Detection and Partial Purification of Salmonella serovar Typhimurium Cytotoxic Protein Affecting Seed Germination

Isolation and characterisation of four distinct cytotoxic factors of

Salmonella Weltevreden

B R Singh & V.D. Sharma

Department of Veterinary Microbiology

College of Veterinary Sciences

G B Pant University of Agriculture & Technology

Pantnagar-263 145, India

Four distinct cytotoxins with different biological, physico-chemical and antigenic

characteristics were isolated from a single Salmonella Weltevreden strain recovered from buffalo meat.

The toxins were purified through salt precipitation, dialysis, gel filtration and ion-exchange

chromatographic methods. Cytotoxin I was dermonecrotic, verocytotoxic and lethal to mouse with 120

µg LD50. It induced accumulation of serosanguinous fluid in rabbit ligated ileal loop (RLIL) and

mucinous fluid in the stomach. It was active within a narrow pH range (5.0 - 8.0) and lost its activity on

autoclaving for 1 min. Cytotoxin II was verocytotoxic, enterotoxic and lethal to mouse (LD50, 1 mg). It

induced delayed vasopermeability in rabbit skin, was active between pH 5.6 and 8.4 only and heat-

sensitive (100°C, 30 min). Cytotoxin III was neither dermatotoxic nor enterotoxic. It induced

vacuolation, multinucleation and formation of syncytia in vero cells. It was sensitive to pH beyond the

range of 4.8 to 8.2. It was completely inactivated on boiling for 30 min. Cytotoxin IV was intensely

necrotizing to rabbit skin within 6 h of inoculation and lysed Vero cells. It also possessed haemolytic and

lecithinase/phospholipase-C activities. The cytotoxin lost its activity on heating at 90°C (30 min) but

remained active between pH 2.5 and 7.5. The PIs of the cytotoxins were estimated to be 9.0, 7.0, 5.6 and

3.0, respectively. All the four cytotoxins were immunogenic in rabbits but antigenically unrelated as the

anticytotoxin neutralized only the homologous cytotoxin and did not cross react with heterologous

cytotoxins.

* Present address: Scientist, National Salmonella Centre (Vet), Division of Bacteriology and Mycology, Indian

Veterinary Research Institute, Izatnagar-243 122 (UP), India.

PDF created with pdfFactory Pro trial version www.pdffactory.com

http://www.pdffactory.com

Introduction

Salmonella, still remains to be one of the most important causes of

foodborne outbreaks. It leads to gastro-intestinal disorders frequentlyresulting to

systemic salmonellosis. Although Salmonella has been most thoroughly investigated and

more than 2400 serovars are known, its viruelence factors and pathogenesis are yet to be

fully delineated11,27 . Invasiveness of Salmonella is considered to be an important

attribute of virulence2, 4. However, all invasive Salmonella do not induce gastroenteritis.

Enterotoxins have been shown to play significant role in enteric diseases and as many as

54-98% of Salmonella strains have been found to be enterotoxigenic1,2,18. Of the two

types of enterotoxins, i.e. cytotonic and cytotoxic, the former one, which is commonly

referred to as enterotoxin, has inconsistent production and is considered to be less

significant than the cytotoxic enterotoxins (cytotoxins)1,24. Cytotoxigenicity of

Salmonella strains has been shown to be more closely related to their pathogenic potential

in experimental studies1,3,13,22. Partially purified cytotoxin is found to produce typhoid

like lesions, viz., enlargement of spleen, pin point haemorrhages in liver, heart, lungs,

kidneys and brain, congestion in thoracic and peritoneal cavites, focal necrosis in liver

and blood tinged contents in small intestine in mice22. Salmonella cytotoxin has been

purified to a variable extent 1,13,22. This paper reports the isolation of four distinct

cytotoxic moieties from a single strain of S. Weltevreden and their physico-chemical,

biological and immunological characterization.

Materials and Methods

A strain of S. Weltevreden (BM 1643) isolated from buffalo meat and identified

as potent cytotoxin producer22 was procured from the Department of Veterinary

Microbiology, College of Veterinary Sciences, Pantnagar. Escherichia coli K12 (J-53)

strain obtained from the National Salmonella Centre (Veterinary), Izatnagar, was used as

non-toxigenic reference strain.



Experimental animals. Adult (3-month-old) New Zealand White rabbits and adult Swiss

albino mice were obtained from Laboratory Animal Section of the College of Veterinary

Sciences, Pantnagar. Animals were maintained on pathogen-free diet supplied from the

Laboratory Animal Section of the College.

Cytotoxin production. Cell-lysate (CL), cell-free culture filtrate (CFCF) and polymyxin B

extract (PBE) were prepared according to the method described by Malik et al.12,13. The

test organism was propagated in brain heart infusion broth (Hi Media, Mumbai) at 37°C

on a shaker (200 rpm) for 18-24 h. The growth was then centrifuged at 10,000 X g for 10

min. The supernatant was separated and filtered through a 0.45 µm membrane filter to

obtain CFCF. The pellet was subjected to sonication at 150 mA for 30 min and

centrifuged at 10,000 X g for 20 min; the supernatant, so collected, was designated as

CL. To prepare PBE, cell pellet from one litre culture material was suspended in 50 ml

PBS (pH 7.2, 0.15 M) and supplemented with 100 IU of polymyxin B/ml. The preparation

was incubated at 37°C on a shaker (200 rpm) for 4-6 h. The supernatant was collected

after centrifugation as described earlier. All preparations were stored at -20°C after

determining their protein concentration11 and adjusting it to 5 mg/ml.

Test for cytotoxicity. Cytotoxicity of different preparations was determined on African

green monkey kidney (Vero) cells and Madin Darby bovine kidney (MDBK) cells

according to the method of Spiers et al.23 in 96 well tissue culture plates. Stock cells

were trypsinized and grown in the minimum essential medium (MEM, Hi-Media,

Mumbai) supplemented with penicillin G (100 IU/ml), streptomycin (100 µg/ml) and

foetal calf serum (10%). Monolayers were exposed to 100 µl of serially diluted (in PBS,

pH 7.2, 0.15 M) test preparation for 30 min and then 200 µl of MEM supplemented as

above was added to each well. Plates were incubated at 37°C and examined up to 72 h at

6 h interval under inverted light microscope from cytopathic effects (CPE). Cytotoxic

units per mg of protein were determined by dilution method21. Minimum quantity of the

test preparation in terms of protein inducing appreciable CPE in about 50% of tissue



culture cells within 30 h of exposure was designated as one cytotoxic unit. Some of the

tissue culture plates were stained with May Grhnwald Giemsa staining method14, using

absolute methanol as fixative and destainer instead of acetone, xylene and methanol

mixture.

Purification of cytotoxin. PBE was used for the purification of Salmonella cytotoxins as it

contained comparatively less impurities13,19. PBE obtained from 100 ml culture was salt

precipitated with ammonium sulphate at different saturation levels ranging from 30 to

80% at an intervals of 10%. Its pH was maintained near neutrality with help of

ammonium hydroxide. Prepcipitate was washed with corresponding ammonium sulphate

salt solution. The pellet was dissolved in distilled water and desalted on Sephadex G10

(Pharmacia) column equilibrated with Tris-HCl buffer (0.037 M, pH 7.2) and tested for

cytotoxicity. PBE from one litre culture was processed to get salt precipitated protein

(SPP) after determining the salt concentration that precipitated maximum amount of the

cytotoxin. After desalting, SPP was concentrated by dialysing the precipitate against

polyethylene glycol 20,000 to a concentration of 10 mg/ml.

For gel filtration, Seralose 6 B column (2 x 50 cm, SRL, Mumbai) was

equilibrated and eluted at a low rate 1 ml/min with Tris HCl buffer. Elution was

monitored on 280 nm with UV monitor (Pharmacia) and peak materials were collected,

tested for cytotoxicity and stored at -20°C. The cytotoxic peak materials were again

filtered through the same column and protein concentration was adjusted to 5 mg/ml

through dialysis as above. For further purification, 0.5 ml of the preparation was loaded

onto Mono-P column of FPLC Pharmacia equilibrated and eluted with Tris-HCl buffer

for 20 min at 0.75 ml/min speed. The salt gradient was raised by 0.5 M at every 20 min

interval up to 5 M. Finally, it was eluted with 0.025 M acetic acid. Eluent was monitored

at 280 nm and peak materials were collected separately, tested for cytotoxicity and stored

at -20°C. Cytotoxic peak materials collected from 20 runs of column P were concentrated

and passed again through the same column as above. For further purification of eluent



passed unbound to the column, the preparation was again eluted with Tris-HCl buffer

(pH, 8.6) and salt gradient (0.1-0.5 M) as above through the same colum equilibrated

with Tris-HCl buffer (0.037 M, pH, 8.6). Concentrated peak materials were subjected to

polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulphate PAGE16. For

PAGE and SDS-PAGE semipurified and purified preparations were loaded at the rate of

100 µg and 40 µg/ lane ,respectively. Gels were stained16 with coomassie blue and/or

periodic acid Schiff (PAS) stain to visualize the protein bands.

Preparation of immune serum. After collecting pre-immune serum, each of the three

rabbits, was inoculated intradermally in the abdominal region with 100 µg of purified

toxin without adjuvant1. Three booster doses, each of 100 µg, were administered at an

interval of 15 days. Serum was collected after 10 days of the last booster and stored at -

20°C.

Characterization of cytotoxin. To further define the purified cytotoxins, the preparations

were characterized physicochemically, biologically and immunologically as follows :

In order to evaluate heat sensitivity of the cytotoxin of the preparation, 0.5 ml of

the test preparation was heated at 60°C (30 min), 90°C (30 min), 100°C and 121°C (1, 5,

15 and 30 min).

To determine the effect of proteolytic enzymes, viz., trypsin, chymotrypsin,

pepsin, and bacterial proteases on cytotoxins, method of Jacks and Wu6 was followed.

The effect of pH on cytotoxicity was assessed by adjusting17 the pH of the toxin

in a range of 2.0 through 10.0 at an interval of 0.2 with 1N HCl or 1N NaOH. The test

material was incubated at 37°C for 4 h and finally the pH was readjusted to neutrality.

Cytotoxicity of treated preparations was determined as described earlier and compared

with that of untreated controls.

The effect of formalin on cytotoxicity was determined by the method of Lallier

and Lariviere9.



Haemolytic potential of various purified cytotoxins was determined by using

freshly collected erythrocytes of rabbit, sheep, guinea pig and humans (O group) after

washing them with PBS (pH, 7.2, 0.15 M). 100 µl of toxin preparation was mixed with

equal volume of 2 % RBC (red blood cells) suspension (in PBS pH, 7.2, 0.15 M) in a 96-

well plate and then incubated at 37°C for 6 h. After recording the results, the plates were

kept at 4°C for overnight and read finally.

Lecithinase activity of different cytotoxins was assessed on egg yolk agar plates7.

Wells (6 mm dia) were cut, sealed and filled with 100 µl of test preparations. The plates

were incubated at 37°C for 18-24 h. Formation of a zone of opacity around wells was

considered as positive reaction.

Mouse foot pad test20 was performed by injecting 0.05 ml of toxic preparation

intradermally in one of the hind foot pads, while the other was kept as negative control

(0.05 ml PBS, pH 7.2, 0.15 M). Observations were recorded after 24 h by measuring

thickness of both the foot pads. An index of ≥ 125 was considered positive.

Dermatotoxicity of test preparations was assayed by injecting 0.1 ml of each

preparation intradermally on abdominal region of New Zealand white rabbits17. The

reaction in skin, if any, was recorded at 6, 18, 24, 48, 72 h and 7 days post-inoculation.

Rabbit ligated ileal loop (RLIL) technique17 was used to detect the enterotoxic

effect of crude and purified cytotoxins. A dilatation index of ≥ 0.5 was considered

positive. Cholera toxin (Sigma Chemical Co., USA) was used as positive control.

Mouse lethality assay22 was performed by injecting 0.1 ml of serially diluted

preparation (in PBS pH 7.2, 0.15 M) intraperitoneally (ip) in 6 mice for determining 50%

lethal dose (LD50) of the toxins. LD50 was considered to be equal to the amount capable

to causing mortality within 10 days of inoculation in 50% of the mice.



To perform neutralisation test, serial dilution of Salmonella cytotoxins and their antisera

were mixed1 in 96 well tissue culture plates. The plates were incubated at 37°C for 4 h.

Contents of each of the wells was tested for cytotoxicity, dermatotoxicity, lecithinase

activity and mouse lethality following the procedures described earlier.

Agar gel precipitation test (AGPT) was performed5 to detect antigenic

relatedness between different S. Weltevreden toxins and their antisera. The effect of

various treatments on antigenicity of cytotoxins was also determined with this technique.

Besides, different purified preparations were also tested with AGPT against antisera to O

(3,10,15 ) and H (r, Z6) factors of Salmonella Weltevreden. To determine endotoxin

contamination in purified cytotoxic preparation, E. Toxate assay (Sigma Chemical Co.,

USA) was performed according to the instructions of manufacturer. Briefly, 0.1 ml of the

test solution (100 µg purified cytotoxins, 0.1 ml pyrogen-free water and 0.1 ml of E.

Toxate was mixed in endotoxin-free tubes, incubated at 37°C and examined after 30 and

60 min for gel formation). Positive control tubes contained 0.1 ml of S. Weltevreden LPS

(1 and 5 µg).

For performing agglutination test6, one drop of fresh 18 h culture was mixed with

a drop of antiserum and agglutination was observed within 5-10 min.

Results

Toxic activities of crude preparations. All the three preparations, i.e., the CFCS, CL and

PBE induced rounding, granulation, vacuolation, degenration, detachment and cell lysis

in the MDBK and Vero cell lines. On rabbit skin, crude cytotoxin caused central white

zone of necrosis surrounded by oedematous hot and red swelling. Necrosed area

sloughed off within 3 days leaving an ulcer behind. However, the CFCS did not cause

early necrosis but other lesions were similar to those induced by CL and PBE including

sloughing of skin. All the 3 preparations induced serosanguinous fluid accumulation in

RLIL loops and distension of the stomach. The mice dying with crude preparations



experienced diarrhoea before death and had distended stomach and urinary bladder with

pin-point haemorrhages on lungs, liver, kidneys and heart.

The amount of cytotoxin varied greatly in different preparations (Table 1). The

PBE was found to be best source of cytotoxin.

Purification of Salmonella cytotoxins. Differential salt fractionation studies revealed that

protein precipitated up to 50% and below salt saturation level did not contain much

cytotoxicity, while precipitates at 60% and 70% salt saturation levels had cytotoxic

activity equivalent to 128 and 256 cytotoxic units/mg of protein, respectively. There was

no appreciable increase in cytotoxic activity on further raising the salt concentration.

Therefore, proteins that precipitated up to 50% salt saturation level were discarded and

then the salt concentration was raised to 70% saturation level to obtain the precipitate

for further purification.

On gel filtration through Seralose 6 B, two peaks (Fig. 1) were observed. The

first one was eluted in 33 to 43 ml and the second between 45 to 67 ml of eluent. The

first peak contained no significant cytotoxic activity, while the second peak contents

displayed marked cytotoxic effects on cell lines as well as in rabbit skin. The second

peak contents yielded more than 6 protein bands on native PAGE.

Concentrated second peak contents from Seralose 6 B column, when eluted

through Mono-P column yielded 6 major peaks (Fig. 2) of which 4 peaks (i.e. 1, 3, 4 and

6) were found to be cytotoxic to variable degrees on Vero and MDBK cells. Peak I was

eluted unbound to column P yielded one thick and two thin protein bands on PAGE and

SDS PAGE (Fig 3); when this peak contents were eluted through Mono-P column

equilibrated with Tris-HCl buffer (0.37 M, pH, 8.6), it yielded single vytotoxic peak at

0.1 M NaCl gradient, while other peaks at 0.0 and 0.5 M salt gradient were non-toxic.

Cytotoxic eluent yielded single protein band at about 67 kDa level in SDS PAGE (Fig 3).

This cytotoxic peak was considered as purified cytotoxin I. In addition to properties

mentioned in Table 2, it also induced hyperaemia, depilation and scab formation away



from intradermal inoculation site in rabbits on either regions (Fig 4), while in mice, whole

of the hind quarters and tail developed areas of frank necrosis (Fig. 5) after 5-7 days of

cytotoxin inoculation. In RLIL loop assay, the toxin induced very little serosanguinous

fluid accumulation yielding RLIL indices ranging from 0.2 to 0.5. However, marked

distention of stomach and urinary bladder in rabbits and mice was invariably observed.

The toxin induced hyperthermia by 1.5 to 2.5°C after 4 to 6 h of intraperitoneal

inoculation. Dose-dependent loss of appetite was evident in all the animals experimented

with toxin. The dead mice and rabbits had extensive haemorrhage in lungs (Fig 6) and

kidneys. Besides, pin-point haemorrhages and small focal areas of necrosis were also

observed in the liver and spleen. Peritoneal cavity, thoracic cavity, meninges and brain

had pin-point to large areas of haemorrhages and extensive congestion. Small intestines

were full of blood tinged contents.

Native PAGE analysis of 3rd cytotoxic peak revealed it as single protein of about

65 kDa and was designated as cytotoxin II. The characteristics of cytotoxin II have been

depicted in Table 2. Apart from these, it also induced diarrhoea in mice after

intraperitoneal inoculation and positive RLIL response with indices ranging from 0.7 to

1.85, depending on dose. Fluid in RLIL was blood tinged (Fig 7). However, other

lesions, viz., accumulation of fluid in stomach, distension of urinary bladder and

haemorrhages in visceral organs, as observed with cytotoxin I were absent.

The 4th and 6th cytotoxic peaks also yielded single protein band both in PAGE

and SDS- PAGE at a level of about 55 and 72 kDa (Fig 3,8) and were designated as

cytotxin III and IV, respectively.

Besides, characteristics shown in Table 2, Type III cytotoxin appeared to be the

least toxic with no effect in RLIL, rabbit skin or mouse foot pads. It could not induce

lethality in mice even at 2 mg dose level. It induced distension in tissue culture cells,

often with massive vacuolation, granulation and multinucleation and eventual death (Fig.

9).



Type IV cytotoxin revealed phospholipase C activity on egg yolk agar and intense

necrotizing effect on rabbit skin (Fig. 10). In dead mice, except haemorrhagic fluid in

peritoneal cavity, no gross lesion was observed in brain, thoracic cavity or visceral

organs. It failed to induce fluid accumulation in RLIL but haemorrhages were present in

the inoculated loop. It invariably haemolysed the erythrocytes of sheep, rabbit, guinea pig

and human (O group) requiring 10, 8, 6 and 6 µg cytotoxin for complete haemolysis of

RBC (100 µl, 1 % v/v) within 6 h.

Serum Studies. Antisera to all the four cytotoxin formed precipitation lines in AGPT with

only homologous purified cytotoxin and crude preparations (PBE and CL). Also,

antisera to cytototxins- I, II and III produced line of precipitation with concentrated CFCF

(10 mg protein/ml). The antisera neutralized toxic effects of homologous toxin only and

no cross neutralization was observed in any of the bioassay models. The antisera except

antitoxin-IV, agglutinated the whole cell culture of S. Weltevreden.

The effect of heat, pH and proteolytic enzymes on cytotoxicity has been presented

in Table 2. These treatments had similar effect on antigenicity except that at pH 9.6

cytotoxicity was lost irreversibly while the treated preparations reacted with antitoxin

serum. None of the four purified cytotoxin preparations reacted with antiserum against

either O, H or common eneterobacteriaceae antigens. E Toxate analysis of purified

cytotoxic preparations revealed absence of endotoxin.

Discussion

Salmonella Weltevreden has frequently been isolated from ready-to-eat meat

products18,22 and has been shown to be a potent cytotoxin producer 12,22. In

the present investigation, crude preparations (CFCS, CL and PBE) of S. Weltevreden

induced similar types of CPE in Vero and MDBK cells and lesions in the skin and

internal organs of rabbit as reported earlier in case of cytotoxic preparations from a

number of other Salmonella serovars1,3,12,13,22..



Partial purification of Salmonella cytotoxin was earlier achieved by means of gel

filtration13, membrane filtration and dialysis1,5,22. In this study, after partial

purification through salt precipitation, dialysis and gel filtration, further purification was

attempted by using ion-exchange chromatography. However, it did not yield promising

results as the proteins were either bound too tightly (DEAE-Sephadex) to be eluted with

buffer or eluted without any binding (CM-Sephadex) to the matrix. Therefore, partially

purified toxin was eluted through column P, which is a weak anion exchanger.

Interestingly, the preparation could be fractionated into 6 major peaks of which four

contained cytotoxic activity. This indicates that the cytotoxic proteins are highly charged.

The elution patterns of cytotoxins I, II, III and IV of S. Weltevreden through

Mono-P column and chromatofocusing of purified preprations indicated that their PI

values were approximately 9.0, 7.0, 5.6 and 3.0, respectively. SDS-PAGE profile

indicated their molecular weight to be approximately 67.65, 55 and 72 kDa, respectively.

Although these appears to be no report on PIs of Salmonella cytotoxin, closely related PIs

of shigalike toxin (SLT) I (7.0), SLT II (4.1-5.1) and SLT IIv (9.0) of E. coli26;

Klebsiella pneumoniae cytotoxin (KCT) II (6.8), KCT III (5.6) and KCT I (9.0)20 suggest

some structural similarity between Salmonella, E. coli and Klebsiella cytotoxins. It has

been claimed that antishiga toxin serum9, which neutralized SLT I of E. coli24 also

neutralized S. typhimurium cytotoxins but further studies did not confirm antigenic

relationship between Salmonella cytotoxins and other enterobacterial cytotoxins.

Formalin treatment of all 4 SCTs resulted in loss of their cytotoxic potential

similar to that observedduring earlier studies18,22 and can be ascribed to protein

denaturing activity of formalin.

Neither pepsin nor protease had any appreciable effect on any of the 4 SCTs but

it was observed that trypsin and chymotrypsin enhanced cytotoxicity of SCT I and II,

while these had adverse effect on SCT III and IV. Enhanced cytotoxicity of SCT I and II

in presence of enteric enzymes is significant as it might contribute to enteropathogenicity



of the organism. Similarly, augmentative effect of toxicity of different cytotoxic

preparations of Salmonella1,22 and Klebsiella19 and of other enteropathogens18 are on

record. This has been ascribed to the disintigration of cytotoxin molecule in smaller

biologically active subunits or hyperactivation of the less active cytotoxin18. Further

studies in this regard are, however, warranted for precise understanding of the actual

mechanism underlying it.

Isolation of 4 distinct cytotoxic moieties from a single Salmonella strain is

significant as there seems to be no previous report in this regard. Singh22 claimed

purification of a single cytotoxin of S. Weltevreden and Ashkenazi et al.1 predicted the

presence of more than one type of cytotoxin in different strains of Salmonella on the basis

of varied responses of partially purified Salmonella cytotoxins to heat, pH and

proteolytic enzymes. However, it appears that they did not investigate the subject further

to confirm it. The presence of four distinct cytotoxic factors with different biological

activities might be responsible for the varied toxic effects i.e., Verocytotoxic, enterotoxic,

dermatotoxic and neurotoxic effects of crude/partially purified preparations of different

Salmonella cystotoxins as reported earlier1,3,13,22. For example, cytotoxic preparations

from certain Salmonella strains failing to induce dermonecrosis in rabbit skin might be

lacking cytotoxin IV. Cytotoxin I, which caused haemmorhages in the intestine along

with fever might be responsible for dysentery like symptoms, while cytotoxin II had

diarrhogenic activity; these symptoms are commonly observed in enteric fever15.

All the four types of cytotoxins were found to be immunogenic in rabbits.

Formation of single precipitation bands in AGPT only between specific cytotoxin and its

homologous antiserum indicates that they do not share antigenic determinant. Negative

results in E-Toxate assay revealed absence of endotoxin in purified cytotoxins. It was also

substantiated by serum neutralization studies. AGPT results by using antisera to O and H

factors and common enterobacteriaceae antigen further revealed absence of contaminants

like LPS or flagellar proteins in purified preparations.



In conclusion, isolation of four distinct cytotoxins from a single Salmonella strain

with varied biological activities, physicochemical characteristics and antigenicity

suggested multiplicity of cytotoxic moieties. This offers possible explanation for

discrepancies that exist in literature with regard to characteristics of crude/partially

purified cytotoxin of different Salmonella strains. Obviously, it will contribute

significantly to the understanding pathogenesis of salmonellosis.

Acknowledgement

The authors are thankful to Indian Council of Agricultural Research, New Delhi,

for providing funds under the National Fellow Scheme on ‘Salmonella enterotoxins and

Council of Scientific and Industrial Research, New Delhi, for awarding the Senior

Research Fellowship to the first author.



References

1. Ashkenazi, S., T.G. Cleary, B.E. Murray, A. Wanger and L.K: Pickering.

Quantitative analysis and partial characterization of cytotoxin production by

Salmonella strains. Infect Immun. 56 (1988) 3089-3094

2. D'Aoust, J.Y. : Salmonella, In : Doyle M.D. (ed) Food borne Bacterial Pathogens,

Marul Dekker Ind; pp. 332-362 (1989)

3. Gonzalez, E.A., J. Blanco, M. Blanco, J.I. Garabal, and M.P. Alonso : Enterotoxic,

cytotoxic, necrotoxic and lethal activities in cell free extracts of Salmonella

strains isolated from humans. Zbl Bakt. 271 (1989) 281-292

4. Hsu, H.S. : Pathogenesis and immunity in murine salmonellosis. Microbiol. Rev.

53 (1989) 390-409

5. Hudson, L. and F.C. Hay : Practical Immunology, 2nd Edn. Blackwell, Oxford. pp.

117-120 (1980)

6. Jacks, T. M. and B. J. Wu : Biochemical properties of Escherichia coli low molecular

weight heat stable eneterotoxin. Infect Immun. 9 (1974) 324-347

7. Joshi, R.K. and V.D. Sharma : Detection of thermolabilie phospholipase-C in the cell

free supernatants of Salmonella. Indian J. Comp. Microbiol. Immunol. Infect.

Dis. 12 (1991) 153-157

8. Kety, I. : Toxins as virulence factors of bacterial enteric pathogens. Acta

Microbiologica Hungarica. 32 (1985) 279-304.

9.Lallier, R. and B. Lariviere : Effect of different treatment on the activity of heat labile

enterotoxin of Escherichia coli. Canadian J. Comp. Med. 42 (1978) 214-218

10. Lax, A.J., P.A. Barrow, P.W. Jones and T.S. Wallis : Current perspectives in

salmonellosis. British Vet. J. 151 (1995) 351-377

11. Lowry, O. H., N. J. Rosenbrough, A. L. Farr and R. J. Randalt: Protein measurement

with Folin phenol reagent. J. Biol. Chem. 13 91951) 265-377



12. Malik P., V.D. Sharma, and R. Chandra : Cytotoxigenicity in Salmonella Serovars.

Indian J. Exp. Biol. 33 (1995) 177-181

13. Malik, P., V. D. Sharma, and D. C. Thapliyal: Partial purification and

characterisation of Salmonella cytotoxin. Vet. Microbiol. 49 (1996) 11-19

14. Negroni, G. : Tissue culture techniques. In : Harris, R.J. (ed) Techniques in

Experimental Virology. Academic Press, London. pp 328-358 (1964)

15. Parker, M.T. : Enteric infections : typhoid and paratyphoid fever. In : Smith, G.R. and

Easman, C.S.F. (eds) Topley and Wilson's Principales of Bacteriology, Virology

and Immunity. 8th Ed. (3) Edwards and Arnold, London. pp 423-446 (1990)

16. Saoji, A.M. and P.M. Khare : Polyacrylamide gel electrophoresis (PAGE) : Disc

electrophoresis. Indian J. Pathol. Microbiol. 28 (1985) 179-186

17. Sharma, V.D., S.P. Singh and A. Taku : Purification of Salmonella stanley enterotoxin

and its immunology and dermatotoxicity. Indian J. Exp. Biol. 30 (1992) 23-25

18. Sharma, V.D., D.C. Thapliyal, S.P. Singh and P. Malik : Cytotonic and cytotoxic

enterotoxins of enterobacteria. Indian J. Microbiol. 32 (1992) 327-356

19. Singh, B.R. : Purification and molecular characterization of Klebsiella pneumoniae

cytotoxins. Ph.D. Thesis, G.B. Pant Univ. of Agric. & Tech., Pantnagar. pp 243

(1997)

20. Singh, B.R. and S.B. Kulshreshtha : Preliminary examinations on the

enterotixigenicity of isolates of Klebsiella pneumoniae from seafoods. Int. J.

Food Microbiol. 16 (1992) 349-352

21. Singh, B.R. and A.K. Tiwari : Toxigenicity of Edwardsiella tarda isolates of fish and

pig origin in experimental models. Indian J. Exp. Biol. 33 (1996) 1254-1256

22. Singh, Y. : Study on Salmonella cytotoxin. Ph.D. Thesis, G.B. Pant Univ. of Agric. &

Tech., Pantnagar. pp 173 (1995)



23. Spiers, J.I., S. Stavric and J. Konowalchuk : Assay of Escherichia coli heat labile

enterotoxin with Vero cells. Infect. Immun. 16 (1977) 617-622

24. Strockbine, N.A., L.R.M. Marques, J.W. Newland, H.W.D. Smith, R.K. Holmes and

A.D.O'Brien : Two toxin converting phages from Excherichia coli 0157:H7

strain 933 encode antigenically distinct toxin with similar biological activities.

Infect. Immun. 53 (1986) 135-140

25. Wallis, T.S., W.G. Starky, J. Stephen, S.J. Hadden, M.P. Osborne, and D.C.A. Candy

: Enterotoxin production by Salmonella typhimurium strains of different

virulence. J. Med. Microbiol. 21 (1986) 19-23

26. Weinstein, D.L., M.P. Jackson, L.P. Parera and J.E. Samuel : In vivo formation of

hybrid toxin comprising shigatoxin and shigalike toxins and role of B subunits in

localization and cytotoxic activity. Infect. Immun. 57 (1989) 3743-3750

27. Wray, C. : Salmonellosis : A hundred years old and still going strong. British Vet. J.

151 (1995) 339-341



Legends for Figures

1. Cytotoxin elution pattern of polymyxin B extract (PBE) of S. Weltevreden

through Seralose 6 B column (2 x 50 cm) eluted with Tris HCl buffer (pH 7.2,

0.037 M).

2. Cytotoxin elution pattern through Mono-P column (Pharmacia) loaded with 2.5

mg of second peak from Seralose 6 B column, eluted with Tris HCl (0.037 M, pH

7.2) NaCl gradient and finally with 0.025 M acetic acid.

3. Sodium dodecyl sulphate polyacrylamide gel electrophoresis profile of different

purified and semipurified preparations of S. Weltevreden cytotoxins.

Lane 1: Cytotoxic peak eluted through Seralose 6B column.

Lane 2: Molecular weight markers.

Lane 3: Cytotoxin I.

Lane 4: Cytotoxin I on re-purification at pH 8.6

Lane 5: Cytotoxin III.

4. Depilation of hair and scab formation at wither region after 5 days of intradermal

inoculation of cytotoxin I (100 µg) at andominal region.

5. Dermonecrosis on hind quarters and tail of mice (T) after 5 days of inoculation of

cytotoxin I (100 µg) compared with healthy control (C ).

6. Haemorrhage in lung of mice died after 18 h of intraperitoneal inoculation of

cytotoxin I (100 µg).

7. Accumulation of haemorrhagic fluid in rabbit ligated ileal loop1 inoculated with

100 µg of cytotoxin II, clear straw-coloured fluid in loop2 and 4 inoculated with

50 µg of Cholera toxin.



8. SDS-PAGE profile of cytotoxin IV revealing single band of it at the level of 72

kDa mol. wt. (Lane 1), Lane 2 shows the mol. wt. markers.

9. MDBK cells showing rounding, enlargement, granulation and vacuolation after

18 h of cytotoxin III exposure, stained with modified May Grhnwald and

Giemsa staining procedure (250 x).

10. Rabbit abdominal skin revealing zone of necrosis surrounded by red margins, 12

h post-inoculation with 50 µg of cytotoxin IV, intradermally.

TABLE 1 : Cytotoxicity of different preparations of S. Weltevreden on cell lines _________________________________________________________________ Type of Preparation Protein Conc. Cytotoxic Units/mg protein (mg/ml) ------------------------------------- MDBK Cells Vero Cells _________________________________________________________________ Cell free culture 0.85 32 32 supernatant Cell lysate 6.25 64 128 Polymyxin B extract 3.20 160 160 _________________________________________________________________



TABLE 2 : Biological and physico-chemical characteristics of four S. Weltevreden cytotoxins ______________________________________________________________________________________________________________________________________________ Characteristics Type of Cytotoxin I II III IV Binding to column P at pH 7.2,0.037M No Yes Yes Yes Salt gradient needed for elution No 0.5M 1.5M >5.0M Effect on Vero/MDBK cells Rounding, detachment Rounding,degeneration, Vacuolation, syncytia- Rapid degradation and lysis detachment, lysis formation, multinucleation Vero cytotoxic units/mg of protein 640 320 1280 1280 Effect on rabbit skin Delayed (72h) hyperaemia, Hyperaemia and oedema No visible effect White zone of necrosis oedema and pus formation in 18 h and detachment with red margins of epidermis in 72 h Effect in mouse food pad Delayed oedema (72 h) Hyperaemia and oedema No effect Necrosis of foot pad Lethality to mice (LD50) Yes (120 µg) Yes (1000 µg) ND Yes (150 µg) Haemolytic action on rabbit, sheep and No No No Yes human erythrocytes PI values 9.0 7.0 5.6 3.0 Inactivation of cytotoxicity with heat treatment 121°C, 1 min 100°C, 30 min 100°C, 30 min 90°C, 30 min Activity in pH range of 5.0-8.0 5.6-8.4 4.8-8.2 2.5-7.5 Irrerversible inactivation at pH <4.6 and >9.6 <4.6 and >9.2 <4.0 and >9.2 <2.0 and >8.6 Effect of trypsin (0.1%, 6 h) 50% increase in 40% increase in 50% loss in activity 90% loss in cytotoxicity cytotoxicity cytotoxicity Effect of chymotrypsin (0.1%, 6 h) 20% increase in 10% increase in 50% loss in activity 76% loss of cytotoxicity cytotoxicity cytotoxicity Effect of 0.05% formalin (12 h) 100% loss of 100% loss of 100% loss of 100% loss of cytotoxicity cytotoxicity cytotoxicity cytotoxicity ______________________________________________________________________________________________________________________________________________ ND : could not be determined because mouse survived after inoculation of >1.0 mg cytotoxin/mouse. Note : Pepsin and protease had no effect on cytotoxicity of any of the 4 cytotoxins







Detection and Partial Purification of Salmonella serovar Typhimurium Cytotoxic Protein Affecting Seed Germination

Documents