J. Appl. Anim. Res. 18 (2000) : 9'7-102

Seroreactivity of an Antigen of Leptospira interrogans Serovar Grippotyphosa

S.K. Srivastava", Vinita Tiwari

Bacteriology and Mycology Division Indian Veterinary Research Institute

Izatnagar-243 122, India

(Received November 22, 1999; accepted May 1, 2000)

Abstract Srivastava, S.K. and Tiwari, V. 2000. Seroreactivity of a n antigen of Lepto- spira iiiterrogaiis serovar grippotyphosa. J. Appl. Anim. Res. , 18: 97-102.

Aritigeii wa,s extmcted from L. interrogans sr. grippotyphosa by sodium taurocliola,te treatment a,iid characterized. Using an ELISA the extracted antigen showed higher reactivity with the hoinologous aiitiserurit tltaii observed with aiitisera. against serova.rs canicola, pomona, pyrogenes, h.ardjo, ballum, patoc and andamana. Fractiomtioii of the extmcted a d g e i i ernployiiig ion-exchange cltroinatograpliy resulted in the sepa.ratioii of' relaiively specific major protein alorig with iioii-rea.ctiw proteiiis. Use of the a.iitigeii for coiiductirig ELISA for serodiagiiosis of leptospirosis i n field ccrttle indicated tlmt, a.s coinpared to microscopic agglutination test, it wa,s slightly less seiisitive but wa.s equally specific.

Key words : Leptospira. iiiterroga.iis, antigen, seroreactivity, sero-diagnosis, ELISA.

In trod uct ion Leptospira cells possess a variety of antigens exposed on their surface, notably the lipopolysaccharide (LPS) and membrane proteins. These antigens have been extracted from the cells and characterized employing different techniques with the purpose of standardization

*I+cscnt Adtlress: National Research Centre on Equines, Sirsa Road, Hisar-125 001, India.

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98 S.K. Sriuastava aiid V. Tiwari

of s e r o d i a g n o s t i c t e s t s , m o s t commonly t h e e n z y m e l i n k e d inimunosorbent assay (Trueba ef al . , 1990, Haalte et al . , 1993, Suruballi, 199'7, Suruballi et al., 1997). Though the use of LPS or proteins as the diagnostic antigen results in the improvement in the diagnosis of the disease, the technique followed for their extraction is usually cumbersome and beyond the scope of moderately equipped diagnostic laboratories. This h a s necessitated the need to look for a n extraction technique which is simple to perform and the resultant antigen is suitable for use as a diagnostic antigen. Sting a n d Dura (19%) extracted antigens from several Leptospii-a s t r a i n s using sodium taurocholate and reported their serovar specificity. I n the present s tudy, characterization a n d application of t h e ant igen, extracted by t reatment of Leptospira cells with sodiuni taurocholate, for serodiagnosis of leptospirosis is reported.

Materials a n d Methods Uac t e i * i a l s t r'a ii 1,: Lep t osp i ra i i i t e i-roga i i s , s e r ov a r s gi. ippo typ hosa, ca I i, ico la, p o iuoi !,a, p y roge ii,es, ic t e rohae i n o rr h.agiae, ha rdjo and L. 0iflc.z-a serovars patoc and aiidaiiiaiia, maintained in B.M. Division were used. The s t ra ins were procured from Leptospirosis Reference C e n t r e , A m s t e r d a m , t h e N e t h e r l a n d s a n d m a i n t a i n e d i n 1l:llinghausen and McCullough (EM) semisolid medium with serial passages .

.!htigeii pvepai.atioii.: Antigen against each of the Leptospira s t ra ins Lvas prepared in rabbi ts by inoculating IV formalin killed loG oi*ganisnis/ml for 3 consecutive days and repeating the same schedule a week later. Serum was collected after a week and stored frozen a t -2oc .

Soii tcatioii of Leptospii-a cells: Sonicated preparation of L. iiiterrogaiis serovar giippotyphosa was prepared a s described by Srivastava et crl. (1989).

Extiuctioii aiid piiwificatioit, of aiitigeii: Surface antigens of serovar giYppofyphosa were extracted using 2 % sodium taurocholate at 50C as described by Sting and Dura (1994). Protein in the antigen w a s measured by the method of Bradford (1976). For purification, column chromatography w a s employed a s described by Srivastava a n d Kumar (1992) using a DEAE sepharose CL-GB (Sigma) column (1.5

cin x 25 cm). Tris-HC1 buffer (30 mM), p H 8.6 was the equilibrating buffer. The gel in column was first washed with 150 in1 of the buffer and 10 mg of the crude antigen was loaded on to the column followed by the elution of the antigen fractions using a soclium chloride gradient start ing from 0. l M , 0.2M and 0.5hI. Approximately 250 nil of each of the sodium chloride solution with different molarity was used to achieve the elution. The eluates were collected in 3 ml fractions at the flow rate of 20 ml/h. Protein peaks were determined a t 280 nm and precipitated at 70% saturation of ammonium sulfate. Proteins in each peak was measured as above.

Eitzyiiie liiilzed i i w i uiiosoi-beiit assay (ELISA): ELISA was performed following the protocol described by Engvall and Perlinann ( l9 i l ) and Bercovich et al. (1990) with slight modifications. Antigen used was either sonicated preparation of grippotyphosa cells or its fractionated or crude extracted antigen, each in the concentration of 5 pg/well. The coating of the antigen was done a t 37C for 24 h followed by blocking using 5% skimmed inilk powder (Parag, India). Species- specific antiglobulin-HRPO conjugate used was gifted by National Insti tute of Immunology, New Delhi a n d the substrate used was orthophenylene diaininodihydrochloride (Sigma. USA). ' I he optical clensity was measured a t 492 n m using ELISA reader (Anthos Lale. Tech., Austria). Seruin dilution exhibiting a n O.D. which u-as double or more than the serum control, was taken a s a posit,ive reaction.

Mici.oscopic agglutiiiatioii test (MAT): MAT was performed using a microtitration procedure as described by Cole cf crL. (19i3). Antigen used was 5 - i days old culture of L. iiitkwogaiis serovar g'i.ippofjpl,oscr grown in EM medium.

Aiiiijz al se1.a: Sera collected or received were from 52 cattle belonging to various farms around Bareilly, Hyderabad and Warangal subjected to ML4T and ELISA and the specificity and sensitivit \. ELISA, as compared to MAT, was calculated as follows:

True positive Sensitivity: x 100

True positive + False negative

True negative

True negative + False positive Specificity: x 100

100 S.K. Srivasfava aiid V. Tiroari

Results and Discussion Treatment of cells of L. intei*i*og~aii.s serovar grippotyphosa resulted in the extraction of a n antigen. Purification of the extracted antigen was achieved using ion-exchange chromatography. The elution profile is shown in Fig. 1. A peak each a t 0.2 M NaCl and 0.5 M NaCl gradient was eluted. To study i ts seroreactivity, ELISA plates were coated with these purified fractions a n d tes ted against re fe re n ce ant is e r u in of se rova r giipp o t y p h osa a n d ca 11 a lzo I La e . Fraction eluted a t 0.2 M NaCl reacted with both the antisera exhibiting a titre of 1:500. The antigen fraction obtained at 0.5M gave,a titre of only 1:50. Obviously the 0.2 M Nacl eluted antigen fraction was more seroreactive, hence used for further studies.

Serospecificity of the crude and the purified fraction (0.2 M NaCl eluted) was studied using various Leptospira antisera. The results are given in Table 1. As compared to the sonicated cell preparation, the crude Sodium taurocholate extracted antigen showed low homologous as well as heterologous ELISA titres. The homologous titres were, however, higher than the heterologous titres. With the purified antigen the heterologous titres further decreased suggesting that the purification of the antigen resulted

Fig. 1: Elution profile of Sodium taurocholate extracted antigen of L. iiiterrogoits SI'. grippotyphosa using a DEAE-sepharose CL-GB column.

Seroreactiuity of leptospira 101

Table 1 Serospecificity of various antigens of serovar grippotyplzosa.

Antigen ELISA titres (x100) observed using antisera'

Giippo Carri Port1 Pyro Ictero patoc orida hor.djo

Sonicated cells 32 8 8 8 8 1 6 1 6 4

Sodium taurocholate extracted:

Crude 1 6 1 4 4 2 8 8 2

Purified 8 1 2 2 1 4 4 1

'Grippo., gr.ippotjjphoso; cani., carricola; pom., portlorlo; pyro.. pyrogeries; ictero., ic/cr.o/roertror~r.hagiae; anda., aridortiaria.

Next, the efficacy of the Sodium taurocholate extracted antigen in detecting cases of leptospirosis among cattle was tested using ELISA. A total of 52 cattle sera when subjected to internationally accepted MAT and ELISA, 30 were positive to MAT whereas only 26 were positive to ELISA. None of the remaining 22 MAT negative sera were positive to ELISA. The sensitivity and specificity of ELISA was thus calculated to be 88.2% and loo%, respectively, which is considered to be of a satisfactory level. The data suggested tha t the use of ELISA using sodium taurocholate extracted preparation as the antigen could be a suitable alternative to the MAT. Considering the ease at which the antigen can be extracted for routine testing of animal se ra the antigen could be used for serodiagnosis of leptospirosis.

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102 S.K. Srivastava arid V. Tiwari

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Smuballi. O.P.. Marenger, R.M., Eaglesome. M.D., Sugden, E.A. 1997. Development and initial evaluation of an indirect ELISA for the detection of Leptospira iiiterrogaiis serovar hardjo antibodies in bovine sera. Can. J. Vet. Res., 61: 260-266.

Trueba, G.A.. Bolin, C.A. and Thoen, C.O. 1990. Evaluation of an enzyme immuno- assay for diagnosis of bovine leptospirosis caused by Leptospira iiiterrogaiis serovar hardjo type Iiardjobovis. J. Vet. Diag. Invest., 2: 323-329.

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