Salmonella typhimurium

I. Organism Information

A. Taxonomy Information
  1. Species:
    1. Salmonella typhimurium. :
      1. GenBank Taxonomy No.: 602
      2. Description: Kauffmann (1960, 1963) and Kauffmann and Edwards (1952) divided species in the genus Salmonella into 'sub-genera' I, II, III and IV based on their biochemical characteristics. Strains which inhabit in the intestinal tract of warm-blooded animals were in 'sub-genus' I. Le Minor and Rhode (1974) described eight types of Salmonella in 'sub-genus' I, e.g. "Salmonella choleraesuis, Salmonella hirschfeldii (Salmonella paratyphi-C), Salmonella typhi, Salmonella paratyphi-A, Salmonella schottmuelleri, Salmonella typhimurium, Salmonella enteritidis and Salmonella gallinarum" ( Ezaki et al., 2000). The genus Salmonella comprises two species: S. enterica, which is subdivided into over 2,000 serovars, and Salmonella bongori. Some serovars of S. enterica, such as S. typhi, cause systemic infections and typhoid fever, whereas others, such as S. typhimurium, cause gastroenteritis. Some serovars, such as S. typhi, are host specialists that infect only humans, whereas others such as S. typhimurium, are host generalists that occur in humans and many other mammalian species (McClelland et al., 2001). Salmonella enterica serotype Typhimurium (S. typhimurium) was discovered in 1892 by Loeffler as the causative agent of an epidemic disease in mice (Tsolis et al., 1999) Salmonella typhimurium strain LT2, the principal strain for cellular and molecular biology in Salmonella, was isolated in the 1940s and used in the first studies on phage-mediated transduction (McClelland et al., 2001). Among the more than 2,500 serotypes of the genus Salmonella described to date, two, Enteritidis and Typhimurium, are predominant in many developed countries. S. enterica serotype Typhimurium was the second most prevalent serotype (Enteritidis ranked first) in Europe during the period 1998 to 2003 (Weill et al., 2006). Synonym: Salmonella choleraesuis serotype typhimurium, Salmonella typhi-murium, Bacillus typhimurium, "Salmonella typhi-murium" (sic) (Loeffler 1892) Castellani and Chalmers 1919, "Bacillus typhimurium" Loeffler 1892, Salmonella typhimurium (Loeffler 1892) Castellani and Chalmers 1919 (NCBI_Taxonomy). Synonyms: Bacillus typhimurium, Bacillus psittacosis, Bacillus kaensche, Bacillus breslaviensis, Bacterium breslaviensis, Bacterium typhi murium, Bacillus Aertrycke, bacillus murium, Bacillus pestis-caviae, Bacillus paraertrycke, Bacterium psittacosis, Salmonella aertrycke, Bacillus paratyphosus B, Pasteurella pestis-caviae, Bacterium aertrycke, Group VII, Bacillus enteritidis B, Typhimurium, Bacterium enteritidis breslaviense, Bacterium enteritidis Breslau, Typus Breslau, Salmonella breslaviensis (Kelterborn et al., 1967).
      3. Variant(s):
        • Salmonella typhimurium DT104. :
          • GenBank Taxonomy No.: 85569
          • Parent: Salmonella typhimurium.
          • Description: In 1984, the first recorded isolations of R-Type ACSSuT StmDT104 were documented in the United Kingdom from specimens obtained from wild and exotic birds (Hollinger et al., 1998). The organisms isolated from exotic birds varied in their susceptibility profiles, but the majority were R-type ACSSuT with additional loss of susceptibility to some other antibiotics. These birds were imported from countries in the Far East, Africa, Germany, and the Netherlands (Hollinger et al., 1998). Multidrug-resistant ACSSuT-type (resistance to ampicillin, chloramphenicol, streptomycin, sulfonamide, and tetracycline) Salmonella enterica serovar Typhimurium definitive type 104 (DT104) has risen to prominence in Europe and North America but has been reported less in Asia (McClelland et al., 2001). Evidence in Europe suggests that the emergence of DT104 in cattle was the precursor to its spread to other animals used for food production. Although DT104 is presently the dominant resistant clone of S ser Typhimurium many other phage types (DT29, DT204, DT193, and DT204C) of this serovar have been associated with multidrug resistance (Sanchez et al., 2002). Of particular concern is the worldwide emergence of a distinct strain of multidrug-resistant S. Typhimurium, characterized as definitive phage type 104 (DT104) that is resistant to at lease five antimicrobials- ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline. All ST104 strains contain a chromosome- and integron-encoded B(beta)-lactamase (pSE-1) that appears to have been acquired from plasmids in Pseudomonas species (Pegues et al., 2002). The ACSSuT-type serovar Typhimurium DT104 is derived from two separate evolutionary events. One is the integration of a 43-kb Salmonella genomic island 1 (SGI1), which carries the following multiple antimicrobial resistance genes: pse for ampicillin resistance (A), floR for chloramphenicol resistance (C), str or aad for streptomycin resistance (S), sulI for sulfonamide resistance (Su), and tetR or tetG for tetracycline resistance (T). The other is the integration of P22-like phage into the chromosome to form prophage PDT17 or ST104, which encodes 64 open reading frames without antibiotic resistance genes within the 41-kb DNA fragment (Chiu et al., 2006). We estimate that of the 40,000 salmonella isolates reported annually, 3400 are typhimurium with the five-drug pattern of resistance (Glynn et al., 1998).
        • Salmonella typhimurium LT2. :
          • GenBank Taxonomy No.: 99287
          • Parent: Salmonella typhimurium.
          • Description: Salmonella typhimurium strain LT2, the principal strain for cellular and molecular biology in Salmonella, was isolated in the 1940s and used in the first studies on phage-mediated transduction (McClelland et al., 2001). Salmonella typhimurium LT2, S. typhi, S. paratyphi A and S. paratyphi B are all in subspecies I of S. enterica, which colonizes mammals and birds and causes 99% of Salmonella infections in human (McClelland et al., 2001). Most strains of S. typhimurium contain a plasmid of about 90 kb. The plasmid of strain LT2 is called pSLT (McClelland et al., 2001).
        • Salmonella typhimurium SL1344. :
          • GenBank Taxonomy No.: 216597
          • Parent: Salmonella typhimurium.
          • Description: SL1344 (the genetically marked virulent grandparent of aro(-) live vaccine strain SL3261), is virulent for mice, given i.p. or by feeding. and for calves by the oral route (Hoiseth et al., 1981). This strain, which is a genetically marked subline of a calf-virulent isolate, has been extensively used for studies on Salmonella pathogenicity and vaccine development (Qi et al., 1996).
    2. Salmonella enterica subsp. enterica serovar Typhimurium :
      1. GenBank Taxonomy No.: 90371
      2. Description: Salmonella enterica subsp. enterica serovar Typhimurium var. Copenhagen is an O:5-negative variant of Salmonella serovar Typhimurium which was primarily reported to be found in pigeons. It is now frequently isolated from cattle, swine, and other animals (Hegde et al., 2005).
      3. Variant(s):
        • Salmonella enterica subsp. enterica serovar Typhimurium var. Copenhagen :
          • GenBank Taxonomy No.: 353544
          • Parent: Salmonella enterica subsp. enterica serovar Typhimurium
          • Description: Salmonella enterica subsp. enterica (S.) serovar Typhimurium is of major zoonotic importance, whereas its O:5-negative variant, designated variant Copenhagen (v.c.), has rarely been detected in connection with diseases in humans. In pigeons however, S. Typhimurium v.c. causes various clinical symptoms depending on the age of the infected animals, such as fatal septicaemia or meningoencephalitis in young pigeons or arthritis affecting joints in wings and legs in older pigeons. Although S. Typhimurium v.c. infections are mainly seen in pigeons, these bacteria have also been isolated from salmonellosis cases in other animals, such as cattle and swine, at high frequencies. Occasionally S. Typhimurium v.c. isolates have also been detected in dogs and cats (Frech et al., 2003).
B. Lifecycle Information :
  1. Bacillus typhimurium :
    1. Size: 2 to 3 u by 0.4 to 0.6 u in size (Pegues et al., 2002).
    2. Shape: Salmonellae are gram-negative, non-spore-forming, facultatively anaerobic bacilli (Pegues et al., 2002).
    3. Picture(s):
      1. SEM Image of Salmonella typhimurium (Image):

        Description: Scanning electron microscope image of Salmonella typhimurium (Image).
    4. Description: The worldwide emergence of a distinct strain of multidrug-resistant S. Typhimurium, characterized as definitive phage type 104 (DT104) that is resistant to at least five antimicrobials- ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracyclines. The DT104 strain has broad host reservoirs and is difficult to control in domestic livestock, leading to its widespread clonal dissemination among food animals, especially cattle and humans in Europe, United States, Canada, and the Middle and Far East (Pegues et al., 2002). They cause disease not only in man, but in cattle, pigs, dogs, cats, rats, field mice, an many birds, including ducks and chicken. In fact, practically every animal may be infected (Huckstep et al., 2002).
C. Genome Summary:
  1. Genome of Salmonella typhimurium LT2.
    1. Description: Salmonella typhimurium strain LT2, the principal strain for cellular and molecular biology in Salmonella, was isolated in the 1940s and used in the first studies on phage-mediated transduction1. Attenuated mutants of S. enterica may be used as live oral vaccines against Salmonella infection, to express antigens from other pathogens, and to deliver proteins to solid tumours (McClelland et al., 2001)
    2. Chromosome:
      1. GenBank Accession Number: AE006468; RefSeq: NC_003197
      2. Size: 4,857,432 nt (McClelland et al., 2001).
      3. Gene Count: 4622 genes, 4425 proteins, 118 RNAs, 39 pseudogenes (McClelland et al., 2001).
      4. Description: Sequenced the 4,857-kilobase (kb) chromosome and 94-kb virulence plasmid of S. typhimurium strain LT2 (McClelland et al., 2001). Lateral transfer of genes is frequent, with 11% of the S. typhimurium LT2 genes missing from S. enterica serovar Typhi (S. typhi), and 29% missing from Escherichia coli K12 (McClelland et al., 2001).
    3. Plasmid pSLT:
      1. GenBank Accession Number: AE006471; RefSeq: NC_003277
      2. Size: 93,939 nt (NCBI_Taxonomy).
      3. Gene Count: 112 genes, 102 proteins, RNAs 1 (NCBI_Taxonomy).
      4. Description: Most strains of S. typhimurium contain a plasmid of about 90 kb. The plasmid of strain LT2 is called pSLT20. Out of 108 annotated CDS and pseudogenes in pSLT, only three have a close homologue in S. typhi, S. paratyphi A or S. paratyphi B, as expected owing to these strains lacking the plasmid. A search through GenBank revealed 50 pSLT genes that have a close homologue in plasmids from other Salmonella serovars. Many homologues of genes in the tra operon of the F-factor of E. coli K12 were identified, which presumably are responsible for the self-transmissibility of pSLT at rates of up to 3 times 10-4 . The copy number of the pSLT plasmid was estimated as 2.75, using the relative sequence coverage of the plasmid versus the chromosome in the shotgun phase of sequencing, and estimated as 1.4-3.1 under a variety of growth conditions, when measured by average signal intensity on microarrays (McClelland et al., 2001)

  2. Genome of Salmonella typhimurium.
    1. Plasmid R64:
      1. GenBank Accession Number: AP005147; RefSeq: NC_005014
      2. Size: 120,826 nt (NCBI_Taxonomy)
      3. Gene Count: 137 genes, 135 proteins (NCBI_Taxonomy)
      4. Description: The 120 826 bp plasmid R64, belonging to the incompatibility group I1, has a very complex transfer system (Komano et al., 2000). The R64 transfer region located within an [less than] 54 kb DNA segment was shown to encode the most complex transfer system so far studied. It contains at least 49 genes and may produce 58 different proteins as a result of shufflon DNA rearrangement and overlapping genes. Among the 49 genes, 23 tra, trb and nik genes have been shown to be indispensable for R64 conjugal transfer in liquid and on surfaces. Twelve additional pil genes are required only for liquid matings (Komano et al., 2000).

  3. Genome of Salmonella typhimurium.
    1. Plasmid SC101:
      1. GenBank Accession Number: X01654; RefSeq:NC_002056
      2. Size: 9,263 nt (NCBI_Taxonomy)
      3. Gene Count: 6 genes, 6 proteins (NCBI_Taxonomy)
      4. Description: pSC101 is a low copy number plasmid isolated from Salmonella, which confers tetracycline resistance to the recipient cell (Bernardi et al., 1984). pSC101 is a nonconjugative plasmid but mobilizable (mob ) at variable frequency according to the R-factors used for the transfer (Bernardi et al., 1984).

  4. Genome of Salmonella typhimurium.
    1. Plasmid U302S:
      1. GenBank Accession Number: NC_006815
      2. Size: 3,208 nt (NCBI_Genome)
      3. Gene Count: 6 genes, 4 proteins, structural RNA 2 (NCBI_Genome).
      4. Description: The multi-antibiotic resistant (MR) Salmonella enterica serovar Typhimurium phage type U302 strain G8430 exhibits the penta-resistant ACSSuT-phenotype (ampicillin, chloramphenicol, streptomycin, sulfonamides and tetracycline), and is also resistant to carbenicillin, erythromycin, kanamycin, and gentamicin. Two plasmids, 3.2- and 84.5-kb in size, carrying antibiotic resistance genes were isolated from this strain, and the nucleotide sequences were determined and analyzed. The 3.2-kb plasmid, pU302S, belongs to the ColE1 family and carries the aph(3')-I gene (Kan(R)) (Chen et al., 2007).

  5. Genome of Salmonella typhimurium.
    1. Plasmid U3O2L:
      1. GenBank Accession Number: NC_006816
      2. Size: 84,514 nt (NCBI_Genome)
      3. Gene Count: 104 genes, 103 proteins (NCBI_Genome).
      4. Description: The 84.5-kb plasmid, pU302L, is an F-like plasmid and contains 14 complete IS elements and multiple resistance genes including aac3, aph(3')-I, sulII, tetA/R, strA/B, bla(TEM-1), mph, and the mer operon (Chen et al., 2007). Sequence analyses of pU302L revealed extensive homology to various plasmids or transposons, including F, R100, pHCM1, pO157, and pCTX-M3 plasmids and TnSF1 transposon, in regions involved in plasmid replication/maintenance functions and/or in antibiotic resistance gene clusters (Chen et al., 2007).

II. Epidemiology Information

The five most commonly occurring Salmonella serotypes in humans reported to the Public Health Laboratory Service in England and Wales during 2000 were S. enteritidis, S. typhimurium, S. hadar, S. Virchow and S. infantis. Although S. enteritidis and s. typhimurium accounted for most than 75 per cent of the cases, these two serotypes have broad host ranges infecting a wide range of birds and mammals ( Liebana et al., 2002). Typhimurium infections are common in the United States; it is the second most commonly identified salmonella serotype and accounted for 25 percent of culture-confirmed, serotyped cases of salmonella in 1996 (Glynn et al., 1998). Three specific serotypes, Enteritidis, Typhimurium and Typhi accounted for more than 76% of all isolates reported in 1995 (Herikstad et al., 2002). Typhimurium infections are common in the United States; it is the second most commonly identified salmonella serotype and accounted for 25 percent of culture-confirmed, serotyped cases of salmonella in 1996 (Glynn et al., 1998). Typhimurium is already prevalent in Europe and the Americas, and is of growing importance in the Southeast Asian, Western Pacific and African region. Typhimurium is present among many animal species, and like other nontyphoidal salmonella serotypes is most likely to infect humans through contaminated foods of animal origin (Herikstad et al., 2002). Non-typhoid Salmonella (NTS) is a major food borne pathogen and the leading cause of hospitalization and death due to food borne infections in industrialized countries. The incidence of NTS has been on the rise since the 1970s. Of the 2500 NTS serotypes capable of infecting humans, only a handful are responsible for the majority of NTS illnesses. The most successful serotypes that became predominant in most geographic locations are Salmonella enteritidis and Salmonella typhimurium. Although there are variations in the composition of the leading NTS serotypes and the trends over time between different locations, it is surprising how much similarity exists globally (Weinberger et al., 2005). The trends in the proportion of isolates that were Typhimurium were more variable. This decreased in the American (20.8%/8.0%) and European region (20.2%/17.6%), and increased in the Southeast Asia (9.5%/19.6%), Eastern Mediterranean (4.5%/4.9%), Western Pacific region (17.4%/26.0%), and African region (12.9%/15.9%) between 1990 and 1995 (Herikstad et al., 2002). USA: Salmonella spp are one of the major bacterial causes of foodborne gastroenteritis. The CDC report approximately 40,000 confirmed cases of salmonellosis annually (Sanchez et al., 2002). S. typhimurium usually contaminates the external surfaces of foods primarily of animal origin (Liebana et al., 2003). Non-typhoidal salmonellosis is one of the leading causes of acute bacterial gastroenteritis in the United States, responsible for an estimated 1.4 million cases of illness annually (Sanchez et al., 2002). United States: Salmonella spp. infect an estimated 1.4 million persons annually in the United States. Although most infections are self-limiting with diarrhea, vomiting, abdominal cramps, and fever, severe infections are not uncommon. Estimates suggest that approximately 15,000 people are hospitalized and >500 deaths occur annually due to Salmonella infections (Wright et al., 2005). In 1998, the overall incidence of Salmonella infections in the United States population was 17.4 cases/100,000 persons; infections in ages 1 through 9 occur at an incidence of 50/100,000. After the age of 9, the incidence decreases to approximately 25/100,000 and remains constant for the other age groups (Sanchez et al., 2002). In 2002, the incidence rate of salmonellosis (17.7 per 100,000 population) was highest among 10 potentially foodborne diseases (Pegues et al., 2002). Two major changes occurred in the United states during the past 2 decades in the epidemiologic characteristics of nontyphoidal salmonellosis. These were the evolution of 2 pandemic serovars, S ser Enteritidis and S ser Typhimurium DT104, that have caused marked increases in the percentage of foodborne human Salmonella infections. S. Typhimurium was the second most commonly reported Salmonella serotype in 1995, accounting for 9702 (24%) of 41,222 Salmonella isolates reported that year. During July-August 1996, the algorithm used by the Public Health Laboratory Information System (PHLIS) to detect Salmonella outbreaks indicated that, in 29 states, the number of S. Typhimurium isolates had substantially increased when compared with a 5-year historical baseline. Although it is unknown whether these increases were associated with the emergence of DT104, the ACSSuT resistance pattern was present in 90 (32%) of the 282 human S. Typhimurium isolates tested at CDC in 1996. This pattern also was present in 273 (28%) of a national sample of 976 S. Typhimurium isolates tested during 1995, compared with 7% in 1990. In 1995, a total of 30 S. Typhimurium R-type ACSSuT isolates were obtained from 10 states and were sent to the UK for phage typing; of these, 25 (83%) were DT104. Nebraska Outbreak (Hosek et al., 1997). In 2000, the two most common serotypes isolated from human sources were S. enterica serotype Typhimurium and S. enterica serotype Enteritidis (Chiu et al., 2004). In 2000, S. Typhimurium and S. Enteritidis were the most common serotypes, together accounting for 42% of all laboratory-confirmed cases of human salmonellosis. The incidence of salmonellosis is highest during the rainy season in tropical climates and soaring May through October in temperate climates, coinciding with the peak in foodborne outbreaks (Pegues et al., 2002). AFRICA: In Africa, multidrug-resistant non-typhoidal salmonellae (NTS) are one of the leading causes of morbidity and high mortality in children under 5 years of age, second in importance only to pneumococcal disease (Kariuki et al., 2006). Zaire and Rwanda: Multidrug-resistant S. Typhimurium causes serious outbreaks. For example, in Zaire and Rwanda, multidrug-resistant S. Typhimurium is the predominant cause of bacteraemic illness in children, while in Kenya this serotype is the predominant isolate in children with salmonellae bacteraemia (Kariuki et al., 2006). It is estimated that the minimum incidence of community-acquired NTS in rural and urban populations of children may be as high as 166 per 100 000 per year for children under 5 years of age (Kariuki et al., 2006). The maximum number of Salmonella typhimurium infections occurred during the month of September although the numbers isolated started to go up from April onwards (Mirza et al.,1989). Nairobi, Kenya: In sub-Saharan Africa community-acquired non-typhoidal Salmonella (NTS) is a major cause of high morbidity and death among children under 5 years of age especially from resource poor settings. The emergence of multidrug resistance is a major challenge in treatment of life threatening invasive NTS infections in these settings (Kariuki et al., 2006). Overall 170 (51.2%) of children presented with bacteraemia alone, 28 (8.4%) with gastroenteritis and bacteraemia and 134 (40.4%) with gastroenteritis alone. NTS serotypes obtained from all the cases included S. Typhimurium (196; 59%), S. Enteritidis (94; 28.3%) and other serotypes in smaller numbers (42; 12.7%); distribution of these serotypes among cases with bacteremia or gastroenteritis was not significantly different. A significantly higher proportion of younger children ([less than] 3 years of age) and those from the slums presented with invasive NTS compared to older children and those from upper socio-economic groups. One hundred and forty-seven (44.3%) NTS were resistant to 3 or more antibiotics, and out of these 59% were resistant to ampicillin, chloramphenicol and tetracycline. There was no significant difference in antibiotic resistance between the two serotypes, S. Typhimurium and S. Enteritidis (Kariuki et al., 2006). Europe, Netherlands: At the end of September 2005, an outbreak of Salmonella Typhimurium DT104 infections was detected in the Netherlands by the Dutch National Salmonella Centre. From 19 September to 7 November 2005, 165 extra cases of S. Typhimurium DT104 infection were recorded. The majority of the bacterial isolates had the same antibiotic resistance profile, i.e. resistant to amoxicillin, tetracycline, chloramphenicol, sulphamethoxazole and sometimes trimethoprim but sensitive to ciprofloxacin and cefotaxime (Kivi et al., 2005). Antarctica: The number of human visitors to Antarctica is increasing rapidly, and with it a risk of introducing infectious organisms to native animals (Palmgren et al., 2002).

A. Outbreak Locations:
  1. Meats poultry, shell eggs, and produce have been implicated as vehicles in salmonellosis outbreaks (Swaminathan et al., 2006). Canada: Dry fermented sausages, such as salami, contain raw, ground meat combined with various spices, curing agents, and salt. Through the processes of fermentation, which lowers the pH of the mixture, and drying, pathogenic bacteria are gradually replaced by non-pathogenic flora over a period of days or months. Salami is considered ready to eat and is not cooked before consumption. Mortadella is a dry sausage that does not undergo fermentation but is smoked at a high temperature before being air-dried. Prosciutto is salted ham that is air-dried to cure. Salami has been previously identified as the food vehicle for S. Typhimurium in two geographically widespread outbreaks in Northern Italy (PT 193) and England (definitive type 124). Investigation of the manufacturing plants in these outbreaks revealed that a lack of microbial starter cultures and/or insufficient ripening time likely contributed to the survival of Salmonella in the fermentation and drying stages. In May 2005, there was a small outbreak of illness in Sweden due to S. Typhimurium PT U302 contamination of German mini salami, resulting in a product recall in Sweden, Finland, and the Netherlands. There have also been reported cases of salmonellosis related to the consumption of prosciutto in Italy (CPHL et al., 2006). Europe, Finland: A rare multiresistant Salmonella Typhimurium DT 104B has caused an outbreak of 60 microbiologically confirmed cases in May 2005, widely distributed across southern and western Finland. The isolates have an identical pulsed field gel electrophoresis (PFGE) and antimicrobial resistance pattern (ACSSuT). Of the 56 cases confirmed so far, 80% were in females and 45% were in people aged between 15-24 years (range 7 to 53). S. Typhimurium DT 104B was detected in samples of salad made from lettuce, other vegetables and/or noodles from three successive days, served between 10 and 12 May. A traceback investigation showed that the nursing school and the restaurant had both purchased iceberg lettuce, the only kind of lettuce served on the implicated days, with a documented trail leading back to a supplier in Spain. Thousands of kilograms of this iceberg lettuce have been imported from Spain and distributed throughout Finland (Takkinen et al., 2002). Asia, Singapore: Multidrug-resistant Salmonella enterica subsp. enterica serotype Typhimurium DT104L was first reported in Singapore from mid-July to mid-October 2000. A total of 33 cases involving mainly infants and toddlers were detected in the 3-month long outbreak. The outbreak strain was of the R-type ACGSTSu, i.e. resistant to ampicillin, chloramphenicol, gentamicin, streptomycin, tetracycline and sulphonamide. PFGE showed all isolates had an indistinguishable pattern, indicating a common source of infection. Consumption of imported dried anchovy was found to be the vehicle of transmission. Imported dried seafood should be properly processed, packed, labelled, and thoroughly cooked to prevent transmission of multidrug-resistant S. Typhimurium (Ling et al., 2002).
  2. Africa, Kenya: Multidrug-resistant S. Typhimurium was the predominant cause of community-acquired bacteraemic illness in both children and in adults. The authors studied NTS isolates from paediatric admissions at two hospitals in Nairobi, Kenya, and followed the index cases to their homes, where rectal swabs and stools from parents and siblings, and from animals in close contact, were obtained. The majority of NTS obtained from cases were Salmonella enterica serotype Typhimurium (106 out of 193; 54.9%) and Salmonella enterica serotype Enteritidis (64; 33.2%), a significant proportion (34.2%) of which were multiply resistant to three or more antibiotics, including ampicillin, tetracycline, cotrimoxazole and chloramphenicol (Kariuki et al., 2006).
  3. Dairy cattle: A dairy cattle-associated outbreak caused by S ser Typhimurium DT104 was reported (Sanchez et al., 2002). In 1997 this strain infected several members of a family that resulted in 1 person hospitalized with invasive disease (Sanchez et al., 2002).
  4. Pasteurized milk (Illinois, USA): There is certainly no evidence to indicate that S. typhimurium is the only serovar, or even the major serovar causing outbreaks of salmonellosis, or that has, however, been implicated in some of the major outbreaks of salmonellosis which have been reported in this country. It has been stated in numerous instances that the largest single outbreak of salmonellosis reported in the U.S. was that which resulted from contamination of pasteurized milk in a plant in Illinois in 1985. That outbreak was found to be caused by a strain of S. typhimurium which was unusually resistant to a number of antimicrobial compounds. It was found that the causative organism involved had been responsible for at least three outbreaks of disease in Illinois over a period of 8 months causing the largest single outbreak ever recorded in this country (Gutherie et al., 1991). During October 1996, the Nebraska Department of Health was notified about an outbreak of diarrheal [sic] illness among elementary school children in Cass County, a farming community in east central Nebraska. During October 12-14, a total of 19 (59%) of 32 children attending an elementary school developed diarrhea (100%), fever (89%), headache (89%), nausea (89%), and vomiting (58%); three reported bloody diarrhea. None required hospitalization, and all recovered. On October 10, during lunch at the school, children had been served cold chocolate milk poured from cartons. Of the 22 children who drank the milk, 18 (82%) developed diarrhea, compared with one (10%) of 10 children who did not drink it (Hosek et al., 1997). A previous milkborne salmonellosis outbreak (raw instead of pasteurized milk was involved) was reported as being caused by a S. typhimurium serovar. That strain was unusual in that it was resistant to chloramphenicol among other drugs, including ampicillin, carbenicillin, kanamycin sulfate, streptomycin, sulfisoxazole, and tetracycline. In this outbreak there was at least one fatality reported as due to infection by this organism (Gutherie et al., 1991).
  5. Animal veterinary clinics (Idaho, USA): In 1999 and 2000, 3 state health departments reported 4 outbreaks of gastrointestinal illness due to Salmonella enterica serotype Typhimurium in employees, clients, and client animals from 3 companion animal veterinary clinics and 1 animal shelter. More than 45 persons and companion animals became ill. Four independent investigations resulted in the testing of 19 human samples and [greater than] 200 animal samples; 18 persons and 36 animals were culture-positive for S. Typhimurium (Wright et al., 2005). An outbreak in an Idaho clinic was caused by multidrug-resistant S. Typhimurium R-type ACKSSuT with 2 isolates demonstrating additional resistance to ceftriaxone, an antimicrobial agent commonly used to treat children with severe Salmonella infections. Outbreaks in 2 Washington clinics and a Minnesota animal shelter were caused by multidrug-resistant S. Typhimurium R-type ACSSuT DT104 (Wright et al., 2005). This report documents nosocomial transmission of S. Typhimurium and demonstrates that companion animal facilities may serve as foci of transmission for salmonellae between animals and humans if adequate precautions are not followed (Wright et al., 2005). In each outbreak discussed, the veterinary facility or animal shelter was the only common exposure for infected persons, which demonstrated that infected animals brought to companion animal veterinary clinics and animal shelters can be foci for nosocomial transmission to other animals and for zoonotic transmission to humans. These outbreaks illustrate 1) the hazards of occupational zoonotic transmission of Salmonella spp. from ill animals to clinic employees, 2) the hazards of zoonotic transmission of Salmonella spp. to clients/pet owners, 3) the risk for nosocomial transmission of Salmonella spp. between animals within veterinary facilities and animal shelters, and 4) the potential for environmental contamination to serve as an ongoing source of infection (Wright et al., 2005). The use of antimicrobial agents prescribed by veterinarians may contribute to increased transmission of multidrug-resistant Salmonella spp. between animals by lowering the infectious dose required for infection to occur or by increasing the duration of illness when an infected animal is treated with an ineffective drug. The risk for Salmonella transmission between animals in veterinary facilities is likely increased by the presence of animals with increased susceptibility to multidrug-resistant Salmonella infection due to treatment with antimicrobial agents for other conditions. Fluoroquinolone antimicrobial therapy did not eliminate fecal shedding of susceptible strains of salmonellae in 3 cats and 1 person from whom follow-up cultures were obtained. This finding is similar to other results in humans, which demonstrate that fluoroquinolone treatment is associated with longer duration of carriage (Wright et al., 2005). All ill employees had eaten meals in the clinic in the days before illness onset. A breakroom was provided for employees, yet all reported eating on work surfaces instead (Wright et al., 2005).
  6. Salmonella infections in newborns are associated with higher morbidity and mortality due to immaturity of their host defense mechanisms. Salmonella typhimurium is one of the most common serotypes responsible for nursery epidemics (Kumar et al., 1995) Children's nursery (Virginia, USA): An outbreak caused by a highly resistant strain of Salmonella typhimurium occurred in a nursery at a university medical center. The outbreak strain, which was resistant to ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole, was apparently imported from the Far East by a Cambodian refugee (Lamb et al., 1984). Highly resistant strains of S. typhimurium imported from the Far East may spread rapidly when introduced into a hospital nursery. Prompt institution of control measures may limit the outbreak and prevent systemic infections for which there are few effective therapeutic agents (Lamb et al., 1984). The index case was in a 7-month-old Cambodian refugee who was born in a hospital in the Philippines and who emigrated, at less than 1 month of age, to the United States in September 1981 (Lamb et al., 1984). The outbreak occurred in the Sick Baby Nursery at the Medical College of Virginia. The SBN was a 16-bed unit, with all patients cared for in a single room without partitions. The age range of infants admitted to this unit was from birth to 9 months (Lamb et al., 1984). The five patients involved had severe underlying diseases, and bacteremia and meningitis developed in one of these patients. The only reservoir identified was the gastrointestinal tracts of the infected patients, and infection was probably transmitted by the contaminated hands of hospital personnel. The outbreak was rapidly brought under control by isolating cases outside of the nursery and by instituting enteric precautions for infants who remained in the nursery (Lamb et al., 1984). India, Varanasi: Thirty-four neonates with S. typhimurium infection born between April and July, 1993, in the University Hospital, Banaras Hindu University, Varanasi, constituted the study material. Twenty-one infants were symptomatic carriers (Kumar et al., 1995). A nursery epidemic caused by multidrug resistant Salmonella typhimurium is reported. In total, 21 infants developed symptomatic illness; of these, 17 had septicemia (7 blood culture positive) and 4 had diarrhea alone. Asymptomatic carrier state was identified in 13 infants. Male sex and birth asphyxia increased the risk for symptomatic illness (Kumar et al., 1995). Amongst the septicemic infants there was no difference in clinical profile whether the blood culture was positive or negative for S. typhimurium. In the symptomatic group, S. typhimurium was isolated from feces in 19 cases and from blood in 7 cases. In both symptomatic and asymptomatic infants, all isolates of S. typhimurium, whether obtained from feces and/or from blood, were resistant to ampicillin, chloramphenicol, and trimethoprim, and a significant number (almost one-fifth) of them also showed resistance to third generation cephalosporins. More than 90% of isolates were sensitive to aminoglycosides and ciprofloxacin. On a combination of third generation cephalosporin (cefotaxime or ceftriaxone) and amikacin, 17 (81%) infants recovered, 2 succumbed to their illness, and 2 failed to improve and required ciprofloxacin. The origin of epidemic was traced to a carrier staff nurse working in nursery (Kumar et al., 1995).
  7. Milk Feeding (China): S. typhimurium infection has been epidemic in China in recent years. It is especially dangerous to the newborn, because the younger the patient , the higher the incidence of sepsis and the death rate (Wu et al., 1993). Seven out of the 12 neonates had positive blood cultures for S. typhimurium, and 2 of them died of severe sepsis (Wu et al., 1993). A pregnant woman who had a slight diarrhea for 1-2 days delivered a full term boy (Wu et al., 1993). Her baby had fever (38.9 degrees C), vomiting and diarrhea on the third day after birth. In the following 3 weeks 11 other neonates suffered from diarrhea, because no strict isolation measures were taken. The patient's ages ranged from 2 to 10 days (Wu et al., 1993). The source of the infection was the index patient's mother who had a slight diarrhea; the mode of transmission was most likely due to the transfer of organisms from infant to infant by the contaminated hands of nurses during milk feeding (Wu et al., 1993). In this report all strains of S. typhimurium were resistant to ampicillin, chloramphenicol, streptomycin, gentamicin, streptomycin and tetracycline, but they became sensitive to these antibiotics after the 98 Kb plasmid was eliminated by plasmid curing agent (Wu et al., 1993). This result indicates that the 98 Kb plasmid was the R plasmid which encoded the multi-resistant gene to the above 5 antibiotics. Only one non-epidemic strain was resistant to amikacin (Wu et al., 1993). Amikacin is not influenced by the R plasmid which makes the bacteria resistant to gentamicin and tobramycin, so it can be used to treat Gram negative bacterial infections which are resistant to tobramycin and gentamicin. Amikacin, therefore is the drug of choice to treat S. typhimurium infection (Wu et al., 1993). Canada: Between 1 March and 31 May, 2005, 47 confirmed cases of S. Typhimurium PT U302 were identified in Ontario (CPHL et al., 2006). Illness was significantly related to the consumption of salami (MOR equal to 3.75, 95% confidence interval [CI]: 1.2 to 11.3). Analysis using combined deli meat exposures showed that 87% of cases (26/30) reported eating either salami, mortadella, or prosciutto, compared with 40% of controls (12/30) (MOR equal to 8.0, 95% CI: 1.8 to 34.8) (CPHL et al., 2006). The common source could likely be a food product with a long shelf life that was widely distributed across southern Ontario. We suspect that it may have been a ready-to-eat item that did not require cooking, since food-borne infection with Salmonella species can usually be prevented with adequate refrigeration and cooking temperatures, and proper handwashing and food preparation practices (CPHL et al., 2006). The time lag between outbreak identification and the investigation's hypotheses-generating interviews (range 7 to 16 days) compounded the investigative delay and made accurate recall difficult for cases (CPHL et al., 2006). S. Typhimurium is the most frequently isolated serovar in Canada, accounting for approximately 20% of all human Salmonella isolates reported to the National Enteric Surveillance Program between 2001 and 2004 (CPHL et al., 2006). Data collected through the Canadian Integrated Program for Antibiotic Resistance Surveillance for the years 2003 and 2004 show that PT U302 accounted for 3.5% of S. Typhimurium isolates. In comparison, PT U302 accounted for 36% of S. Tyhimurium cases (9/25) identified in Ontario in March and 62% of cases (34/55) reported in April 2005 (CPHL et al., 2006).
  8. Antarctica: The number of human visitors to Antarctica is increasing rapidly, and with it a risk of introducing infectious organisms to native animals (Palmgren et al., 2002). Salmonella serotypes known to be human pathogens have also been found in penguins (Palmgren et al., 2002). The aim of this study was to investigate the presence of salmonella in a population of sub-Antarctic seabirds and seals and to characterize further the isolates found in terms of serotype, phagetype and genotype (Palmgren et al., 2002). To study the occurrence of salmonella serotypes in sub-Antarctic wildlife, faecal samples were collected from gentoo penguins, macaroni penguins, gray-headed albatrosses, black-browed albatrosses and Antarctic fur seals on Bird Island in the South Georgian archipelago during the austral summer of 1996 and 1998. In 1996, S. havana, S. typhimurium and S. enteritidis were isolated from 7% of gentoo penguins and 4% of fur seals (Palmgren et al., 2002). However, human derived micro-organisms have been detected in sewage outlets and waste dumps from Antarctic stations and damage to the fauna has been caused by accidental pollution from sewage. Low water temperature allows these bacteria to survive in the marine environment for long periods, where transfer to seabirds and seals could take place (Palmgren et al., 2002).
  9. Australia: Nosocomially-acquired salmonellosis outbreaks are most commonly linked to contaminated food. Person-to-person spread can readily occur in the hospital environment (Alam et al., 2005). Salmonella enterica serotype Typhimurium (DTm) is the most frequently notified serotype in Australia. The most commonly reported phage type (PT) among S. Typhimurium is PT170 (Alam et al., 2005). Nosocomially-acquired salmonellosis is uncommonly reported in Australia. We report a cluster of gastroenteritis caused by Salmonella Typhimurium phage type 170 (STm 170) centred on a tertiary paediatric hospital in Sydney, New South Wales from 8 to 19 May 2004. A total of 12 children had STm 170 isolated from faecal specimens. Of the 12 cases, seven were acquired in hospital and five in the community. The mean age of the cases was 4.1 years (range: 2 months to 11.2 years) (Alam et al., 2005). We inspected preparation, storage and handling of food in the hospital, including the preparation of infants formula and observed general food handling and cleanliness cleanliness in the food preparation areas (Alam et al., 2005). Person-to-person spread can readily occur in the hospital environment (Alam et al., 2005). Salmonellosis outbreaks in paediatric hospitals are of particular concern due to the increased susceptibility and associated high morbidity in this group (Alam et al., 2005).
  10. Africa, Kenya: Salmonella infections at Kenyatta National Hospital (KNH) are on the increase and multi drug resistant strains are becoming common (Mirza et al.,1989). A total of 560 Salmonellae species were isolated from Jan-Dec 1985. Of these, 347 (62%) were from blood cultures, 180 (32%) from stools and 33 (6%) were from cerebrospinal fluid (CSF) and other body aspirates. S. typhimurium were the highest isolated. These were, 291 (52%) from blood cultures, 94 (17%) from stool cultures and 32 (6%) from CSF. S. typhimurium was also multi-drug resistant. More than 50% strains of S. typhimurium were resistant to ampicillin, tetracycline, kanamycin and chloramphenicol (Mirza et al.,1989). Total typhimurium isolated from all the specimens were 417 (74%) (Mirza et al.,1989). Infections in this hospital indicate that they may have been both hospital and community acquired and that mortality may have been high (Mirza et al.,1989).
B. Transmission Information:
  1. From: Animals To: Human
    Mechanism: Human Infection From Animal Products: Food animals are the primary reservoir for human nontyphoidal Salmonella infections. Person-to-person transmission of nontyphoidal salmonellae is uncommon in the United States. Transmission of salmonellae to humans typically occurs by ingesting meat, dairy products, and other foods contaminated by animal feces or by cross-contamination from foods contaminated with salmonellae (Wright et al., 2005). Nontyphoid Salmonella serotypes causing gastroenteritis in humans are most often transmitted through the food chain by contamination of poultry and eggs, pork, beef and dairy products, and, increasingly in the United States by vegetables and fruits that are irrigated with Salmonella-contaminated water (Chiu et al., 2004). The most logical means of diminishing the transmission of Salmonella through the food chain to humans would be to vaccinate farm animals on a routine basis with live attenuated Salmonella vaccine. The problem is that other than Germany, there is no requirement to do so in any other part of the world. Agriculturally important animals are commodities, and therefore producers are not willing to invest in the cost of a vaccine and a vaccination program unless they are required to do so or unless failure to vaccinate results in a severe problem, such as being unable to market their products. In this regard, it is permissible in the United States to have up to 20% of broiler carcasses fecally contaminated with Salmonella at the time of slaughter (Chiu et al., 2004).

  2. From: Animals To: Humans
    Mechanism: Zoonotic Infection: Companion animal veterinary facilities can serve as foci of transmission of salmonellae to animals and humans (Wright et al., 2005). On December 2, 1999, five S. Typhimurium isolates from cats originating from the same county, submitted by a regional animal shelter (shelter A), were subtyped at MDH; all had the same PFGE pattern. The following day, a human S. Typhimurium isolate routinely submitted to MDH was determined to have the same PFGE pattern as the cat isolates (Wright et al., 2005). Veterinary Clinic, Idaho: In September 1999, several kittens being treated for diarrhea at a veterinary clinic (clinic A) died; stool specimens were not collected. Within 2 days of the kittens' deaths, an employee who had cared for the kittens became ill with diarrhea. Days later a second employee who cared for the kittens became ill, as did other employees who had no direct contact with the kittens. Within 2 weeks, 10 (50%) of 20 employees of clinic A had experienced diarrhea and abdominal cramps (Wright et al., 2005). Animal Shelter, Minnesota: On December 2, 1999, five S. Typhimurium isolates from cats originating from the same county, submitted by a regional animal shelter , were subtyped at MDH; all had the same PFGE pattern (Wright et al., 2005). Zoonotic transmission of Salmonella spp. can also occur through direct exposure to the feces of reptiles, farm animals, pets, pet treats, and other animals (Wright et al., 2005).

  3. From: Human To: Human
    Mechanism: Contamination of prepared food: The vehicles linked with food-borne transmission include chicken, pork sausage and meat paste, contaminated beef, turkey and raw-milk cheese (Ling et al., 2002). Salmonella is one of the most prevalent enteric pathogens encountered in seafood in Thailand. It has been isolated from ready-to-eat imported seafood such as cooked shrimp, shellfish, fish paste, smoked fish, salted dried fish and caviar in the United States. Outbreaks of salmonellosis caused by ingestion of contaminated cuttlefish chips as snacks have also been reported in Japan (Ling et al., 2002). Salmonella is one of the most prevalent enteric pathogens encountered in seafood in Thailand. It has been isolated from ready-to-eat imported seafood such as cooked shrimp, shellfish, fish paste, smoked fish, salted dried fish and caviar in the United States. Outbreaks of salmonellosis caused by ingestion of contaminated cuttlefish chips as snacks have also been reported in Japan (Ling et al., 2002).

  4. From: Human To: Human
    Mechanism: Human Infection from Human Fecal/Oral Transmission: The foods most likely to be contaminated are meats. Since the contamination for transmission must come after the food has been processed or prepared for consumption, those foods which are allowed to stand for some considerable period of time after being cooked before they are served or the foods which are not heated before service are the ones most likely to be involved in this transmission. Food may be contaminated by persons who are carriers and who use less than desirable hygienic methods in preparation or who use contaminated ingredients, including contaminated water used as an ingredient or used for cleaning utensils. When these contaminations are followed by inadequate or no cooking, and the food is then allowed to stand, multiplication of bacteria in the food will be sufficient to provide an infectious dose to the consumer (Gutherie et al., 1991).

  5. From: Human To: Human
    Mechanism: Person-to-person transmission: Although person-to-person transmission of nontyphoidal Salmonella spp. is rare in the United States, this mode of transmission appears likely in the Minnesota outbreak in which an ill child apparently infected classmates in a daycare center (Wright et al., 2005).

  6. From: Human To: Human
    Mechanism: Nosocomial infection: Several instances of probable secondary transmission to animals within client households after apparent primary nosocomial infection were demonstrated during these outbreaks. The isolation of Salmonella spp. from client-owned vacuum cleaner bags illustrated the potential for such secondary transmission. Additional isolation of the outbreak strain from environmental surfaces in the Washington clinic B investigation reinforces the findings of previous studies, which demonstrated the potential to transmit salmonellae through environmental contact (Wright et al., 2005). A 10-year (1978-1987) study of 248 outbreaks of nosocomially-acquired salmonellosis in the United Kingdom found 30 per cent of infections were linked to person-to-person spread (Alam et al., 2005).

  7. From: Human To: Human
    Mechanism: Milk Feeding: Transmission of S. typhimurium from infant to infant took place by way of oral or fecal route, most likely via the contaminated hands of nurses especially during the milk feeding (Wu et al., 1993).

C. Environmental Reservoir:
  1. Food :
    1. Description: Salmonella is a cause of food borne disease in humans and domestic animals, even though some animals and humans can also be apparently healthy carriers. Wild birds are known to be carriers of the genus (Palmgren et al., 2002). Food animals are the primary reservoir for human nontyphoidal Salmonella infections (Wright et al., 2005). In humans, nontyphoidal Salmonella infections are most often associated with food products and are the most frequently identified agent of foodborne disease outbreaks. Food of animal origin, including meat, poultry, eggs, or dairy products, can become contaminated with Salmonella (Pegues et al., 2002). Eating uncooked or inadequately cooked food or foods cross-contaminated with these products may lead to human infection (Pegues et al., 2002). Salmonellosis associated with exotic pets is a resurgent public health problem, with an estimated 3% to 5% of all cases of salmonellosis in humans associated with exposure to exotic pets, especially reptiles. As many as 90% of reptiles may be carriers of Salmonella (Pegues et al., 2002). Exposure to pet birds, pet rodents, dogs, and cats also is a potential source of salmonellosis, and outbreaks with small animal veterinary facilities have been reported (Pegues et al., 2002). Serotypes Typhimurium and Enteritidis, have a broad host range and can infect a wide variety of animals (Chiu et al., 2004). The DT104 strain has broad host reservoirs and is difficult to control in domestic livestock, leading to its widespread clonal dissemination among food animals, especially cattle and humans in Europe, the united States, Canada, and the Middle and Far East (Pegues et al., 2002). Animals are a primary reservoir for nontyphoidal salmonellae associated with human infections, and contact with animal feces either directly through animal handling or manure or indirectly through fecal contamination of foods are principal vehicles of human infection (Sanchez et al., 2002). Domestic animals act as a reservoir for the food-borne spread of host-generalist serovars, which accounts for the high incidence of non-typhoid Salmonella infections worldwide (McClelland et al., 2001). The use of protein supplements in animal fee to increase weight gain has helped to increase the likelihood of S. typhimurium colonization of the animal's intestinal tract. S. typhimurium can be spread form one animal to another. Adult farm animals colonized with S. typhimurium usually do not appear to be ill, so it is difficult to identify colonized animals (Salyers et al., 2002). During slaughter, the bacteria can easily contaminate the carcass and enter the food supply (Salyers et al., 2002). Eggs can be contaminated on the outside or the inside of the shell. If eggs that have been contaminated with salmonellae are left at room temperature for long periods of time, the bacteria have a chance to increase in number, this increase is not accompanied by changes in smell or appearance of the white or yolk (Salyers et al., 2002). Reptiles have long been recognized as sources of Salmonella spp for humans. The FDA banned the sale of pet turtles in 1975 because of the associated risk of infections in children (Sanchez et al., 2002). In most cases, outbreaks of NTS infection are caused by Salmonella enterica serotype Typhimurium (S. Typhimurium) and Salmonella enterica serotype Enteritidis (S. Enteritidis). A variety of foods have been implicated as vehicles transmitting salmonellosis to humans, including poultry, beef, pork, eggs, milk, cheese, fish, shellfish, fresh fruit and juice, and vegetables (Kariuki et al., 2006). Poultry, meat products, and eggs are most commonly identified as food sources responsible for outbreaks of salmonellosis; however, many other food-stuffs such as ice cream, vegetables and fruits, breakfast cereal, milk, juices, herbs, and spices have also been vehicles of large outbreaks (Sanchez et al., 2002). The salmonella organisms grow in food products, and cause an active endotoxin. Preserved meats such as meat pies, sausages and brawn, have proved a potent source; the meat may be contaminated with salmonellae from the animal's gut, or by human carriers during the course of preparation. Infected eggs have also been responsible for many epidemics, especially dried eggs. Synthetic creams have been an especially potent source in the past, as have unripe cheeses, milk and ice-cream (Huckstep et al., 2002).
    2. Survival Information: Many animals, both domestic and wild, are colonized by Salmonella spp, usually harboring the bacteria in their gastrointestinal tracts with no apparent signs of illness. Hence, salmonellae are often present in feces excreted by healthy animals and frequently contaminate raw foods of animal origin through fecal contact during production and slaughter (Sanchez et al., 2002). Most Salmonella strains can survive for long periods of time in water and in dry materials such as dust. Low numbers of micro-organisms surviving in the environment in a dormant state can multiply rapidly if suitable conditions are present (Liebana et al., 2003). This special ability, together with the contribution of wildlife vectors represents a real problem to the control of this infection in poultry environments (Liebana et al., 2003). Salmonella are also susceptible to the heat applied in pasteurization of milk at 71.2 degrees C for 15 s and are resistant to drying (may survive in dust for long periods of time, even for years) (Gutherie et al., 1991). The incubation period is usually between 10-30 hours, during which time the ingested organisms multiply in the gastrointestinal tract (Huckstep et al., 2002).
  2. Human :
    1. Description: The only reservoir identified was the gastrointestinal tracts of the infected patients, and infection was probably transmitted by the contaminated hands of hospital personnel (Lamb et al., 1984).
    2. Survival Information:
  3. Mammals and birds :
    1. Description: Salmonella is an important pathogenic organism in both humans and animals (Guerin et al., 2005). Subclinical infection is common and many animals become asymptomatic carriers, intermittently shedding the bacteria into the environment for variable periods of time (Guerin et al., 2005). It has a large animal reservoir, including farm animals, pets, and wild animals (Weill et al., 2006). The outbreak of DT104 infections in Nebraska suggests possible associations with animal reservoirs (e.g., through milk or contact with animals) (Hosek et al., 1997). A case-control study of farms with infections identified contact with animals, particularly ill farm animals, as a risk factor for disease. Transmission of an organism carrying multiple-drug resistance to those classes of drugs commonly used in human medicine may result in failure when treating humans with disease, particularly if that person is treated with antibiotics from the small class of drugs (Hollinger et al., 1998). The R-Type strain is currently epidemic in other food animal species and domestic animals such as dogs and cats. It also has been isolated from wild birds and rodents. Cattle may be the main reservoir in domestic animals from the epizootics in other animal species because incidents in other food animals and domestic animals have paralleled the number of incidents in cattle, and these incidents in other species appear to lag temporally behind the epidemic in cattle, with initial isolations of the R-Type ACSSuT from sheep, chickens, and turkeys in 1990, 1992 and 1993 (Hollinger et al., 1998). There is strong evidence that Salmonella enterica serovar Typhimurium has established reservoirs in wild-living birds and hedgehogs in Norway. Wild-living birds and hedgehogs may function as effective spreaders of Salmonella bacteria to humans and to different animal species through contamination of the environment ( Refsum et al., 2002). In Norway, sporadic indigenous cases and a national outbreak of human salmonellosis, caused by serovar Typhimurium, have been related to infections in small passerines ( Refsum et al., 2002). In a waterborne serovar Typhimurium infection outbreak in northwestern Norway in 1999, gulls were suggested to be the most likely source of infection ( Refsum et al., 2002). Moreover, two human outbreaks in 1996 and 2000 have been associated with infected hedgehog populations ( Refsum et al., 2002). There were 1767 isolates between October 1990 and December 2001 comprising 63 different serovars, including 961 isolates from chickens, 418 from cattle, 108 from pigs, 102 from turkeys, and 178 from all other species combined. Salmonella Typhimurium, Heidelberg, Hadar, Kentucky, and Thompson were the 5 most frequently isolated serovars (Guerin et al., 2005). Approximately 60% of the S. Typhimurium were isolated from cattle (Guerin et al., 2005). Short-duration clusters may imply point source infections, whereas long-duration clusters may indicate an increase in the prevalence of the serovar, farm-to-farm transmission, or a wide-spread common source. A higher concentration of clusters in the winter months may reflect greater confinement, reduced ventilation, stressors, or increased exposure to wildlife vectors that are sharing housing during the winter. Detection of large clusters of Salmonella may have public health implications in addition to animal health concerns (Guerin et al., 2005).
    2. Survival Information:
D. Intentional Releases:
  1. Intentional Release information :
    1. Description: The most famous, and successful use of bioterrorism on U.S. soil occurred during September 1984, when followers of the Bhagwan Shree Rajneesh contaminated salad bars in The Dalles, Oregon with Salmonella Typhimurium. Over 750 cases of salmonellosis were determined to be caused by the salad bar contamination. It was later discovered that the Rajneeshpuram cult wanted to influence the local county commissioners election, so as to form their own township. The September bioterrorism act was a trial run for the planned November election attack, which was later canceled, as the plan seemed to be ineffective. The cult members obtained the Salmonella strain through the mail from the American Type Culture Collection (ATCC) (Public Health Services).
    2. Containment: The agent may be present in feces, blood, urine, and in food, feed, and environmental materials. Ingestion or parenteral inoculation are the primary laboratory hazards. The importance of aerosol exposure is not known. Naturally or experimentally infected animals are a potential source of infection for laboratory and animal care personnel, and for other animals (CDC). Biosafety Level 2 practices, containment equipment and facilities are recommended for activities with clinical materials and cultures known or potentially containing the agents. Animal Biosafety Level 2 practices, containment equipment and facilities are recommended for activities with experimentally or naturally infected animals (CDC).

III. Infected Hosts

  1. Homo sapiens: (NCBI_Taxonomy:
    1. Taxonomy Information:
      1. Species:
        1. Human. (NCBI_Taxonomy):
          • GenBank Taxonomy No.: 9606
          • Scientific Name: Homo sapiens (NCBI_Taxonomy).
          • Description: Salmonella species are enteric pathogens which must cross the mucosa of the small bowel to initiate disease. The passage of these bacteria through mammalian cell membranes occurs via a process which induces dramatic rearrangements of the host cell cytoskeleton (Jones et al., 1994) Pathogenic Salmonella species initiate infection of a host by penetrating the intestinal mucosa, primarily at the lymphoid follicles of the Peyer's patches. Recent data suggest that these bacteria pass through the epithelium of a host by preferentially invading and destroying the M cells of Peyer's patches (Jones et al., 1994). The host range for Salmonella spp varies from narrow (S ser Pullorum: avian species only) to broad-host adapted serovars (S ser Typhimurium: avian and mammalian species) (Sanchez et al., 2002). Salmonella organisms exists as intracellular pathogens in diseased animal hosts (Sanchez et al., 2002). The organism is usually shed in the feces for several weeks (10 to 15% of patients with nontyphoidal gastroenteritis will shed the organism in the stool for 1 to 2 months), but this shedding persists for approximately 4 months. No patient acute or recovering, should be considered to be free of the pathogen until three successive fecal samples obtained three weeks apart result in negative cultures (Gutherie et al., 1991). In a small percentage of cases (1 to 3%), an infected person can continue to shed the bacteria for over a year (chronic carrier) (Salyers et al., 2002).

    2. Infection Process:
      1. Infectious Dose: In studies involving administration of laboratory Salmonella strains to healthy human volunteers, the median dose required to produce disease was approximately 10(6) bacteria. Investigations of point source outbreaks suggest that as few as 200 bacteria may produce nontyphidal gastroenteritis in many of those exposed (Pegues et al., 2002). A dose of 10 million cells was necessary to cause disease in 50% of normal volunteers, and with other serovars of Salmonella contaminating other foods, it has been suggested that fewer than 10 viable cells can cause an infection (Gutherie et al., 1991). Between 1 and 6 cells of S. typhimurium from cheddar cheese were calculated as the infectious doses for six patients who became ill after consuming the cheese (Gutherie et al., 1991).
      2. Description: An important component of Salmonella pathogenesis is the ability of the bacteria to transport themselves through the membranes of host cells, including M cells, enterocytes, lymphocytes, and professional killing cells (Jones et al., 2001). The penetration of the intestinal mucosal barrier is an essential step in Salmonella pathogenesis (Jones et al., 2001). invasive S. typhimurium, introduced into the stomachs of mice, became associated primarily with the lymphoid follicles, or Peyer's patches, of the small intestine rather than with the intestinal wall (Jones et al., 2001). Invasive S. typhimurium specifically invaded the M cells of the lymphoid follicles within 30 min of introducing microorganisms into murine intestinal loops; there was no apparent interaction with adjacent enterocytes during this early phase of infection. Bacterial entry of the M cells occurred by rearrangement of the apical membrane of the cell (ruffling) in a manner identical to that seen for invasion of the guinea pig ileum and for tissue culture cells. However, in contrast to other cell types, M cell internalization of the invasive organisms had a destructive effect on the cytoskeletal architecture that was permanent (Jones et al., 2001). Jones et al also examined the interactions between the bacteria and the Peyer's patch tissue at later times. Disintegrating M cells were often seen detaching from the epithelial surface 60 min after the addition of bacteria to the ligated loops. The invading bacteria do not seem to replicate to any significant extent within the M cells, but they subsequently interact with the underlying cell populations of the follicle dome. Virulent Salmonella are well known for their ability to induce their own uptake into macrophages, and these bacteria are also capable of invading B cell and T cell tissue culture lines. While adjacent enterocytes are spared at early times after infection, by 2 h post-infection there is significant invasion of enterocytes, and the invading microorganisms can be seen moving unhindered into deeper tissue structures. Enterocyte invasion is a classical feature of Salmonella infection of laboratory animals and, of course, of Salmonella food poisoning. It is not clear at this time whether nonhost-adapted Salmonella serovars of the type that cause common food poisoning of humans actually initiate infection by the M cell route (Jones et al., 2001). The essential feature of M cell invasion in the pathogenesis of Salmonella infection is seen from the observation that two independent S. typhimurium mutants, selected because they could no longer invade cultured epithelial cells, could not cross the epithelial barrier in the ligated loop model; they were also avirulent when fed to animals. Yet, these same mutants when injected intraperitoneally were perfectly virulent (Jones et al., 2001).

    3. Disease Information:
      1. Gastroenteritis :
        1. Pathogenesis Mechanism: When Salmonella are ingested they must survive the acid pH of the stomach to set up infection. If surviving in adequate numbers, the bacteria reaching the small intestine may penetrate the mucosa of the intestine to the midlayer of this membrane where they are engulfed into the epithelial cells. Salmonella penetrate epithelial cells, causing an inflammatory response in the small bowel and the colon. The presence of the bacteria in this location results in an inflammatory reaction, and depending on the serovar involved, the infection may progress past this tissue into the deeper layers of the mucosa of the intestinal wall. The salmonellosis, the diarrheal symptoms result from the inflammatory reaction which has been elicited in the small intestine. Some strains, at least , are capable of producing an enterotoxin which is important in the production of diarrhea (Gutherie et al., 1991). Following ingestion, after the food has passed through the acid conditions of the stomach, the organisms multiply in the small intestine and the infection is established. S. typhi as well as other serovars are resistant to the alkaline conditions found in the intestinal tract (Gutherie et al., 1991). The multi-resistance of S. typhimurium has become a worldwide problem. The chief cause of the increase of bacterial resistance is due to wide transmission of multi-resistant (R) plasmids and transposon carrying the resistant genes among different species of bacteria (Wu et al., 1993).

        2. Incubation Period: The incubation period is commonly said to be 6 to 48 h. This rather lengthy incubation period is an indication that this disease is an actual infection, rather than an intoxication or food poisoning as it is often called. The longer incubation period results because the bacteria must increase in number in the intestinal tract before symptoms of the disease begin (Gutherie et al., 1991). The observed low attack rates and long incubation periods (median 4 days, range 1 to 27 days) indicated low-grade contamination of foods, and microbiological investigation revealed S. Typhimurium in food samples from the buffet (CPHL et al., 2006).

        3. Prognosis: Although most infections are self-limiting with diarrhea, vomiting, abdominal cramps, and fever, severe infections are not uncommon. Estimates suggest that approximately 15,000 people are hospitalized and >500 deaths occur annually due to Salmonella infections (Wright et al., 2005) S. typhimurium infection has been epidemic in China in recent years. It is especially dangerous to the newborn, because the younger the patient , the higher the incidence of sepsis and the death rate (Wu et al., 1993). The mortality rate being 16.7% (Wu et al., 1993). Mortality was 18% (Mirza et al.,1989). The next largest group affected also consisted of children from 1 year to 5 years (21%) (Mirza et al.,1989). Nontyphoid Salmonella serotypes are major causes of food-borne infections worldwide. They still seriously affect human health and cause morbidity and mortality. Infections with nontyphoid Salmonella serotypes most often result in self-limited acute gastroenteritis that does not require antimicrobial therapy. Nevertheless, approximately 5% of individuals with gastrointestinal illness caused by nontyphoid Salmonella serotypes develop bacteremia. Children with certain underlying conditions are at increased risk of bacteremia, which may lead to extraintestinal focal infections. Such conditions include very young age (babies), AIDS, malignancies, immunosuppressive therapy, hemolytic anemia, and inflammatory bowel disease. Nontyphoid Salmonella bacteremia is even more serious in adult patients with underlying diseases; these patients are more likely to develop focal infections such as meningitis, septic arthritis, and osteomyelitis (Chiu et al., 2004). Mortality rates are highest in outbreaks that affect high numbers of children or the elderly; the incidence of invasive disease is approximately 10%, but us higher in the elderly (18 %) (Sanchez et al., 2002). The overall mortality is about 1 per cent (Huckstep et al., 2002). In the United States, nontyphoidal Salmonella infections result in an estimated rate of hospitalization of 2.2 per 1 million population, and 582 deaths per year (Pegues et al., 2002). The median duration of illness was 8 days (range 5-11 days) (Wright et al., 2005). Salmonella gastroenteritis is usually a self-limited disease, and therapy primarily should be directed to the replacement of fluid and electrolyte losses. In a large meta-analysis, antimicrobial therapy for uncomplicated Salmonella gastroenteritis, including short -course or single-dose regimens with oral fluoroquinolones, amoxicillin, or trimethoprime-sulfamethoxazole, did not significantly decrease the length of illness, including duration of fever or diarrhea, and was associated with an increased risk of relapse, positive culture after 3 weeks, and adverse drug reactions. Therefore, antimicrobials should not be used routinely to treat uncomplicated nontyphoidal Salmonella gastroenteritis or to reduce convalescent stool excretion (Pegues et al., 2002). The vast majority of the doctors do not use antibiotics to treat salmonella gastro-enteritis in older children and adults, because antibiotic therapy can shorten the clinical course and, on the contrary, it prolongs the duration of the convalescent carrier state and leads to the increase of resistant strains. Salmonella gastro-enteritis in neonates, however, is easy to cause sepsis, so it is advisable to treat them with amikacin or the third generation cephalosporins (Wu et al., 1993). Infection with multidrug resistant S. typhimurium is associated with high mortality ranging from 77.7% to 100% (Kumar et al., 1995).

        4. Diagnosis Overview: Symptoms of gastroenteritis appear within 6 to 24 h after ingestion of contaminated food or water and last as long as a week (Salyers et al., 2002).

        5. Symptom Information :
          • Syndrome -- Salmonellosis, non-typhoid:
            • Description: Salmonellosis in the human occurs in a wide variety of forms presenting a broad clinical spectrum. The disease may occur solely as an intestinal infection, termed salmonellosis or salmonella gastroenteritis, as a focal infection in any organ of the body, or as a systemic febrile infection. The clinical symptoms of the intestinal infections vary from asymptomatic, or no symptoms, to a most severe diarrhea with fever and nausea (Gutherie et al., 1991). Entry into mucosal tissues is a hallmark of enteric salmonellosis. Invasion of intestinal epithelial and transepithelial transport by dendritic cells have been implicated in penetration of the epithelial barrier by serovar Typhimurium (Suar et al., 2006). The onset is abrupt, with diarrhoea and vomiting, headache and abdominal pain. The temperature is usually slightly raised, but seldom over 100 (degrees) F. The symptoms, as a rule, gradually abate over a period of one to four days, but occasionally there may be complications, and even death, due to dehydration, and electrolyte imbalance from diarrhoea and vomiting (Huckstep et al., 2002).
            • Observed: Most often, salmonellosis begins with a sudden headache, abdominal distress, diarrhea, nausea, and sometimes vomiting approximately 36 to 72 h after the organisms have been ingested (Gutherie et al., 1991). Symptoms of gastroenteritis appear within 6 to 24 h after ingestion of contaminated food or water and last as long as a week. Initial symptoms include nausea and vomiting, which subside after a few hours. These symptoms are followed by abdominal pain, diarrhea, and sometimes fever. The severity of the pain and diarrhea vary widely from one person to another, ranging form mild pain and barely detectable diarrhea to pain resembling appendicitis and severe, even bloody, diarrhea. After the symptoms subside, the infected person with continue to excrete bacteria for up to 3 months (Salyers et al., 2002). Clinically salmonellosis in newborns manifests like any other illness caused by Gram-negative organisms. We also found a higher incidence of septicemia (81%0 in this series (Kumar et al., 1995). In this study certain factors like male sex, and birth asphyxia were more frequently associated with symptomatic illness than with asymptomatic carrier state. Males are well known to have a higher incidence of sepsis (Kumar et al., 1995).

            • Symptoms Shown in the Syndrome:

            • Diarrhea:
              • Description: Diarrhea is usually self-limited, typically lasting for 3 to 7 days (Pegues et al., 2002). Although most infections are self-limiting with diarrhea, vomiting, abdominal cramps, and fever, severe infections are not uncommon (Wright et al., 2005). The stools are watery, and without blood (Huckstep et al., 2002). When diarrhea occurs, the stools are typically watery and blood specked (Gutherie et al., 1991). Microscopic examination of stools shows neutrophils and, less frequently, red blood cells (Pegues et al., 2002). There was no difference in clinical features of culture positive and culture negative septicemic infants. Symptoms appeared between 3 and 7 days of life in all infants. Diarrheal stools were watery, yellow-green to green in color, and often contained mucus but not visible blood (Kumar et al., 1995).
              • Observed: Clinical information was available for 93% of confirmed cases (42/45). All but one reported diarrhea, and 52% (22/42) reported bloody diarrhea (CPHL et al., 2006).
          • Vomiting:
            • Description: Although most infections are self-limiting with diarrhea, vomiting, abdominal cramps, and fever, severe infections are not uncommon (Wright et al., 2005). Vomit in salmonella food poisoning is usually watery, and may be bile stained and occasionally blood stained (Huckstep et al., 2002).
            • Observed: Vomiting (24%) (CPHL et al., 2006) Vomiting (52.6%) (Ling et al., 2002)
          • Abdominal cramps:
            • Description: Although most infections are self-limiting with diarrhea, vomiting, abdominal cramps, and fever, severe infections are not uncommon (Wright et al., 2005). The abdomen shows generalised tenderness without guarding, and the patient may show signs of severe dehydration (Huckstep et al., 2002).
            • Observed: Abdominal cramping (79%) (CPHL et al., 2006). Abdominal cramps (26.3%) (Ling et al., 2002).
          • Fever:
            • Description: Fevers (38 degrees to 39 degrees C), abdominal cramping, nausea, vomiting and chills frequently are reported (Pegues et al., 2002). If fever is present, it usually resolves within 48 to 72 hours (Pegues et al., 2002). Although most infections are self-limiting with diarrhea, vomiting, abdominal cramps, and fever, severe infections are not uncommon (Wright et al., 2005).
            • Observed: Fever is present in at least 50% of the cases (Gutherie et al., 1991). Fever (88%) (CPHL et al., 2006).
          • Dehydration:
            • Description: Dehydration resulting from vomiting and diarrhea may be severe, particularly in small children (Gutherie et al., 1991). Occasionally, patients require hospitalization because of dehydration and death occurs infrequently (Pegues et al., 2002).
            • Observed: Dehydration (61.2%) (Saporito et al., 2007).
          • Nausea:
            • Description: Symptoms in humans consist of diarrhea, fever, headache, nausea, abdominal pain, vomiting, and, less frequently, blood in the stool (Poppe et al., 1998).
            • Observed: Nausea (36%) (CPHL et al., 2006).
          • Headache:
            • Description: Symptoms in humans consist of diarrhea, fever, headache, nausea, abdominal pain, vomiting, and, less frequently, blood in the stool (Poppe et al., 1998).
            • Observed: Headache (45%) (CPHL et al., 2006).

        6. Treatment Information:
          • Antibiotics: The worldwide emergence of a distinct strain of multidrug-resistant S. Typhimurium, characterized as definitive phage type 104 (DT104) that is resistant to at least five antimicrobials- ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracyclines (Pegues et al., 2002). Antimicrobial agents should not be used routinely to treat uncomplicated nontyphoid Salmonella gastroenteritis (Chiu et al., 2004). Antimicrobial resistance among nontyphoid Salmonella serotypes has been a serious problem worldwide. Conventional antimicrobial agents, such as ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole, had been the drugs of choice in the treatment of salmonellosis before the 1980s. However, multidrug resistance, with rates of resistance to these antimicrobial agents of more than 50%, has been reported in many areas of the world. Extended-spectrum cephalosporins and fluoroquinolones have been suggested as appropriate alternative agents in the treatment of infections caused by such multidrug-resistant Salmonella serotypes; however, since 1991, outbreaks or cases of infections caused by Salmonella serotypes resistant to extended-spectrum cephalosporins or fluoroquinolones have been increasingly reported (Chiu et al., 2004). The emergence of antimicrobial resistance in Salmonella is complicated because the use of antibiotics for therapeutic purposes in veterinary medicine and as growth promoters in animal feed may promote the emergence of resistance, thus presenting a potential risk to public health from zoonotic infections (Chiu et al., 2004). Transmission from swine to humans of a DT104 strain that was resistant to nalidixic acid and had reduced susceptibility to ciprofloxacin has been reported from Denmark. These studies raise substantial concern that the ongoing use of fluoroquinolones as growth promoters in livestock may select for ciprofloxacin-resistant Salmonella among food animals and humans (Pegues et al., 2002). Treatment should consist of an oral or intravenous antimicrobial administered for 48 to 72 hours or until the patient becomes afebrile. Longer treatment may result in a higher rate of chronic carriage and relapse. For susceptible organisms, treatment with an oral fluoroquinolone, trimethoprime-sulfamethoxazole, or amoxicillin is adequate (Pegues et al., 2002).
            • Applicable: Fewer than 5% of all patients with Salmonella gastroenteritis develop bacteremia, certain patients are at increased risk for invasive infection and may benefit from preemptive antimicrobial therapy (Pegues et al., 2002). As with most salmonella infections, treatment of these infections depends on the severity of illness. Antimicrobial-drug therapy is usually not essential in cases of uncomplicated salmonella gastroenteritis; a fluoroquinolone, such as ciprofloxacin, or an extended-spectrum cephalosporin, such as ceftriaxone, remains the recommended treatment for invasive typhimurium infections with the five-drug pattern of resistance (Glynn et al., 1998). An appropriate antimicrobial-drug therapy is necessary in these severe infections. Two antimicrobial classes are used for that purpose, ciprofloxacin and, when ciprofloxacin is contraindicated, extended-spectrum cephalosporins (ESC) (Pegues et al., 2002). In these DT104 isolates, multiple antibiotic resistance was due to chromosomal integration of a 43-kb structure called Salmonella genomic island 1 (SGI1), comprised of the genes coding for resistance to ampicillin (blaPSE-1), streptomycin and spectinomycin (aadA2), chloramphenicol and florfenicol (floR), sulfonamides (sul1), and tetracycline [tet(G)] . Additional resistance to nalidixic acid resistance (with reduced susceptibility to ciprofloxacin) has increasingly been reported in DT104 isolates recovered from humans and animals in England . A few reports have also described multidrug-resistant serotype Typhimurium isolates highly resistant to ciprofloxacin (MIC [greater than] 8 mg/liter) in humans and animals from Europe and Asia (Pegues et al., 2002). Although fluoroquinolones are not recommended for administration to children younger than 10 years of age, they may have a role in treating severe nontyphoidal salmonellosis in this age group. In one sample study, seven children with severe typhoidal or nontyphodial salmonellosis who failed conventional therapy improved rapidly when treated with oral pefloxacin (12 mg/kg daily for 7 days). In addition, a double-blind, placebo-controlled trial from Turkey demonstrated that intravenous immunoglobulin (500 mg/kg on days 1, 2, 3 and 8) in combination with cefoperazone when administered to preterm neonates, with S. Typhimurium infection, reduced the rate of mortality, complications, and duration of antimicrobial therapy compared to treatment with cefoperazone alone (Pegues et al., 2002). The proportion of stool isolates showing sensitivity to various drugs were as follows: streptomycin (94.2%), gentamicin (89.4%), amikacin (1005), netromycin (89.4%), ciprofloxacin (94.2%), cefotaxime (84.2%), ceftriaxone (98.9%), furazolidone (47.35), nalidixic acid (21.1%). All the strains were resistant to ampicillin, trimethoprime, and chloramphenicol (Kumar et al., 1995). All symptomatic cases were treated with antibiotics, given intravenously, for 10-14 days except in a case of meningitis where the duration of treatment was 21 days. Sixteen infants received cefotaxime and amikacin; of these, 13 recovered, 1 failed to respond and required ciprofloxacin to improve whereas two died within 2 days of starting antibiotics because of fulminant illness (Kumar et al., 1995). Four of the 5 infants treated with ceftriaxone and amikacin recovered while one did not respond to this combination and required ciprofloxacin (Kumar et al., 1995).
            • Contraindicator: Although fluoroquinolones are not recommended for administration to children younger than 10 years of age, they may have a role in treating sever nontyphoidal salmonellosis in this age group (Wright et al., 2005).
            • Success Rate: The median duration of illness was 8 days (range 5-11 days) (Wright et al., 2005). One of the ill persons was treated with ciprofloxacin but continued to shed Salmonella spp. in stool for at least 214 days after illness onset (Wright et al., 2005).
            • Drug Resistance: In 1998, the first reported case of ceftrixone-resistant Salmonella infection acquired in the United States occurred in a child in Nebraska and was associated with exposure from on his family's ranch that harbored S. Typhimurium with a 160-kb plasmid encoding CMY-2 AmpC beta-lactamase (Pegues et al., 2002). Although R-type ACSSuT is the most common antimicrobial resistance pattern of DT104 isolates (present in 54%-67% of DT104 isolates in the UK during 1992-1995), resistance of DT104 isolates to trimethoprim and fluoroquinolones is emerging. In the UK, from 1993 to 1995, trimethoprim-resistant DT104 (R-type ACSSuTTm) increased from 1% to 27% of isolates, and ciprofloxacin-resistant DT104 (R-type ACSSuTCp) increased from 0 to 6% of isolates. Acquisition of trimethoprim resistance may have resulted from use of this agent to treat DT104 R-type ACSSuT infections in cattle. In addition, the emergence of fluoroquinolone resistance may be related to veterinary use (Hosek et al., 1997). NTS from the age group [less than] 3 years showed the highest level of resistance to all the commonly used antibiotics. Although we were unable to obtain data on the use of antibiotics without prescription in the community, a general survey indicated that sale of commonly available antibiotics such as ampicillin, cotrimoxazole, chloramphenicol over the counter without prescription and mainly for use in treatment of acute respiratory infections and childhood diarrhoea was common in the urban population (Kariuki et al., 2006). The most commonly used antibiotic for treatment of NTS infections at the hospitals was cefuroxime or a combination of ampicillin and gentamicin. The high level of usage of orally administered ampicillin and cefuroxime in the clinics and health centres and their availability over-the-counter in some chemist shops in urban centres was likely to be related to high levels of resistance observed. In addition the high levels of resistance to cotrimoxazole could be attributed to the widespread use of sulphamethoxazole-pyrimethamine (SP) for malaria treatment, which has previously been associated with increased resistance to cotrimoxazole (Kariuki et al., 2006). Although it is well established that antimicrobial treatment of this condition does not add value and may even prolong carriage of NTS in patient, it is a common practice in Kenya to prescribe an antibiotic in almost all cases gastroenteritis due to belief that this may prevent progression into the bloodstream, thus leading to massive antibiotic misuse (Kariuki et al., 2006)

    4. Prevention:
      1. Food handler safety precautions:
      2. Cooking:
        • Description: Salmonella are sensitive to heat (Gutherie et al., 1991). The organisms are killed at temperatures of 70 degrees C or above (Gutherie et al., 1991). Because of this characteristic, ordinary cooking is sufficient to destroy Salmonella cells if applied for times sufficiently long enough to reach this temperature throughout the food being cooked (Gutherie et al., 1991). Salmonella are also susceptible to the heat applied in pasteurization of milk 71.2 degrees C for 15 seconds and are resistant to drying (Gutherie et al., 1991).
      3. Hygiene:
        • Description: Salmonella outbreaks with illness in animals and humans have been reported in both equine and companion animal veterinary facilities. These reports commonly describe poor hand washing by employees, eating in work areas, and prior antimicrobial drug therapy in humans or animals (Wright et al., 2005). Nosocomial transmission of Salmonella from patients to health care workers as been associated with handling soiled linen (Pegues et al., 2002). Non-compliance with barrier precautions (Pegues et al., 2002). The risk of nosocomial transmission to neonates and infants from acutely or chronically infected family members is high. Neonates are at high risk for fecal-oral transmission of Salmonella because of relative gastric achlorhydria and the buffering capacity of ingested breast milk and formula (Pegues et al., 2002). Anyone who like raw eggs (e.g., in traditional Caesar salad dressing) or undercooked eggs (e.g., in egg-thickened sauces is well advised to refrigerate eggs immediately after purchase (Salyers et al., 2002). Control of outbreaks in daycare centers may be difficult because of the need for frequent diaper changing and the higher rate and longer duration of convalescent carriage seen in the preschool age group (Pegues et al., 2002). The elderly are at increased risk for Salmonella bacteremia and extraintestinal infection as a result of debility, underlying illnesses, and waning immunity (Pegues et al., 2002).

    5. Model System:
      1. Mouse model:
        1. Model Host: Mus musculus: Salmonella typhimurium virulence factors have been identified and characterized using experimental infection of mice. While the murine typhoid model has been used successfully for Salmonella typhi vaccine development and to infer virulence mechanisms important during typhoid fever, information derived from infection of mice has been of limited value in elucidating the mechanisms by which S. typhimurium causes enteritis in humans (Tsolis et al., 1999). The mouse, which has been used for most of the studies addressing the various aspects of host-pathogen interaction in S. typhimurium infection, develops a systemic disease when infected with S. typhimurium, but no diarrhea. Murine infection with S. typhimurium results in a disease that is similar to human typhoid fever caused by infection with serotypes Typhi, Paratyphi A, B, and C, which are host-restricted serotypes that infect only man and few other primate species. Thus, murine infection with S. typhimurium has been employed as a model for human typhoid fever, but not Salmonella-induced diarrhea. In contrast, calves infected with S. typhimurium develop a diarrheic disease with clinical manifestations similar to those observed in human infections, which also result in diarrhea with a low mortality rate (Santos et al., 2003). In genetically susceptible mouse strains, oral infection with S. typhimurium leads to spread via the gut-associated lymphoid tissues to systemic sites. Bacteremia and lesions in systemic organs are reminiscent of typhoidal salmonellosis in humans - hence this disease is known as mouse typhoid. This mouse typhoid model relies on a surrogate pathogen and a surrogate host: S. typhimurium and mice are used instead of S. typhi and humans or other primates. Nevertheless, the mouse typhoid model has been extensively used to study immune responses to S. typhimurium and bacterial virulence factors required for systemic infection (Hapfelmeier et al., 2005). In genetically resistant inbred mouse strains (e.g. 129SvEv; Nramp1+/+) S. typhimurium causes chronic infection of systemic organs. These mice provide a useful animal model for persistent systemic infection. In fact, mice are thought to form a natural reservoir for Salmonella spp. and have been implicated in transmission to, and infection of, livestock (Hapfelmeier et al., 2005) In all of these typhoid mouse models, S. typhimurium neither efficiently colonizes the intestine [less than or equal to] 10(5)cfu per g of faeces, in orally infected calves that develop diarrhea faecal excretion reaches 10(6)-10(8) cfu per g) nor causes acute intestinal inflammation. Thus, these models have not been useful for studying acute S. typhimurium enterocolitis (Hapfelmeier et al., 2005). Recently, it was found that antibiotic pretreated mice develop acute intestinal inflammation in response to oral S. typhimurium infection upon oral pretreatment of mice with a single high dose of streptomycin (20 mg), S. typhimurium efficiently colonizes the large intestine [10(8)-10(10)] cfu per g) and triggers severe acute diffuse inflammation of cecum and colon (colitis) as early as 8 h post-infection. In this colitis model, both the pathogen and host are well-studied (Hapfelmeier et al., 2005). Mice pretreated with streptomycin develop inflammation of the cecum and colon (Hapfelmeier et al., 2005). This murine serovar Typhimurium colitis model can help to explore molecular mechanisms of the pathogen-host interaction which lead to acute intestinal inflammation (Suar et al., 2006). The streptomycin-pretreated mouse model is a surrogate host model because mice with an intact intestinal microflora do not normally get overt intestinal inflammation from any S. enterica serovar (Suar et al., 2006). Diarrhea is a frequent symptom in human S. typhimurium enterocolitis (Hapfelmeier et al., 2005). The mouse, which does not develop diarrhea, responds to the infection mostly with a mononuclear infiltrate in the intestine (Santos et al., 2003).
        2. Model Pathogens:
          • Salmonella typhimurium. . Salmonella species infect a broad range of animals and can cause different diseases in different hosts (Finlay et al., 2002). Salmonella typhi causes typhoid fever in humans, an invasive disease that can be fatal. In contrast, S. typhimurium usually causes a self-limiting gastroenteritis in humans but induces a systemic disease in mice that is similar to typhoid fever (Finlay et al., 2002).
        3. Description: Salmonella enterica subspecies I, serovar Typhimurium (S. typhimurium), is a leading cause of human gastroenteritis, and is used as a mouse model of human typhoid fever (McClelland et al., 2001). Salmonella typhimurium has emerged as a model pathogen that manipulates host cells in a complex fashion, thus causing disease. In humans, S. typhimurium causes acute intestinal inflammation (Hapfelmeier et al., 2005).
      2. Calf model:
        1. Model Host: Bos taurus: Calves are regarded as the animal model of choice to study S. typhimurium-induced enterocolitis and diarrhea. Cattle are natural hosts for this pathogen and, most importantly, clinical and histological manifestations of S. typhimurium enterocolitis are similar to the human disease (Hapfelmeier et al., 2005). The mouse, which has been used for most of the studies addressing the various aspects of host-pathogen interaction in S. typhimurium infection, develops a systemic disease when infected with S. typhimurium, but no diarrhea. Murine infection with S. typhimurium results in a disease that is similar to human typhoid fever caused by infection with serotypes Typhi, Paratyphi A, B, and C, which are host-restricted serotypes that infect only man and few other primate species. Thus, murine infection with S. typhimurium has been employed as a model for human typhoid fever, but not Salmonella-induced diarrhea. In contrast, calves infected with S. typhimurium develop a diarrheic disease with clinical manifestations similar to those observed in human infections, which also result in diarrhea with a low mortality rate (Santos et al., 2003). Infections with Salmonella serotypes are a major cause of food-borne diseases worldwide. Animal models other than the mouse have been employed for the study of nontyphoidal Salmonella infections because the murine model is not suitable for the study of Salmonella-induced diarrhea (Santos et al., 2003). An important aspect of the pathology of Salmonella-induced enteritis is the same pattern of inflammatory reaction developed by calves after S. typhimurium infection, which is characterized by a marked infiltration of neutrophils, also observed in non-human primates in experimental infections, and in human infections. In sharp contrast, mice infected with S. typhimurium develop an inflammatory response with predominance of mononuclear leukocytes, which is not associated with diarrhea (Santos et al., 2003). Milk-fed, 28-day old Friesian/Holstein bull calves have been used to model S. typhimurium induced enteritis. After oral infection with a dose of 10(6) CFU of S. typhimurium ATCC14028, calves develop diarrhea within 12-48 hours (Tsolis et al., 1999). This dose, the disease is self-limited and does not result in mortality. Lethal infections can be induced by inoculation with 10(9)-10(10) CFU/animal and are caused by dehydration and intestinal lesions. Similar dose responses were obtained using other S. typhimurium isolates. S. typhimurium initially invades M cells of the follicle associated epithelium of bovine Peyer's patches. This initial interaction is followed b entry into enterocytes of absorptive villi in the terminal ileum. Necroscopy of calves with severe diarrhea reveals enteritis over areas of Peyer's patches (Tsolis et al., 1999). Histopathological examination of the intestine shows destruction of the mucosal epithelium, purulent inflammation, and depletion of lymphocytes in the germinal centers of intestinal lymphoid follicles (Tsolis et al., 1999). Between 2 and 5 days post infection, bacteria can be recovered in similar numbers ( up to 10[6] to 10[8] CFU/g tissue) from ileal tissue samples containing Peyer's patches or villous intestine. S. typhimurium can be recovered in 10-fold lower numbers from the ileal mesenteric lymph nodes and the cecum. The majority of S. typhimurium infections in calves remain localized to the intestine and mesenteric lymph node. We found that even after oral inoculation with high doses (10[9]-10[10] CFU/animal) S. typhimurium colonizes the spleen in only 50% of infected animals where it is found in low numbers (10[2] to 10[5] CFU/tissue) (Tsolis et al., 1999). Infection of calves with S. typhimurium results in enteritis in which neutrophils are the primary inflammatory cells involved (Santos et al., 2003). In calves, inflammation is observed in the terminal ileum and the proximal colon (enterocolitis) (Hapfelmeier et al., 2005).
        2. Model Pathogens:
        3. Description: Diarrhea, the main sign of disease in calves and man, does not develop in mice. The different disease manifestations in mice limit the degree in which data from the murine typhoid model an be extrapolated to diarrheal disease caused by S. typhimurium in calves and man (Tsolis et al., 1999).
  2. Mammals: (Mammals and birds:
    1. Taxonomy Information:
      1. Species:
        1. Mammals. (NCBI_taxonomy):
          • Scientific Name: Mammalia (NCBI_taxonomy)
          • Description: S. enterica subspecies 1 serovars Typhimurium and Enteritidis infect a broad range of host animals. Interestingly, they cause different diseases in different animal species. In calves, serovar Typhimurium (and rarely Enteritidis]) causes enterocolitis, and the animals can succumb to dehydration . In newly hatched chicks, serovars Enteritidis and Typhimurium cause systemic disease and diarrhea, whereas older chickens are asymptomatic carriers. In immunocompetent humans, serovars Enteritidis and Typhimurium cause localized self-limiting enterocolitis. Systemic disease may develop in immunocompromised individuals . Finally, serovars Enteritidis and Typhimurium cause a systemic typhoid fever-like disease in susceptible mouse strains, but no diarrhea. The mechanisms determining which type of disease is caused in which host by serovars Enteritidis and Typhimurium are still poorly understood (Suar et al., 2006). In the case of serovar Typhimurium infections, the intestinal microflora is an important factor (termed "colonization resistance" or "microbial interference) in determining whether enteric disease can develop or not. This was demonstrated with germfree mice lacking the entire microflora and with streptomycin-pretreated mice which have a severely disrupted intestinal microflora . In the absence of an intact intestinal microflora, serovar Typhimurium not only causes the well-known systemic disease but also colonizes the murine cecum and colon and causes pronounced colitis. This streptomycin-pretreated mouse model has proven useful to study key aspects of the molecular pathogenesis of serovar Typhimurium enterocolitis . Here, we have extended our studies and tested the virulence of several host-restricted and further broad-host-range S. enterica subspecies 1 serovars, including 6 strains whose genomic sequence has been or will soon be completed. Our data establish that the streptomycin-pretreated mouse model is not restricted to serovar Typhimurium (Suar et al., 2006). There is strong evidence that Salmonella enterica serovar Typhimurium has established reservoirs in wild-living birds and hedgehogs in Norway. Wild-living birds and hedgehogs may function as effective spreaders of Salmonella bacteria to humans and to different animal species through contamination of the environment ( Refsum et al., 2002). In Norway, sporadic indigenous cases and a national outbreak of human salmonellosis, caused by serovar Typhimurium, have been related to infections in small passerines ( Refsum et al., 2002). Moreover, two human outbreaks in 1996 and 2000 have been associated with infected hedgehog populations ( Refsum et al., 2002). A significantly higher carrier rate of S. Typhimurium occurred among hedgehogs sampled at feeding places, compared to those caught elsewhere. The salmonella infected hedgehog populations most likely constituted the primary source of infection both of the human disease outbreaks, and the Norwegian hedgehog is suggested as a reservoir host of S. Typhimurium ( Handeland et al., 2002).
        2. Birds. (NCBI_Taxonomy):
          • Scientific Name: Aves (NCBI_taxonomy).
          • Description: There is strong evidence that Salmonella enterica serovar Typhimurium has established reservoirs in wild-living birds and hedgehogs in Norway. Wild-living birds and hedgehogs may function as effective spreaders of Salmonella bacteria to humans and to different animal species through contamination of the environment ( Refsum et al., 2002). In a waterborne serovar Typhimurium infection outbreak in northwestern Norway in 1999, gulls were suggested to be the most likely source of infection ( Refsum et al., 2002). Moreover, two human outbreaks in 1996 and 2000 have been associated with infected hedgehog populations ( Refsum et al., 2002).

    2. Infection Process:

      No infection process information is currently available here.

    3. Disease Information:

      No disease information is currently available here.

    4. Prevention:

      No prevention information is currently available here.

    5. Model System:

      No model system information is currently available here.

IV. Labwork Information

A. Biosafety Information:
  1. General biosafety information (CDC):
    • Biosafety Level: Biosafety Level 2 practices, containment equipment and facilities are recommended for activities with clinical materials and cultures known or potentially containing the agents (CDC).
    • Applicable: Primary reservoir hosts include a broad spectrum of domestic and wild animals, including birds, mammals, and reptiles, all of which may serve as a source of infection to laboratory personnel (CDC).
    • Precautions:
      • Contact with animals and animal products is also a risk factor for Salmonella infections in humans. Contact with sick livestock is not an uncommon method of exposure for farm workers (Sanchez et al., 2002). Veterinarians should expect, at least occasionally, to evaluate animals infected with Salmonella spp. Following these outbreaks, recommendations for infection prevention and control were formulated to help prevent future outbreaks of salmonellosis in association with companion animal facilities. Recommendations include wearing gloves while cleaning cages and treating animals, then immediately removing the gloves and washing hands when the task is completed. No eating or drinking should be allowed in animal treatment and holding areas, and feces-contaminated areas should be immediately cleaned and disinfected. Clear warnings of the risk for transmission of Salmonella spp. should be given when pets with probable salmonellosis are encountered. Veterinarians should consider culturing the stools of animals with diarrhea and should be aware of the increased risk for infection with multidrug-resistant salmonellae in animals who are given antimicrobial drugs for other conditions. Because use of antimicrobial agents contributes to increasing resistance and facilitates transmission of multidrug-resistant salmonellae, promoting guidelines aimed at improving appropriate use of antimicrobial agents may help prevent transmission of multidrug-resistant Salmonella infections in veterinary facilities (Wright et al., 2005).
      • Laboratory hazards: The agent may be present in feces, blood, urine, and in food, feed, and environmental materials. Ingestion or parenteral inoculation are the primary laboratory hazards. The importance of aerosol exposure is not known. Naturally or experimentally infected animals are a potential source of infection for laboratory and animal care personnel, and for other animals (CDC).
    • Disposal:
      • Animal Biosafety Level 2 practices, containment equipment and facilities are recommended for activities with experimentally or naturally infected animals (CDC).
B. Culturing Information:
  1. Blood culture :
    1. Description: Blood cultures and stool specimens were processed using standard techniques. Briefly, blood cultures were incubated in 5 % Carbon dioxide at 37 C for 18 h, and if signs of bacterial growth were observed (air bubbles, turbidity, or both) they were subcultured on sheep blood agar and chocolate agar. The remaining blood cultures were reincubated for a further 7 days or until positive (Kariuki et al., 2006).

    2. Medium:
      1. Sheep blood agar and chocolate agar (Kariuki et al., 2006)
    3. Optimal Temperature: 37 C (Kariuki et al., 2006)
  2. Stool culture :
    1. Description: Stool specimens or rectal swabs were collected from all patients in the SBN and transported to the laboratory for processing. Rectal swabs for cultures were also taken from hospital staff; these swabs were collected in tubes of selenite F broth (Difco Laboratories) for transport to the laboratory. Cultures were taken from environmental surfaces with sterile cotton swabs moistened in sterile water. Rectal swabs were incubated overnight at room temperature in selenite F broth and were then subcultured to salmonella-shigella agar and xylose-lactose-glucose agar. Swabs from environmental surfaces were plated on blood agar. Fluids for culture were added to an equal volume of brain heart infusion broth. All cultures were incubated at 37 C for 48 h. Isolates of S. typhimurium were tested for their susceptibility to antibiotics by a disk diffusion technique at CDC and by an agar dilution technique at the Medical College of Virginia. Tube dilution susceptibility tests were performed by a previously described technique (Lamb et al., 1984) Stools were processed by direct plating onto selective media (XLD and brilliant green agar) (Oxoid) and by overnight enrichment in selective Selenite F broth (Oxoid) followed by plating onto XLD and brilliant green agar, and incubated in air at 37 C for 18 h. NTS were identified using agglutinating antisera (Murex Biotech), and their identification was confirmed biochemically using API 20E strips (API System). Environmental samples were initially cultured in Rappaport-Vassiliadis soya broth (Oxoid) for enrichment. The broth culture was then subcultured onto XLD and brilliant green agar. NTS were identified as for blood and stool cultures. All NTS isolates were stored at -70 C on Protect beads (Technical Service Consultants) until analysed (Kariuki et al., 2006). Samples were collected from various seabird species and Antarctic fur seals (Arctocephalus gazella) on the sub-Antarctic island of Bird Island, South Georgia. In February and March 1996, faecal swabs were taken from 40 pups of A. gazella, 30 adult gentoo penguins (Pygoscelis papua), 50 macaroni penguin chicks (Eudyptes chrysolophus), 50 blackbrowed albatross chicks (Diomedea melanophrys) and 50 grey-headed albatross chicks (D. chrysostoma). Sampling was repeated in February and March 1998, when swabs were collected from 206 Antarctic fur seal pups, 100 macaroni penguin chicks, 100 gray-headed and 40 black-browed albatross chicks. Faecal samples were collected using cotton wool swabs inserted into the rectum. Samples were stored in a charcoal transport medium at 5 (plus or minus) 10 C and transported to Sweden, where they were cultured within 3 weeks from the date collected. Isolation and identification of bacteria Each sample was enriched in selenite broth and incubated at 37 C for 18-24 h. This was subcultured on xylin-lysine-deoxycholate agar and hydrogen sulphide positive colonies were verified as salmonella by their reaction in fermentation tests. Serotyping was carried out according to the Kauffmann-White scheme and phage typing of S. typhimurium was performed according to Anderson and S. enteritidis according to Ward (Palmgren et al., 2002).

  3. Direct culture :
    1. Description: In clinical-disease situations, whether systemic or enteric samples may be inoculated directly on to plating media. Samples of internal organs, which are normally sterile, should be inoculated on to non-selective (e.g. blood agar, nutrient agar) or at least weakly selective media (e.g. MacConkey agar), in addition to the more selective plating media (e.g. brilliant green agar, xylose lysine deoxycholate agar) (Waltman et al., 2000).

    2. Medium:
      1. Plating media for the isolation of salmonella can be subdivided into three groups according to the selective agents used. These are the bile salt agars, the brilliant green agars and bismuth sulphite agar (Busse et al., 1995).
  4. Enrichment culture :
    1. Description: Typically in chronic infections, carrier animals or environmental samples, the numbers of Salmonella are low, especially relative to the high numbers of other bacteria. These samples should be inoculated into selective enrichment media for optimal recovery of Salmonella (Waltman et al., 2000). Direct plating and direct selective enrichment of certain types of samples were often unsuccessful for the detection of Salmonella. Typically, samples that have been dried, heated, irradiated, or otherwise processed require the use of non-selective pre-enrichment for optimal recovery of Salmonella. In these samples, Salmonella may be present but are 'damaged' or sublethally injured. These organisms although still viable and able to cause disease under the right conditions, are easily killed if placed into the harsh environment of most selective-enrichment broths, especially when incubated at higher temperatures (Waltman et al., 2000). Pre-enrichment is useful for isolating Salmonella from faeces and environmental samples (Waltman et al., 2000). Selective-enrichment broths are formulated to selectively inhibit other bacteria while allowing Salmonella to multiply to levels that may be detected after plating (Waltman et al., 2000).

    2. Medium:
      1. Pre-enrichment media: Lactose broth (LB) was perhaps the first to receive widespread use (Waltman et al., 2000). Other pre-enrichment: Buffered peptone water (BPW), M9 and universal pre-enrichment (Waltman et al., 2000). Several studies have found that BPW was better than LB for isolating Salmonella (Waltman et al., 2000). BP has been the pre-enrichment broth of choice for use in conjunction with rappaport-Vassiliadis (RV) enrichment media (Waltman et al., 2000). Selective enrichment media: There are currently three major types of selective-enrichment media: tetrathionate, selenite and RV (Waltman et al., 2000). Pre-enrichment of the sample, regardless of the type or source, is advocated with RV broth (Waltman et al., 2000). The inoculation ratio commonly used for tetrathionate and selenite enrichment broths is 1:10; with RV broth, however, it is 1:100. Some investigators have even advocated a ratio of 1:1000 (Waltman et al., 2000). The enrichment is normally plated after 24 h incubation (Waltman et al., 2000). Generally, suspect Salmonella colonies are selected and inoculated into tubes of composite media, e.g. triple sugar iron (TSI) and lysine iron (LI) agar. These tubes are incubated overnight at 37 C and the resulting biochemical reactions observed (Waltman et al., 2000).
    3. Optimal Temperature: The recommended incubation temperature for pre-enrichment is 35-37 C. Since the purpose for pre-enrichment is the resuscitation of damaged Salmonella, a higher temperature should be avoided (Waltman et al., 2000). The recommended incubation temperature (for enrichment) is 41.5 C (Waltman et al., 2000). Some studies advocate temperatures as high as 43 C (Waltman et al., 2000). Generally, samples such as internal organs or tissues, have low levels of background flora are incubated at 35-37 C (Waltman et al., 2000). Intestinal and environmental samples, which generally have higher levels of competing bacteria, may be incubated at higher temperatures (40-43 C), because Salmonella are more tolerant to the higher temperature. The optimal growth temperature of Salmonella is about 37 C (Waltman et al., 2000). However, with some enrichment media, especially when incubated at higher temperatures, Salmonella begin to die between 24 and 48 h (Waltman et al., 2000). If the initial subculture is negative for Salmonella, the enrichment broth is left at room temperature for 5-7 days; then 0.5-1.0 ml of the original selective-enrichment broth is transferred into 10 ml of fresh selective-enrichment broth, which is incubated at 37 C for 18-24 h and then subcultured on to plating media (Waltman et al., 2000). DSE (delayed secondary enrichment) cultures are held at room temperature, not incubation temperatures of 35 C or higher (Waltman et al., 2000).
    4. Optimal pH: The pH of the LB cultures after incubation ranged from 4.8 to 5.5, whereas the ranges for BPW and M9 were 5.8-6.4 and 5.9-6.2, respectively (Waltman et al., 2000).
C. Diagnostic Tests :
  1. Organism Detection Tests:
    1. Antimicrobial susceptibility testing:
      1. Time to Perform: 1-to-2-days
      2. Description: Disk diffusion technique (CDC) (Lamb et al., 1984) Prior to testing, each frozen stock was streaked for isolation on MacConkey agar plates and incubated at 37 C for (approximately) 16 h. The antimicrobial resistance of serovar Typhimurium strains was tested using the disk diffusion assay with diameters of the inhibition zones as the outcome variable used in cluster analysis. Briefly, a single isolated colony of each isolate was transferred from the agar plate to brain heart infusion broth and incubated for 5 h at 37 C. Following incubation, the inoculum was diluted to 0.5 McFarland units with 0.85% sterile NaCl solution. A sterile, nontoxic swab was used to streak to a cation-adjusted Mueller-Hinton agar plate (150 by 15 mm) to form a uniform lawn of bacterial growth. Drug-impregnated disks were placed on the agar surface using a disk dispenser and incubated for 16 h at 37C. Following incubation, the diameters of the inhibition zones around the antibiotic disks ("clear zones") were measured and recorded (Adaska et al., 2006). Agar dilution technique (Medical college of Virginia): Results of agar dilution susceptibility tests with the epidemic strain of S. typhimurium: Moxalactam: 1.0 (MIC ug/ml); Amikacin: 2.0 (MIC ug/ml); Cefoxitin:5.0 (MIC ug/ml); Cefoxitin:5.0 (MIC ug/ml); Gentamicin: 5.0 (MIC ug/ml); Penicillin: >5.0 (MIC ug/ml); Ampicillin >25.0; Nafcillin: >10.0 (MIC ug/ml); Carbenicillin: >250.0 (MIC ug/ml); Cefazolin: 25.0 (MIC ug/ml); Tetracycline: >5.0 (MIC ug/ml); Chloramphenicol: >25.0 (MIC ug/ml); Erythromycin: >5.0 (MIC ug/ml); TMP-SMX: >8-152 (MIC ug/ml) (Lamb et al., 1984). Tube dilution susceptibility tests (Lamb et al., 1984) Susceptibilities to various antimicrobials, ampicillin, coamoxiclav, tetracycline, cotrimoxazole, chloramphenicol, gentamicin, nalidixic acid, ciprofloxacin, cefuroxime and ceftriaxone, were determined by both controlled disc diffusion and measuring MICs using E-test strips (AB BIODISK) according to the manufacturer's instructions. Escherichia coli ATCC 25922 (with known MICs) was used as a control for potency of antibiotic discs and E-test strips. Disc diffusion susceptibility tests and MICs were interpreted according to the guidelines provided by the National Committee for Clinical Laboratory Standards (Kariuki et al., 2006). MIC results using the E-Test of 10 antimicrobial agents for 193 NTS isolates from paediatric wards at two hospitals in Nairobi, Kenya (2002-2004). Ampicillin: Range; 0.25-256 MIC (ug/ml); percentage resistant; 54 Coamoxiclav: Range; 0.75-256 MIC (ug/ml); percentage resistant; 8 Cefuroxime: Range; 2-256 MIC (ug/ml); percentage resistant; 30 Ceftriaxone: Range; 0.094-16 MIC (ug/ml); percentage resistant;0 Gentamicin: Range; 0.06-256 MIC (ug/ml1); percentage resistant; 16 Cotrimoxazole: Range; 0.064-32 MIC (ug/ml); percentage resistant; 46; Chloramphenicol: Range; 0.19-256 MIC (ug/ml); 26; percentage resistant; 26; Tetracycline: Range; 0.064-256 MIC (ug/ml); percentage resistant; 51; Nalidixic acid: Range; 1.5-256 MIC (ug/ml); percentage resistant; 12; Ciprofloxacin: Range; 0.064-4 MIC (ug/ml); percentage resistant; 0 (Kariuki et al., 2006)
    2. PFGE of macrorestricted chromosomal DNA:
      1. Time to Perform: 1-hour-to-1-day
      2. Description: Chromosomal DNA from NTS isolates was prepared in agarose plugs as described previously. DNA in agarose plugs was digested using 25 U each of XbaI or SpeI (Roche Diagnostics). PFGE of agarose plug inserts was then performed on a CHEF-DR III system on a horizontal 1 % agarose gel for 20 h at 120 V, with a pulse time of 1-40 s at 14 C. A lambda DNA digest consisting of a ladder (approximately 22 fragments) of increasing size from 50 to approximately 1000 kb was included as a DNA size standard. The gel was stained with ethidium bromide and photographed on a UV transilluminator. The restriction endonuclease digest patterns were compared, and their similarities were scored by the method of Tenover et al. and by using the Dice similarity coefficient formula 2h/(a(plus)b), where h is the number of matching bands, and a(plus)b is the total number of bands including matching and non-matching. Isolates that differed in their PFGE fragment patterns by one or two bands were regarded as closely related, as minor mutational changes would result in such patterns (Kariuki et al., 2006). Pulsed-field gel electrophoresis: Restriction enzyme digests for pulsed-field gel electrophoresis (PFGE) were performed with SpeI, BlnI and XbaI. Each salmonella isolate was analysed with all three enzymes. The isolates were grown on blood agar at 37 C for 18-20 h and 4 colonies were dispersed in 1 ml TEN-buffer (1 m NaCl, 10 mm Tris pH 8.0, 10 mm EDTA) and centrifuged at 6000 rpm. The bacteria were suspended in 250 ul lysis buffer (1 mNaCl, 10 mm Tris pH 8.0, 200 mm EDTA, 0.5% Sacrosyl, 0.2% Sodium deoxycholate) and embedded in 2% Low Melt Prep Agarose with 35 ul (20 mg}ml) lysozyme per agarose slice. The slices were incubated in 2.5 ml lysis buffer with 85 ul proteinase K (1 mg/ml final concentration) at 56 C for 36 h, and then washed 6 times in 1xTE-buffer. Half of each agarose slice was incubated for 18 h with 20 U of respective restriction enzyme in 100 ul enzyme buffer at 37 C. One mm of each slice was run on a 1% agarose gel (Pulse field Certified Agarose, Bio-Rad) in 10% PFGE buffer at 10 C, on an automated PFGE apparatus. Standard programmes for fragment sizes 50-400 kb (SpeI and XbaI) and 50-700 kb (BlnI) were used and a standard lambda DNA ladder was run alongside the samples. The gel was stained with 0.2% ethidium bromide, washed in tap water, and photographed using a DS34 Polaroid camera. Analysis of clonality between isolates belonging to the same serotype was based on 30-35 restriction fragments per serotype. Fragments sized 100 kb were excluded to minimize the effect of plasmid fragments. Gels were analysed visually except for S. newport isolates which were compared using GelCompar version 4.0. Polaroid photographs of macrorestriction probes were scanned with a UMAX Vista-S6E scanner and digitalized using Adobe Photoshop 3.0.5 for Windows, and saved in TIFF format. Banding patterns of combined gels were compared by the UPGMA (Unweighted Pair Group Method with Arithmetic averages) clustering method using the Dice coefficient, according to the manufacturer's instruction. A band position tolerance of 1.2% was applied (Palmgren et al., 2002).

  2. Immunoassay Tests:
    1. ELISA:
      1. Time to Perform: 1-hour-to-1-day
      2. Description: In this study, the levels of IgG, IgM, and IgA antibodies against serovar Enteritidis LPS and serovar Typhimurium LPS were measured in sera from 303 patients diagnosed by fecal culturing with either serovar Enteritidis or serovar Typhimurium (Strid et al., 2007). The ELISAs were based on serovar Enteritidis lipopolysaccharide (LPS) and serovar Typhimurium LPS. The antibody levels were assessed approximately 1, 3, 6, and 12 months after the onset of salmonellosis. Sera from 164 healthy blood donors were analyzed to establish cutoff values for each analysis (Strid et al., 2007). One month after the onset of symptoms, the sensitivities of the assays were 95% for patients recovering from a serovar Enteritidis infection and 89% for patients recovering from a serovar Typhimurium infection. Three months after the onset of symptoms, these values had decreased to 85% and 55%. At 6 months they were 62% and 40%, and at 12 months they were 40% and 16%, respectively. The specificities of the assays were 97% for the serovar Enteritidis LPS ELISA and 94% for the serovar Typhimurium LPS ELISA. All antisera were supplemented with 0.01% sodium azide and stored at -20 C. In the analyses of sera from patients diagnosed with either serovar Enteritidis or serovar Typhimurium infections, the detection rate was defined as the percentage of sera producing detectable antibodies against Salmonella LPS, i.e., generating an OD value in the ELISA above the cutoff value (Strid et al., 2007). An indirect ELISA was developed for the determination of the contents of antibodies against Salmonella LPS in human sera. For the determination of antibodies against serovar Enteritidis LPS, commercially available LPS from serovar Enteritidis was used. Likewise, for the determination of antibodies against serovar Typhimurium LPS, commercially available LPS from S. enterica serovar Typhimurium was used. A number of different batches and lot numbers were tested and gave the same results. LPS preparations were checked for protein impurities by silver stain polyacrylamide gel electrophoresis (Strid et al., 2007). The ELISA was performed with microtiter plates using a BioMek 2000 robot. Wells were coated overnight at 4 C with 0.1 ug antigen in 100 ul coating buffer (carbonate buffer at pH 9.60 containing 0.2% phenol red). Wells were washed four times with wash buffer (i.e., phosphate-buffered saline at pH 7.40 with 0.1% Tween 20) and thereafter blocked with 250 ul wash buffer per well for 30 min at room temperature. The wells were subsequently washed (four times). 100 ul of serum diluted 1:400 in dilution buffer (phosphate-buffered saline at pH 7.40 with 0.1% Tween 20 and 0.2% phenol red) was added per well in duplicate, and then the plates were incubated at room temperature for 30 min. After the wells were washed (four times), 100 ul of horseradish peroxidase-conjugated rabbit anti-human IgG, IgM, or IgA was added per well (anti-IgG, 1:2500; anti-IgM, 1:1000; and anti-IgA, 1:500) and incubated for 30 min at room temperature. After the wells were washed, 100 ul of a 3,3',5,5'-tetramethylbenzidine solution was added per well and incubated for 15 min at room temperature. The reaction was stopped with 100 ul 1 M sulfuric acid, and the OD at 490 nm (650 nm was used as a reference) was finally determined. Each serum sample was analyzed for the presence of IgG, IgM, and IgA anti-LPS antibodies. Day-to-day variations were minimized by including the dilution series of a standard for each assay. Reproducibility was tested using duplicates of sera both within the plate and between different days, and a coefficient of variation under 10% was observed (Strid et al., 2007). Cutoff values were determined on the basis of sera from 33 healthy Danish blood donors and calculated by the "mean plus 2 standard deviations" method. These cutoff values for IgG, IgM, and IgA, respectively, were 0.65, 0.84, and 0.74. Using these cutoffs, the specificity of the assay was 100%, since all of the analyzed blood donor sera produced OD values below the cutoffs (Strid et al., 2007). An LPS-based ELISA may be useful for both acute-phase and postinfection diagnoses of gastrointestinal salmonellosis (Strid et al., 2007).
    2. Widal tube agglutination test: tube dilution and slide agglutination:
      1. Time to Perform: 2-to-7-days
      2. Description: The differences within Salmonella serovars are based on the surface antigen differences of O and H antigens. The O antigens are derived from the polysaccharide domain of lipopolysaccharide (LPS) in the cell wall, while the H antigens are derived from the flagellin protein in the flagella. O and H antigens are used for the identification of Salmonella serovars by slide and tube agglutination tests using O and H antigen-specific anti-sera, and Salmonella serovars are determined by the combination of O and H antigen types (Lim et al., 2002). A standard tube agglutination assay (the Widal test) was performed for the determination of antibody titers against Salmonella. Whole-cell antigens were prepared from overnight cultures (SSI standard strains) either by formalin or alcohol fixation. O (LPS) and H (flagella) whole cell-antigen preparations were controlled by including standards and positive controls each time a new preparation was made as well as each time the assay was performed (Strid et al., 2007). All patient sera were analyzed for agglutination to S. enterica serovar Typhi O antigen, S. enterica serotype Paratyphi A O antigen, and serotype Paratyphi B O antigen. The O antigens from serovar Typhi (O:9,12) are very similar to the O antigens from serovar Enteritidis (O:1,9,12), and the O antigens from serotype Paratyphi B (O:1,4,5,12) are identical to the O antigens from serovar Typhimurium (O:1,4,5,12). Furthermore, sera from patients diagnosed with serovar Enteritidis infections were analyzed for agglutination to serovar Enteritidis H antigen, and sera from patients diagnosed with serovar Typhimurium infections were analyzed for agglutination to serovar Typhimurium H antigens (i:1,2).Specific (Strid et al., 2007). A mixture of suspended antigen and antibody is incubated for up to 20 h at 37 C in a water bath. Agglutinations are visualised in the form of pellets, clumped together at the bottom of the test tube. Results are scored from 0 to 4 (+) positive agglutination (Olopoenia et al., 2000). Serum was considered positive for Widal test when anti-O or anti-H titer was (>or equal to) 160 (Mekara et al., 1990). The levels are measured by using serial dilutions of sera. Usually, O antibodies appear on days 6-8 and H antibodies on days 10-12 after the onset of the disease. The test is usually performed on acute and convalescent sera to detect the rising titers. The test has only moderate sensitivity and specificity (Ismail, 2006). Classic and inexpensive. Despite mixed results in endemic areas, still performs well for screening large volumes. May need standardisation and quality assurance of reagents (Bhutta, 2006). Of the indirect tests available for the diagnosis of enteric fever, the most established is the Widal which has enjoyed widespread use as an adjunct to clinical assessment and bacterial isolation (Choo et al., 1999). The Widal test was carried out using antigens from Wellcome Diagnostics and standard serial dilutions of serum in normal saline starting at 1:40. A positive Widal test was taken as a titre of at least 1 in 80 (Choo et al., 1999). The Widal test does not provide results for some 2 days after blood sampling (Choo et al., 1999). Approximate cost (U.S. dollars): 0.50, No. of tests/kit: 55; Reaction time: 5 minutes, Temperature for storage: 2-8 C; Amount of serum needed: 300 ul (plus/minus) (two dilutions) (Olsen et al., 2000)
      3. False Positive: Widal 1:80, positive predictive value 19.6% (Nizami et al., 2006). Sensitivity: 47-77 %; Specificity: 50-92 % (Bhutta, 2006).
      4. False Negative: Widal 1:80, negative predictive value 90.2% (Nizami et al., 2006).

  3. Nucleic Acid Detection Tests: :
    1. Phage type identification:
      1. Time to Perform: 1-hour-to-1-day
      2. Description: An epidemic strain of Salmonella enterica serotype Typhimurium definitive type 104 (DT104) rose to prominence when this multidrug-resistant (R-type ACSSuT) pathogen was identified as a major cause of salmonellosis in people and farm animals in Britain and in people in the United States. The epidemic strain of DT104 was defined by British researchers based upon Salmonella serovar Typhimurium phage type 104, the R-type ACSSuT, and a plasmid profile consisting of a single (approximately) 60-MDa plasmid (Pritchett et al., 2002). We describe in this note the identification of a DNA sequence that is unique to the DT104 and U302 phage types among S. enterica serotype Typhimurium isolates, and the development of a PCR assay to identify isolates containing this sequence. This PCR assay can be used to rapidly screen suspect samples or isolates for further testing and identification (Pritchett et al., 2002). The PCR amplification of the 16S-to-23S spacer region of bacterial rRNA genes has been used to detect polymorphisms in bacterial species and to identify strains of S. enterica serotype Typhimurium associated with disease outbreaks. Using this technique on bovine isolates of S. enterica serotype Typhimurium, the DT104 isolates demonstrated a unique band compared to other phage types (Pritchett et al., 2002). To evaluate the multiplex PCR, 239 Salmonella isolates representing a wide range of serotypes and sources were selected from a bank of Salmonella isolates (Pritchett et al., 2002). Cell lysates for each isolate were prepared in duplicate by suspending a single bacterial colony in 300 ml of sterile distilled water in a microcentrifuge tube and boiling for 20 min. Cell lysates were stored at -20 C until amplified. Aliquots (5 ml) of cell lysates were each amplified in a 25-ml reaction mixture with 1 mM (each) primers INVA-1, INVA-2, DT104F, and DT104R; 200 mM (each) deoxynucleoside triphosphates (dNTP), 2 mM MgCl2, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, and 1.25 U of Taq DNA polymerase. Amplification was performed in 0.2-ml microreaction tubes in as follows: denaturation at 96 C for 1 min; 30 cycles of 96 C for 30 s, 60 C for 30 s, and 72 C for 35 s; and 1 final extension cycle at 72 C for 30 s. The PCR products were visualized on ethidium bromide-stained agarose gels (Pritchett et al., 2002). All 21 DT104 isolates tested by this multiplex PCR produced the 162-bp amplification product. In addition, three isolates with phage type U302 produced the 162-bp amplification product, and U302 is considered an offspring of the DT104 strain. None of the 15 other defined phage types of S. enterica serotype Typhimurium tested produced the 162-bp product following PCR. Three isolates of multidrug-resistant S. enterica serotype Typhimurium submitted for phage typing could not be assigned a defined phage type, and these isolates also did not produce the 162-bp product following PCR (Pritchett et al., 2002).
      3. Primers:
    2. Multiplex PCR for multidrug resistant strains of Salmonella typhimurium:
      1. Time to Perform: 1-hour-to-1-day
      2. Description: Developed a multiplex PCR method to detect the ACSSuT-type serovar Typhimurium strains from 104 multidrug-resistant clinical isolates of serogroup B Salmonella (Chiu et al., 2006). A 50 ul reaction mixture contained six different primer pairs of different concentrations, 200 uM deoxynucleoside triphosphates, 1 U of Taq DNA polymerase (Promega), 1.5 mM MgCl, and 2 ul of DNA templates. An aliquot of overnight bacterial culture (100 ul) was collected and boiled at 100 C for 10 min. The bacterial lysate was centrifuged at 13,000 x g for 10 min, and the supernatant was used as the DNA template. The multiplex PCR was performed for 35 cycles as follows: denaturation at 95 C for 45 s, annealing at 56 C for 45 s, and extension at 72 C for 1 min. A final step of extension at 72 C for 5 min was performed. To further identify the integrons of these isolates, PCR using primers CS-F and CS-R was performed to amplify the class 1 integron resistance gene cassettes (Chiu et al., 2006).
      3. Primers:
    3. Multiplex polymerase chain reaction assay:
      1. Time to Perform: minutes-to-1-hour
      2. Description: A multiplex polymerase chain reaction (PCR) assay was developed for the identification of Salmonella enterica serovar Typhimurium. Three sets of primers were designed for detecting O4, H:i, and H:1,2 antigen genes from the antigen-specific genes rfbJ, fliC, and fljB. These were evaluated in a multiplex PCR assay by using DNAs from S. enterica serovar Typhimurium, 15 other Salmonella serovars, and 8 non-Salmonella enteric pathogens. Multiplex PCR proved to be capable of identifying S. enterica serovar Typhimurium specifically and differentiating it from other Salmonella serovars in addition to non-Salmonella enteric pathogens. Thus, this multiplex PCR assay can be practically applied to the identification of S. enterica serovar Typhimurium. The 35 clinical isolates of serovar typhimurium used for multiplex PCR were isolated from patients who had food-borne diseases and were selected from independent food-borne cases. In the present study, three sets of primers that targeted the rfbJ, fliC, and fljB genes in a tube were used. This allowed for making a specific identification of O4, H:i, and H:1,2 antigenic properties, because, only S. enterica serovar Typhimurium has the antigenic structure combination of O4, H:i, and H:1,2 out of about 2,000 Salmonella serovars. The products amplified from S. enterica serovar Typhimurium using these primers were sufficiently specific to allow for the detection of the serovar Typhimurium. The sensitivity of this multiplex PCR was 500 ng dNA per tube or 10(5) bacteria per tube. These results clearly show that multiplex PCR is indeed very useful for making a specific detection of S. enterica serovar Typhimurium, and three sets of primers specifically detected serovar Typhimurium strains. These results also show that Salmonella strains can be identified when the strains possess any antigenic property of O:4, H:I, or H:1, H:2 (Lim et al., 2002).
      3. Primers:
      4. False Positive: The sensitivity of this multiplex PCR was 500 ng DNA per tube or 10(5) bacteria per tube (Lim et al., 2002).

    4. Molecular markers:
      1. Time to Perform: 1-hour-to-1-day
      2. Description: We aimed to develop DNA markers suitable for a molecular typing scheme that correlates with phage typing scheme. We see a distinctive advantage for such a scheme as it provides a continuity of epidemiological surveillance which has been done using phage typing (Lan et al., 2007). Amplified Fragment Length Polymorphism (AFLP) was initially applied to 46 Typhimurium isolates of Australian origin comprising of 9 phage types and grouped most isolates by phage type, enabling differentiation of phage types (Lan et al., 2007). We observed 84 phage-type specific polymorphic AFLP fragments for which presence or absence correlated with phage types, and 18 of these fragments were cloned and sequenced. Fifteen are of known genes or have a homologue in the databases (Lan et al., 2007). Three of these are plasmid related, eight are phage related and only four relate to chromosomal genes. This gave the proof in principal for use of these fragments being present or absent as the basis for a PCR based molecular phage-typing scheme for Typhimurium (Lan et al., 2007). The strength of this approach is based on the fact that the majority of the genomic differences found are mobile elements (mainly of phage origin), greatly facilitating PCR or microarray based detection (Lan et al., 2007). Some of the markers may directly be correlated with phage type or phage acquisition (Lan et al., 2007). Chromosomal DNA (0.1 ug) was used for digestion and ligation of adaptors, which were performed simultaneously. The ligation mix was denatured, diluted 1:10, and 1 ul was used for preamplification. Preamplification was done in a 20 ul volume with a PCR mix containing 0.2 um of each dNTP, 4ug/ul BSA, 10mM Tris-HCL (ph 8.3), 50 mM KCL, 2 mM Mgcl(2), and 0.5 U AmpliTaq Gold plus 0.3 uM preamplification primers. The cycling profile was 20 cycles of denaturation at 94 C for 15 s except for 10 min for the first cycle, annealing at 56 C for 30 s, and extension of 72 C for 1 min. For final amplification 1 ul diluted product (1:10) from preamplification was mixed with 19 ul of PCR mix as for preamplification, but containing 0.3 uM unlabelled selective primer of 0.1 uM (22)P labeled selective primer. Basic cycling parameters were denaturation at 94 C for 15 s except for 10 min for the firs cycle, annealing at temperatures specified below for 30 s, extension at 72 C for 1 min. The first 10 cycles were touch-down from 66 to 56 C decreasing by 1 (degree) C/cycle, and then 20 cycles at 56 C. AFLP products were run on standard 6% polyacrylamide sequencing gels and visualized by exposure to Kodak BioMax-MR film overnight (Lan et al., 2007). Polymorphic AFLP fragments were excised for a 6% polyacrylamide gel after radioactive AFLP, with AFLP patterns visualized by autoradiography. The excised fragments were cloned into the pGEM-T Easy plasmid cloning vector and transformed into E. coli K-12 strain JM109 by electroporation using a Bio-Rad Gene Pulser. The recombinant plasmid was extracted (Lan et al., 2007). PCR typing of each marker was done separately (Lan et al., 2007). The annealing temperature for each was determined by running a gradient PCR. PCR product was visualized by ethidium bromide staining after electrophoresis on a 1.5% agarose gel (Lan et al., 2007). The band patterns from manual scoring of PCR typing were converted to distance between isolates according to the Dice coefficient of similarity. An in-house program MLEECOMP was used to generate the Dice coefficient which was used to draw relationships using programs in the PHYLIP package (Lan et al., 2007). The PCR typing scheme using 22 markers had high discriminatory power and significant correlation with phage typing (Lan et al., 2007). GenBank with accession numbers from DQ835568-DQ835581 (Lan et al., 2007).
    5. Bacteriophage typing:
      1. Time to Perform: 1-hour-to-1-day
      2. Description: Phage typing has a long history with regard to the differentiation of Salmonella serovars of human and animal origin (Jones et al., 2000). Phage-typing schemes for S. typhi, S. paratyphi and B, S. typhimurium and S. enteritidis are used in most reference laboratories (Jones et al., 2000). Many phages occur in sewage, sewage-contaminated water and faeces and, once isolated, they have to be purified by repeated single-plaque isolation. Preliminary identification consists of establishing the host range and plaque morphology. Typing-phage preparations can be produced by growing equal concentrations of purified phage particles and host cells in their logarithmic phase of growth in pre-warmed nutrient broth at 37 C for as long as lysis visibly continues and usually no longer than 7 h. The mixture is then heated at 60 C for 30 min to kill the host cells; most typing phages for Salmonella survive such treatment. If the phage is not thermostable, the host cells may be killed by the addition of a few drops of chloroform. The lysates are then centrifuged to remove the killed bacteria (Jones et al., 2000). The phage preparation is then titrated on its host strain to determine the routine test dilution (RTD), which is defined as the highest dilution that produces confluent or, in some cases, semi-confluent lysis of the host strain that was used for propagation (Jones et al., 2000). The Salmonella to be typed is grown in 5 ml nutrient broth in an incubator at 37 C. The period of time and conditions depend on the serovar (Jones et al., 2000). Nutrient agar plates that have been dried at 37 C for 180 min are flooded with the Salmonella culture, so that a tin film results (Jones et al., 2000). The plates are dried again for a maximum of 15 min at 37 C and the phage preparations added (Jones et al., 2000). Various mechanical devices such as the Lidwell apparatus, may be used to spot the phage on to the Salmonella lawn. The plates are then read after being incubated overnight at 37 C (Jones et al., 2000).

  4. Other Types of Diagnostic Tests:
    1. Plasmid pattern analysis:
      1. Time to Perform: 1-hour-to-1-day
      2. Description: Plasmid profiling was the first genotypic method used for strain separation within Enterobacteriaceae. When the bacterium divides, copies of resident plasmids will be distributed between the two daughter cells, and members of the same clonal line are expected to carry the same plasmids. Strains of Salmonella often carry plasmids ranging in size from 2 to 150 kb, but frequencies and size distributions vary between serovars (Olsen et al., 2000). Useful results can be obtained in less than 2 h from an overnight broth culture or colonies on an agar plate (Olsen et al., 2000). Certain resistance (R) plasmids in S. typhimurium have been shown to undergo rearrangement or deletions if the selection pressure is not in favour of maintaining the R factors, and the way the cultures are treated prior to analysis may influence the plasmid profiles, because plasmid loss may occur in stab cultures during storage (Olsen et al., 2000) Thirty epidemic strains of S. typhimurium belonging to phage type 12 had the same plasmid profiles (98.0, 6.7 and 3.8 Kb) and identical restriction digest patterns (23.0, 20.4, 15.0, 9.6, 8.2, 7.4, 5.8, 4.3, 3.8, 2.0 and 1.8 Kb) which were different from those of the 2 non-epidemic strains (Wu et al., 1993). In the environment of calf dealers in the UK, used plasmid profiling in combination with phage typing and biotyping to demonstrate routes of transmission between farms (Olsen et al., 2000).
    2. Microsphere-based Fiber-optic DNA microarrays:
      1. Time to Perform: 1-to-2-days
      2. Description: A fiber-optic DNA microarray using microsphere-immobilized oligonucleotide probes specific for the Salmonella invA and spvB genes was developed for detection of Salmonella spp. Microarrays were prepared by randomly distributing DNA probe-functionalized microspheres (3.1 um diameter) into microwells created by etching optical fiber bundles. Hybridization of the probe-functionalized microspheres to target DNA from Salmonella was performed and visualized using Cy3-labeled secondary probes in a sandwich-type assay format. In this study, 10(3)-10(4) cfu/mL of the target organism could be detected after 1 h hybridization without any additional amplification. The DNA microarray showed no cross-reactivity with other common food pathogens, including E. coli and Y. enterocolitica, and could even detect Salmonella spp. from cocktails of bacterial strains with only moderate loss of sensitivity due to nonspecific binding. This work suggests that fiber-optic DNA microarrays can be used for rapid and sensitive detection of Salmonella spp (Ahn et al., 2002). The DNA fiber-optic microarrays were prepared by loading oligonucleotide probe-functionalized microspheres into microwells created by selectively etching the distal end of optical fiber bundles. A sandwich-type assay was performed in which target DNA samples were hybridized to the capture probes attached to the microspheres, followed by labeling through a second hybridization with Cy3-conjugated signal probes. The resulting Cy3 fluorescence signals from all the probe-functionalized microspheres were observed simultaneously using a charge-coupled device (CCD) camera. This simple approach can rapidly detect low concentrations of Salmonella spp. with a high degree of sensitivity and selectivity (Ahn et al., 2002). To evaluate the use of DNA microarrays for detecting Salmonella spp., oligonucleotide probes were designed to target the bacterial virulence genes: invA, invE, spvB, and agf (Ahn et al., 2002). These genes were chosen because they have demonstrated high specificity for detection of Salmonella spp. in previous studies. Additionally, these genes are useful for identifying highly pathogenic Salmonella serotypes, as they are essential for pathogenesis. For each target gene except invA, a capture probe and signal probe pair were designed. For the invA gene, two capture probes were used. Five types of DNA probe-functionalized microspheres, InvA1, InvA2, InvE, SpvB, and Agf, were prepared by attaching amino-terminated oligonucleotides of each respective capture probe sequence to glutaraldehyde-activated microspheres (Ahn et al., 2002). Various hybridization times were used depending on the target concentration; 5, 20, and 30 min were used for synthetic target solutions of 10 nM, 10 pM, and 100 fM (Ahn et al., 2002) For each array, an average of 70 microspheres was loaded on the microarray (Ahn et al., 2002).
      3. False Positive: Both InvA2 and SpvB probes had the lowest detection limits of 10(3) cfu/mL, compared to 10(5) and 10(8) cfu/mL for InvA1 and Agf, respectively. While the detection limits of InvA2 and SpvB probes were satisfactory, the detection limits of InvA1 and Agf probes were high. In particular, Agf probes showed very low signal intensities over the tested concentration range (Ahn et al., 2002). The microarray was able to detect 10(3)-10(4) cfu/mL of various strains of Salmonella spp. in pure culture or 10(4)-10(5 )cfu/mL in a mock sample in the presence of interfering organisms. Detection assays on this system can be completed within 1 h, which is much faster than other conventional detection assays. Detection was very specific for Salmonella spp., and no cross-reactivity was observed for genealogically close organisms including E. coli and Y. enterocolitica (Ahn et al., 2002).

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D. Thesis References:

No thesis or dissertation references used.

VI. Curation Information