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Epidemiology of Salmonella and Salmonellosis

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International Letters of Natural Sciences Vol. 47 (2015) pp 54-73 Online: 2015-09-30
© (2015) SciPress Ltd., Switzerland
doi:10.18052/www.scipress.com/ILNS.47.54

Epidemiology of Salmonella and Salmonellosis

Nwabor, Ozioma Forstinus1, Dickson, Ihenriochi Dickson2,
Ajibo, Queensley Chinyere3
1, 2,3
Department of Microbiology, University of Nigeria, Nsukka, Nigeria.
1
([email protected], +2348038781993),
2
([email protected], +234836468556),
3
([email protected], +2348069298655).

Keywords: Salmonella, Salmonellosis, Prevalence, Epidemiology

ABSTRACT
The prevalence of enteritis and its accompanying diarrheal and other health challenges linked
to infections with Salmonella has continuously plagued sub Saharan Africa. In Nigeria, typhoid
fever is among the major widespread diseases affecting both young and old as a result of many
interrelated factors such as inadequate sanitaion, indiscriminate use of antibiotics and fecal
contamination of water sources. Morbidity associated with illness due to Salmonella continues to
increase with untold fatal consequences, often resulting in death. An accurate figure of cases is
difficult to arrive at because only large outbreaks are mostly investigated whereas sporadic cases are
under-reported. A vast majority of rural dwellers in Africa often resort to self medication or seek no
treatment at all, hence serving as carries of this disease. Non typhoidal cases of salmonellosis
account for about 1.3 billion cases with 3 million deaths annually. Given the magnitude of the
economic losses incurred by African nations in the battle against salmonella and salmonellosis, this
article takes a critical look at the genus Salmonella, its morphology, isolation, physiological and
biochemical characteristics, typing methods, methods of detection, virulence factor, epidemiology
and methods of spread within the environment.

1. INTRODUCTION
The study of Salmonella began with Eberth’s first recognition of the organism in 1880, and
the subsequent isolation of the bacillus responsible for human typhoid fever by Gaffky (Le Minor,
1991). Further investigations by European workers characterized the bacillus and developed a
serodiagnostic test for the detection of this human disease agent (Tindall et al., 2005). Thereafter,
D.E. Salmon isolated the bacterium then thought to be the etiological agent of hog cholera, but later
disproved. The genus was named Salmonella by Lignieres in 1900 in honour of D.E. Salmon.
Further investigations led to the isolation of other Salmonella species (Su and Chiu, 2006). An
antigenic scheme for the classification of Salmonella was first proposed by White and subsequently
expanded by Kauffmann into Kauffmann-White scheme, which currently includes more than 2540
serovars (Popoff and Le Minor, 2005). Salmonella nomenclature is very complex and scientists
used different systems to refer to and communicate about this genus. Unfortunately, current usage
often combines several nomenclature systems that divide the genus into species, subspecies,
subgenera, groups, subgroups, and serotypes (serovars), and all these usages caused lots of
confusion among researchers (Rakesh et al., 2009). Salmonella nomenclature has progressed
through a succession of taxonomical and serological characteristics and on the principles of
numerical taxonomy and DNA homology (Tindall et al., 2005). The nomenclature for the genus
Salmonella has evolved from the initial one serotype-one species concept proposed by Kauffmann
in 1966 on the basis of somatic (O), flagellar (H) and capsular (Vi) antigens. In the early
development of the taxonomic scheme, biochemical reactions were used to separate Salmonella into
subgroups and the Kauffmann-White scheme was the first attempt to systematically classify
Salmonella using scientific parameters. The scientific development in Salmonella taxonomy
occurred in 1973 when Crosa et al. (1973) demonstrated using DNA-DNA hybridization that all
serotypes and sub-genera I, II, and IV of Salmonella and all serotypes of Arizona were related at the

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 International Letters of Natural Sciences Vol. 47 55

species level. Thus, they belonged to a single species, and the exception later described was called
Salmonella bongori, previously known as subspecies-V. Other taxonomic proposals have been
made based on the clinical role of a strain and biochemical characteristics that divided the serovars
into subgenera (Brenner et al., 2000; Ezaki et al., 2000). The antigenic formulae of Salmonella
serovars are defined and maintained by the World Health Organization (WHO) Collaborating
Centre for Reference and Research on Salmonella at the Pasteur Institute, Paris (Popoff et al.,
2004). Presently, Salmonella genus consists of two species: Salmonella enterica and Salmonella
bongori. Salmonella enterica is further divided into six subspecies; S. enterica subsp. enterica (I),
S. enterica subsp. salamae (II), S. enterica subsp. arizonae (Illa), S. enterica subsp. diarizonae
(lllb), S. enterica subsp. houtenae (IV), and S. enterica subsp. indica (VI) (Popoff and Le Minor,
2005).

2. CHARACTERISTICS OF SALMONELLA

Salmonella are Gram negative, facultative anaerobic, rod shaped bacteria belonging to family
Enterobacteriaceae. Members of this genus are motile by peritrichous flagella, except Salmonella
enterica serovar Pullorum and Salmonella enterica serovar Gallinarum. Salmonella are 2-3 x 0.4-
0.6 µm in size and they are chemoorganotrophs, with ability to metabolize nutrients by both
respiratory and fermentative pathways (D’Aoust et al., 2001; Popoff and Le Minor, 2005).
Hydrogen sulphide (H2S) is produced by most Salmonella but a few serovars like Salmonella
paratyphi A and Salmonella choleraesuis do not produce H2S. Most Salmonellae are aerogenic;
however, Salmonella typhi does not produce gas. Members of the genus have a % G+C content of
50-53. They are urease and Voges-Proskauer negative and citrate utilizing (Montville and
Matthews, 2008). Most Salmonella do not ferment lactose and this property has been the basis for
the development of numerous selective and differential media for culture and presumptive
identification of Salmonella spp; they include xylose lysine decarboxycholate agar, Salmonella-
Shigella agar, brilliant green agar, Hektoen enteric agar, MacConkey’s agar, lysine iron agar and
triple sugar iron agar. Isolation of Salmonella from food and environmental samples with culture
method utilizes the multiple steps of pre-enrichment and enrichment on the selective and
differential media in order to increase the sensitivity of the detection assay (Andrews and
Hammack, 2001; Anderson and Ziprin, 2001). Isolation of Salmonella often involves pre-
enrichment, a process in which the sample is first cultured in a non-selective growth medium such
as buffered peptone water or lactose broth with the intent of allowing the growth of any viable
bacteria, and the recovery of injured cells. Subsequently, pre-enriched samples are cultured on
enrichment media to restrict the growth of undesirable bacteria. Enrichment media commonly used
include tetrathionate broth, selenite cystine broth and Rappaport Vassiliadis broth. Following the
enrichment period, the enriched cultures are spread onto selective and differential agar plate and
then typical colonies for Salmonella has to be identified. Final confirmation of typical colonies is
determined by series of biochemical and serological tests (Rakesh et al., 2009). A few Salmonella
serovars do not exhibit the typical biochemical characteristics of the genus and these strains pose
problem diagnostically because they may not easily be recovered on the commonly used differential
media. About 1% of the Salmonella serovars submitted to Centre for Disease Control (CDC)
ferment lactose; hydrogen sulphide production too was quite variable (Ziprin, 1994). Salmonella
chrome agar medium has been described very promising for detection of both lactose positive and
lactose negative Salmonella isolates from food samples (Dick et al., 2005).

3. PHYSIOLOGY AND BIOCHEMICAL CHARACTERISTICS

The biochemical properties of Salmonella spp show that almost all Salmonella serovars do
not produce indole, hydrolyze urea, nor deaminate phenylalanine or tryptophan. Most of the
serovars readily reduce nitrate to nitrite and most ferment a variety of carbohydrates with the
production of acid, and have been reported to be negative for Voges-Proskauer (VP) reaction. Other
56 Volume 47

prominent characteristics of this genus include the ability of most serovars to produce hydrogen
sulfide (H2S) and decarboxylate lysine, arginine and ornithine with a few exceptions (Popoff and Le
Minor, 2005). Most Salmonella serovars utilize citrate with a few exceptions such as Salmonella
Typhi, Salmonella paratyphi A and a few Salmonella choleraesuis serovars. Dulcitol is generally
utilized by all serovars except Salmonella enterica subsp. arizonae (Illa) and Salmonella enterica
subsp. diarizonae (lllb) (Popoff and Le Minor, 2005). Lactose may not be utilized by most
Salmonella serovars, however, it has been reported that less than 1% of all Salmonella spp ferment
lactose (Ewing, 1986). Furthermore, Salmonella isolation from different sources with routine
selective and differential media utilizes non-lactose fermentation as a key biochemical property and
commonly used differential plating media for isolation of Salmonella contains lactose. Salmonella
serovars are considered resilient microorganisms that readily adapt to extreme environmental
conditions. Optimum temperature for growth is in the range of 35 – 37oC but some can grow at
temperatures as high as 54oC and as low as 2oC (Gray and Fedorka-Cray, 2002). Salmonella grow
in a pH range of 4 - 9 with the optimum being 6.5 – 7.5. They require high water activity (aw) for
growth (> 0.94) but can survive at aw of < 0.2 such as in dried foods. Inhibition of growth occurs at
temperatures < 7oC, pH < 3.8 or aw < 0.94 (Hanes, 2003; D’Aoust and Maurer, 2007). The outer
membrane (OM) of Salmonella, as with almost all Gram-negative bacteria, is composed of outer
membrane proteins (OMPs) and lipopolysaccharides (LPS). LPS plays an essential role in
maintaining the cell’s structural integrity and protection from chemicals. In the host organisms, they
act as endotoxins and as a pyrogen displaying a strong immune response. Structurally, they are
composed of three distinct components: lipid A, core oligosaccharide and O-polysaccharide (Bell
and Kyriakides, 2002; Raetz and Whitfield, 2002).

4. RAPID DETECTION METHOD FOR SALMONELLA

Polymerase chain reaction (PCR)

Nucleic acid (DNA or RNA) based methods have become very popular for rapid detection of
pathogens. The first in vitro amplification of mammalian genes using the Klenow fragment of
Escherichia coli DNA polymerase was carried out by Kary Mullis (Mullis and Faloona, 1987). This
assay is now popularly known as polymerase chain reaction (PCR).
The polymerase chain reaction is a method which produces multiple copies of a target DNA.
The PCR method uses a thermostable polymerase enzyme (Taq polymerase) to create multiple
copies of target DNA. Detection of target DNA is achieved through the use of short sections of
synthetic, single stranded DNA known as oligonucleotide primers. These primers can be designed
to be specific for an individual organism, or for a group of organisms (Simon, 1999).
PCR also works by using a cycling of different temperatures. It also requires the target
template DNA, primers, dNTPs and Taq polymerase (Tenover et al., 1997). This large number of a
target DNA segment can then be detected using standard detection methods such as agarose gel
electrophoresis or membrane hybridization. The ability of PCR to produce extremely large numbers
of copies of a specific nucleic acid segment provides the requirements for the rapid, very sensitive
and specific detection of desired microorganisms in a water sample.
PCR holds great potential for the direct detection of microbial pathogens and detection of
virulence genes in water and wastewater (Malorny et al., 2003a; del Cerro et al., 2002). Specific
nucleic acid primers already exist for most of the major waterborne pathogens and have been
proven to be specific for these organisms. It is both highly specific and sensitive and is capable of
detecting very small numbers of microorganisms in a sample. In addition, multiple primers can be
used to detect different pathogens in one multiplex reaction (Ziemer and Steadham, 2003;
Moganedi et al., 2007; El-Lathy et al., 2009).
PCR based methods have been found to be very sensitive for detection of Salmonella spp in
environmental water samples and other sources (Way et al., l993; Pathmanathan et al., 2003;
Aurelie et al., 2005). PCR based detection assays for rapid and specific detection of Salmonella in
wastewater were compared with conventional method and reported; PCR method was comparable
to the culture method (Fricker and Fricker, 1995; Simon, 1999).
 International Letters of Natural Sciences Vol. 47 57

PCR does not require the culturing of microorganisms and therefore can improve detection
efficiency, time and labor. It negates the requirement for indicator organisms as pathogenic
microorganisms can be directly detected from a wastewater sample. PCR also has the advantage of
being able to be used to determine the viability of a microorganism and thus, is not restricted by
dormancy status or the ability to culture the microorganism (Simon, 1999).
The use of PCR as a routine surveillance tool in the water industry still remains a potential for
the future. At present, the costs and expertise required to use these techniques remain prohibitive for
most laboratories. Rapid advances, however, have recently been made in a number of these problem
areas, promising the potential for viable solutions in the near future.

5. SALMONELLA TYPING METHODS

Biotyping
Salmonella strains in a particular serovar may be differentiated into biotypes by their utilization
pattern of selected substrates such as carbohydrates and amino acids. In many serovars there are few
biochemical tests in which significant numbers of strains behave differently and so the number of
identifiable biotypes within a serovar can be obtained. The organisms expressing different
phenotypes of a given serotype are considered a different biotype, and these differences can be
associated with differences in virulence properties (Anderson and Ziprin, 2001).
Duguid et al. (1975) developed a scheme for biotyping to study the epidemiology of infections with
Salmonella typhimurium. This scheme was based on the use of 15 biochemical characters. Thirty-
two potential primary biotypes were defined by the combinations of positive and negative reactions
shown in 5 tests (d-xylose, m-inositol, l-rhamnose, d- tartrate and m-tartrate) most discriminating in
Salmonella typhimurium. These primary biotypes were designated by numbers (1-32) and the full
biotypes were developed by an additional 10 secondary tests and finally a total of 24 primary and
184 full biotypes have been identified.
Recently, de la Torre (2005) used the biochemical kinetic data to determine strain relatedness
among Salmonella enterica subsp. enterica isolates. Different biochemical tests results were used in
the determination of strain relatedness among different serovars of Salmonella enterica subsp.
enterica (59 Salmonella typhimurium strains, 25 Salmonella typhimurium monophasic variant
strains, 25 Salmonella anatum strains, 12 Salmonella tilburg strains, 7 Salmonella virchow strains, 6
Salmonella choleraesuis strains, and l Salmonella enterica (4,5,l2::) (Hoszowski and Wasyl, 2001).

Serotyping
The basis of Salmonella serotyping depends upon the complete determination of the different
antigens; somatic (O), flagellar (H) and capsular (Vi) antigens.
The aim of the serological testing procedure is to determine the complete antigenic formula of the
individual Salmonella isolate. Commercially available polyvalent somatic antisera kits consist of
mixtures of antibodies specific for major antibodies (Herikstad et al., 2002).
Antigen-antibody complexes are formed (agglutination) when a bacterial culture is mixed with a
specific antiserum directed against bacterial surface components. The complexes are usually visible
to the naked eye which allows for easy determination of O and H antigens by slide agglutination.
Some cultures are monophasic and may be directly H-typed, whereas the second phase in a diphasic
culture is determined after phase inversion. After full serotyping of the Salmonella culture the name
of the serotype can be determined by using the Kauffmann-White Scheme. The serological typing
of Salmonella has led to identification of large number of Salmonella serovars. Currently,
Kauffmann-White scheme recognizes 2610 Salmonella serovars, the majority (2587) belongs to S.
enterica, while the remaining (23 serovars) are assigned to S. bongori (Guibourdenche et al., 2010).
Somatic (O) antigens
These are heat stable antigens which are composed of phospholipid-polysaccharide complexes.
Analysis of O antigens revealed polysaccharide (60%), lipid (20 to 30%), and hesomione (3.5-
4.5%). The nature of terminal groups and the order in which they occur in the repeating units of the
58 Volume 47

polysaccharide chain provide the specificity to the numerous kinds of O antigens (Hu and Kpoecko,
2003).
Somatic antigens are resistant to alcohol and dilute acid. Different variants (smooth, rough) are
prevalent in Salmonella spp and these variations affect the serological typing of Salmonella. In
addition, smooth (S) to rough (R) variations occur in Salmonella (Yousef and Carlstrom, 2003).
The heat stable O antigen consist of lipopolysaccharide-protein chain exposed on the cell surface
and are classified as major and minor antigens. The major category consists of antigens such as
somatic factors O:4 and O:3, which are specific determinants of serogroups like B and E
respectively. In contrast, minor somatic antigenic components, such as O:l2 are nondiscriminatory,
as evidenced by their presence in different serogroups (D’Aoust et al., 2001). These are
heterogeneous structures and the antigenic specificity is determined by the composition and the
lineage of the O group sugars and sometimes mutation affect the sugars leading to new O antigen
(Grimont and Weill, 2007).

Flagellar (H) antigens

H-antigens are heat labile proteins associated with the peritrichous flagella and can be expressed in
one of two phases. These are heat labile antigens that are present in the flagella of Salmonella are
proteineous in nature and are called flagellin (Yousef and Carlstrom, 2003). The flagellin is a
keratinomyosin epiderm-fibrinogen group protein of 40 kDa in molecular weight. The amino acid
content and the order in which these acids present in the flagellins determine the specificity of the
different H antigens. The flagellar agglutination occurs very rapidly and the aggregates formed are
loosely knit and floccular forms (Raetz and Whitfield, 2002).
The phase 1 H-antigens are specific and associated with the immunological identity of the particular
serovars, whereas phase 2 antigens are non-specific antigens containing different antigenic subunit
proteins which can be shared by many serovars. These homologous surface antigens are
chromosomally encoded by the H1 (phase 1) and H2 (phase 2) of the vh2 locus. By convention each
serotype has been denoted by an antigenic formula with the major O antigen, followed by phase l
H-antigen(s), and then phase 2 H-antigen(s). The phase l H-antigens are designated by lowercase
letters and then phase 2 H-antigens by Arabic numerals or some instances by components of e or z
series (Brenner et al., 2000; Grimont and Weill, 2007).

Capsular (Vi) antigens

The capsular antigens are present in Salmonella Typhi, Salmonella dublin and Salmonella paratyphi
A. The Vi antigen could be purified by chemical method. The thermal solubilization of capsular
antigen (Vi) antigen is necessary for the immunological detection of serotypes containing capsular
antigens (Fluit, 2005).
More than 99% of Salmonella strains causing human infections belong to Salmonella enterica
subspecies enterica. Although not common, cross-reactivity between O antigens of Salmonella and
other genera of Enterobacteriaceae do occur. Therefore, further classification of serotypes is based
on the antigenicity of the flagellar H antigens which are highly specific for Salmonella (Scherer and
Miller, 2001). Officially recognized by the World Health Organization (WHO), the Kauffmann-
White diagnostic scheme involves the primarily subdivision of Salmonella into serogroups and
further delineated into serotypes based on the O, H and Vi antigenic formula (Popoff and LeMinor,
2005).
Phage typing
Bacteriophages are the most abundant entities on earth and have contributed a lot to the field of
molecular biology and biotechnology. Many mysteries of molecular biology were solved using
bacteriophages. Bacteriophages are getting enormous amount of attention due to their potential to
be used as antibacterials, phage display systems, and vehicles for vaccines delivery. They have also
been used for diagnostic purposes (phage typing) as well (Clark and March, 2006).
These bacterial viruses have genetic material in the form of either DNA or RNA, encapsulated by a
protein coat (Clark and March, 2006). The capsid is attached to a tail which has fibers, used for
 International Letters of Natural Sciences Vol. 47 59

attachments to receptors on bacterial cell surface. Most of the phages have polyhedral capsid except
filamentous phages (Ackerman, 1998).
Phages infect bacteria and can propagate in two possible ways; lytic life cycle and lysogenic life
cycle. When phages multiply vegetatively they kill their hosts and the life cycle is referred to as
lytic life cycle. On the other hand, some phages known as temperate phages can grow vegetatively
and can integrate their genome into host chromosome replicating with the host for many generations
(Inal, 2003). If induction to some harsh conditions like ultraviolet (UV) radiations occurs then the
prophage will escape via lysis of bacteria (Inal, 2003).
The specificity of phages for bacterial cells enables them to be used for the typing of bacterial
strains and the detection of pathogenic bacteria. Phage typing is also known as the use of sensitivity
patterns to specific phages for precisely identifying the microbial strains. The sensitivity of the
detection would be increased if the phages bound to bacteria are detected by specific antibodies. For
the detection of unknown bacterial strain its lawn is provided with different phages, and if the
plaque (clear zones) appears then it means that the phage has grown and lysed the bacterial cell,
making it easy to identify the specific bacterial strain (Clark and March, 2006).
There are certain other methods which can be employed to detect pathogenic bacteria such as the
use of phages that can deliver reporter genes (e.g. lux) specifically (Kodikara et al., 1991) or using
green fluorescent protein (Funatsu et al., 2002) that would express after infection of bacteria.
Similarly, phages having a fluorescent dye covalently attached to their coats can be used to detect
specific adsorption (Hennes et al., 1995; Goodridge et al., 1999). The detection of some of the
released components such as adenylate kinase (Corbitt et al., 2000) after the specific lysis of
bacteria and the use of antibodies and peptides that are displayed by phages which bind to toxins
and bacterial pathogens specifically can also be used, (Petrenko and Vodyanoy, 2003).
Dual phage technology is another application of phages in detection of bacteria, in which phages are
used to detect the binding of antibodies to specific antigens (Sulakvelidze and Kutter, 2005). Phage
amplification assay can also be used to detect pathogenic bacteria. The technique has most
extensively been used for the detection of Mycobacterium tuberculosis, E.coli, Pseudomonas,
Salmonella, Listeria, and Campylobacter species (Barry et al., 1996).
The applications of phages range from the diagnosis of the disease, through phage typing, and its
prevention (phage vaccine), to the treatment (phage therapy).There is the hope that phages could be
useful to humans in many ways.
6. MOLECULAR TYPING OF SALMONELLA

Conventional culture methods have been popularly employed in identifying and isolating
microorganisms present in wastewater. Unfortunately, as a result of inconsistently expressed
phenotypic traits, these classical typing approaches are often unable to discriminate between related
outbreak strains.
The ability to characterize and determine the genetic relatedness among bacterial isolates involved
in a waterborne outbreak is a prerequisite for epidemiological investigations. Detailed strain
identification is essential for the successful epidemiological investigation of Salmonella outbreaks.
Investigations have relied traditionally on serological and antibiogram techniques. In contrast,
modern typing methods are based on characterization of the genotype of the organism. Thus,
molecular typing or fingerprinting of Salmonella isolates is an invaluable epidemiological tool that
can be used to track the source of infection and to determine the epidemiological link between
isolates from different sources (Rakesh et al., 2009).
Some molecular typing systems can distinguish among epidemiologically unrelated isolates based
on genetic variation in chromosomal DNA of a bacterial species (Swaminathan and Matar, 1993).
Usually, this variability is high, and differentiation of unrelated strains can be accomplished using a
variety of fingerprinting techniques.
The genotypic methods are those methods, which are based on the genetic structure of an organism
and include polymorphisms in DNA restriction patterns based on cleavage of the chromosome. The
digestion of the chromosomal DNA provides variable number of the DNA fragments, thus revealing
60 Volume 47

genetic variations. Genotyping methods are less subject to natural variation, though various factors
may be responsible for genetic variants such as insertions or deletions of DNA into the
chromosome, the gain or loss of the extra chromosomal DNA, and random mutations that may
create or eliminate restriction sites (Tenover et al., 1997).
There is currently no gold standard typing system available for Salmonella fingerprinting, however,
the combination of different genotyping methods such as plasmid profile analysis, ribotyping,
characterization of virulence factors in Salmonella serovars, enterobacterial repetitive intergenic
consensus sequence analysis (ERIC-PCR), random amplified polymorphic DNA (RAPD) and
pulsed field gel electrophoresis methods have been evaluated for more precise subtyping of
Salmonella serovars (Mohand et al., 1999; Lagatolla et al., 1996; Shangkuan and Lin, 1998).

Random amplified polymorphic DNA (RAPD)-PCR

Over the last decade, polymerase chain reaction has become a widespread technique for several
novel genetic assays based on selective amplification of DNA (Bardakci, 2001). The popularity of
PCR is primarily due to its apparent simplicity and high probability of success. Unfortunately,
because of the need for DNA sequence information, PCR assays are limited in their application.
The discovery that PCR with random primers can be used to amplify a set of randomly distributed
loci in any genome facilitated the development of genetic markers for a variety of purposes
(Williams et al., 1900; Welsh and McClelland, 1990).
Random Amplification of Polymorphic DNA (RAPD) is a modification of the PCR in which a
single, short and arbitrary oligonucleotide primer, able to anneal and prime at multiple locations
throughout the genome, can produce a spectrum of amplification products that are characteristics of
the template DNA. No knowledge of the DNA sequence for the targeted gene is required, as the
primers will bind somewhere in the sequence, but it is not certain exactly where. This makes the
method popular for comparing the DNA of biological systems that have not had the attention of the
scientific community, or in a system in which relatively few DNA sequences are compared (Senthil
Kumar and Gurusubramanian, 2011).
The simplicity and applicability of the RAPD technique have captivated the interest of many
scientists. Perhaps the main reason for the success is the gain of a large number of genetic markers
that require small amounts of DNA without the requirement for cloning, sequencing or any other
form of the molecular characterization of the genome of the species in question.
The standard RAPD technology utilises short synthetic oligonucleotides (about 10 bases long) of
random sequences as primers to amplify nanogram amounts of total genomic DNA under low
annealing temperatures by PCR. Amplification products are generally separated on agarose gels and
stained with ethidium bromide (Bardakci, 2001).
Welsh and McClelland (1990) independently developed a similar methodology using primers about
15 nucleotides long and different amplification and electrophoretic conditions from RAPD and
called it the arbitrarily primed polymerase chain reaction (AP-PCR) technique. PCR amplification
with primers shorter than 10 nucleotides known as DNA amplification fingerprinting (DAF) have
also been used to produce more complex DNA fingerprinting profiles (Caetano-Annoles et al.,
1991). Although these approaches are different with respect to the length of the random primers,
amplification conditions and visualization methods, they all differ from the standard PCR condition
in that only a single oligonucleotide of random sequence is employed and no prior knowledge of the
genome subjected to analysis is required.
At an appropriate annealing temperature during the thermal cycle, oligonucleotide primers of
random sequence bind several priming sites on the complementary sequences in the template
genomic DNA and produce discrete DNA products if these priming sites are within an amplifiable
distance of each other. The profile of amplified DNA primarily depends on nucleotide sequence
homology between the template DNA and oligonucleotide primer at the end of each amplified
product.
Nucleotide variation between different sets of template DNAs will result in the presence or absence
of bands because of changes in the priming sites. Recently, sequence characterized amplified
regions (SCARs) analysis of RAPD polymorphisms showed that one cause of RAPD
 International Letters of Natural Sciences Vol. 47 61

polymorphisms is chromosomal rearrangements such as insertions/deletions. Therefore,

amplification products from the same alleles in a heterozygote differ in length and will be detected
as presence and absence of bands in the RAPD profile (Bardakci and Skibinski, 1999).
RAPD technique has found a wide range of applications in gene mapping (Hemmat et al., 1994),
population genetics (Chalmers et al., 1992; Kambhampati et al., 1992), molecular evolutionary
genetics (Fani et al., 1993; Naish et al., 1995), and plant and animal breeding (Russel et al., 1993).
This is mainly due to the speed, cost and efficiency of the technique to generate large numbers of
markers in a short period compared with previous methods. Therefore, RAPD technique can be
performed in a moderate laboratory for most of its applications. It also has the advantage that no
prior knowledge of the genome under research is necessary.
Although the RAPD method is relatively fast, cheap and easy to perform in comparison with other
methods that have been used as DNA markers, the issue of reproducibility has been of much
concern. In fact, ordinary PCR is also sensitive to changes in reaction conditions, but the RAPD
reaction is far more sensitive than conventional PCR because of the length of a single and arbitrary
primer used to amplify anonymous regions of a given genome. This reproducibility problem is
usually the case for bands with lower intensity. The reason for bands with high or lower intensity is
still not known. Perhaps some primers do not perfectly match the priming sequence, amplification
in some cycles might not occur, and therefore bands remain faint. The chance of these kinds of
bands being sensitive to reaction conditions of course would be higher than those with higher
intensity amplified with primers perfectly matching the priming sites. The most important factor for
reproducibility of the RAPD profile has been found to be the result of inadequately prepared
template DNA. Differences between the template DNA concentration of two individual’s DNA
samples result in the loss or gain of some bands (Welsh and McClelland, 1994; Bardacki, 1996).
Since RAPD amplification is directed with a single, arbitrary and short oligonucleotide primer,
DNA from virtually all sources is amenable to amplification. Therefore, DNA from the genome in
question may include contaminant DNA from infections and parasites in the material from which
the DNA has been isolated. Special care is needed in keeping the DNA to be amplified from other
sources of DNA.
7. PATHOGENICITY AND VIRULENCE

The nature of pathogenicity of an organism lies in the virulence genes or virulence factors.
However, these terms are still not strictly defined (Wassenaar and Gastraa, 2001). The possible
virulence factors of Salmonella have been understood with the gain in the knowledge on the
molecular mechanism behind the pathogenicity of Salmonella. Recently, the involvement of
effector proteins in the survival and replication of Salmonella in host cells has been elucidated. The
majority of virulence genes of Salmonella are clustered in a region distributed over the
chromosome, called Salmonella Pathogenicity Islands (SPI) (Groisman and Ochman, 1996; Marcus
et al., 2000). Until recently, five SPIs (SPI 1-5) have been identified on the Salmonella
chromosome at centisome 63, 31, 82, 92 and 25 cs, respectively (Blanc-Portard and Groisman,
1997; Hayward and Koronakis, 2002).
Each SPI was responsible in various cellular activities towards the virulence factor of the
organism (Wong et al., 1998; Wood et al., 1998). On completion of genome sequence of
Salmonella Typhi strain CT 18, five more regions were identified and designated as SPI-6, SPI-7,
SPI-8, SPI-9 and SPI-10.
SPI-6 encodes for saf and tcf fimbrial operon and SPI-7 encodes for Vi biosyntheis genes and
also for the IV fimbrial operon (Parkhill et al., 2001; Pickard et al., 2003). The 6.8 kb large SPI-8
encodes for genes conferring resistance to bacteriocin, SPI-9 for type 1 secretion system, whereas
SPI-10 encode for sef fimbrial operon (Galan et al., 1992; Parkhill et al., 2001). The flagella
mediated bacterial motility accelerates but is not required for Salmonella enteritidis invasion in
differentiated Caco-cells (van Asten et al., 2004).
Salmonella virulence factors were also detected in virulence plasmids in certain Salmonella
serovars namely Salmonella abortusovis, Salmonella cholerasuis, Salmonella dublin, Salmonella
62 Volume 47

enteritidis, Salmonella gallinarum, Salmonella pullorum and Salmonella typhimurium, although not
all isolates of these serotypes carry the virulence plasmid (Rotgar and Casadesus, 1999). All
plasmids contain the 7.8 kb Salmonella plasmid virulence (spv) locus. This locus harbored five
genes designated spv RABCD and expressions of spv genes which may play a role in the
multiplication of intracellular Salmonella (Chu et al., 2001). The results showed that spvB together
with spvC conferred virulence to Salmonella typhimurium when administered subcutaneously to
mice (Matsui et al., 2001).
Salmonella Typhi CT 18 exhibited a 106 kb large cryptic plasmid with some homology to a
virulence plasmid of Yersinia pestis. However, the majority of Salmonella typhi tested did not
harbor this plasmid. Cryptic plasmid has also been reported for Salmonella paratyphi C, Salmonella
derby, and Salmonella copenhagan, Salmonella durban, Salmonella give and Salmonella infantis
(Rotgar and Casadesus, 1999). Hybridization analysis has shown a few other serotypes such as
Salmonella johannesburg, Salmonella kottbus and Salmonella newport found to bear the virulence
plasmids.
Salmonella produces both endotoxin and exotoxin and virulence due to these toxins are well
documented. The endotoxin, lipid portion (lipid A) of the outer lipopolysaccharide (LPS) membrane
of Salmonella elicits a variety of in vitro and in vivo biological responses. The best studied exotoxin
of Salmonella was the heat labile Salmonella enterotoxin (stn) of approximately 29 kDa encoded by
stn gene (Prager et al., 1995; Portillo, 2000). A study on 90 kDa heat labile enterotoxins of
Salmonella typhimurium was also reported by Rahman and Sharma (1995). The role of fimbriae and
the flagella of Salmonella have been well identified in the attachment and movement of the
organism but their roles in pathogenesis are still not properly understood (Folkesson et al., 1999;
Edwards et al., 2000; Portillo, 2000).
Characterization of different virulence factors in Salmonella serotypes have been carried out
by amplifying different gene sequences responsible for specific phenotypic properties. The
amplification of invA gene by PCR indicates the presence of invasion gene in Salmonella serovars.
A PCR based study demonstrated that stn gene was present in all Salmonella enterica serovars,
whereas it was absent in Salmonella bongori (Prager et al., 1995). The cumulative effects of
virulence by these genes were found to be responsible for invasion to the epithelial cells of intestine
and thereafter leading to gastrointestinal disorder. PCR assays for several virulence (inv, him) and
functional (iroB, fimY) genes were developed for detection of Salmonella in the environment, in
food or faeces samples (Bej et al., 1994; Baumler et al., 1998; Yeh et al., 2002; Malorny et al.,
2003a). The fliC gene also has been successfully used for molecular typing studies on Salmonella,
based on high variability of the central region (Dauga et al., 1998).
8. SALMONELLA; RESERVOIRS AND EPIDEMIOLOGY

The primary reservoir of Salmonella is the intestinal tract of birds and animals, particularly of
poultry and swine. The organisms are excreted in faeces from which they may be transmitted by
insects and other creatures to a large number of places such as water, soils and kitchen surfaces.
There are host adaptations patterns among serovars, namely; highly host adaptive, less host adaptive
and non-host adaptive (Ecuyer et al., 1996). Human host adaptive serovars include Salmonella typhi
(causative agent of typhoid fever); in contrast, the highly host adaptive chicken pathogens viz.,
Salmonella pullorum and Salmonella gallinarum are not human pathogen. There is no report of
Salmonella typhi host range extending beyond human beings. Hence, isolation of Salmonella typhi
from food or water must be indication of contamination from human beings. Other Salmonella
serovars are found to be host adapted animal pathogens and sources of zoonotic infections (Ziprin
and Hume, 2001).
Salmonella choleraesuis is a pathogen of swine but sometime causes severe systemic
infections in humans (Ziprin, 1994; Wang et al., 1996). Similarly, Salmonella dublin may cause
septicemia in cattle and can be transmitted to humans from milk and milk products (Reher et al.,
1995). Salmonella enteritidis and Salmonella senftenberg are host adapted to chicken and turkey
respectively. Some Salmonella serovars are not host adapted and also tend to be less virulent than
 International Letters of Natural Sciences Vol. 47 63

the host-adapted serotypes, but they are found to be responsible for an overwhelming number (90%)
of incidents of human salmonellosis (Webber, 1996; Hunter, 1997).
Typhoid cases are stable with low numbers in developed countries, but non typhoidal
salmonellosis has increased worldwide. Typhoid fever usually causes mortality in 5 to 30% of
typhoid-infected individuals in the developing world. The World Health Organization (WHO)
estimates 16 to 17 million cases occur annually, resulting in about 600,000 deaths. The mortality
rates differ from region to region, but can be as high as 5 to 7% despite the use of appropriate
antibiotic treatment (Scherer and Miller, 2001).
In Nigeria, typhoid fever is among the major widespread diseases affecting both young
children and young adults as a result of many interrelated factors such as inadequate facilities for
processing human wastes and indiscriminate use of antibiotics. Morbidity associated with illness
due to Salmonella continues to be on the increase, in some cases resulting in death (Talabi, 1994;
Akinyemi et al., 2005). A more accurate figure of salmonellosis is difficult to determine because
normally only large outbreaks are investigated whereas sporadic cases are under-reported (Scherer
and Miller, 2001; Parry, 2006). On the other hand, non typhoidal cases account for 1.3 billion cases
with 3 million deaths (Hanes, 2003; Hu and Kopecko, 2003).
The infectious dose of Salmonella depends upon the serovar, bacteria strain, growth condition
and host susceptibility. On the other hand, host factors controlling susceptibility to infection
include; the condition of the intestinal tract, age and underlying illnesses or immune deficiencies.
The infectious dose of Salmonella is broad, varying from 1-109 cfu/g. However, single-food-source
outbreaks indicate that as little as 1 to 10 cells can cause salmonellosis with more susceptibility to
infection by YOPI (Young, Old, Pregnant and Immunocompromised) groups (Yousef and
Carlstrom, 2003; Bhunia, 2008).
Information about incidence and serovars distribution of Salmonella in domestic animal
populations is essential for understanding the relationships within and among reservoirs of
Salmonella in animals and humans that are ultimately responsible for zoonotic disease transmission
(Gast, 1997). Salmonella infection is usually acquired by the oral route, mainly by ingesting
contaminated food or drink. Salmonella can be transmitted directly from human to human or from
animal to human without the presence of contaminated food or water, but this is not a common
mode of transmission.
9. MECHANISM OF SPREAD

Transmission of Salmonella to humans traditionally has been attributed to contaminated

animal-product foods, but epidemiological studies have demonstrated that cases are sporadic and
may more likely involve environmental sources than previously thought. It has been suggested that
contaminated soils, sediments and water as well as wildlife may play a significant role in
Salmonella transmission (Schutze et al., 1998). Moreover, geographic clusters of cases in which no
verifiable food source have been determined, such as those recently caused by S. javiana in the
southeastern US which do not follow the same geographic patterns as cases which have been linked
to a known food source but rather mimic amphibian distribution patterns (Srikantiah, 2004). S.
typhi, which is only transmitted from human to human, is most common in developing nations
where access to safe drinking water may be limited and waste disposal and treatment may be
inadequate (Velema et al., 1997).
First, environment contaminated with Salmonella serves as the infection source because
Salmonella can survive in the environment for a long time. Subsequently, Salmonella is transmitted
to vectors such as rats, flies and birds where Salmonella can be shed in their faeces for weeks and
even months. Following the direct transmission, moving animals such as swines, cows and chickens
act as the important risk factor for infection. These animal reservoirs are infected orally because
Salmonella normally originates from the contaminated environment and also contaminated feed.
Human get infected after eating food or drinking water that is contaminated with Salmonella
through animal reservoirs. However, Salmonella typhi and Salmonella paratyphi A do not have
animal reservoir, therefore infection can occur by eating improperly handled food by infected
64 Volume 47

individuals (Newell et al., 2010). Besides, transmission of Salmonella to the food processing plants
and equipments for food preparation are also of great importance. Once carried by vectors or
transferred to food, consumption by human can result in the risk of salmonellosis. The Salmonella
cells can attach to food contact surfaces such as plastic cutting board which may develop into
biofilm once attached and hence cause cross-contamination. Consequently, Salmonella can enter the
food chain at any point from livestock feed, through food manufacturing, processing and retailing as
well as catering and food preparation in the home (Wong et al., 2002).
Spread of Salmonella may be facilitated in water storage tanks in a building, from wild animal
feces or even from carcasses. Poor sanitation, improper sewage disposal and lack of clean water
system cause the transmission of typhoid fever. In areas where typhoid fever is endemic, water from
lakes or rivers which are used for public consumption and are sometimes contaminated by raw
sewage are the main sources of infection. The consumption of unboiled water during 1997 typhoid
outbreak in Dushanbe, Tajikistan caused 2200 cases of illness and 95 deaths (Penteado and Leitão,
2004; Bordini et al., 2007).
Salmonella contamination of fresh produce could be due to the entry of Salmonella through
scar tissues, entrapment during embryogenesis of produce, natural uptake through root systems and
transfer onto edible plant tissues during slicing. The human health risk is increased further by
Salmonella preference to grow on fresh produce during retail display at ambient temperature. In
2000, cantaloupe from Mexico resulted in a Salmonella Poona outbreak in USA (Penteado and
Leitão, 2004; Bordini et al., 2007). The table below shows the prevalence of predominant
Salmonella serotypes from different sources (Table 1).

Table 1: Prevalence of Salmonella species from different sources

Country Sample/source Prevalence Predominant Reference
serotypes
Iran Chicken 86/190 (45.3%) Thompson Dallal et al., 2010
Beef 38/189 (20.1%)
Brazil Poultry carcass 0/127 (0%) Enteritidis Freitas et al., 2010
Poultry viscera 2/73 (2.7%)
Spain Pig 43/804 (5.3%) Anatum, Gomez-Laguna et al., 2010
Herd 22/67 (32.8%) Typhimurium
China Chicken 276/515 (53.6%) Enteritidis, Yang et al., 2010
Pork 28/91 (30.8%) Typhimurium,
Beef 13/78 (16.7%) Shubra, Indiana,
Lamb 16/80 (20%) Derby, Djugu
Morocco Slaughter house 75/105 (71%) Infantis, Bredeney, Bouchrif et al., 2009
Sea food 10/105 (9.5%) Blokley,
Typhimurium,
Mbandaka,
Branderup II,
Kiambu
Bangladesh Chick egg 4/80 (5%) Typhimurium Hasan et al., 2009

Republic of Retail pork 13/500 (2.6%) Typhimurium Prendergast et al., 2009

Ireland
Turkey Chicken part 1/168 (0.6%) Typhimurium Centinkaya et al., 2008
Minced meat 0/45 (0%) Infantis
Ready-to-eat-salad 0/100 (0%)
Raw vegetable 0/78 (0%)
Raw milk 0/25 (0%)
Iran Raw poultry 24/134 (17.9%) Enteritidis, Jalali et al., 2008
Cooked poultry 3/56 (5.4%) Baibouknown
Raw meat 8/101 (7.9%)
Cooked meat 2/118 (1.7%)
Turkey 1/3 (33.3%)
Quail 2/5 (40%)
Vegetable 3/38 (7.9%)
Lithuania Faeces 28/85 (32.9%) Enteritidis, Pieskus et al., 2008
Caecum 12/52 (23.1%) Typhimurium
Dust 5/34 (14.7%)
Water 1/10 (10%)
Turkey Tulum cheese 6/250 (2.4%) Colak et al., 2007
 International Letters of Natural Sciences Vol. 47 65

Vietnam Pork 32/50 (64%) London, Havana, Van et al., 2007

Beef 31/50 (62%) Anatum, Hadar,
Chicken 16/30 (53.3%) Albany,
Shell fish 9/50 (18%) Typhimurium
Malaysia Street food 12/129 (9.3%) Biafra, Braenderup, Tunung et al., 2007
Fried chicken 1/18 (5.6%) Weltevreden
Kerabu jantung 3/5 (60.0%)
pisang 6/9 (66.7%)
Sambal fish 2/5 (40%)
Mix vegetable
Brazil Chicken abattoir 29/288 (10.1%) Enteritidis, Cortez et al., 2006
Typhimurium
South India Egg 38/492 (7.7%) Enteritidis Suresh et al., 2006
Jordan Chicken, meat 25/93 (26.9%) Enteritidis, Malkawi and Gharaibeh, 2004
Typhimurium
Malaysia Selom 16/43 (37.2%) Weltevreden, Salleh et al., 2003
Pegaga 8/26 (30.8%) Agona,
Kangkong 8/25 (32%) Senftenberg,
Kesum 8/18 (44.4%) Albany
Albania Chicken meat 30/461 (6.5%) Enteritidis Beli et al., 2001
sample
India Fish 104/730 (14.3%) Weltevreden, Hatha and
Crustacean 48/276 (17.4%) Typhi, Paratyphi B, Lakshamanaperumalsamy, 1997
Mgulani,
Typhimurium
Malaysia Retail poultry 158/445 (35.5%) Enteritidis, Rusul et al., 1996
Litter 8/40 (20.0%) Muenchen,
Poultry farm 2/10 (20.0%) Kentucky,
Blockley
Malaysia Chicken portion 13/33 (39.4%) Blockley, Arumugaswamy et al., 1995
Chicken liver 6/17 (35.3%) Enteritidis,
Chicken gizzard 8/18 (44.4%) Chincol,
Cooked meat, Muenchen, Agona
chicken 4/28 (14.3%)
Vegetable 14/60 (23.3%)
Safay 4/16 (25%)
Prawn 2/19 (10.5%)
Oriental shrimp
paste
(Source: Pui et al., 2011)
10. CONCLUSION

The prevalence of diseases caused by salmonella has indeed assumed a public health
dimension, salmonellosis is one of the leading cause of diarrhea diseases globally and is directly
associated with poor water hygiene and availability coupled with contamination of food. In Nigeria,
Typhoid fever caused by Salmonella spp is a major cause of death, second only to malaria. Current
clinical diagnosis of typhoid fever using widal test relying on the antigen-antibody agglutination is
often not reliable and hence leading to false test results. Given the high incidence of diseases caused
by Salmonella, it is necessary to understand wholly this highly virulent and pathogenic organism,
its epidemiology and pattern of spread with a view to promoting better and early detection which
certainly would spare mankind the stress and burden of these diseases. Molecular techniques which
will guarantee quick and reliable diagnosis should also be introduced and adopted by hospitals,
diagnostic laboratories and other health professionals as a means for better health service delivery.
It is also recommended that Salmonella research institutes and organizations be established as a
means of broadening general understanding of this organism and the various diseases it causes.
66 Volume 47

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