I "eterinary Microbiology, 27 ( 1991 ) 277-282 277 Elsevier Science Publishers B.V., Amsterdam

Antibody to Pseudomonas pseudomallei exotoxin in sheep exposed to natural infection

G. Ismail ", R. Mohamed b, S. Rohana b, H.S.M. Sharifah c and N. E m b i b

~Faculty {)f Science and Natural Resources and t't"acu/ty ~lLil~" Science, UKM Bangi, Universiti Kebangsaan Malaysia Sabah ('ap~lpt~s, Locked Bag

62, Kota Kinabalu, Sabah, Malaysia "l ~,terinary Research Institute, lpoh, Malaysia

(Accepted 22 October 1990)

ABSTRACT

Ismail, G., Mohamed, R., Rohana, S., Sharifah, H.S.M. and Embi, N., 1989. Antibody to l'seudo- monas pseudomallei exotoxin in sheep exposed to natural infection. Vet. Microbiol., 27: 277-282.

Specific antibody to Pseudomonas pseudomallei exotoxin was detected in sheep sera exposed to natural infection. An enzyme-linked immunosorbent assay (ELISA) was used. Serum antitoxin was present in 49.3% ofsera obtained from a flock of sheep naturally exposed to P. pseudomallei infection. Among these sera, 17.0% gave titers of 10 000. In contrast, serum antitoxin was present in only 6.0% of sera collected from sheep kept on a melioidosis-free farm. The ELISA reactivity of all positive sera could be completely absorbed with purified P. pseudomallei exotoxin. Similarly, preincubalion of the exotoxin-coated wells with specific antiserum inhibited the ELISA reactivity of sheep sera. The results indicate that exotoxin is produced in vivo during infection by P. pseudomallei.

INTRODUCTION

In Southeast Asia and tropical Australia, melioidosis is an important cause of morbidity and mortality, particularly among sheep (Thomas et al., 1988; Mustaffa and Leong, 1989). Pseudomonas pseudomallei produces a variety of extracellular products which may assume a critical role in the pathogenesis of infection in laboratory animal models (Ismail et al., 1988 ). The lethal ex- otoxin is the most toxic substance known to be produced by P. pseudomallei. The toxin is produced as a 36 000-dalton polypeptide chain, which is able to inhibit intracellular protein synthesis by the same mechanism as diphtheria toxin (Collier, 1975) and the exotoxin A of Pseudomonas aeruginosa (Ig- lewski et al., 1977). It catalyzes the transfer of adenosine-5-diphosphate ri- bose from oxidized nicotinamide adenine dinucleotide to elongation factor 2, an enzyme involved in eucaryotic protein synthesis (Mohamed et al., 1989a). The toxin can cause damage to various cell culture systems (Maheswaran et

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al.. 1987; Mohamed cta l . , 1989b) and may cont,-ibutc to cellula! damage during infection with 1'. i~.~eudotmd/ei. Protection has been achicxcd againsl experimentally induced septicemic melioidosis in ,nice preimnaunizcd ~ith heat-inactivated exotoxin purified from a cell-free extract o1 the o,-ganism ( Razak et al.. 1987 ). These observations indicate that the cxotoxin possesses considerable biological significance and may be of major importance in the pathogenicity of P. i~.seztdoma//ei. In this study, we prescnt cx idcncc thai an- tibody to P. l~seudoma//ei exotoxin is present in sheep naturally exposed to the organism.

M A T E R I A L S A N D M E T H ( ) I ) S

P. pseudomallei exoto.vin Exotoxin was purified as previously described (Wasiman et al., 1987 ) from

a culture of P. pseudomallei originally obtained from a male patient who died of melioidosis at the Microbiology Department, Faculty of Medicine, Univ- ersiti Kebangsaan Malaysia. The exotoxin migrated as one major band on analytical polyacrylamide gel electrophoresis, and had a 50% lethal dose in laboratory mice of approximately 30-50/~g.

Rabbit immzmizalioH New Zealand White rabbits weighing 3-5 kg were given a total of approxi-

mately 100 l~g of purified exotoxin in four intravenous and one or two sub- cutaneous injections in incomplete Freund's adjuvant. The rabbits were bled from the marginal ear veins before each injection, and exotoxin antibody was checked quantitatively by enzyme-linked immunosorbent assay ( ELISA ).

Serolo,~ica/ survey deteclion antiloxin antibody The surveys were carried out on two sheep farms, A and B. Farm A, at Ijok,

Selangor, Malaysia, was known to be contaminated with P. pseudomallei, based on the results of the isolation of the organism from three animals from the flock that had died after showing clinical signs diagnosed as melioidosis. Farm B, at Serdang (Faculty of Veterinary Science, Universiti Pertanian Ma- laysia), is approximately 100 km from farm A, and was considered to have been free of P. pseudomallei infection, since no clinical evidence of melioi- dosis was detected in animals in the flock. The sheep from the two farms were of the same breed, and were aged 1½-2 years. Exotoxin antibody was deter- mined in 364 sera obtained from sheep presumed to have been naturally ex- posed to P. pseudornallei (farm A ) and 130 sera from the control group (farm B ). The sera were from single samples obtained from each animal.

ANTI BODY TO PSIfUDOMO,V. IS PSEL'I)OMtLLEI EX()TIN IN SHEEP 279

ELISA procedure The detection of specific antibody to P. pseudomallei exotoxin was per-

formed according to a method described previously (Ismail et al., 1987a). Sera were tested at dilutions of 10 -2, 10 -3 and 10 -4. To determine the anti- genic similarity of exotoxins produced during infection and in laboratory cul- ture conditions, a competitive ELISA was employed with slight modifications of the method previously reported (Ismail et al., 1987b). The sheep sera were preincubated at 4 ° C overnight with increasing concentrations of purified ex- otoxin, and tested in the ELISA. The decrease in absorbance value resulting from antigenic competition afforded by purified exotoxin was expressed as a percentage of the optical density reading for a negative control. To check whether the sheep antisera possessed reactivity to exotoxin similar to that of antisera of rabbits actively immunized with the toxin, exotoxin-coated wells were preincubated with rabbit antisera. After incubation, the plates were washed and then used in the ELISA.

R E S U L T S A N D D I S C U S S I O N

All sera giving titers of 100 or above were considered positive for antibody to the exotoxin (Ismail et al., 1987a). Antibody was detected in 49.3% of the sera obtained from the exposed flock (Fig. 1 ), while only 6% of the control sera obtained from the melioidosis-free farm gave a positive result. Titers of 10 000 or greater were observed in 17.0% of sera from the exposed flock, whilst the highest titer for control sera was only 1000. Notably, among the exposed flock, sera from three sheep that had succumbed to melioidosis gave

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Fig. 1. Prevalence of antitoxin antibody and comparison of antibody liters determined by ELISA in sheep naturally exposed to P. pseudomallei (farm A) and in the control group (farm B ).

either low titers (100) or were negative lot antitoxin antibod\ . r h e ELLS,\ reactivity of each positive serum could be completely absorbed with cxotoxin obtained from laboratory cultures (Fig. 2 ). Similarly, the EI,ISA reactivity could be substantially abolished by incubating the system wilh antisera to the exotoxin (Fig. 3 ).

The development of serum antibody suggests that exotoxin is produced during infection by P. lZveudomallei. Antitoxin antibody could have several influences on the development and progression of P. pseudoma//ei infection. First, it may protect the affected animals from death due to melioidosis through toxin-neutralizing antibodies. Second, preexisting antitoxin anti- body may modify melioidosis when it occurs, resulting in a subacute rather than an acute disease. The fatal outcome in three sheep that died due to t'. pseudoma/lei infection may have been due to a low level or absence of anti- toxin antibody in their serum. Alternatively, a low level or absence of detect- able serum antitoxin in fatal cases could be explained by early death allowing insufficient time for an adequate antibody response. The origin of exotoxin activity in 6% of the control sheep is not clear. The low antitoxin titers de- monstrable in this control flock may have been due to the ubiquity of t'. p.~et¢-

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Fig. 2. Quanti tat ive inhibition of ELISA reactivity of sheep sera by increasing concentrations of exotoxin purified from a laboratory culture of P. lZveudoma/lei.

~.NTIBODY TO PSEUDOMONAS PSEUDOMALLEI EXOTIN IN SHEEP 281

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Fig. 3. Inhibition of ELISA reactivity of sheep sera with dilutions of antisera from rabbits im- munized with purified exotoxin ofP. pseudomallei ( • ) and normal rabbit sera ( O ).

domallei in the soils of Malaysian farms, resulting in a high frequency of col- onization of healthy sheep by the organism.

Of late, the relatively frequent infections with P. pseudomallei of farm ani- mals in endemic areas have fueled the search for rapid diagnostic procedures and more effective modes for prevention of melioidosis. The importance of exotoxin as one of the most potent virulence factors produced by P. pseudom- allei has been established in laboratory mice (Razak et al., 1984). Our pres- ent data suggest an in vivo pathogenic role of exotoxin during natural infec- tion by P. pseudomallei. For veterinary and clinical purposes, identification of serum antibody specific against such a virulent determinant could be of much value in the diagnosis of both recent and older infections. For example, in the case ofP. pseudomallei infection, a sudden rise in previously low anti- exotoxin titer to a significantly higher titer may represent a serological mark distinguishing between chronic or asymptomatic melioidosis and an acute form. Data obtained from competitive ELISA procedures employing purified exotoxin as well as rabbit antitoxin sera demonstrate the immunogenicity and antigenic similarity of P. pseudornallei exotoxin produced in sheep in re- sponse to infection and the lethal exotoxin produced in vitro from laboratory

culturcs. These a t t r i b u t e s o f the t o x i n h a v e made the c o n c e p t ~ I immum)h)g~v

intervention in 1'./~,~clldoma//ci infection attractive and more Ik'asiblc.

\ ( K N ( )WI_EI)( ;EMEN 1~

This study was supported by IRPA Grant 07-03-012 and EEC (3rant TS2- 0230-UK. The authors gratefully acknowledge the kind support of Dr. Zainri Saad from the Faculty of Veterinary Science and Breeding, UPM. and P.J. Diagnostic Laboratory, Department of Animal Services, Ministry of Ag- riculture, for the gift of serum samples. We are indebted to Ahmad Mansor and Ruhayah Jamal of UKM and M. Karuppiah of VRl for their skilful tech- nical assistance. We thank Mrs. Noraini Abdul Hamid for the typing and her secretarial assistance during preparation of the manuscripl.

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