Indian Journal of 'Ex peri menial Biology Vol. 43, July 2005, pp. 626-630

Novel haemolysins of Salmonella enterica spp. enterica serovar Gallinarum

Ravi Kant Agrawal, B R Singh*, N Babu & Mudit Chandra

National Salmo1lella Centre (Vet), Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar 243 122, India

Received 29 November 2004; revised 29 March 2005

Haemolysins of Salmo1lella are important due to their probable role in pathogenesis of systemic salmonellosis and use in sub-serovar level typing. The present study was undertaken to determine haemolytic potential of Salmo1lella Gallinarum strains through phenotypic and genotypic methods. Amplification of haemolysin gene (clyA) and cytolysin gene (slyA) was attempted in order to determine their role in haemolysin production. Study on 94 strains of S. Gallinarum revealed the production of two types of haemolysis viz., beneath the colony haemolysis (BCH) or contact haemolysis and clear zone haemolysis (CZH). Haemolysis was observed on blood agar prepared with blood of cattle, buffalo, sheep, goat, horse, rabbit, guinea pig, fowl, and human blood group A, B, AB and O. Although, haemolysis was also observed on blood agar prepared with whole blood, clarity of zone was more evident on blood agar made from washed erythrocytes. Clear zone haemolysis was best observed on blood agar prepared with washed erythrocytes of goat and a total of 12% (II of 94) S. Gallinarum strains under study produced CZH on it. The clyA gene could not be detected in any of the 94 strains under study, while slyA gene could be amplified uniformly irrespective of haemolytic potential (CZH) and haemolytic pattern (BCH) of the strains. The study suggested that the two types of haemolysis (CZH and BCH) observed among S. Gallinarum strains may not be due to either slyA or clyA gene products and thus there may be some other gene responsible for haemolytic trait in Gallinarum serovar. Different haemolytic patterns of strains under study indicated multiplicity of haemolysins in S. Gallinarum

Keywords: Haemolysins, Salmo1lella ellterica, Serovar Gallinarum

Salmonella, a well known, ubiquitous pathogen of man and animals remained uncontrollable even after 110 years of its discovery, probably because of its large number (>2500) of serotypes and rapid adaptability to different hosts causing variety of syndromes in different animal and human beingsl . Fowl typhoid organism, Salmonella enterica

. subspecies enterica serovar Gallinarum (Salmonella Gallinarum), first reported by Klein2 after an out break in England in 1888, is found almost in all ' poultry producing areas of the world3. In India, though the pathogen is widely prevalent throughout pOUltry rearing belts, clinical disease is limited to a few pockets mostly in Bengal, Assam, Southern Bihar and Uttar Pradesh4. Salmonellosis in poultry is a highly devastating disease and mortality in natural infection is often high5

• It affects many different kinds of birds irrespective of their age and sex; however, young growing birds are more susceptible6

• The pathogen is known to possess many virulence factors,

*Correspondent author-Phone: 0091-581-2301865(0), -231531O(R), Fax: 0091-581-2303284 E-Mail : singh_br1762@rediffmail.com;brsI762@ivri.up.nic.in

but the role of haemolysins in pathogenesis is poorly ' understood7

. Among the Salmonella haemolytic factors, SeT IV, present in about 16% strains of zoonotic Salmonella and about 4% of Salmonella Gallinarum strains and characterized as a cell bound phospholipase, is important8,9. Besides, one of the cytotoxin genes encoding a 16 kDa cell associated contact haemolysin has been shown to be necessary for virulence and survival of pathogen within macrophages 10. Another poorly expressed salmolysin having roles in killing of M cells and perpetuation of systemic salmonellosis is encoded by slyA gene l1,l2, Recently, another haemolysin similar to E. coli cytolysin A (clyAEd and also named cytolysin A (clyA) , a pore forming cytotoxic protein, has been shown to be produced by only Salmonella Typhi and Salmonella Paratyphi A serovars l3 , More recently, haemolysins active on washed erythrocytes, produced by strains of host adapted Salmonella serovars has been shown to be important for invasion and survival of Salmonella in mononuclear cells 7, The present study was undertaken to evaluate haemolytic potential of S. Gallinarum isolates of Indian origin and to

AGRAWAL et al: NOVEL HAEMOLYSINS OF SALMONELLA GA,LLINARUM 627

determine probable role of different genes in encoding haemolysins, the probable cause of haemolytic anaemia has been reported consistently in fowl typhoid.

Material and Methods Bacterial strains---Frozen stocks of a total of 94 S.

Gallinarum, one Salmonella Typhi and tWQ Salmonella Paratyphi A strains available at National Salmonella Centre (Veterinary), Division of Bacteriology & Mycology, IVRI, Izatnagar, India were revived and confirmed serologically by slide agglutination test using somatic factor '9' specific

. 14 d al b antiserum an so y Salmonella group '0' specific PCRI5

. All the strains were maintained on nutrient agar slants (HiMedia, Mumbai) and Dorsett egg medium at 4°C till tested.

Haemolysin assay---In order to check the production of haemolysins, blood agar plates were made with washed erythrocytes (5% v/v) of different animals including cattle, buffalo, sheep, goat, horse, rabbit, guinea pig, chicken and of human beings (blood group A, B, AB and 0)7. Blood agar plates were also made with defibrinated blood of different animals and human beings (5% v/v). To evaluate haemolysin production, all test and control cultures were spot inoculated on blood agar plates and incubated at 37°C. Plates were observed regularly at 12 hr interval for the development of zone of haemolysis up to 72 hr.

PCR amplification of cytolysin gene (slyA)-l'o amplify cytolysin gene (slyA), standard PCR protocol lO was followed using Sal L 1 (5' AGG AGA TGA AAT TGG AAT CGC CA 3') and Sal L 2 (5' TGC CCC TGC ACC TCA ATC GTG AG 3') primers. The PCR product was characterized by submarine gel electrophoresis on 1.5% agarose gel.. Gene Ruier™ 1oo bp DNA ladder plus (MBI Fermentas, USA) was used as molecular weight marker.

PCR amplification of haemolysin gene (clyA)-l'o amplify clyA gene in S. Gallinarum isolates standard protocol was followed using stmOl (5' CGC AGG TIC TGA ATG CGC AA 3') and stm02 (5' TAA TAC CTG CTG TAG CAA GG 3') primers 13. The PCR products (2650 bp amplicon indicating presence of gene and 420/433 bp amplicon indicating absence of gene) were characterized by submarine gel electrophoresis on 1.5% agarose gel. S. Paratyphi A and S. Typhi chromosomal DNA were used as

positive control in the PeR reaction. Gene Ruler™ 100 bp DNA ladder plus (MBI Fermentas, USA) was used as molecular weight marker.

Results All the 94 isolates of S. Gallinarum agglutinated

with '0' 9 specific factor serum and PCR for rfb gene for 0 '9' antigen, resulted in amplification of 720 bp amplicon specific to group 0 with all the 94 isolates. Salmonella strains produced two types of haemolysis on blood agar made with washed erythrocytes viz., beneath the colony haemolysis (BCH) and clear zone haemolysis (CZH). BCH became evident in 48-72 hr at 37°C and was more clearly visible after removal of the colony from the plates. Clear zone haemolysis became evident within 24 hr of incubation at 37°C but zone of clearance went on increasing up to 72 h; of incubation (Fig. 1). All the 94 strains of S. Gallinarum produced beneath the colony haemoly­sis, but the haemolytic pattern was found to vary with washed erythrocytes of different animals. On the basis of haemolysis on · different blood agars, 94 strains could be classified into 19 haemolytic types (hly types

Fig. l~lear zone haemolysis (CZH) on blood agar plate made with washed erythrocytes of goat. [G44-Salmonella Gallinarum strain No. 44; G90-Salmonella Gallinarum strain No. 90; G95-Salmonella Gallinarum strain No. 95; G20-Salmonella Gallinarum strain No. 20; PTA44b-Salmonella Paratyphi A strain No.44b; PTA345-Salmonella Paratyphi A strain No.345; PT A345NalR-Salmonelia Paratyphi A strain No.345 Nal R; TI06-Salmonella Typhi strain No.206]

628 INDIAN J EXP BIOL, JULY 2005

Table 1). In contrast, clear zone haemolysis was evident on blood agar made with washed erythrocytes of goat, sheep and calf only with the best result on blood agar made with goat erythrocytes. Only 11 of the 94 Gallinarum strains (No. 42, 44, 49, 50, 58, 89, 90, 94, 95, 96 and 97) produced clear zone haemolysis (CZH), similar to that produced by S. Typhi and S. Paratyphi A strains on blood agar made with washed goat erythrocytes. Haemolysis (CZHlBCH) by S. Gallinarum strains was least evident on blood agar made with washed erythrocytes of chicken and 45 of the 94 strains could not induce any kind of haemolysis on chick erythrocytes. PCR amplification of haemolysin gene (clyA) revealed that both the haemolytic (CZH) and non-haemolytic S. Gallinarum strains yielded 420/433 bp product (the indicator for absence of clyA gene), while all the three

Table i-Haemolytic types of Salmonella ellterica spp. enterica serovar Gallinarum

Haemolytic type (Hly)

Hly I Hly II HlyIII HlyIV

HlyV

Hly VI

HlyVII '

Hly VIII

Hly IX Hl y X

Hly XI

Hly XII ,

Haemolytic activity detected against erythrocytes of

All types of erythrocytes All but horse erythrocytes All but chicken erythrocytes

. All but chicken and horse \ , -erythrocytes ~AII but chicken, horse and ~. guinea pig erythrocytes ;, All but chicken horse and

rabbit erythrocytes All but horse and guinea pig erythrocytes All but chicken and guinea pig erythrocytes All but goat erythrocytes All except goat and chicken erythrocytes All but chicken, guinea pig and sheep erythrocytes All but horse, guinea pig and

, goat erythrocytes Hly XIII : '~, All but chicken, horse, goat and

, ' sheep;~rythrocytes Hly XIV : ~11 but ~orse, goaumd cattle

ery'throcytes . , " ,

Hly XV All but sheep, goat and horse

Hly XVI

Hly XVII

Hly XVIII

Hly XIX

erythrocytes Only human erythrocytes except AB+ group Only human and cattle erythrocytes Only human. cattle and rabbit erythrocytes Only cattle, guinea pig and rabbit erythrocytes

Total number of strains

13 10 4

44

6

2

2

2

" ,

control positive strains of S, Typhi and S. Paratyphi A, yielded specific 2650 bp product (Fig. 2). PCR for cytolysin gene (slyA) on S. Gallinarum, S. Typhi and S. Paratyphi A resulted into amplification of specific 470 bp product (Fig. 3), irrespective of haemolytic activity (CZH, BCH) of the strains on erythrocytes of different animals and humans.

Discussion Fowl typhoid, caused by Salmonella enterica ssp

enterica ser Gallinarum, one of the major devastating problems of poultry industry, leads to heavy economic losses every year all over the world barring

Fig, 2-Amplicon of haemolysin (dyA) gene. [Lane I-Salmonella Gallinarum Strain No, 44; Lane 2-Salmollella Gallinarum Strain No, 90; Lane 3-Salmonella Paratyphi A Strain No,44b; Lane 4-Salmonella Paratyphi A Strain No.345; Lane 5-Salmonella Typhi Strain No,206; Lane M- 100 bp DNA ladder plus (MBI Fermentas,USA)]

470

Fig, 3--Amplification product (470bp) of slyA gene, [Lane I-Salmonella Gallinarum Strain No, 44; Lane 2-Salmonella Gallinarum Strain No, 90; Lane 3-Salmonella Paratyphi A Strain No.44b; Lane 4-Salmonella Typhi Strain No,206; Lane M-lOO bp DNA ladder plus (MBI Fermentas,USA)]

AGRA WAL et al: NOVEL HAEMOLYSINS OF SALMONELLA GALLINA RUM 629

only a few developed nations. Once the pathogen is established in a population it is destined to persist until the flock lasts leading to variety of syndromes due to numerous virulence factors of S. GaUinarum, viz., different colonization factors, adhesin molecules, and different enterotoxins and cytotoxins8

. However, haemolytic anaemia often observed in subacute infections is not explicitly understood. In recent past, a few haemolysins have been detected in strains of S. Typhi and S. Paratyphi A serovars 13 but, no such haemolysin has yet been reported in any of S. Gallinarum strains. This study on 94 strains of S. Gallinarum isolated from birds in different parts of the country revealed that all S. Gallinarum strains can induce beneath the colony haemolysis (BCH) on blood agar made with washed erythrocytes of one or other animals. However, they varied in their potential to lyse different types of erythrocytes leading to classification of strains into 19 different haemolytic types indicating the multiplicity of haemolysins produced by S. Gallinarum. In addition, clear zone haemolysis (CZH) was also produced by 11 strains on BA made from washed erythrocytes of goat, which further suggested that S. Gallinarum may produce multiple haemolysins, however, it needs further investigations to characterize them.

Clear zone haemolysin (CZH) produced by S. Gallinarum appears to be different from those produced by S. Typhi and S. Paratyphi A as CZ haemolysin of S. Gallinarum was more active on washed erythrocytes, while haemolysins of serovars Typhi and Paratyphi A have been reported to haemolyse unwashed erythrocytes too 13. Moreover, none of the S. Gallinarum could amplify a 2650 bp product reported to be encompassing haemolysin gene (clyA) of S. TyphilParatyphi A. Instead, a 420/433 bp product, revealing absence of clyA gene in the locus, was observed, similar to the earlier studies on non­Typhi and non- Paratyphi A nonhaemolytic strains 13

•

Therefore, further studies are essential to identify the gene responsible for CZ haemolysin of S. Gallinarum.

Both CZ and BC haemolysins of S. Gallinarum are found to be much different from the haemolytic cytolysins of Salmonella serovars reported earlier9

.

Whereas, 470 bp PCR product indicating the presence of slyA encoding for a contact haemolysin could be detected in all of the 94 S. Gallinarum strains, irrespective of their haemolytic patterns indicating that either slyA coded product is not responsible for haemolysis induced by S. Gallinarum isolates or the

gene has got lot of variability leading to production of haemolysin with varying haemolytic potential. But this should be confirmed only after the cloning and sequencing of the amplified slyA gene from different S. Gallinarum strains. Failure of 45 strains to lyse chicken erythrocytes is also unexplainable which may be either due to lack of receptors on chicken erythrocytes for haemolysins of S. Gallinarum or might be part of some adaptation mechanism to permit the pathogen to persist in the specific host system as proposed earlier on the basis of studies on S. Abortusequi (equine specific) or S. Paratyphi (human specific), reporting failure of lyses of equine and human erythrocytes by respective strains7 and the phenomenon may be having some association with host. specificity of the Salmonella serovars. The observations of the present study indicated the existence of some other novel haemolysins in Salmonella Gallinarum strains, which might be the real cause of haemolytic anaemia observed in fowl typhoid infections in poultry birds.

Acknowledgement Thanks are due to the Director, IVRI, Izatnagar for

financial assistance.

References I Popoff M Y & LeMinor L, Antigenic formulas of the

Salmonella serovars, [WHO Collaborating Centre for Reference on Salmonella, Institute Pasteur, 28 Rue du Dr. Roux, 75724 Paris, France] 2001, 6.

2 Klein E, Uber eine epidemische krankheit der Huhner, Verursacht durch einer Bacillus-Bacillus gallinarum, Zentralbl Bakteriol Parasitenkd Abt I Orig, 5 (1889) 689.

3 Pomeroy B S, Fowl typhoid, Diseases of Poultry, 8th edition edited by: M S Hofstad, H J Barnes, B W Calnek, W M Reid & H W Yoder, (Iowa State University Press, Allies, Iowa) 2001,79.

4 Chauhan H V S & Roy Sushovan, Poultry disease diagnosis and treatment. 2nd edition, (New Age International (P) Limited, Publishers, New Delhi) 2001, 54.

5 Smith H W & Tucker J F, The virulence of Salmonella strains for chickens; their excretion by infected chicken. J Hyg, Cambridge, 84 (1980) 479.

6 Rao S B V, Narayanana S, Ramnami D R & Das J, Avian Salmonellosis : Studies on Salmonella Gallinarum, Indian J Vet Sci, 22 (1952) 199.

7 Singh B R, Singh V P, Agarwal M, Sharma G & Chandra M, Haemolysins of Salmonella, their role in pathogenesis and sUbtyping of Salmonella serovars, Indian J Exp Biol, 42 (2004) 303.

8 Singh B R & Sharma V D, Isolation and characterization of four distinct cytotoxic factors of Salmonella Weltevreden, Zbl Bakt (International J Med Microbiol), 289 (1999) 457.

630 INDIAN J EXP BIOL, JULY 2005

9 Singh B R & Sharma V D, Purification and characterization . of phospholipase C of Salmonella Gallinarum, Indian J Exp Bioi, 36 (1998) 1245.

10 Libby S J, Goebel W, Ludwig A, Buchmeier M, Bowe F, Fang F C, Guiney D G, Songer J G & Heffron F, A cytolysin encoded by Salmonella is required for survival within macrophages, Proc Natl Acad Sci, USA, 91 (1994) 489.

11 Daniels J J D, Autenrieth I B, Ludwig A & Goebel W, The gene SlyA of Salmonella Typhimurium is required for destruction of M cells and intracellular survival but not for invasion or colonization of the small intestine, Infect Immun, 64 (1996) 5075.

12 Watson P R, Pauline S M, Bland A P, Libby S J, Jones P W

& Wallis T S, Differential regulation of enteric and systemic salmonellosis by SlyA, Infect Immun, 67 (1999) 4950.

13 Oscarsson J, Westermark M, Lotdahl S, Olsen B, Palmgren H, Mizunoe Y, Wai S N & Uhlin B E, Characterization of a pore forming cytotoxin expressed by Salmonella enterica serovar Typhi and Paratyphi A, Infect Immun, 70 (2002) 5759.

14 I;:wing, W H, Edwards and Ewing's identification of enterobacteriaceae, 4th edition, (Elsevier Science New York) 1986.

15 Hoorfar J, Baggesen D L & Porting P H, A PCR based strategy for simple and rapid identification of rough presuvvmptive Salmonella isolates, J Microbiol Methods, 35 (1999) 77.