Indian Journal of Experimental Biology Vol. 38, November 2000, pp. 1152-1158

Isolation and characterization of Salmonella Gallinarum cytotoxic factors

B R Singh* & V D Sharma

Department of Veterinary Microbiology and Immunology,College of Veterinary Sciences, G.B. Pant University of Agriculture & Technology,Pantnagar 263 145, India

Received 6 July 1999; revised 26 July 2000

Two distinct cytotoxic factors isolated from a Salmonella Gallinarum strain recovered from a bird died during an outbreak of fowl typhoid were purified to homogeneity through ciprofloxacin extraction, salt precipitation, dialysis, gel­filtration, ionexchange chromatography and chromatofocusing. These were designated as Salmonella Gal lin arum cytotoxin I (GCT-I) and II (GCT-II). GCT-I was a glycoprotein having mol.wt and pi of Ca 70 kDa and 8.8, respectively. It was lethal to birds (LDso ,150 Ilg) inducing fowl typhoid like lesions. GCT-II, a protein with Ca 55 kDa mo\.wt., was not lethal but caused haemorrhagic dialThoea on intraperitoneal inoculation in birds. Both the cytotoxins induced cytopathic effects (CPE) in Vero and Madin Darby bovine kidney (MDBK) cells, enterotoxicity in rabbit ileal loop, dermatotoxicity in the rabbit skin and specific neutralizing antibodies in rabbits. These were active only between a narrow pH range of 6 to 8.5 and thermostable at 90°C (\ min) but lost their activities on boiling. Trypsin and chymotrypsin enhanced their cytotoxicity, while pepsin, papain, protease, lipase and urea (5 M) had no appreciable effect on their cytotoxicity. Sodium carbonate (0.05 M) and formaldehyde (0.05%) had no effect on antigenicity of both the cytotoxic factors but rendered them nontoxic. Identification and characterization of cytotoxic moieties of S. Gallinarum not only reveals the important virulence factor but also indicates about the use of toxic factors as a candidate for toxoid vaccine and immunodiagnostics.

About 100 years ago, member of the genus Salmonella currently designated as Salmonella enterica subsp enterica serovar Gallinarum1 was identified as cause of fowl typhoid in domestic and wild birds affecting both young and adults leading to heavy mortality and colossal economic loss specially on large organized farms with poor hygiene2

,3.

Though S. Gallinarum is a host adapted serovar, its aSSOCiatIOn with occasional incidents of acute gastroenteritis in human beings is also on record2

. A large plasmid has been shown to be associated with its invasive ability and survival in reticuloendothelial cells2

,3 which are thought to be its major virulence markers, but absolutely very little is known about the pathogenesis of salmonellosis in birds. Although cytotoxigenicity has been reported in most of the strains belonging to different Salmonella serovars4

,5

and their cytotoxins have been demonstrated 'to play major role in pathogenesis of salmonellosis6

,7,

information on S. Gallinarum cytotoxins is lacking. This communication reports purification and characterization of two distinct cytotoxic factors of S.

*Present address: Scientist, National Salmonella Centre (Vet), Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar 243 122, India,

Gallinarum contributing significantly to its pathogenecity.

Materials and Methods Bacterial straills - Salmonella Gallinarum was

isolated8 as pure culture from the liver, kidney and spleen of a freshly dead birds obtained from a poultry farm having an outbreak of fowl typhoid. Shigatoxin producer strain of Shigella dysenteriae (E-91) and Shiga like toxin-II (SLTII) producer Escherichia coli (E-4l) procured from National Salmonella Centre (Vety.) Indian Veterinary Research Institute, Izatnagar, were used as positive controls.

Antisera-Antisera to Salmonella Weltevreden cytotoxin (SCT) I, II, III, cholera toxin, Klebsiella pneumoniae cytotoxin (KCT) I, II and III and phospholipase-C available in the laboratory were used.

Experimental animals - Adult (3 month old) New Zealand white albino rabbits and adult White Leghorn birds (1.2-1.5 kg) were procured from the Laboratory Animal Section and Poultry Research Centre of G.B. Pant University of Agriculture and Technology, Pantnagar, respectively and were maintained on Salmonella free diet in indi vidual cages.

Crude cytotoxin preparations - Cell-free culture supernatant (CFCS), cell sonicates and polymyxin B

SINGH & SHARMA :ISOLATION & CHARACTERIZATION OF SALMONELLA CYTOTOXIC FACTORS 1153

extract (PBE) of Salmonella Gallinarum and reference strains (E-91 and E-41) were prepared according to Malik et al.9 Similar to polymyxin B extract, other agents viz. amikacin, cephazolin, carbenicillin, ciprofloxacin, enrofloxacin, nalidixic acid, ofloxacin, pefloxacin, acridine orange, sodium desoxycholate (each at 100 Ilg/ml concentration) and urea (5 M) were used for extraction of cytotoxic moieties of S. Gallinarum. All the crude cytotoxin preparations were stored at -20° C till tested.

Purification of cytotoxin( s) - For purification of cytotoxins, ciprofloxacin extract (CE) of S. Gallinarum was concentrated by ammonium sulphate salt precipitation at different levels of saturation. Cytotoxic fractions were pooled, dialysed for desalting and concentration (10 mg/ml). It was then loaded on a pre-equilibrated Seralose-6B (SRL, Mumbai) column (70 cm x 2 cm) and eluted with the same buffer (Tris HCI, 0.037 M; pH, 7.2) at a flow rate of 0.75 ml/min and monitored on FPLC-UV monitor (Pharmacia) at 280 nm to collect protein peaks. Cytotoxic fractions (5 mg proteinlml) were subjected to ion-exchange chromatography on Mono­P FPLC column (pharrnacia) using Tris-HCI (0.037 M, pH 7.2) buffer for equilibration and elution along with NaCI salt gradient from 0.0 to 2.5 M by raising it by 0.5 M at a regular interval of 20 min. The cytotoxic peaks were chromatofocussed on Mono-P column following the protocol of MIS Pharmacia (FPLC). The chromatofocussed purified cytotoxins were electrophoresed on 10 % polyacrylamide gel (PAGE) and filtered through Sephadex G-100 (Sigma) column (70 cm x 2 cm) along with markers of known molecular weight (mol.wt.) to determine the mol.wt and apart from Coomassie blue, gels were also stained with periodic acid Schiff (PAS) method to determine the nature of proteins 10 • In different cytotoxin preparations, protein was estimated with Lowry's method 1 I.

Cytotoxicity testing - To determine the cytotoxic units, serially diluted preparations were tested on Vero and Madin Darby bovine kidney (MDBK) cell lines (procured from the Centre for Animal Disease Research and Diagnosis, Indian Veterinary Research Institute, Izatnagar) in 96-wel1 ti<,;slIe culture plates l2

•

Enterotoxicity assay - Ell i :1"\.lf<' Ixicity of various cytotoxic preparations was dClc'llIlined in rabbit ileal loops 1 3 by inoculating serially diluted preparation containing 20, 40, 60, 80, 100 and 200 Ilg of toxin in 1 ml of phosphate buffered saline (PBS, pH 7.2). A

preparation yielding dilatation index (DI) of 0.4 or above was considered as enterotoxic.

Dermatotoxicity assay - It was performed in adult rabbits on abdominal skinl4 by inoculating 100 III of the test preparation containin~ varying amount of cytotoxin (2.0 to 128 Ilg protein). The minimum quantity of cytotoxin producing 10 mm zone of hyperemia within 36 hr of inoculation was considered as one dermatotoxicity unit.

Lethality to adult birds - Serially diluted toxin in 0.5 ml PBS (PH 7.2) was injected intraperitoneally in adult White Leghorn birds and observed for 21 days for feed and water consumption and signs of disease and mortality. Birds died during experimentation were necrospsied.

Preparation of hyper immune serum - After collecting preimmune serum, 3 rabbits were inoculated intradermally at multiple (10-12) sites (without adjuvant) each with 100 Ilg of purified cytotoxin preparation contained in 1 ml PBS (though 100 Ilg toxin was dermatotoxic when inoculated at one site,it had insignificant toxicity when inoculated at more than 5 sites). Two boosters, each of 100 Ilg were given at an interval of 15 days through subcutaneous route. After 10 days of the 2nd booster, 50 ml blood was drawn from heart of each rabbit and serum was collected and stored at -20°C in 2 ml aliquotes.

Immunological tests - Agar gel precipitation test (AGPT)15 and serum neutralization test4 were performed to determine cross reactivity and antigenicity

Physico-chemical characterization - To determine heat resistance of different purified cytotoxins, preparations were heated at 60, 70, 90 and 100° C for 1 min and 10 min separately and tested for cytotoxicity.

Effect of enzymes viz. trypsin, chymotrypsin, pepsin, papain, bacterial protease and lipase (Sigma) on cytotoxins was assessed 16 by allowing the enzymes to react for 6 hr at 37°C.

Effect of pH on different cytotoxinns was asssessed by adjusting the pHI7 at 2 to 10 at a regular interval of 0.5.

Effect of formaldehyde (0.05%), sodium carbonate (Na2C03, 0.05 M) and urea (5 M) on cytotoxins was determined by treating test preparations for 6 hr at 37°CI4

.

Results and Discussion Cell-free-culture-supematant (CFCS) of S.

Gallinarum (S-28) was found non-cytotoxic and exposed

1154 INDIAN J EXP BIOL, NOVEMBER 2000

cells remained healthy (Fig. Ia) but concentrated (lOx) CFCS, cell sonicate (CS) and different antibiotic/chemical extracts induced cytopathic effects (CPE) similar to that caused by CS of Shiga-toxigenic (E-91 ) and SLT II produicing strain (E-41). CPE were characterized by initial rounding, granulation, conglomeration, detachment and cell lysis in Vero and MDBK cells (Fig. Ib). The number of cytotoxic units (CTU) in each mg protein of different preparations varied greatly from 1 in CFCS to 64 in ciprofloxacin and enrofloxacin extracts. Pefloxacin, ofloxacin, polymyxin B and nalidixic acid extracts had 48 CTUs, while urea, cephazolin and carbencillin extracts contained 20 CTUs. The CS, amikacin extract

Fig. l- (a) : Healthy monolayer of MDBK cells (l50x) (b): rounding, conglomeration and detachment ofMDBK cells 18 hr after exposure of cytotoxic preparation (Ciprofloxacin extract) (l50x)

and sodium desoxycholate extracts had 36 erUs/mg of protein. Mitomycin-C, urea, polymyxin B sulphate and sodium desoxycholate have been shown earlier to enhance the extracellular release of Salmonella enterotoxins and cytotoxins5

,9,18, 19 but so far effect of other antibiotics has not been explored. Further, what may be the effect of different antibacterials in pathogenesis of clinical salmonellosis is yet to be studied. On the basis of maximum release of cytotoxin augmented by ciprofloxacin and its availability in market, it was selected for further work. In precipitates obtained through salt precipitation at 20, 30 and 40 % saturation level, no cytotoxic activity was detected. At higher saturation levels, i.e., 50, 60, 70 and 80 % of ammonium sulphate, each mg precipitate had 80, 160,256 and 80 CTUs, respectively. Thus, results of fractional salt precipitation revealed that cytotoxin is . salt precipitable between 50 to 80 % saturation levels with maximum at 80 % salt saturation level, as reported earlier for cytotoxins of Salmonella Weltevreden6

, S. Nchanga, S. Virchow9 and Klebsiella pneumoniae17. However, total CTUs precipitated were maximum at 80 % saturation level, erus per mg of protein precipitate at 80 % saturation were less than at 60 or 70 % saturation, which may be due to precipitation of lot of non-toxigenic proteins along with cytotoxins. The elution profile of salt precipitated cytotoxic contents revealed three peaks (Fig. 2), of which only 2nd peak eluted in 48th to 78th ml of eluate was cytotoxic, having 248 CTUs/mg of protein. In the other two peaks, cytotoxicity could not be established even after concentration to 5 mg/mI.

Polyacrylamide gel electrophoresis (PAGE) analysis of the 2nd peak contents revealed the presence of at least 5 protein bands similar to that reported earlier in Seralose-6B eluted cytotoxic peak of S. Weltevreden9

• Ion exchange chromatography of 2nd peak contents of Mono P column yielded 4 major (PI to P4) and a few minor peaks (Fig. 3). The 1st (PI) and 3rd (P3) peaks had cytotoxic activity on Vero and MDBK cells producing CPE similar to that caused by ciprofloxacin extract. On chromatofocussing, pI of PI and P3 were determined to be 8.8 and 6.8, respectively, which were quite similar to that of ser­I and SCT-II of S. Weltevreden reported earlier7

•

Therefore, purified cytotoxic peaks PI and P3 were designated as GCT -I and GCT -II, respectively. Each mg protein of GCT-I and GCT-II contained 160 and 640 erus, respectively. GCT-I was equally effective

SINGH & SHARMA :ISOLATION & CHARACTERIZATION OF SALMONELLA CYTOTOXIC FACTORS 1155

on Vero and MDBK cells but GCT-II was twice more detrimental to Vero cells than MDBK cells (Table 1).

On intradermal inoculation, 100 Jlg GCT -I caused delayed (>18 hr) hyperaemia followed by central zone

0.8

0.7

0.6

E 0.5 c 0 110

0.4 N

~ C 0 0.3

0.2

0.1

0 0 20 40

II

60

Elute (mil

III

80 100 120

Fig. 2 - Purification of Salmonella cytotoxins through Seralose 6-8 column (70 cm x 2 cm) equilibrated and eluted with Tris HCI (PH 7.2, 0.037 M)

3

Pl P2 P3 P4

0.9 ,OOline

---,Sail gradient 2.5

Q8

0.7 ..--- fo2

' ,.

E 0.6 C c: GI 0 '6 0>

0.5 1.5 III

N .--- .. 10

Cl

U C III 0 0.4 z

0.3 I;~

; 0.2

,:-'-- ,..: 0.5

0.1

\ A Ak f LA. 0 u

20 40 60 80 100 120 140

Elute (mi)

Fig. 3 - Ion exchange chromatography of peak II (cytotoxic peak) of Seralose 6-8 column eluate, on Mono-P ·column, with Tris-HCI buffer (PH 7.2, 0.037 M) and NaCl salt gradient.

of necrosis, sloughing and scar formation. After one week of GCT-I inoculation, inoculated rabbits developed hyperemic lesions at shoulder region too, i.e., away from the site of intradermal inoculation. The hyperemic areas eventually get depilated with underlying brown scab. Similar lesions also developed after inoculation with CE but not with GCT -II. The latter, however, induced hyperemia at the inoculation site, persistent up t036 hr and a thin membrane was sloughed off without leaving any scar tissue behind, usually after 5-7 days of inoculation. Intradermal inoculation of 31.25 IJg and 8 Jlg of GCT­I and II, respectively induced a unit hyperemic reaction in rabbit skin. Similar type of skin lesions have been reported earlier with semipurified6

•7

,20 and purified Salmonella and Klebsiella cytotoxins 17

•21

..

In the rabbit ileal loop assay, both the cytotoxins were found to be enterotoxic causing enterohaemor­rhages. The maximum DI for GCT-I (80 Jlg) and GCT-II (100 IJg) were 0.56 and 0.87, respectively. In earlier studies too, Salmonella cytotoxin has been shown to induce accumulation of serosanguinous fluid in RLIL 4.6.7.22, similar to that caused by SLTs of E. coli23

•

On intraperitoneal inoculation, GCT -I proved lethal to birds with LDso of 150 IJg. The dead birds on necropsy revealed pin-point necrotic foci in liver and kidney, enlarged spleen and haemorrhages in lungs, brain, meninges and intestine. The birds who died early due to acute toxicity had distended gall bladder and urinary bladder with focal haemorrhage in fundi of the organs. Small intestines were full of, blood-

Table 1 - Physico-chemical and biological characteristics of S. Gallinarum cytotoxins (GCTs)

Biological characteristics GCTI GCTII Vero cytotoxic units/mg of toxin 160 (6.25 j.Jg) 640 (1.56 j.Jg) MOBK cell cytotoxic units/mg of 160 (6.25 Ilg) 320 (3.12 j.Jg) toxin LOso to adult birds 150j.Jg >1000 j.Jg Oermatotoxic units/mg toxin 32 (31 .25 j.Jg) 125 (8 Ilg) Maximum dilatation index in 0.56 0.87 RLIL Parenteral dose for inducing NO IOOllg diarrhoea Molecular weight 70kDa 55-60 kOa PI 8.8 6.8 Chemical nature Glycoprotein Protein

Figures in parentheses show minimum detectable amount with the assay LOso, lethal dose just sufficient to kill 50 % of the inoculated birds; MOBK, Madin Darby bovine kidney; RLIL, rabbit ligated ileal loop; NO, not detectable because lethal dose was less than the diarrhoeagenic dose.

1156 INDIAN J EXP BIOL, NOVEMBER 2000

tinged yellowish fluid similar to that reported due to SCf-1 of S. Weltevreden in mice7

• The birds who died after 3 days of inoculation had neither distended urinary/gall bladder nor fluid in intestine. However, pus like material was present in all the three organs and the birds also developed paralysis before death. Paralytic response of Salmonella toxin of other serovars is well documented5,6,9,19,23,24, although

Table 2 - Effect of different physico-chemical treatments on S. Gallinarum cytotoxins (GCTs)

Treatments C~totoxic* Units/mg erotein GCTI GCTII

No treatment 160 640 80°C for I min 160 640 90°C for 1 min 160 640 90°C for 10 min 80 20 100°C for I min 8 0 100°C for 10 min 0 0

pH 2.0 to 5.0 for 6 hr 0 0 pH 5.5 for 6 hr 80 320 pH 6.0 to 8.0 for 6 hr 160 640 pH 8.5 for 6 hr 160 160 pH 9.0 for 6 hr 160 0 pH 9.5for 6 hr 20 0 pH 10.0 for 6 hr 0 0 0.1 % Trypsin for 6 hr 180 960 0.1 % Chymotrypsin for 6 hr 240 720 0.1 % PepsinlPapainlProtepase 160 640

for 6 hr Sodium carbonate (0.05 M) for 0 0 6hr Formaldehyde (0.05 %) for 6 hr 0 0 Urea (5 M) for 6 hr 128 640

*Cytotoxic units determined on Vero cells.

production of neurotoxic cytotoxin by S. Gallinarum could not be traced in literature . The birds that recovered after receiving LD50 of ocr -I started taking feed after 3-4 days of anorexia but most of them developed skin lesions and had ruffled feathers. Although ocr-II did not cause death in birds even in large doses (1 mg), it induced haemorrhagic diarrhoea and transient anorexia in doses as low as 100 Ilglbird. The duration and severity of diarrhoea was dose­dependent as 100 Ilg toxin caused only 1-2 bouts of diarrhoea in 8-12 hr post-inoculation, while 200 Ilg toxin took only 2 hr to induce severe diarrhoea leading to prostration. The birds could recover only after 2-3 days of inoculation. Similar type of response in mice on parenteral inoculation of E. COU25 and Salmonella cytotoxin6

,7 has been reported earlier. Thus, it seems that the two cytotoxins in concert contribute significantly to the pathogenesis of fowl typhoid. GCf-1 appears to be mainly responsible for producing septicaemic changes in visceral organs and

Table 3 -Neutralization of Salmonella. Gallinarum cytotoxins (GCTs) with different antisera

Toxin preparation and activity neutralized Antisera used for neutralization --~~~~--~~~~~~~~~~~~~~~~~~~--~~--=-

Anti-GCT-I Anti-GCT-II Anti-SCT-I Anti-SCT-II Anti-phospholipase-C A. Crude Extract (Ciprofioxacin extract) I) Derrnatotoxicity

a) Necrosis b) Hyperemia

2) Cytotoxicity 3) Enterotoxicity 4) Lethality to birds B. GCTI I) Derrnatotoxicity

a) Necrosis b) Hyperemia

2) Cytotoxicity 3) Enterotoxicity 4) Lethality to birds C. GCTII 1) Dermotoxicity- hyperemia 2) Cytotoxicity 3) Enterotoxicity

P (50%) P(70 %)

P (40 %) P(30 %) P (25 %) P (75 %) P(75 %)

+ + + + +

+ + +

-, not neutralized; P-partially neutralized; +, completely neutralized.

P(50%)

P (40 %) P (30 %) P (75 %)

+ + + + +

Figures in parentheses indicate approximate % reduction in biological activity of crude preparation.

P (50 %) P (60 %) P (30 %) P (30 %) P (75 %)

P (25 %)

+ + +

Antisera against SCT -m, KCT -I, II and m and Cholera toxin had no effect on toxicity of either crude or purified preparations. None of the antisera could neutralize crude ereearation of Shiga or Shiga like toxin used in the study.

SINGH & SHARMA :ISOLATION & CHARACTERIZATION OF SALMONEUA CYTOTOXIC FACTORS 1157

eventual death, while haemorrhagic diarrhoea often seen in fowl typhoid may be due to GCf-II.

Molecular weight of GCf-I was found to be Ca 70 kDa while that of GCf -II around 55 kDa. PAS stainidg of polyacrylamide gel revealed glycoproteinous nature of GCf -I, while GCf -II appeared to be simple protein. Further, physico-chemical characterization of GCfs (Table 2) revealed that these could withstand heat treatment at 90°C for 1 min but lost their cytotoxicity on boiling. Both the cytotoxins retained their full cytotoxicity between pH 5.6 to 8 but GCf-I was comparatively more resistant to alkaline pH (9). However, GCf -II was more stable in 5 M urea than the GCf -I. Formaldehyde and Na2C03 were detrimental to both the cytotoxins, while pepsin, protease, papain and lipase had no effect on their activities. Both trypsin and chymotrypsin enhanced cytotoxicity of S. Gallinarum cytotoxins similar to that of E. coli SLT-II26. The exact mechanism underlying enhancement in cytotoxicity is yet to be delineated.

Both the cytotoxins were immunogenic inducing the production of specific antibodies in rabbits. Serum neutralization titres ranged from 240 to 720 varying from animal to animal. Serum neutralization studies (Table 3) revealed that homologous antiserum neutralized all the biological activIties of the corresponding cytotoxin, while crude cytotoxin was not neutralized completely by either of antisera. Lethality to birds and dermonecrotoxicity of crude cytotoxins were neutralized completely only when anti-GCf -I serum was used along with anti-phospholipase-C. Anti-GCf-II neutralized the enterotoxicity of crude cytotoxin considerably. Anti-GCf-1 antiserum neutralized lethal effect of crude cytotoxins significantly (75 %), it had no effect on its diarrhoegenic activity. Thus, the results of serum neutralization further substantiated the aforesaid deductions that GCf-Iplayed a major role in causing mortality, while GCf-II was mainly responsible for diarrhoeagenic activity. Antisera to Salmonella Weltevreden cytotoxin I and II (SCf-1 and SCf-m also neutralized completely the biological activities of GCf-1 and II, respectively, while none of these antitoxins could neutralize cytotoxicty of Shiga toxin or SLT -toxin of E-91 and E-41 strains. Complete failure to cross neutralise the cytotoxins of other enterobacteria with anti­Salmonella cytotoxin or vice-versa indicated that cytotoxins of different enterobacteria, albeit biologically similar, are antigenically unrelated.

The observation of agar gel precipitation test (AGPT) with homologous, crude and heterologous

cytotoxins and their anti-cytotoxins also revealed antigenic ally distinct identity of GCf -I, II and phospholipase-Co

In conclusion, identification of two distinct cytotoxins with different biological, physico-chemical and immunological characteristics proved the multiplicity of cytotoxic factors in S. Gallinarum similar to that reported in other Salmonella serovars4

,7. Production of fowl typhoid like lesions in visceral organs on inoculation of GCf-1 and haemorrhagic diarrhoea by GCf -II suggested their major role in the pathogenesis of fowl typhoid. Besides, the findings have also paved the way for future investigations on exploitation of cytotoxins to develop toxoid vaccine and improved diagnostics.

Acknowledgement The authors thank the ICAR, New Delhi, for

financial help under the National Fellow Scheme on Salmonella enterotoxins and the Council of Scientific and Industrial Research, New Delhi, for Senior Research Fellowship to the first author.

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