Evaluation of a recombinant LipL41 antigen of Leptospira interrogans serovar Canicola in ELISA for serodiagnosis of bovine Leptospirosis

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Comparative Immunology, Microbiology & Infectious Diseases 29 (2006) 269–277 www.elsevier.com/locate/cimid

Evaluation of a recombinant LipL41 antigen of Leptospira interrogans serovar Canicola in ELISA for serodiagnosis of bovine Leptospirosis R. Mariya, Pallab Chaudhary, A.A. Kumar, E. Thangapandian, R. Amutha, S.K. Srivastava,1 Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh 243122, India Accepted 14 June 2006

Abstract The efficacy of a recombinant leptospiral lipoprotein LipL41 as an antigen for conducting enzyme-linked immunosorbent assay (ELISA) for diagnosis of bovine leptospirosis was evaluated. Using known positive and known negative cattle sera the recombinant antigen was found to be highly reactive in the concentration of 100 ng/well. Using a total of 321 field cattle sera the sensitivity of ELISA as compared to microscopic agglutination test (MAT) was calculated to be 100% whereas the specificity was 85.3%. The seropositivity of leptospirosis among bovine population was found to be 21.18% having the predominance of serovars Sejroe and Pomona. It was concluded that rLipL41 protein could be a putative diagnostic candidate for serodiagnosis of bovine leptospirosis. r 2006 Elsevier Ltd. All rights reserved. Keywords: Leptospirosis; Bovine; rLipL41ELISA

Corresponding author. Tel.: +91 0581 2301865; fax: +91 0581 2303284.

E-mail address: [email protected] (S.K. Srivastava). Present address: Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar, Bareilley, Uttar Pradesh 243 122, India. 1

0147-9571/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.cimid.2006.06.007

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Re´sume´ L’efficacite´ d’un LipL41 de lipoprote´ine de leptospiral de recombinant comme un antige`ne pour diriger l’essai de immunosorbent d’enzyme-relie´ (ELISA) pour le diagnostic de leptospirose bovine a e´te´ e´value´. L’utilisation sera de be´tail connue positive et connue ne´gative l’antige`ne de recombinant a e´te´ trouve´ pour eˆtre extreˆmement re´actif dans la concentration de 100 ng/well. L’utilisation d’un total de 321 sera de be´tail de champ la sensibilite´ de ELISA comme en comparaison du test d’agglutination Microscopique (le TAPIS) a e´te´ calcule´ pour eˆtre 100% tandis que la spe´cificite´ e´tait 85,3%. Le seropositivity de leptospirose parmi la population bovine a e´te´ trouve´ pour eˆtre 21,18% avoir la pre´dominance de serovars Sejroe et de Pomona. Il a e´te´ conclu que rLipL41 prote´ine pourrait eˆtre un candidat diagnostique putatif pour serodiagnosis de leptospirose bovine. r 2006 Elsevier Ltd. All rights reserved. Mots-Cle´s: La leptospirose; Bovin; rLipL41ELISA

1. Introduction Leptospirosis of animals and man is considered as an important reemerging infectious disease worldwide. In domestic animals it is an important cause of abortion, stillbirth, infertility, decreased milk production and death [1,2]. In dairy cattle it is responsible for great economic losses as a consequence of agalactia, abortion, stillbirth, birth of weak calves and reduced fertility [3,4]. The standard serological test, such as MAT is hazardous to perform as live Leptospira cultures are used as an antigen. The demonstration of seroconversion by using this test requires paired serum samples collected at acute and convalescent phase. Thus the use of the test is limited to a few established laboratories only [5]. The other available serological methods require whole cell antigen preparations [6,7] for diagnosis as well as for screening of the cases of leptospirosis. The whole-cell antigen preparations possess broadly reactive immunodominant epitopes [8] present in pathogenic as well as non-pathogenic leptospires [9]. Recently, recombinant antigens based serodiagnostic strategies have been developed to conduct seroprevalence studies in human leptospirosis using different outer membrane proteins [10]. The important protein antigens residing in the outer membrane of Leptospira cells reported in recent years are LipL32, LipL41 and ompL1. These are also associated with infection in animals and man as ample evidences are available to show that these proteins are expressed in vivo during the infection [11,12]. The LipL41 is one of the important and surface exposed leptospiral outer membrane protein expressed by pathogenic Leptospira species only [13]. In the present study, an ELISA using the LipL41 recombinant protein as an antigen was developed for carrying out the serodiagnosis of bovine leptospirosis. 2. Materials and methods 2.1. Microscopic agglutination test The microscopic agglutination test (MAT) was carried out according to Faine [14], using reference strains of 8 different leptospiral serovars viz., Leptospira interrogans

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serovars Hardjo (Hardjoprajitno), Pomona, Pyrogenes, Icterohaemorrhagiae, L. borgpetersenii serovars Tarassovi, Javanica, Sejroe and Ballum being maintained at the Leptospira Laboratory of Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar, India. Reciprocal agglutination titres of greater than or equal to 100 were considered positive. 2.2. Bovine serum samples A total of 321 bovine serum samples obtained from various parts of the country were screened for the presence of anti-leptospiral antibodies. These animals had the history of reproductive problems like abortion, repeat breeding, weak calves and mastitis. 2.3. Extraction and purification of crude outer membrane protein The Leptospires were cultured at 28 1C in 500 ml liquid EMJH medium to log phase of growth (5–7 days incubation) and harvested by centrifugation at 40 000 g for 20 min at 4 1C and washed thrice in 0.01 M phosphate buffered saline (PBS, pH 7.2). The cells were suspended in 10 mM HEPES (N-2 hydroxy ethylpiperazine—N1-2 ethane sulfonic buffer (Sigma, USA) and were disrupted by sonication at 60 m for 15 s each for 4 times interrupted by a 5 s pause, while cooling in ice bath. Cellular debris was removed by centrifugation at 2000 g for 10 min at 4 1C. The supernatant was collected and centrifuged at 100 000 g for 60 min at 4 1C. The pellet was resuspended in 2 ml of 1% sodium lauryl sarcosinate (Sigma, USA) prepared in 10 mM HEPES buffer, pH 7.4 and incubated at room temperature overnight with gentle rotation. The sarcosyl insoluble fraction was sedimented by centrifugation at 100 000 g for 60 min at 40 C and suspended in 125 ml of Distilled water. The supernatant (sarcosyl soluble) and the sarcosyl insoluble fractions were stored at
20 1C. The protein estimation in all the preparation was made by using the Lowry technique based protein estimation kit (Genei, Bangalore) 2.4. Production of recombinant LipL41 antigen In brief, the primers were designed with restriction sites from the previously reported gene sequences of L. kirschneri serovar Grippotyphosa [13]. The gene encoding LipL41 was amplified from the genomic DNA of Leptospira interrogans serovar Canicola and cloned into pDrive (Qiagen) cloning vector and sequenced. The nucleotide sequence was submitted to GenBank under the accession number AY642287. The LipL41gene was subcloned into the pPROEXHTb expression vector (Life Technologies) and transformed into Escherichia coli DH5a cells. The recombinant clones were screened using 1 mM IPTG (Isopropyl-a-D-thiogalactopyranoside). The polyhistidine (6X-His) tagged fusion protein LipL41 was purified under denaturing conditions by Nickel chelating affinity chromatography (Qiagen).

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The recombinant protein was dialyzed and the concentration was determined by Lowry’s method (BangaloreGenei, India) 2.5. Indirect enzyme-linked immunosorbent assay Optimum concentration of the recombinant protein antigen, Crude OMP and various reagents was determined by checkerboard analysis and the test was performed according to the method of Engvall and Perlmann [15] with certain modifications. An antigen concentration of 100 ng/well for recombinant protein and 1.5 mg/well for Crude OMP was found to be most suitable. Flat-bottomed polystyrene microtitre plates (Nunc) were coated with 100 ml of the coated buffer having desired concentration of the purified recombinant LipL41 antigen and incubated overnight at 4 1C. The plates were washed five times with PBS (pH 7.4,0.01 M) containing 0.05% Tween-20 (PBS-T) and incubated with a blocking solution of 5% (w/v) skim milk powder in PBST for 1 h at 37 1C and again washed as earlier. Now 100 ml of 1:100 dilutions of test bovine serum samples in blocking buffer were added to the wells. Each plate had the following controls: conjugate, known positive and negative control sera. After an incubation of 1 h at 37 1C, the plates were again washed five times with PBS-T and 100 ml of 1:5000 rabbit antibovine IgG peroxidase conjugate (Sigma) were added to the wells and the plates were incubated for 1 h at 37 1C. Finally, after further five washes, 100 ml of OPD solution (Fasta, Sigma) were added to each well and plates were incubated at room temperature for 10 min in dark for colour development. The reaction was stopped by adding 100 ml 1 M H2SO4. The absorbance was measured at 492 nm using ELISA reader (Multiscan Ex, Lab Systems). The infected calves sera (n ¼ 10) and an equal number of known negative sera were used in the initial trials to determine optimal serum dilution for the test. The MAT positive (a titre of X100) and MAT negative sera obtained from field animals were diluted in blocking buffer upto dilutions of 1:50–1:6400 and used. The best performance was obtained with sera samples diluted at 1:100. Individual serum samples were tested in duplicate on five different plates. The mean per cent positivity (PP) value of the negative control group was calculated and double of this value was taken as the cut-off PP value as described by Wright et al. [16]. The field bovine serum samples were classified as positive or negative for bovine leptospirosis based on the per cent PP values as described by Wright et al. [16]. 2.6. Evaluation of MAT and ELISA The relative sensitivity, specificity and accuracy of the recombinant LipL41 antigen based ELISA for serodiagnosis of bovine leptospirosis were evaluated in comparison to the MAT as described below. Sensitivity ¼ a/(a+c)  100, where, ‘a’ is the number of sera positive by ELISA and MAT, ‘c’ the number of sera positive by MAT but negative by ELISA.

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Specificity ¼ d/(b+d)  100, where ‘d’ is the number of sera negative by ELISA and MAT, ‘b’ the number of sera negative by MAT but positive by ELISA. Accuracy ¼

aþd  100. aþbþcþd

3. Results 3.1. Production of rLipL41 antigen The purification of the rLipL41 was carried out by using the polyhistidine (6X-His) system of Qiagen. The final elution was deduced by SDS-PAGE. The elute 3 and 4 had the maximum concentration of the protein (Fig. 1). From 5-liter culture, a total of 19.6 mg of rLipL41 protein was purified. 3.2. Optimum antigen concentration The optimum concentration of the purified rLipL41 antigen (Fig. 1) which showed maximum difference between the positive and negative sera was determined to be 100 ng/well (Fig. 2), where as for OMP a concentration of 1.5 mg/well was most suitable. 3.3. Determination of ELISA cutoff value Using 100 ng/well of rLipL41 antigen, the mean OD value of control negative serum samples at 1:100 dilution was 0.208 and that of control positive serum samples it was 1.091. Based on these OD values, the mean PP value of the negative control group was calculated as 19.06. Therefore, any sample showing a PP of 38.00 (twice the mean negative PP value) or more was considered as positive for leptospirosis. PP values using crude OMP ELISA was not calculated. 3.4. rLipL41 ELISA results Out of 321 bovine sera examined, 105 samples (32.7%) showed a PP value of equal to or more than 38 (cut-off PP value) and they were classified as positive for leptospirosis. The frequency distribution of PP values obtained in these sera is shown in Table 1. Maximum numbers of negative sera (138) showed PP values in the range of 0–18. Among the positive sera, majority were in the range of 57–75. Sera showing more than this range was considered to be highly positive. 3.5. Evaluation of MAT and ELISA The sensitivity, specificity and accuracy of ELISA as relative to the MAT were calculated to be 100%, 85.3% and 88.4%, respectively (Table 2).

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M

1

2

3

4

5

M

kDa 116.0

66.2

45

41 kDa

35

25

18.4

Fig. 1. Purification of recombinant LipL41 protein (41 kDa) using Nickel column chromatography. Lane M: Molecular weight marker; Lane 1: elute 1; Lane 2: elute 2; Lane 3: elute 3; Lane 4: elute 4; Lane 5: elute 5.

1.4

Known negative

OD (492 nm)

1.2

Known positive

1

Ratio

0.8 0.6 0.4 0.2 0 2µg

1.5µg

1µg

500 ng 250 ng 100 ng

50 ng

25 ng

Concentration of antigen Fig. 2. Determination of optimum rLipL41 antigen concentration for conducting ELISA using a serum dilution of 1:100.

4. Discussion In this study, we tested the efficiency of the recombinant LipL41 antigen for the detection of bovine anti-leptospires antibodies using ELISA. It was observed that using the crude OMP as antigen higher concentration i.e. 1.5 mg/well is required to

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Table 1 Distribution of PP values obtained on bovine serum samples using ELISA Range of PP values

No. samples tested

Result

0–18 19–37 38–56 57–75 76–94 95–113

138 78 28 38 29 10

Negative Negative Positive Positive Highly positive Highly positive

Total

321

 Cut-off value.

Table 2 Evaluation of ELISA to detect anti-leptospiral antibodies in bovine sera as compared to MAT MAT

Total

Positive

Negative

ELISA Positive Negative

68 —

37 216

105 216

Total

68

253

321

Sensitivity ¼ 100%; Specificity ¼ 85.3%; Accuracy ¼ 88.4%.

obtained satisfactory results similar to that obtained using rLipL41 protein. Moreover, crude antigen showed higher OD values in known negative sera as compared to the recombinant protein. Hence, for routine testing only rLipL41 based ELISA was performed. The relative sensitivity of recombinant LipL41 protein based ELISA was calculated to be 100% suggesting that it was as effective as Mat in detecting True positive animals. However, the specificity was 85.3% signifying the inability of the ELISA to detect all the MAT negative animals as ELISA negative too. This could be explained on the basis that all those sera which turned out to be MAT negative may not actually be negative as only 8 Leptospira serovars were used as antigens for antibody detection. Furthermore, ELISA being a more sensitive test [17,18], was able to detect antibodies in the serum samples which MAT failed to detect in the given dilution of the test sera. The advantage of using this rLipL41 antigen in ELISA is that bulk amount of the purified antigen can be obtained without culturing Leptospira organisms. It is also safe than MAT since it eliminates the handling of live leptospires. Another advantage with this antigen is that it can detect genus-specific antibodies, which MAT does not. The conserved nature of LipL41 among the pathogenic Leptospira spp. suggests that rLipL41 ELISA may exhibit similar performance regardless of the locally

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predominant serovars. Thus, use of the recombinant LipL41 antigen in ELISA has the potential to become a useful tool for serodiagnosis of bovine leptospiral infection.

Acknowledgements The authors are thankful to Director, IVRI for providing necessary funds and facilities for conducting the study.

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