INTRODUCTION

Salmonella has been long recognsized as an important pathogen of

human, animals and poultry. The organism has its global distribution and

usually causes enteric disease in human and animals through ingestion of

contaminated food/feed mostly of origin (Sharma and Singh, 1997). Non-

typhoidal salmonellosis is an important food borne infection with an estimated

incidence of 1.3 billion cases and 3 million deaths all over the world (Thong et

al., 1995).

Enteric Salmonella infection is a global problem both in human beings

and animals. Newborn and young animals commonly suffer from enteric

infection within 15 days of their birth (Kaura et al., 2001). Enteric disorders

that occur at organized animal farms in this country account for 10 to 30% of

annual mortaility and thus are responsible for great economic losses. In human,

diarrhoeal syndrome caused by non-typhoidal salmonella is common in tropical

and developing countries. Available data on food poisoning indicate that

poultry meat is frequently involved in cases of human salmonellosis (Galbraith,

1990).

Although, many of routine epidemiological features viz. host specificity,

sources of infection, the forms of disease manifestation and carrier status of the

organisms have been documented (Wray and Sojka, 1997; Thapliyal, 1979).

Some new epidemiological parameters are being studied in recent years to

elucidate the disease process of salmonellosis. Among these, antimicrogram

1

studies and plasmid profile are of major importance. On study of

epidemiological feature of salmoellosis, plasmids have been reported to play an

important role in virulence and antibiotic resistance of Salmonella (Aarestrup

et al., 1997).

Bacteria are among the chief causal agents of acute diarrhoea and the

majority of the bacterial enteropothogens appear to cause fluid lose by

stimulation enterocytes to actively secrete electrolytes (Rao and Field, 1985).

Elaboration of one or more enterotoxins is recognized as an important

pathological attribute of some non-invasive diarrhoea including bacteria in

elucidating of their enteropoathogenic activity (Rao and Field, 1985; Harne et

al., 1994). Various Salmonella serotypes have been shown to produce

enterotoxins (Baloda et al., 1983; Rahman et al 1991a). The enterotoxic moiety

from salmonella serotypes has been purified and characterized by several

workers (Finkalstein et al., 1987; Rahman et al., 1994). Salmonella

pathogenesis is a complex and multifactorial phenomenon. Salmonella species

interact with ileal mucosa and disrupt normal intestinal functions, which result

in acute inflammatory cell influx, fluid secretion and enteritis (Wood et al.,

1998). Many genes are required for full virulence but only a few of these have

been shown to be necessary for the induction of enteritis (Wallis and Galyov,

2000). Like many other gram-negative pathogenic bacteria, Salmonella

possesses a dedicated protein secretion system denoted as Type III Secretion

System (TTSS), which plays a central role in virulence (Huech, 1998).

Salmonella serovars have been reported to produce several Type III secretions

2

including SIP (Salmonella Invasion Protein) and Sop (Salmonella outer

protein). Of the different Sop proteins, Sop B is associated with enteritis and

diarrhoea. The gene encoding production of Salmonella outer protein (Sop B)

is located on Salmonella specific DNA fragment representing a pathogenicity

island, SPI 5 (Wood et al., 1998) and is found to be widely distributed among

Salmonella (Rahman et al., 2001).

Another important type III secretion is Sop E, which is associated with

invasion by stimulating membrane ruffling (Hard et al., 1998). Unlike Sop B

gene, the Sop E is not widely distributed among Salmonellae and diarrhoea and

as the cases of this kind is frequently observed in livestock and poultry as well

as in human.

Apart from these outer proteins an important pathogenicity is exhibited

by Salmonella species i.e. Salmonella enterotoxin. Although most of the

Salmonella serovars were shown to produce enterotoxins, enterotoxigenicity of

Salmonella has been found difficult to detect by different assays since

Salmonella produce very level of enterotoxin when cultured by conventional

methods (Baloda et al., 1983). Moreover, a major portion of Stn is retained as

cell-bound content, which is not released in the extrcellular milieu (Kaura and

Sharma, 1988). These are some Salmonella strains, which produce enterotoxin

but do not release extracellularly and thus may escape different tests and

detected to be non-enterotoxigenic if only entracellular occurrence of Stn is

tested (Rahman et al., 1991a).

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Production of the enterotoxin (Stn) has been found to be mediated by the

presence of Stn gene and it has been cloned and sequenced (Chary et al., 1993;

Chopra et al., 1994). More recently, the Polymerase Chain Reaction (PCR) has

been used for the detection of Stn gene (Prager et al., 1995; Rahman, 1999). Its

detection by the PCR in field isolates of various Salmonella can indicate the

protential of Stn production. Use of the standard PCR technique to study the

presence of Stn gene among the field isolates can an important tool in the

molecular characterization of field Salmonella isolates.

The epidemiology and pathogenic process in salmonellosis is dictated

by an array of factors that act in tandem and ultimately manifest into the typical

symptoms of salmonellosis. In recent years, a number of virulence-associated

fimbriae including Salmonella Enteritidis Fimbriae designated as SEF and

Plasmid Encoded fimbriae designated as PEF have been identified and cloned

(Clouthier et al., 1994). However, information on the occurrence of these

fimbriae among the field isolates is still meagre (Rahman et al., 2000a). Both

the above types of fimbriae are genetically encoded and its characterization can

help in understanding the role of these genes in expression of these virulence

factors.

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AIM AND OBJECTIVES

The aim of the project was undertaken with the following objectives

with given 50 samples:

I. To study the Antimicrogram patterning of the given isolates.

II. To study some of the molecular characteristics of the given

isolates such as

III. Detection of Sop B gene.

IV. Detection of fimbrial genes (Sef and Pef).

V. Detection of salmonella enterotoxin gene (Stn)

VI. Detection of Sop E protein by Dot-ELISA.

VII. Study on plasmid profiles.

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REVIEW OF LITERATURE

Salmonella

The importance of bacteria of the genus Salmonella as potential

pathogens of human and animals needs no emphasis. Salmonella are widely

distributed in nature. Currently, there are more than 2400 Salmonella serotypes

prevalent in the world. A few of them are highly host specific, while majority

of them are unadapted and can cause infection in a wide variety of animals

species (Gupta and Verma, 1993). They are the chief cause of diarrhoeal

disease all over the world (Gianella, 1980). Newborn and young animals

commonly suffer from enteric infections within 15 days of their birth (Kaura et

al., 2001). Enteric disorders that occur at organized animal farms account for

10 to 30% of annual mortality in our country and are thus responsible for great

economic loss Kaura et al., (2001).

The genus name Salmonella has been adopted in the honor of Salmon,

who isolated the “hog cholera bacillus” considered to be the causal agent of

swine plague (Salmon and smith, 1885) but subsequently the organism was

found to be only a secondary invader and named as Salmonella Choleraesuis.

The typhoid bacillus was first seen by Eberth (1880) in the spleen and

mesenteric gland of patient that died of typhoid and the organism was isolated

by Gaffkey (1884) and Schotmuller (1901) differentiated Salmonella

Paratyphi A and Salmonella Paratyphi B.

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The genus Salmonella of the Enterobacteriaceae family consists of

Gram-negative bacilli, fermentative, aerobic or facultative anaerobic bacteria

that are generally motile with peritichous flagella except Salmonella pullorum

and Salmonella gallinarum. The organisms of the genus are non-lactose

fermenters, oxidase negative, urease negative acetyl carbinol negative,

potassium cyanide negative but citrate positive (Sleigh and Duguid, 1989).

Nomenclature of Salmonella species

Nomenclature and classification of Salmonella are ever changing. In

recent years, the Kauffman- white Antigenic scheme as proposed by Ewing

(1986) has been modified and updated and this updated version is being

accepted by majority of the laboratories worldwide.The members of of the

genus Salmonella have been grouped in two species, viz. Salmonella bongori

containing 18 serovars and Salmonella enterica consisting of more than 2400

serovars (Popoff and lallinov, 1997). Salmonella enterica has been further

divided into subspecies, viz. Salmonella enterica subsp. arizone (IIIa or 3a),

Salmonella enterica subsp. diarizone (IIIb or 3b), Salmonella Enterica subsp.

houtenae (IV or 4) and Salmonella Enterica subsp. indica (VI or 6). According

to the latest nomenclature, the names of the serovars are not italicized (Popoff

and Lellinov, 1997) and the first letter of the serovar is written in capital hand.

For example, the strain that used to be identified, as Salmonella typhimurium is

now known as Salmonella typhimurium

Antimicrogram patterning

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Until the fifties, nearly all Salmonella were sensitive to a wide range of

antimicrobial agents. However, due to indiscriminate use of antibiotics

worldwide both in humans and animals as medication or in animals feeds,

plasmid mediated resistance has appeared in them throughout the world. Huey

and Edwards (1958) demonstrated resistance to Tetracyline in Salmonella

strains of Salmonella Typhimurium isolated before 1948, when antibiotics were

not in animal feeds and after 1956 against Tetracyline. It was observed that 5%

of human and 9% of fowl strains exhibited resistance to the antibiotic since the

time Tetracycline was incoprated in the feed. Similarly, Yurack (1964) showed

that 54 of 644 Salmonella cultures exhibited resistance to one or more of

Tetracyline, Chloramlhenicol and Ampicillin. Further, it was found that

Salmonella Typhimurium showed much higher resistance to Chloramphrnicol.

Sojka et al., (1972) made a survey on drug resistance of 2143 Salmonella

strains comprising of 44 serotypes isolated from animals (1329 from cattle, 511

from poultry, 183 from sheep, 74 from pigs and 46 from other species of

animals and miscellaneous sources) in England and Wales. The most common

serotype tested was Salmonella Dublin (925 strains) and Salmonella

Choleraesuis (483 strains). Of these strains, 7.8 % were sensitive to all the

drugs under test. Majority of the strains (91.3%) were resistant to

Sulphadimidine, one strain to Chloramphenicol, 0.2 per cent to Furazolidone,

13.4% to Sulphathiazole, 3.5 percent to Oxytentracycline, 1.2 percent to

Ampicillin, 0.6 % to kanamycin and 0.5 % to polymyxin B. But none of the

strains isolated from poultry was resistant to Streptomycin. In another study,

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Sojka et al., (1974) isolated Salmonella organisms from animals in England

which showed resistance to Streptomycin (80.9%) Sulphonamide (54.5%),

Ampicillin ((3.7%), Furazolidone (1.7%), Chlortetracycline (1.3%),

Chloramphenicol and Neomycin (0.45 each). However, none of the strains

were resistant to Trimethoprim. Sethi et al., (1976) studied the resistance

pattern of 104 Salmonella strains to Chloramphenicol and reported that strains

isolated from animals and poultry did not develop resistance to

Chloramphenicol in India as compared to some other parts of the world.

In India, Kaliannan and Gupta (1977) studied the drug resistance pattern

of 90 Salmonella strains comprising of 11 serotypes isolated from human and

animals against Chloramphenicol, Tetracycline, Kanamycin, Streptomycin and

Ampicillin and stated that 4 out 10 Chloramphenicol resistant strains were also

resistant to Tetracycline and streptomycin. Mc Garr et al, (1977) studied the

drug sensitivity of 219 Salmonella strains from poultry to 16 antimicrobial

agents. All the strains were sensitive to Ampicillin, Trimethoprim-

Sulphamethoxazole complex, Furazolidone, Cephaloridine, Amoxycillin and

resistant to Tetracycline and Streptomycin. Upadhyay and Misra (1978)

reported that Salmonella Weltevreden isolated from buffalo, goat, pigs and

poultry were sensitive to Ampicillin, Tetracycline, Oxytetracyline,

Streptomycin, Chloramphenicol, Kanamycin and Nitrofurantoin, but were

resistant to Bacitracin. Ishiguro et al., (1979) reported Salmonella strains

resistant to Tetracycline, Streptomycin and sulphadimethoxine from faecal

samples of pigs. Further (Ishiguro et al., 1980) studied that 59% of 84

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Salmonella isolates were resistant to one or more antibiotics such as

Tetracycline, Streptomycin, Sulphadimethoxine and Cholramphenicol. Anon

(1980) reported resistance of Salmonella to Chloramphenicol, Streptomycin,

Chlortetracyline, Neomycin, Ampicillin and Furazolidone. Gyurov (1980)

demonstrated the drug resistance of 326 Salmonella strains isolated from

poultry. No strain was found to be resistant to Gentamicin, whereas, only one

was resistant to Kanamycin. Of the 326 strains, 1.57% was resisitant to

Carbenicillin, 2.45 percent to Chloramphenicol, 2.76 percent to Streptomycin,

27.6% to Tetracycline and 5.85% to Furazolidone. All were resistant to

Oxacillin and majority to Erythromycin, Penicillin and Ampicillin.

Koshi (1981) reported that strain of Salmonella Typhimurium isolated in

Southern India showed alarming increase in multiple drug-resistances. The

overall drug resistance of Salmonella Typhimurium increased from 59.6 % in

1978 to 90.6 % in 1979 and to 97.1 % in 1980. But the resistance against 5 to 9

drugs (Ampicillin, Chloramphenicol, Gentamicin, Kanamycin, Neomycin,

Streptomycin, Sulfonamide, co-trimoxazole and Tetrcycline) increased

alarmingly from 38.9 percent in 1978 to 83 % in 1979 and 95.5 % in 1980.

Nabburt et al., (1981) reported susceptibility of 333 strains of Salmonella to

eight antimicrobial agents. It was observed that 99.4 % of the strains were

sensitive to Cephalothin, 99 % to Furadione, 98.8 percent to Chloramphenicol,

98.2 % to Ampicillin, 96.7 % to Polymxin, 95.8 % to Kanamycin and 91 % to

Streptomycin. Only 50.4 % of the strains were susceptible to Tetracycline,

whereas 44.1 percent were intermediate sensitive and 5.4 % were resistant to

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this drug. Gupta et al. (1983) revealed that 44.0 % of 261 Salmonella strains

were resistant to Tetracycline, 9.2 % to Chloramphenicol and 3.8 % to

Ampicillin. In all, 59.7 % and 97.6 % strains were senitive to Chlormphenicol

and Nitrofurantoi, respectively. Hsu et al., (1983) reported that out of 98

isolates of pig including Salmonella Choleraesuis and Salmonella

Typhimurium examined for drug susceptibility, 94 (95.5%) were resistant to

two or more drugs. The percentage of isolates resistant to individual drugs were

as follows: 95.9 percent to Sulphathiazole, 89.8 percent to Tetracycline, 76.5

percent to Streptomycin, 56.1 percent to Kanamycin, 53.1 percent to

Chloramphenicol and 45.9 percent to Ampicillin. However, all the isolates

were sensitive to Colistin, Gentamicin and Nalidixic acid. Blackburn et al.,

(1984) observed multiple resistance in 80 % Salmonella recovered from

animals. The percentage was higher in the isolates from swine. Similarly, Pohl

et al., (1984) found that multiple drug resistance was common in Salmonella

Typhimurium isolates from animals, meat and feeds.

Kim et al., (1984) studied drug sensitivity of Salmonella isolated from

chickens in Korea and reported that 65 isolates were sensitive to Ampicillin,

Colistin, Chloramphenicol, Gentamycin, Kanamycin and Trimethoprim-

Suplhamethoxazole, while 70.8 % were resistant to Streptomycin, 5.39 % to

Sulphonamides, 41.5 % to Tetracycline and 4.6 % to Nitrofurantoin. Siddique

et al., (1985) reported that of 111 strains of salmonella isolated from poultry,

majority showed sensitivity to Ampicillin, Kanamycin. Gentamycin, Neomycin

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and Streptomycin, while most of them were resistant to Tetracyline, Tylosin,

Trimethoprim, Sulmethoxazole and Furazolidone.

Verma et al., (1993) tested the in-vitro drug sensitivity of 38 Salmonella

Virchow strains identified at the National Salmonella Center. All the strains

were sensitive to Contrimoxazole, 97.37 percent to Chloramphenicol, Nalidixic

acid and Streptomycin. All the strains were resistant to Bacitracin,

Chlortetracycline, Oxytetracycline and Sulphamethoxazole. High levels of

resistance were found to Tetracycline (97.37 %), Penicillin (81.58 %),

Erythromycin (78.94 %), Nitrofurantoin (74.74 %) and high High degree of

resistance was due to extensive use of antibiotics. Borah (1994) tested the in-

vitro drug sensitivity of 19 Salmonella strains and reported that overall

susceptibility of the strains was highest (100 %) to Gentamycin,

Chloramphenicol, Nalidixic acid and Norfloxacin. Less sensitivity was shown

to Doxycycline, Furazolidone, Tetracycline and oxytetracycline, while all the

strains were resistant to Amoxycillin.

Narayanswamy et al. (1996) studied the drug sensitivity of Salmonella

isolated from pigs. Six isolates of Salmonella representing two serotypes, viz.

Salmonella Choleraesuis and Salmonella Enteritidis were tested. All the

isolates were highly sensitive to Chloramphenicol, Gentamycin, Nalidixic acid

Doxycycline; five were sensitive to Sulphadiazine, Nitrofurantoin and three to

Tetracycline, Oxytetracycline and Erythromycin.

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In Assam, Rahman et al., (1997) studied the drug sensitivity of eight

strains of Salmonella representing two serotypes, viz. Salmonella Gallinarum

(6) and Salmonella Indian (2). All the strains were sensitive to

Chloramphenicol, Gentamycin and Norfloxacin, seven to Nalidixic acid, six

strains each to Trimethoprim, Streptomycin and Kanamycin, and three strains

to Tetracycline.

Plasmid profile

Plasmids are circular DNA molecules, which exist in the bacterial cells

independent to the chromosome. Plasmids have been known to carry genes

which determine a wide range of biological functions and also impart upon

individual organisms certain characteristic features. Due to this, the role of

plasmids in pathogenesis and epidemiology becomes multifactoral. Conferment

of antibiotic resistance is one such feature induced by plasmid genes. Most

antibiotic resistant bacteria isolated from the field are not chromosomal

mutants but carry the resistance genes on plasmids and many of these plasmids

can be transferred, particularly between different genera of enteric bacteria.

Moreover, many of themspecify resistance to more than one drug and in some

cases, to as many as six or seven unrelated drugs (Williams, 1978). Corpet

(1996) has opined that the use of Antimicrobial Growth Promoters (AGPs) in

animals production is fraught with danger as genetic resistance then the

specific AGPs may be carried on the plasmids and may transfer from animals

to man. In addition to its established role in conferment of antibiotic resistance,

plasmids have been known to play an important role in increased virulence of

13

Salmonella.many serotypes including, Samonella Abortusovis, Salmonella

Choleraesuis, Salmonella Dublin, Salmonella Tyhimurium and Salmonella

Enteritidis have been repoted to possess large virulence-associated plasmids

that range in size from 50 kb to 96 kb (Jones et al., 1998, Popoff et al., 1997).

Most field strains of Salmonella are known to carry plasmids differing

in size and number. The use of plasmids profiles has gained increasing

acceptance in recent years as an alternative means for typing Salmonellae.

Plasmids profile however is not always helpful and sometimes the information

derived from the profile is limited. The demonstration that several strains

harbour and indistinguishable plasmid cannot always be taken as evidence of

their relatedness. In an extensive study carried out by Mohan et al., (1995) on

plasmid profile and antimicrogram pattern of Salmonella Typhimurium strains

encountered in different parts of India, they reported that all except two of the

190 strains were plasmid negative. Among the remaining strains the plasmid

profile could be grouped into as many as 52 different profiles. They found the

considerable variation in the plasmid profiles on regional basis with northern,

central and southern region of India showing 24,12 and 16 different plasmid

profiles respectively. They couldn’t establish any comprehensive correlation

between plasmid profiles and also between antibiotic resistant patterns

Salmonella enterotoxin (Stn)

Non-typhoidal salmonellosis, a disease caused by Salmonella other than

Salmonella Typhi, is an important food-borne infection with worldwide

distribution causing gastroenteritis and diarrhoea (Chalker and Blaser, 1988).

14

Until middle of 1960’s endotoxins of Salmonella were incriminated in the

pathogenesis salmonella infections (Kohler and Bohl, 1966). Taylor and

Wilkins (1961) first observed that strains of Salmonella caused fluid

accumulation in the rabbit intestinal loops but they left the Cell-Free Culture

Supernatant (CFCS) unassayed in the test. Gienella et al., (1974) found that

although strains of Salmonella Typhimurium evoked fluid accumulation

observed in the ligated rabbit ileum, their CFCS and its concentrate were

unable to induce fluid secretion. Sakazaki et al., (1974) observed that out of 13

strains, culture filtrates of 11 and only 4 live cultures evoked fluid secretion in

Rabbit Ligated Ileal Loops (RLIL). But reason for the variability remained

unexplained. Koupall and Deibel (1975) demonstrated that oral administration

of enterotoxic factor of Salmonella Enteritidis in infant mice caused fluid loss.

Rahman (1989) tested CFCS, PDP and purified toxin of Salmonella

Typhimurium for the presence of Permeability Factor (PF). Although delayed

PF were present in all the preparations, rapid PF could not be detected in any of

them. Permeability factor along with induration of rabbit skin was observed

with preparations from Salmonella Typhimurium and Salmonella Enteritidis

(Baloda et al., 1983), and Salmonella Weltevreden (Kaura and Sharma, 1983.

Besides indurations, necrosis was also observed in concentrated CFCS of

Salmonella Enteritidis and Salmonella Typhimurium (Baloda et al., 1983) PDP

and purified Stn of Salmonella Typhimurium (Rahman, 1989). The presence of

enterotoxic activity, CHO cell elongation factor, delayed PF in the same peak

(SG-200 FPC) which yielded a sigle protein band in polyacrylamide gel

15

electrophoresis and the observation that these activities were neutralized by the

antiserum to the purified enterotoxin of Salmonella Typhimurium indicated

that the three were due to a single moiety (Rahman et al., 1994).

Detection of Stn gene by PCR

In recent years, nucleic based diagnostic techniques have been very

popular due to their high sensitivity, rapidity and specificity. The same

techniques are now also being introduced for detection of various gene encoded

virulence factor. Production of the Stn has been found to be mediated by the

presence of Stn gene. The chromosomal Stn gene has been cloned and

sequenced (Chary et al., 1993; Chopra et al., 1994). The PCR has been used for

the detection of Stn gene (Prager et al., 1995, Rahman, 1999b). Its detection in

field Salmonella isolates indicates the potential of Stn production. Use of the

standard PCR technique to study on the presence of Stn gene among the field

isolates can be an important tool in the molecular characterization of field

Salmonella isolates. In a study carried out in 46 strains of 2 species of

Salmonella i.e. Salmonella Typhimurium and Salmonella Enterititidis, Rahman

(1999b) observed that Stn gene could be detected with the help of PCR using

known primers amongst all the serovars of Salmonella Typhimurium and

Salmonella Enteritidis whereas the E. coli serovars negative for Stn gene. He

16

also reported that only 34.6% of the Stn positive organisms as detected by PCR

expressed phenotpically in the CHO assay.

Salmonella outer protein (Sop) B and (Sop) E

Salmonella pathogenesis is a complex phenomenon involving several

pathogenic mechanisms. Salmonella sp. interact with illeal mucosa and disrupt

normal intestinal function, which results in an acute inflammatory, cell influx,

secretion and enteritis (Wood et al., 1998). Galyov et al., (1997) reported that

interactions between intestinal epithelial cells and the pathogens play a key in

mediating the inflammatory response. It has been shown that enteropathogenic

Salmonella strains are able to induce intact intestinal epithelia to recruit sub

epithelial neuttrophils ((Mc Cormick et al., 1995a, 1995b). This strains

epithelial signalling to neutrophils requires the adhesion of Salmonella to the

epithelial signalling to neutrophils requires the adhesion of Salmonella to the

epithelial apical membrane and is dependent on protein synthesis in both

bacteria and eukaryotic cell.

Salmonella entry into nonphagocytic epithelial cells requires several

chromosomal genes (inv/spa) clustered in pathogenicity island termed SPI 1

(Salmonella Pathogenicity Island 1). SPI 1 encodes a (TTSS) type III secretion

system, a dedicated protein secretion system of Salmonella, which plays a

central role in the virulence.

17

Salmonella serovars have been reported to produce several type III

secretions including Sip and Sop are found to contribute to virulence by

directing secretion and translocation of several bacterial affector protein into

the cytoplasm of the host cells (Wood et al., 1998).

Sop B (Salmonella outer protein B) a novel enterotoxin of Salmonella

Dublin is a major Sop protein involved in enteropathogenesis and exhibits

extensive sequence similarity to the Ipg D protein of Shogella Flexeri (Galyov

et al.,1997).

Wood et al., (1998) reported characterization of Sop B and presented

evidence that Sop B is translocated into the eulkaryotic cells via, a Sip

dependent mechanism to promote fluid secretion and inflammatory response.

Translocation of intracellular activities of Sop B affects cellular response

leading to influx of neutrophils into the intestinal epithelium and induction of

fluid secretion. Jones et al., (1998) investigated the enteropathogenicity of

another Salmonella Dublin effector protein Sop D. The ability of mutant

Salmonella Dublin strains to induce fluid secretion and influx of

polymorphonuclear leukocytes (PMN) in bovine ligated ileal loops was

investigated. Comparing with the type strain, Sop D mutant Salmonella Dublin

induced less secretion and PMN influx. The Sop B mutant strain also induced

lower secretory and inflammatory responses than the wild type strain. When

both Sop B and Sop D mutations were introduced into Salmonella Dublin, it

resulted in even lower secretory and inflammatory responses than either of the

single mutants. Thus it was suggested that Sop D had a role in the induction of

18

enteritis and that Sop b and Sop D acted in concert to promote fluid secretion

and inflammatory responses in the infected ileum.

Another important type III secretion is Sop E and it is associated with

invasion by stimulating membrane ruffling (Hardt et al., 1998). Unlike Sop B

gene, Sop E is not widely distributed among Salmonellae (Rahman et al.,

2001). The Sop B has been reported in several serovars of Salmonella recently

((Rahman et al., 2001).

Detection of Sop E antigen by serological assay (Dot-ELISA)

The Enzyme Linked Immunosorbent Assay(ELISA) was applied for

assaying cholera toxin for the first time by Holmgren and Svennerholm (1973).

The level of sensitivity of this ELISA technique was calculated to be

approximately 0.09 µg/ml for cholera toxin. Yolken et al., (1977) employed

this technique for detection of E.coli LT enterotoxin. Saxena and Sharma

(1987) also used this test to detect salmonella enterotoxin and they compared it

to that of Rrabbit Ligated Illeal Loop (RLIL) technique.

A different type of ELISA called GM1 ganglioside ELISA was also

widely used for detection of enterotoxin of E.coli (Svennerholm and Holmgren,

1978). The test is based on the fact that ganglioside is a specific receptor of LT

enterotoxin of E.coli. This test is also used for detection of heat stable toxin

(ST) of E. coli and as low as 1 to 2 mg the toxin in crude culture filtrates was

detected (Urban et al., 1990). Dot-ELISA is one of the solid phase

immunoassay developed by Hawkes et al., (1982) to detect either antigen or

19

antibody. Panigrahi et al., (1987) developed an immuno-dot-blot assay for

detection of cholera related enterotoxin of Salmonella Typhimurium where he

used nitrocellulose paper for absorption of the enteroxin instead of polystyrene

plates and they could detect also was 0.02 ng of purified enterotoxin.

Rahman et al., (1991) used ELISA and GM1 ELISA to test for

enterotoxigenicity of Salmonella isolated from food of animals origin. ELISA

could detect as low as 15 ng/100 ml of purified Salmonella enterotoxin and 10

ng/100 ml of cholera toxin when tested with their homogeneous antisera. Gaol

and Liw (19930 identified 329 serotypes of E.coli isolated from diarrhoeic

piglets based on antigenic difference of adhesins by using Dot-ELISA.

Rahman (1999) examined 19 strains of Salmonella enterica belonging to

seven serovars and two strains of Salmonella Bongori for enterotoxin

production by using Chinese Hamster Ovary (CHO) cell assay and Dot-ELISA.

The number of strains, which showed positive reactions in CHO cell assay and

Dot-ELISA, were 13 (64.42 %) and 16 (84.21 %) respectively.

Roychoudhury (1999) studied the production of heat labine enterotoxin

(LT) of E.coli strains using two immunological testes, viz. Biken test and Dot-

ELISA and found that 80.89 % strains were positive for LT in Dot-ELISA,

while 43.82 % were positive in Biken test.

Detection of fimbrial genes (Sef and Pef) by PCR

All members of the family Enterobactericiae express fimbriae, which

allow them to attach to the host surfaces. This attachment is the first step in the

20

colonization of their preferred host niche. Depending upon their

haemagglutinating properties, fimbriae were initially classified into three types:

Type 1 fimbriae were those whose agglutination is prevented by co incubation

with D- Mannose (Old, 1972); Type 2 were the non agglutination types

(Lockmann and Curtiss III, 1992); Type 3 were those which agglutinated with

animal erythrocytes only if they were coated with tannic acid (Duguid et al.,

1996). An additional thin fimbriae with a unique curly morphology in members

of the Enterobacteiaceae has also been described (Arnqvist et al., 1992) and

these show a high degree of sequence conservation. In addition, a fifth

enterobacteial class of fimbriae, termed type 4 fimbriae, has been shown to use

a different export systems for translocation of its fombrial subunits across the

cytoplasmic membrane (Strone and Lory, 1993).

Serovar Enteritidis produces at least five distinct fimbriae, namely

Salmonella Enteritidis Fimbriae (SEF) 14, SEF 17, SEF 21, (all type 1

fimbriae) LPF and PEF (Rajashekara et al., 2000). The information regarding

the role of these fimbriae is still very ambiguous. Of all serovar Enteritidis

fimbriae, SEF 14 has been studied the most. SEF 14 has been known to consist

of 3 major protein subunits sefA (14kd), sefB (28 kd) and sefC (90 kd), which

are arranged sequentially in the fimbrial structure and sefC forms the last and

largest component of fimbriae. sefA,sefB,sefC are encoded by fimbrial operons

sefA, sefB, sefC respectively (Clouthier et al., 1994). Subsequently Clouthier

et al., (1994) also reported one more fimbrial structureb enconded by sefD in

the same 14 fimbrial gene cluster of Salmonella Enteritidis. Amongst the

21

remaining fimbriae, SEF 17 is encoded by agf BAC (Collinnson et al., 1993)

and SEF 21 by fimA (Muller et al., 1991) genes.

Rahman et al., (2000) conducted a PCR amplification study to observe

the occurrence of Sef and Pef genes among different serovars of Salmonella

using specific primers of internal fragments of sefC and pefA fimbrial operons.

They found that strains of Salmonella Enteitidis harboured both sef and pef

genes irrespective of their source. Strains of Salmonella Typhimurium and

Salmonella Gallinarum only per and sef genes respectively. Salmonella

Newport. Salmonella Kentucky, Salmonella Weltevreden and Salmonella

Indiana were found to be negative for both Pef and Sef genes.

22

MATERIALS AND METHODS

BACTERIAL STRAINS

Standard strains

The enterotoxigenic strain of Salmonella Enterica subspecies Enteric

belonging to server Salmonella Typhimurium and Salmonella. Enteritidis were

used for the experimental study. These strains were already serotyped in

National Salmonella and Escherichia Center, Central Research Institude,

Kasauli, Himachal Pradesh (India).

Maintenance of bacterial cultures

All the 50 bacterial cultures were collected from the different sources

such as pig, poultry, cattle, humans and quail by different methods and

maintained through sub-culturing on buffered nutrient agar slants at regular

interval of 3 weeks. The cultures were checked for their purity by streaking on

Brilliant Agar (BGA) and their morphological, cultural and biochemical

characteristics were confirmed (Edwards and Ewing, 1986).

Antimicrogram patterning

In vitro susceptibility of the given Salmonella strains to various

antibiotics was determined by the disc diffusion technique (Kim et al., 1984).

Pure cultures of each isolate was inoculated into 5 ml of nutrient broth and

incubated for 6-8 hrs. Purity of the cultures was checked by microscopic

examination. About 2 ml of each culture was spread uniformly on the surface

nutrient agar (BBL laboratories, USA) plates and kept undisturbed for 5 min.

23

The excess inoculums was sucked out from the plate with the help of sterile

Pasteur pipette. The inoculated plates were allowed to dry in an incubator at 370

C for 5 min. The antibiotic discs were placed gentle on the inoculated agar

surface maintaining adequate distance from each other. The discs were pressed

gentle with the help of a pair of sterilized forceps to ensure complete contact

with the medium. The plates were then incubated at 370C for 18-24 hr. The

zone of inhibition around the discs was measured

PLASMID PROFILE

Extraction of plasmid DNA ( Alkaline Lysis Method)

Extraction of Plasmid DNA was carried out by alkaline lysis method as

per the modified procedure of Kado and Liu (1981) as described by Kinde et

al., (1996). The reagents and buffers required for Plasmid extraction are given

in appendix. A loop full of culture was grown in 3ml of Luria broth (Hi-Media)

and incubated at 370C for 16-18 hr. After overnight incubation, the bacteria

were harvested by centrifugation incubation at 8000rpm for 10 min. The

washed pellet was resuspended in 100μ of (solution I) freshly prepared

lysozyme buffer and incubated at room temperature for 10 min. 200μl of

(solution II) SDS reagent [0.2 N NaOH, 1% Sodium Dodecyl Sulphate (SDS)]

was added to the resuspended bacteria and after gentle shaking, the suspension

was left on ice for 10 min. 100μl of (solution III) (pH 5.6) 3 M potassium

acetate was added to the suspension. The suspension was then vortexed

thoroughly and left on ice for 30 min. The suspension was then centrifuged at

10,000rpm for 10 min at 40. The supernatant was carefully separated and then

24

mixed with equal volume of phenol: chloroform: isoamyl alcohol solution

(25:24:1) and after careful mixing the solution was centrifuged at 10,000rpm

for 10minutes at 40C. The supernatant was removed and left at room

temperature for 5 minutes after thorough mixing. The precipitated DNA was

harvested by centrifugation at 15,000rpm for 10 minutes. The DNA pellet was

then washed with 70% ethanol to remove the isopropanol and air dried for 15

min. The glassy pellet was then resuspended in about 20μ of TE buffer.

Agarose gel electrophoresis of plamid DNA

The plasmid DNA was visualized by carrying out agarose gel

electrophoresis in a horizontal submarine electrophoresis (Pharmacia) as the

procedure described by Sambrock et al., (1989). For electrophoresis, 0.7%

agarose gel in TBE buffer was used. About 7μl of Plasmid DNA mixed with

2μl of gel loading buffer as loaded into the gel with the standard markers.

Electrophoresis was carried out at 80 V for 2 hr minutes till the bromophenol

blue of the gel loading buffer migrated more than fourth and fifth the length of

the gel. At the end of the electrophoresis, the gel was stained with ethidium

bromide (0.5mg/ml) and was visualized in the gel documentation system

(Image Master-VDS, Amersham Pharmacia, Sweden). Molecular weight of the

Plasmid was determined with the help of AAB image software (Amersham

Pharmacia, Sweden).

DETECTION OF SALMONELLA OUTER PROTEIN (SOP) E

Isolation of Sop E secreted by different strains of Salmonella

25

The Sop E was isolated from all the strains of Salmonella as per the

method described by Rahman et al., (2001). Bacteria were grown on Luria

Bertani (LB) agar (Difco, UK) overnight at 370C. One colony from agar plate

was inoculated in 5 ml of Luria Bertani broth (Difco, UK) containing 0.3 M

NaC1 and incubated at 370C for 6 hr (OD,0.7 to 0.8) on a rotary shaker (100

rpm). The culturer was then diluted 4 times in fresh LB broth (final volume 20

ml) in a 100 ml conical flask and incubated at 370C for 18 hr on rotary shaker

(100 rpm). Then the culture was cooled in ice bath for 30 min and centrifuged

at 20,000rpm at 40C for 1 hr. The culture supernatant was collected and filtered

(0.45µm, Sartorius, Goettingen, Germany). The protein present in the

supernatant was precipitated with 10 % (v/v) trichloroaceticacid (Sigma, USA)

for 1 hour and then centrifuged at 20,000rpm at 40C for 1 hr. The sediment was

collected and dissolved in 0.4 ml of NaOH (0.1 M) to which 2.0 ml ice-cold

acetone (- 200C) was added and incubated at - 200C for 20 min. The suspension

was centrifuged at 20,000rpm at 40C for 15 min. The sediments were

redissolved in 20 ml of acetone (- 200C) and incubated and centrifuged as

above. The sediments were dried at room temperature and dissolved in 0.1ml of

PBS and stored at 40C.

DOT-ELISA

The Dot-ELISA for detection of Sop E was carried out as described by

Rahman (1991) with modification. Standard strains of Salmonella Tyhimurium

and Salmonella Enteritidis were used as test isolates a single isolate of E. coli

was used as negative control.

26

Assay Procedure

Two μl of Sop E preparation of 20 randomly selective strains of

Salmonella were dotted on polyvinyldine membrane (PVD) strip (Sigma,

USA), which was prewetted with methanol and dried at 40C for overnight. The

unsaturated sites were blocked by immersing the strips in 3% of bovine serum

albumin, fraction V (Sigma) in PBS for 1 hr at 370C. The strips were washed

three times in wash buffer (0.01 M PBS, pH 7.2 with 0.3% Tween-20 and

0.05% Triton X 100) for 5 minutes. The strips were dipped in the Anti-Sop E

antisera diluted 1: 1000 in wash buffer and incubated at 370C for 1 hr. After

incubation, the strips were washed thrice in wash buffer and incubated with

goat antirabbit IgG horse radish peroxidise ((HRPO) conjugate (Boehringen,

Germany) at a dilution of 1: 1000 for 1 hr at 370C. The strips were then washed

thrice in wash buffer and immersed in freshly prepared substrate solution

(sigma fast-TM DAB/ H2O2, Sigma). Washing the strips in running tap water

stopped the enzymatic reaction and a position reaction was indicated by the

appearance of deep purple dot against a while background within 10-15

seconds.

DETECTION OF VIRULENCE GENES OF SALMONELLA

TYPHIMURIUM AND SALMONELLA ENTERITIDIS BY MULTI PCR

Deterction of Stn gene by PCR

The PCR analysis for the deterction of Stn gene was carried out as per

the method followed by Prager et al., (1995) and Rahman (1999). Bacterial

cells from the overnight cultures were suspended in 350 μl distilled water and

27

boiled at 1000C for 10 min. After boiling, the cell suspension were cooled and

were immediately tasted for Stn gene by PCR analysis. Primers used for PCR

reaction were Stn P1 5-TTG TGT CGC TAT CAC TGG CAA CC-3 (lower

primer) and Stn M13 5- ATT CGT AAC CCG CTC TCG TCC-3 (lower

primer). These primers flank a 617-bp segment in Stn sequence. The PCR

mixture (25 μl) included 12.5μl master mix (QIAGEN) containing 2.5 U Tag

DNA Polymerase, 200μM each of dATP dCTP dTTP dGTP and PCR buffer, 5

μl (lμM) each of upper primer and lower primer and 2.5μl of template DNA

(bacterial cell suspension). PCR incubation was performed in a thermocycler

(Perkin-Elmer, USA) in 25 cycles of denaturation (940C for 1 min), primer

annealing (590C for 1 min.) and extension (720C for 1min.) followed by

incubation at 720C for 10 min. A 15 μl aliquot of each primer mixture was

electrophoretically separated on agarose gel (1 % containing 0.5 μl/ml ethidium

bromide, pharmacia) and the PCR produced were visualized in the gel

documentation system (Pharmacia)

Detection of Sef gene by PCR

The PCR analysis for the detection of Sef gene was carried out as per

the methods described by Clouthier et al., (1994) and Rahman et al., (2000).

Bacterial cells from the overnight cultures were suspended in 350μl distilled

water and boiled at 1000C for 10 min. After boiling rhe cell suspension were

cooled on ice and immediately tested for Sef C gene by PCR analysis. Primers

used for the PCR reaction were Sef C 5-GCG AAA ACC AAT GCG ACT

GTA -3 (upper primer) and Sef C 5- CCC ACC AGC AAC ATT A=CAT

28

CCC-3 (lower primer). These primers flank a 1103 bp segment in sef sequence.

The PCR mixture (25 μl) included 12.5 μl master mix (QIAGEN) containing

2.5 U Tag DNA polymerase, 200μM each of dATP, dCTP, dTTP and dGTP

and PCR buffer, 5 μl (1μM) each of upper and lower primers and 2.5μl of

template DNA (bacterial cell suspension). PCR incubation was performed in a

thermocycler (Perkin-Elmer, USA) in 25 cycles of denaturation (940C for 1

min.) following by incubation at 720C for 10 min. A 15μl aliquot of each

primer mixture was electrophoretically separated on agarose gel (1 %

containing 0.5μl/ml ethidium bromide, Pharmaca) and the PCR products were

visualized in the gel documentation system (Amersham Pharmacia biotech,

Sweden).

Detection of Pef gene by PCR

The PCR analysis for the detection of Pef gene was carried out as per

the methods described by Baumler and Heffron, (1995) and Rahman et al.,

(2000). Bacterial cells from the overnight cultures were suspended in 350 μl

distilled water and boiled at 1000C for 10 min. After boiling the cell suspension

were cooled on ice and immediately tested for Pef gene by PCR analysis.

Primers used for PCR reactions were Pef A1 5 TGT TTC CGG GCT TGT

GCT -3 Upper primer and Pef A2 5- CAG GCC ATT TGC TGA TTC TTC

C-3 lower primer these primers flank a 700 bp segment in Pef sequence. The

PCR mixture (25μl) included 12.5μl master mix (QIAGEN) containing 2.5 U

Tag DNA polymerase, 200μM each of dATP,dCTP, dTTP and dGTP and PCR

buffer, 5μl (1μM) each of upper and lower primers and 2.5μl of template DNA

29

(bacterial cell suspension). PCR incubation was performed in a thermocycler

(Prkin-Elmer, USA) in 25 cycles of denaturation (940C for 55 sec) followed by

incubation at 720C for 10 min. A 15μl aliquot of each primer mixture was

electrophoretically separated on agarose gel (1%containing 0.5μl/ml ethidium

bromide, pharmacia) and the PCR products were visualized in the gel

documentation system (Amersham Pharmacia biotech, Sweden). Tag DNA

polymerase, 200 μM each of dATP, dCTP, dTTP and dGTP and PCR buffer,

5μl (1μM) each of upper and lower primers and 2.5μl of template DNA

(bacterial cell suspension). PCR incubation was performed in a thermocycler

(Perkin-Elmer, USA) in 25 cycles of denaturation (940C for 55 sec), primer

annealing (550C for 55 sec) and primer extension (720C for 55 sec) followed by

incubation at 720C for 10 min. A 15μl aliquot of each primer mixture was

electrophoretically separated on agarose gel (1% containing 0.5μl/ml rthidium

bromide, Pharmacia) and the PCR products were visualized in the gel

documentation system (Amarsham Pharmacia biotech, Sweden).

Detection of Sop B gene by PCR

The PCR analysis for the detection of Sop B was carried out as per the

method described by Rahman (1999). Bacterial calls from boiled at 1000C for

10 min. After boiling, the cell suspensions were cooled on ice and were

immediately tested for Sop B gene by PCR analysis. Primers used for PCR

30

reaction were Sop P1 5- CAA CCG TTC TGG GTA AAC AAG AC-3 (upper

primer and) and SOP M13 5- AGG ATT GAG CTC CTC TGG CGAT -3

(lower primer). These primers flank a 397-bp segment in Sop sequence. The

PCR mixture (25μl) included 12.5μl master mix (QIAGEN) contatiningb 2.5 U

Tag DNA Polymerase, 200μM each of dATP dCTP dTTP dGTP and PCR

buffer, 5μl (1μM) each of upper primer and lower primer and 2.5μl of template

DNA (bacterial cell suspension). PCR incubation was perfomed in a

thermocycler (Perkin-Elmer, USA) in 25 cycles of denaturation (940C for 1

min.), primer annealing (550C for 1 min) and primer extension (720C 2 min.)

followed by incubation at 720C for 10 min. A 15 μl aliquot of each primer

mixture was electophoretically separated on agarose gel (1 % containing 0.5 μl/

ml ethidium bromuide, Pharmacia) and the PCR products were visualized in

the gel documentation system (Pharmacia).

31

RESULTS

A total number of 50 Salmonella isolates were used for the present

study. They were plated on the BGA medium, which showed pink colour

colonies and were sub cultured in the nutrient agar slants they showed white

translucent colonies.

By Gram staining it was found to be pink colour rods under 100X object

and they were undergone some of the biochemical tests. In which they showed

some of the positive and some of the negative results, according to their

biochemical activity.

A total of 50 Salmonella isolates collect from various sources is given in

Table 1. Among these 50 isolates, 18 are Salmonella Typhimurium and 32 are

Salmonella Enteritidis. All the cultures were tested for their sensitivity to 10

different antibiotics using disc diffusion method and the species-wise results

are present in the Table 2.

The Salmonella strains isolated from animals were found to be resistant

to Ampicilin and were sensitive to Nalidixic acid, Norfloxacin, Streptomycin,

Ciprofloxacin, Chloramphenical, Gentamycin, Cotrimaxazole and tetracycline,

six of which belongs to Salmonella Enteritidis and four to Salomonella

Typhimurium.

Among the antibiotics to which comparatively lower resistance was

observed. The isolates were found to be resistant to Nitrofurantoin (12%)

32

belongs to Salmonella Typhimurium, ampicillin (12%) belongs to Salmonella

Typhimurium and Steptomycin (12%) belongs to Salmonella Typhimurium.

Salmonella strains isolated from community infection showed highest

sensitivity 95% to all antibiotics in which all isolates are Salmonella

Typhimurium. In which, 83% showed sensitive to Nalidixic acid, 100%

Norfloxacin, 100% Streptomycin, 100% Ciproflaxacin, 83% Chloramphenicol,

100% Gentamycin, 100% Contrimaxazole, 100% Tetracycline, 100%

Nitrofurantoin and 83% Ampicillin showed sensitivity. The resistance was

showed least effect (5%) to all isolates. Among 6 isolates one isolate was

found to be resistant to Streptomycin (16.6%). One isolate (16.6%) showed

resistant to Ampicillin and one isolate (16.6%) showed resistant to

Cholramphenicol.

The Salmonella strains isolated from hospital infections showed high

sentitivity (88%) to all antibiotics.In which, 77% of isolates showed sensitivity

to Nalidixic acid, 92% showed to Norfloxacin, 92% showed to Streptomycin,

100% showed to Ciproflaxacin, 92% showed to Chloramphenical, 92% to

Gentamycin, 84% showed to Cotrimaxazole, 84% showed to Tetracycline,

100% showed to Nitrofurantoin and 61% showed to Ampicillin .The

percentage of sensitivity of these antibiotics to both Salmonella Typhimurium

and Salmonella Enteritidis (Table 3).

33

The resistance to these antibiotics is least effect with 12%. Almost all

antibiotics are resistant to some isolates except Ciprofloxacin and

Natrofurantoin.

In overall percentage of sensitive against these antibiotics, it showed

different results. For Nalidixic acid it showed 86% for both Salmonella

Typhimurium and for Co- Ttrymaxazole 91% Tetracycline 91%, Norflaxcin

08%, Ciproflaxacin 100%, Nitrofurantoin 98%, Chloramphenicol 89%,

Ampicillin 52, Stretomycin96% and Gentamycin showed 98% (Table 4).

Plasmid profile study carried out for the randomly selected isolates

revealed a distinctly varied plasmid profile. Plasmid profile analysis of a single

isolate of Salmonella Typhimurium revealed the presence of 4 different

plasmid profiles. And another one only revealed the presence of different

plasmid profiles. A single isolate of Salmonella Typhimurium harboured a 2

different plasmid profiles (Fig. 4).

Dot-ELISA test was carried out with sop E preparation of randomly

selected 19 isolates in which 2 were Salmonella Typhimurium and 17 were

Salmonella Enteritidis as test isolates, the E.coli was used as negative control.

All the 17 test isolates were positive for Sop E, and the Salmonella

Typhimurium showed negative results (Fig. 1).

Among the various known virulence genes of Salmonella Typhimurium

and Salmonella Enteritidis, studies on the detection of enterotoxin gene (Stn),

34

fimbrial gene (Sef and Pef) and Sop B gene were carried out by multiple

polymerase chain reaction (Multi PCR) using their specific primers.

The Stn gene was found to be present in 9 among the 10 isolates (Table

5) in which E. coli 33 was used as negative control. All these 9 positive strains

were found to give 617 bp product in the Stn gene segment. The negative

control of E. coli 33 was found to be negative for Stn gene (Fig. 5).

The Sef gene was found to be present in only 5 isollates (Table 5) in

which one isolate was belongs to Salmonella Typhimurium and four belongs to

Salmonella Erteritidis. All these strains were found to give rise to an 1103 bp

product in the sef gene segment. The Sef gene was found to be absent in the E.

coli 33 strain negative control (Fig. 5).

The Pef gene was found to be present in 4 of the 10 isolates (Table 5).

All of these strains were found to give rise to a 700 bp product in the Pef gene

segment. In 4 Salmonella Typhimurium, 2 showed the presence of this gene

and in 5 Salmonella Enteritidis, 2 showed positive for Pef gene. The control of

the E.coli 33 strain showed negative result (Fig. 5).

The Sop B gene was found to be present in all 9 positive controls (Table

5). of these strains were found to give rise to 317 bp in the Sop gene segment.It

was found to be present in both Salmonella typhimurium and Salmonella

enteritidis isolates and the negative control of E.coli 57 was found to be absent

for Sop B gene (Fig 4).

35

Table 1. Details of samples which were used.

36

Sl. No. Sources of the given samples No. of samples

% of samples

1. Hospital infections 30 60

2. Community infections 10 20

3. Animal infections 10 20

Table 2. Antimicrobial agent their concentration used for antibiogram

studies of the Salmonella isolates.

S.No Antimicrobial Concentration of disc/µg

1. Norfloxacin (Nx) 100

2. Chloramphenicol (C) 80

3. Nalidixic acid (Na) 70

4. Ciprofloxacin (Cf) 60

5. Co-Trimaxazole (Co) 55

6. Tetracycline (Tc) 90

7. Streptomycin (S) 50

8. Nitrofrantoin (NF) 70

9. Ampicillin (Am) 90

10. Gentamycin (Gm) 70

Table 3. Antimicrobial sensitivity pattern of Salmonella Typhimurium

and Salmonella Enteritidis.

S.No Culture Antimicrobial agents (%)

Na Co Tc Nx Cf Nf C Ap S Gm

1. Salmonella 78.5 100 86 100 100 93 78.5 57 93 100

37

Typhimurium

2. Salmonella

Enteritidis

87.5 87.5 94 97 100 100 94 50 97 97

Table 4. Overall percentage of antimicrobial sensitivity pattern of

Salmonella Typhimurium and Salmonella Enteritidis.

38

Table 5. Detection of Virulence genes of Salmonella Typhimurium and

Salmonella Enteritidis.

39

Culture Antimicrobial agents (%)

Na Co Tc Nx Cf Nf C Ap S Gm

Salmonella Typhimurium

and Salmonella Enteritidis

98 91 91 98 10

0

98 89 52 96 98

S.No Virulence

Genes

No.of

Salmonella

Typhimurium/9

isolates

No.of

Salmonella

Enteritidis/9

isolates

Molecular

Weight

1. Stn 4 5 617bp

2. Sef 1 4 1103bp3

3. Pef 2 4 700bp

4. Sop B 4 5 317bp

40

41

42

43

FIG.2 ANTIBIOTIC SENSITIVITIES OF SALMONELLA TYPHIMURIUM & SALMONELLA ENTERITIDIES

44

FIG.3 ANTIBIOTIC SENSITIV ITIES OF SALMONELLA TYPHIMURIUM & SALMONELLA ENTERITIDIES

45

46

47

48

FIG.9 ANTIBIOTIC SENSITIVITIES OF SALMONELLA TYPHIMURIUM & SALMONELLA ENTERITIDIES

DISCUSSION

All the 50 cultures were tested for antimicrobial patterning, with 10

different antimicrobial agents. Gupta et al., (1983) tested 261 Salmonella

isolates with chloramphoenical and they got the result of 91.6% of sensitive but

in the present study showed 97.8% sensitive.

In overall percentage of sensitive to all antimicrobial agents for both

organisms, such as Salmonella Typhimurium and Salmonella Enteritidis, it

showed that 98% sensitive to Nitrofurantoin, Norfloxacin and Streptomycin.

Salmonella Typhimurium showed more sensitive than the Salmonella

Enteritidis. In addition 100% sensitive to Gentamycin and Norfloxacin.

The present study showed very less resistance percentage in both

organisms. Among all the antimicrobial agents Ampicillin showed higher

resistance than the others. Salmonella Enteritidis showed 50% of resistance

against Ampicillin. Especially, poultry isolates showed 92.3% of resistance.

Similar observation done by Blackburn et al.,(1984) reported 80% of resistance

against the culture, isolated from different animals.

Plasmid profile showed different results based on the species and was

found that a single isolate of Salmonella Typhimurium showed high molecular

plasmid. Mohan et al., (1995) reported that except 2 of the 190 strains were

plasmid negative. But the present study showed 100% result for the presence

of plasmid and their different plasmid profiles also i.e. a single isolate of

49

Salmonella Typhimutium showed 4 different plasmid profiles and in

Salmonella Enteritidis also showed 4 different plasmid profiles. It was found

that 3,2 and single plasmid profiles also.

Rahman et al., (1991) examined 29 Salmonella strains for the detection

of enterotoxin and they found 84.21% of positive result by Dot-ELISA. The

present study showed positive result for the presence of Sop E protein in all

the Salmonella Enteritidis isolates and sop E protein is absent in Salmonella

Typhimurium isolates.

Among the various known virulence genes of Salmonella, the detection

of enterotoxin gene (Stn), fimbrial genes (Sef and Pef) and Sop gene were

carried out by multi PCR using their specific primers.

The present study showed that all the isolates of Salmonella Enteritidis

and Salmonella Typhimurium were present in (Salmonella enterootoxin gene)

and found that this gene containing 617 bp by PCR technique. Rahman et al.,

(1991) observed this gene in 58 Salmonella isolates belongs to Salmonella

Typhimurium and Salmonella Enteritidis by the PCR technique. The present

study concludes that the Stn gene contains 617.

Similar type of results were observed by Galyov et al., (1997) observed

that sop B (a 60 Kda protein) encoded sop B genes from a sip B mutant of

Salmonella enterica, and this gene contains 317 bp. The present study is

confirmed that the Sop B gene contain 317 bp with their results.

50

Rahman et al., (2000) conducted a PCR amplification study to observe

the occurrence of Sef and Pef genes among different serovars of Salmonella.

The present study also confirmed that the presence of Sef and Pef genes in

Salmonella Typhimurium and Salmonella Enteritidis and Sef gene is having

1103 bp and Pef gene is having 700 bp.

51

SUMMARY

The present study was aimed to study some of the molecular

characteristics of Salmonella and to evaluate the performance of some of the

available methods for detection of Salmonella enterotoxin (Stn) gene, fimbraial

genes (Sef and Pef), Sop B genes and sop E antigen. Apart from that the

plasmid profile and antibiotic sensitivity were also studied. The evaluation of

the various direct (Dot- ELISA) and indirect methods detection of Salmonella

enterotoxin (Stn) gene by PCR, presence of Stn gene, fimbraial genes (Sef and

Pef and Sop B genes) were carried out in Salmonella Typhimurium and

Salmonella Enteritidis were also studied.

Fifty given isolates which were isolated from various sources, such as

hospital infections, community infections and animals were used for the

present study. These were subjected to in-vitro antibiotic resistance and

plasmid profiling studies.

The antimicrogram revealed varying antimicrobial sensitive patterns.

Most of the antimicrobial agents, which were used, are very effective to all the

isolates especially Norfloxacin, Gentamycin, Nalidixic acid Ciproflaxacin,

Chloramphenicol and Nalidixic acid to be showed a reasonably good efficacy.

On the other hand the highest number of isolates were showed resitance to

Ampicillin and Tetracycline. Salmonella Typhimurium showed higher

sensitivity to most of antibiotics than the Salmonella Enteritidis.

52

Plasmid profile showed a multilateral distribution of plasmid amongst

both serotypes. A total of 2 plasmid pictures were observed. All the isolates

showed different plasmid profiles. Higher plasmid number (three or more) was

observed in both isolates (one one each).

Detection of Salmonella enterotoxin (Stn) was carried out by PCR

(indirect method). Dot-ELISA was found to be the most suitable in terms of its

accuracy, simplicity and rapidity. In the molecular assay, Stn gene was found

to be present in all the 9 isolates that were detected by the presence of a 617 bp

product following PCR with primers. Considering its percent detection level

and this method was found to be most sensitive.

Detection of sop E was carried out by the immunological assay Dot-

ELISA. Twenty isolates were used for this test which 3 isolates belongs to

Salmonella Typhimurium and 17 belongs to Salmonella Enteritidis. Since the

procedure was very easy and it will be more useful for some other

immunological assays.

Detection of fimbrial genes (Sef and Pef) was carried out by PCR using

known primers. Sef gene, detected by the presence of 1103bp product and

found to be present of Salmonella Typhimurium isolate. The Pef gene was

found to be presence in both Salmonella Typhimurium and Salmonella

enteritidis isolates, which was detected by the presence of a 700bp product.

Detection of Sop B was carried out by PCR using known primers. Sop

B, gene detected by the presence of 1348bp product, was found be present in

53

both the isolates of Salmonella Typhimurium and Salmonella Enteritidis.

Considering its percent detection level, this method was found to be most

sensitive. By these detections by PCR amplification technique, it was

concluded that technique is more sensitive since its percent detection level.

54

CONCLUSION

It can thus be concluded that the antimicrogram pattern of the

given 50 salmonella isolates was performed in determine in order to

determine this species type. From the derived antimirogram, it can be

confirmed that the Salmonella Typhimurium isolates are more sensitive

to all the given antimicrobial agents than Salmonella Enteritidis.

Typhoid being the major disease, the antimicrogram helped to

determine the highest sensitive to Gentamycin, Co-Trymaxazole,

Norfloxacin and Ciprofloxacin and it conformed the best applicable

agents for the present day.

This finding was in agreement with the respective plasmid profile

studies; the PCR technique was successfully applied to amplify the

virulence genes (Sef and Pef; Sop B) of both species and its plasmid

profile study.

Identification and determination of various molecular

characteristics of two species enhances the knowledge towards the

vaccine production.

55