thus, the factors that impact the ability of Salmonella to colonize a particular host species, such as chickens, are complex and multifactorial, involving the host, the pathogen, and extrinsic pressures. It is the interplay of these factors which leads to the differences in host ranges that we observe today.     

Limited structure and widespread diversity suggest potential buffers to genetic erosion in a threatened grassland shrub Pimelea spinescens (Thymelaeaceae)

Pimelea spinescens is a critically endangered species of the temperate grasslands of southeastern Australia. Two subspecies are recognised. Subspecies, P. spinescens subsp. spinescens, formerly common and widespread, is found in isolated remnants of previously extensive grasslands. The second subspecies, subsp. pubiflora, is thought to have been historically rare with only two geographically-isolated extant populations. The grassland communities exist now as fragmented remnants representing <1 % of their extent prior to European settlement in the early 1800s. Conservation management strategies for species in these critically endangered ecosystems rely on an understanding of genetic diversity and population structure to ensure long-term evolutionary potential. We used chloroplast DNA (cpDNA) and microsatellite markers to examine the population genetic structure of both subspecies. Analysis of cpDNA revealed 14 haplotypes with high divergence between the single haplotype found in subsp. pubiflora and most remaining haplotypes restricted to subsp. spinescens. Microsatellites also indicated high genetic differentiation between subspecies but little evidence of sub-structuring within either subspecies. Results suggest that seed dispersal has not been as limited as previously thought. In this fragmented habitat, a lack of genetic structure suggests buffers to genetic erosion, with current patterns reflecting genetic diversity prior to fragmentation. Plant longevity and the presence of seed banks may contribute to the maintenance of these patterns, resulting in a lag between fragmentation and genetic erosion. Whilst factors such as longevity and seed banks may be preserving historic genetic diversity, management is required to ensure the maintenance of this diversity into the future.     

Interaction of Phytophagous Insects with Salmonella enterica on Plants and Enhanced Persistence of the Pathogen with Macrosteles quadrilineatus Infestation or Frankliniella occidentalis Feeding

Recently, most foodborne illness outbreaks of salmonellosis have been caused by consumption of contaminated fresh produce. Yet, the mechanisms that allow the human pathogen Salmonella enterica to contaminate and grow in plant environments remain poorly described. We examined the effect of feeding by phytophagous insects on survival of S. enterica on lettuce. Larger S. enterica populations were found on leaves infested with Macrosteles quadrilineatus. In contrast, pathogen populations among plants exposed to Frankliniella occidentalis or Myzus persicae were similar to those without insects. However, on plants infested with F. occidentalis, areas of the infested leaf with feeding damage sustained higher S. enterica populations than areas without damage. The spatial distribution of S. enterica cells on leaves infested with F. occidentalis may be altered resulting in higher populations in feeding lesions or survival may be different across a leaf dependent on local damage. Results suggest the possibility of some specificity with select insects and the persistence of S. enterica. Additionally, we demonstrated the potential for phytophagous insects to become contaminated with S. enterica from contaminated plant material. S. enterica was detected in approximately 50% of all M. quadrilineatus, F. occidentalis, and M. persicae after 24 h exposure to contaminated leaves. Particularly, 17% of F. occidentalis, the smallest of the insects tested, harbored more than 10² CFU/F. occidentalis. Our results show that phytophagous insects may influence the population dynamics of S. enterica in agricultural crops. This study provides evidence of a human bacterial pathogen interacting with phytophagous insect during plant infestation.     

Genomic Insights into Bifidobacteria

Summary: Since the discovery in 1899 of bifidobacteria as numerically dominant microbes in the feces of breast-fed infants, there have been numerous studies addressing their role in modulating gut microflora as well as their other potential health benefits. Because of this, they are frequently incorporated into foods as probiotic cultures. An understanding of their full interactions with intestinal microbes and the host is needed to scientifically validate any health benefits they may afford. Recently, the genome sequences of nine strains representing four species of Bifidobacterium became available. A comparative genome analysis of these genomes reveals a likely efficient capacity to adapt to their habitats, with B. longum subsp. infantis exhibiting more genomic potential to utilize human milk oligosaccharides, consistent with its habitat in the infant gut. Conversely, B. longum subsp. longum exhibits a higher genomic potential for utilization of plant-derived complex carbohydrates and polyols, consistent with its habitat in an adult gut. An intriguing observation is the loss of much of this genome potential when strains are adapted to pure culture environments, as highlighted by the genomes of B. animalis subsp. lactis strains, which exhibit the least potential for a gut habitat and are believed to have evolved from the B. animalis species during adaptation to dairy fermentation environments.     

Specific Discrimination of Three Pathogenic Salmonella enterica subsp. enterica Serotypes by carB-Based Oligonucleotide Microarray

It is important to rapidly and selectively detect and analyze pathogenic Salmonella enterica subsp. enterica in contaminated food to reduce the morbidity and mortality of Salmonella infection and to guarantee food safety. In the present work, we developed an oligonucleotide microarray containing duplicate specific capture probes based on the carB gene, which encodes the carbamoyl phosphate synthetase large subunit, as a competent biomarker evaluated by genetic analysis to selectively and efficiently detect and discriminate three S. enterica subsp. enterica serotypes: Choleraesuis, Enteritidis, and Typhimurium. Using the developed microarray system, three serotype targets were successfully analyzed in a range as low as 1.6 to 3.1 nM and were specifically discriminated from each other without nonspecific signals. In addition, the constructed microarray did not have cross-reactivity with other common pathogenic bacteria and even enabled the clear discrimination of the target Salmonella serotype from a bacterial mixture. Therefore, these results demonstrated that our novel carB-based oligonucleotide microarray can be used as an effective and specific detection system for S. enterica subsp. enterica serotypes.     

Bacteriophage Therapy To Reduce Salmonella Colonization of Broiler Chickens

Acute enteric infections caused by salmonellas remain a major public health burden worldwide. Poultry, particularly chickens, are known to be the main reservoir for this zoonotic pathogen. Although some progress has been made in reducing Salmonella colonization of broiler chickens by using biosecurity and antimicrobials, it still remains a considerable problem. The use of host-specific bacteriophages as a biocontrol is one possible intervention by which Salmonella colonization could be reduced. A total of 232 Salmonella bacteriophages were isolated from poultry farms, abattoirs, and wastewater in 2004 and 2005. Three phages exhibiting the broadest host ranges against Salmonella enterica serotypes Enteritidis, Hadar, and Typhimurium were characterized further by determining their morphology and lytic activity in vitro. These phages were then administered in antacid suspension to birds experimentally colonized with specific Salmonella host strains. The first phage reduced S. enterica serotype Enteritidis cecal colonization by ≥4.2 log₁₀ CFU within 24 h compared with controls. Administration of the second phage reduced S. enterica serotype Typhimurium by ≥2.19 log₁₀ CFU within 24 h. The third bacteriophage was ineffective at reducing S. enterica serotype Hadar colonization. Bacteriophage resistance occurred at a frequency commensurate with the titer of phage being administered, with larger phage titers resulting in a greater proportion of resistant salmonellas. The selection of appropriate bacteriophages and optimization of both the timing and method of phage delivery are key factors in the successful phage-mediated control of salmonellas in broiler chickens.     

O-Antigen Delays Lipopolysaccharide Recognition and Impairs Antibacterial Host Defense in Murine Intestinal Epithelial Cells

Although Toll-like receptor (TLR) 4 signals from the cell surface of myeloid cells, it is restricted to an intracellular compartment and requires ligand internalization in intestinal epithelial cells (IECs). Yet, the functional consequence of cell-type specific receptor localization and uptake-dependent lipopolysaccharide (LPS) recognition is unknown. Here, we demonstrate a strikingly delayed activation of IECs but not macrophages by wildtype Salmonella enterica subsp. enterica sv. (S.) Typhimurium as compared to isogenic O-antigen deficient mutants. Delayed epithelial activation is associated with impaired LPS internalization and retarded TLR4-mediated immune recognition. The O-antigen-mediated evasion from early epithelial innate immune activation significantly enhances intraepithelial bacterial survival in vitro and in vivo following oral challenge. These data identify O-antigen expression as an innate immune evasion mechanism during apical intestinal epithelial invasion and illustrate the importance of early innate immune recognition for efficient host defense against invading Salmonella.     

Effects of Norspermidine and Spermidine on Biofilm Formation by Potentially Pathogenic Escherichia coli and Salmonella enterica Wild-Type Strains

Polyamines are present in all living cells. In bacteria, polyamines are involved in a variety of functions, including biofilm formation, thus indicating that polyamines may have potential in the control of unwanted biofilm. In the present study, the effects of the polyamines norspermidine and spermidine on biofilms of 10 potentially pathogenic wild-type strains of Escherichia coli serotype O103:H2, Salmonella enterica subsp. enterica serovar Typhimurium, and S. enterica serovar Agona were investigated. We found that exogenously supplied norspermidine and spermidine did not mediate disassembly of preformed biofilm of any of the E. coli and S. enterica strains. However, the polyamines did affect biofilm production. Interestingly, the two species reacted differently to the polyamines. Both polyamines reduced the amount of biofilm formed by E. coli but tended to increase biofilm formation by S. enterica. Whether the effects observed were due to the polyamines specifically targeting biofilm formation, being toxic for the cells, or maybe a combination of the two, is not known. However, there were no indications that the effect was mediated through binding to exopolysaccharides, as earlier suggested for E. coli. Our results indicate that norspermidine and spermidine do not have potential as inhibitors of S. enterica biofilm. Furthermore, we found that the commercial polyamines used contributed to the higher pH of the test medium. Failure to acknowledge and control this important phenomenon may lead to misinterpretation of the results.     

Production of Autoinducer 2 in Salmonella enterica Serovar Thompson Contributes to Its Fitness in Chickens but Not on Cilantro Leaf Surfaces

Food-borne illness caused by Salmonella enterica has been linked traditionally to poultry products but is associated increasingly with fresh fruits and vegetables. We have investigated the role of the production of autoinducer 2 (AI-2) in the ability of S. enterica serovar Thompson to colonize the chicken intestine and the cilantro phyllosphere. A mutant of S. enterica serovar Thompson that is defective in AI-2 production was constructed by insertional mutagenesis of luxS. The population size of the S. enterica serovar Thompson parental strain was significantly higher than that of its LuxS⁻ mutant in the intestine, spleen, and droppings of chicks 12 days after their oral inoculation with the strains in a ratio of 1:1. In contrast, no significant difference in the population dynamics of the parental and LuxS⁻ strain was observed after their inoculation singly or in mixtures onto cilantro plants. Digital image analysis revealed that 54% of S. enterica serovar Thompson cells were present in large aggregates on cilantro leaves but that the frequency distributions of the size of aggregates formed by the parental strain and the LuxS⁻ mutant were not significantly different. Carbon utilization profiles indicated that the AI-2-producing strain utilized a variety of amino and organic acids more efficiently than its LuxS⁻ mutant but that most sugars were utilized similarly in both strains. Thus, inherent differences in the nutrients available to S. enterica in the phyllosphere and in the chicken intestine may underlie the differential contribution of AI-2 synthesis to the fitness of S. enterica in these environments.     

A proposed core genome scheme for analyses of the Salmonella genus

The salmonellae are found in a wide range of animal hosts and many food products for human consumption. Most cases of human disease are caused by S. enterica subspecies I; however as opportunistic pathogens the other subspecies (II-VI) and S. bongori are capable of causing disease. Loci that were not consistently present in all of the species and subspecies were removed from a previously proposed core genome scheme (EBcgMLSTv2.0), the removal of these 252 loci resulted in a core genus scheme (SalmcgMLSTv1.0). SalmcgMLSTv1.0 clustered isolates from the same subspecies more rapidly and more accurately grouped isolates from different subspecies when compared with EBcgMLSTv2.0. All loci within the EBcgMLSTv2.0 scheme were present in over 98% of S. enterica subspecies I isolates and should, therefore, continue to be used for subspecies I analyses, while the SalmcgMLSTv1.0 scheme is more appropriate for cross genus investigations.     

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus. However, there are two species, six subspecies, and over 2,500 different Salmonella serovars, and not all are equal in their significance to public health. For example, finding S. enterica subspecies IIIa Arizona on a table egg layer farm is insignificant compared to the isolation of S. enterica subspecies I serovar Enteritidis, the leading cause of salmonellosis linked to the consumption of table eggs. Serovars are identified based on antigenic differences in lipopolysaccharide (LPS)(O antigen) and flagellin (H1 and H2 antigens). These antigenic differences are the outward appearance of the diversity of genes and gene alleles associated with this phenotype.We have developed an allelotyping, multiplex PCR that keys on genetic differences between four major S. enterica subspecies I serovars found in poultry and associated with significant human disease in the US. The PCR primer pairs were targeted to key genes or sequences unique to a specific Salmonella serovar and designed to produce an amplicon with size specific for that gene or allele. Salmonella serovar is assigned to an isolate based on the combination of PCR test results for specific LPS and flagellin gene alleles. The multiplex PCRs described in this article are specific for the detection of S. enterica subspecies I serovars Enteritidis, Hadar, Heidelberg, and Typhimurium.Here we demonstrate how to use the multiplex PCRs to identify serovar for a Salmonella isolate.