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Development of rapid flow-through-based dot-immunoassay for serodiagnosis of leptospirosis in dogs
Comparative Immunology, Microbiology and Infectious Diseases 34 (2011) 17–22
Contents lists available at ScienceDirect
Comparative Immunology, Microbiology and Infectious Diseases journal homepage: www.elsevier.com/locate/cimid
Development of rapid flow-through-based dot-immunoassay for serodiagnosis of leptospirosis in dogs M. Subathra, T.M.A. Senthilkumar *, P. Ramadass, G. Dhinakar Raj Department of Animal Biotechnology, Madras Veterinary College, TANUVAS, Chennai 600007, India
A R T I C L E I N F O
A B S T R A C T
Article history: Accepted 5 October 2009
An IgG-ELISA used recombinant antigen and a rapid flow-through enzyme immunoassay were developed for rapid screening of leptospiral antibodies in dogs using recombinant LipL41, which is one of the conserved outer membrane proteins in pathogenic leptospires as the coating antigen. Results from this study were compared with the standard microscopic agglutination test and found that the sensitivity and specificity of the enzyme-linked immunosorbent assay were 75.46% and 93.29% and whereas that of flowthrough-based dot-immunobinding assay were 87.73% and 89.63%, respectively. Relative merits of these tests were also assessed. The flow-through-based dot-immunobinding assay was thus proved to be a valid screening test for canine leptospirosis. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: Canine leptospirosis Enzyme-linked immunosorbent assay Flow-through-based dot-immunobinding assay Microscopic agglutination test Recombinant LipL41
1. Introduction Leptospirosis is an anthropozoonosis of ubiquitous distribution, caused by spirochaetes of the pathogenic Leptospira species. Leptospirosis affects a wide range of hosts including humans, domestic and wild animal species [1,2]. Laboratory confirmation of leptospirosis is obtained when either the pathogen is isolated or a positive serological result is obtained. The microscopic agglutination test (MAT) is considered as the reference test for leptospirosis. The result of MAT is considered consistent with leptospirosis when either a four-fold increase in titre is observed between paired serum samples or a significantly increased titre for a single serum sample [3]. Both culture and the MAT seem to be laborious as well as expensive. Several methods for detecting specific IgM antibody including, enzyme-linked immunosorbent assay (ELISA) [4,5], Dipstick ELISA [6], and indirect immunofluorescent assay [7] are widely used and some of them are available commercially. As all the existing tests have advantages as well as limitations, the development of new diagnostics assumes
* Corresponding author. Tel.: +91 44 2536 9301; fax: +91 44 2536 9301. E-mail address: [email protected] (T.M.A. Senthilkumar). 0147-9571/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.cimid.2009.10.003
greater significance since leptospirosis has become an important public health problem in most countries of the world with many outbreaks reported in the recent past [8– 12]. Proteins located on the leptospiral outer membrane are of the greatest interest, as outer membrane proteins (OMPs) are potentially exposed to the host immune system. Antigenic conservation of leptospiral proteins was demonstrated in immunoblot studies that showed that sera from rabbits hyperimmunized with one strain recognized numerous proteins of a wide variety of serovars [13,14]. Several of these protein antigens have been identified, including the outer membrane lipoprotein LipL41. In the present study, a simple dot-immunobinding assay was developed based on the flow-through principle [15] utilizing the recombinant LipL41 (rLipL41) protein expressed in Escherichia coli as capture antigen. 2. Materials and methods 2.1. Bacterial strains and media The panel of eight Leptospira interrogans serovars included in MAT was Australis, Autumnalis, Canicola, Javanica, Pomona, Icterohaemorrhagiae, Grippotyphosa,
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M. Subathra et al. / Comparative Immunology, Microbiology and Infectious Diseases 34 (2011) 17–22
and Pyrogenes. They were grown and maintained in EMJH liquid and semi-solid medium (Difco, USA) at 29–30 8C. The concentration of organisms was assessed regularly by dark field microscopy. These reference leptospiral strains were obtained from Koninklijk Institutut voor de Tropen (KIT), Amsterdam, The Netherlands, and National Leptospirosis Reference Center, Port Blair, Andaman and Nicobar Islands, India. E. coli DH5a cells (F f80DlacZDM15D (lacZYA-argF) U169 recA1 endA1 hsdR17 (rk, mk+) phoA supE44 l-thi-1 gyrA96 relA1) (Invitrogen, USA) were used as host cells for the expression of recombinant antigen and were grown and maintained in LB broth and LB agar.
tion. The polyhistidine tag (6X-His) of the fusion protein was utilized for purification of fusion protein by Ni2+-NTA affinity chromatography (Qiagen). The purified protein was analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The concentration of the protein was determined by Lowry’s method and stored in aliquots at 80 8C until used. 2.5. IgG-enzyme-linked immunosorbent assay (IgG-ELISA)
The gold standard serodiagnostic test for leptospirosis is MAT was performed as per the method of Cole et al. [16]. Briefly, the sera from the patients were serially diluted from 1:50 to 1:3, 200 in phosphate buffered saline (PBS), pH 7.2 and reacted with live antigen suspensions of the panel of eight reference leptospiral serovars. After 2 h incubation at 37 8C, the serum–antigen mixtures were examined by dark field microscope for the presence of agglutination/clearance and the titres were determined. Reciprocal agglutination titres of greater than or equal to 100 were considered as positive reactions.
IgG-ELISA was carried out initially to evaluate the purified rLipL41 protein as antigen for serodiagnosis of leptospirosis. The optimum concentration of the antigen was determined by the checkerboard titration [18]. Flatbottom polystyrene microtitre plates (MaxiSorp, Nunc) were coated with purified antigen diluted in 0.06 M carbonate–bicarbonate buffer (Na2CO3 1.59 g and NaHCO3 2.93 g in 1 l of distilled water, pH 9.6) overnight at 4 8C. The plates were washed thrice with phosphate buffered saline, pH 7.2 with 0.05% Tween 20 (PBST) and blocked with 2% bovine serum albumin (BSA) in PBST for 1 h at 37 8C. The plates were washed thrice with PBST and incubated at 37 8C with 100 ml of two-fold serially diluted serum samples to be tested in PBST from 1:100 to 1:3, 200 dilutions for 1 h. After three washes with PBST, wells were added with 1:15,000 dilution of rabbit anti-dog IgGperoxidase (HRP) conjugate (Bangalore Genei, India) and incubated for 1 h at 37 8C. The substrate solution ABTS (2,20 -Azino di-ethyl benz-thiozoline-6-sulphonic acid) (Sigma, USA) was prepared and added to the wells at 100 ml volume and allowed to react for 10 min. When the colour developed, the absorbance at 405 nm was measured in an ELISA reader (Biotek instruments Inc., USA). The mean absorbance of three negative control wells was calculated. Double the values of mean negative control optical densities (ODs) were considered as positive OD, and the reciprocal dilution of the sample showing positive OD was observed as the titre value of the sample.
2.4. Recombinant leptospiral antigen LipL41
2.6. Flow-through-based dot-immunoassay
The outer membrane protein (OMP) gene LipL41 was partially amplified from the reference L. interrogans serovar Icterohaemorrhagiae (strain RGA) by polymerase chain reaction (PCR). The primers with built in restriction enzyme sites were selected based on the published LipL41 primer sequence of L. krischneri species [17]. The PCR amplicon size of 1077 bp (GenBank accession no. DQ132992) was cloned into an expression vector pPROEXHT‘b’ (Invitrogen, USA). The expression of recombinant fusion protein (rLipL41) was achieved by adding isopropyl b-D thiogalactoside (IPTG) at 1 mM final concentration. Purification of the expressed protein was carried out by sequential addition of buffer A (50 mM Tris–HCl, pH 8.0, 7.5% glycerol, 0.1 mM EDTA and 50 mM NaCl), buffer B (50 mM EDTA, 10% Triton X-100, 2.5 mg/ml lysozyme and 2 mM PMSF or b-Mercaptoethanol) and 5 mM MgCl2 to the cell pellet and incubation in ice for 1 h. Then the cells were sonicated at constant pulse twice for 30 s. The lysate was clarified at 17,950 g for 30 min at 4 8C. The supernatant was subjected for affinity purifica-
The flow-through assay was developed as per the method of Paepens et al. [15] with minor modifications. Briefly, NCMs (0.45 mm, Advanced Microdevices Pvt. Ltd., Ambala Cantt, India) were cut into small squares (2 cm 2 cm). The test was performed in an immunofiltration device, a small rectangular plastic module with test window holding water absorbing material and a nitrocellulose membrane (NCM). The NCM was pressed tightly over the water absorbing material in the test window. One microlitre of the antigen (rLipL41) diluted in PBS, pH 7.2 was dotted on the NCM in the ‘T’ side of the test window and one ml of 1:100 diluted healthy dog serum (Bangalore Genei, India) in PBS, pH 7.2 was dotted in the ‘C’ side of the test window to serve as reagent control. The modules were incubated at 37 8C for 1 h and stored in a sealed plastic bag at room temperature. The assay was performed at room temperature. Hundred microlitres of wash buffer (1 M Na2HPO4, 1 M NaH2PO4, pH 6.0, 0.9% NaCl, 0.5% Triton X-100, and 1% BSA) was added twice to the test window to wet and 2%
2.2. Canine serum samples Three hundred and twenty-two blood samples were collected from the dogs suspected for leptospirosis with pyrexia, jaundice and other malaise, irrespective of their age, breed and sex in the Small Animal Clinics, Madras Veterinary College Hospital, Chennai, India. Blood samples were also collected from five apparently healthy dogs that were brought for general health check up with vaccination details to serve as negative controls. Sera were separated on the same day and stored at 20 8C until used. 2.3. Microscopic agglutination test (MAT)
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Fig. 1. Evaluation and standardization of the rLipL41 concentration (5, 10, 25, 50, 100, 200, 400 and 800 ng) at a serum dilution of 1:100. P 0.01.
BSA in PBS, pH 7.2 was added to block the NCM. The antigen concentration for dotting the NCM was assessed using checkerboard titration by comparing the intensities of colour developed with different antigen concentrations and serum dilutions. The suspected dog serum samples were diluted to 1:10 with PBS, pH 7.2 and 100 ml was added to the test window. The unbound antibodies were washed by adding 100 ml of wash buffer twice. The goat anti-dog IgG-peroxidase conjugate (Bangalore Genei, India), 100 ml was added in the dilution of 1:500 in PBS, pH 7.2. After washing with wash buffer twice, the substrate Diamino benzidine (DAB) (Sigma, USA) was added and allowed for absorption. Immediately the NCM was rinsed with 100 ml of distilled water twice. The appearance of two brown dots in the ‘T’ and ‘C’ sides of test window indicated a positive reaction, and the absence of dot in the ‘T’ side of test window indicated a negative reaction. If there were no dots in the test window, the test was considered invalid. The entire protocol could be completed within 5–10 min. 2.7. Relative sensitivity, specificity and concordance The relative sensitivity, specificity and concordance (in percent) of the IgG-ELISA and flow-through-based dotimmunoassay for the detection of leptospiral antibodies in dog serum samples were determined in comparison to the MAT as described below: a sensitivity ¼ 100 aþc where ‘a’ is the number of serum samples positive by the test and MAT and ‘c’ is the number of serum samples positive by MAT but negative by test: specificity ¼
3. Results 3.1. IgG-enzyme-linked immunosorbent assay The optimum concentration of the purified rLipL41 antigen, which showed maximum difference in optical density (OD) values, between the positive and negative serum samples was determined to be 10 ng/well (Fig. 1). Though all the concentrations of the antigen are significant (P 0.01), the concentration at which three-fold difference between the positive OD and negative OD was taken as the coating antigen concentration of LipL41. Among 327 canine serum samples, 134 samples were found positive. The sensitivity, specificity and concordance of the assay in relation to the standard MAT are shown in Table 1. 3.2. Flow-through-based dot-immunoassay A series of experiments were performed to determine the optimal conditions and to adjust the test sensitivity to an appropriate level. Checkerboard titrations were carried out with various concentrations of antigen (10, 5, 2 mg/ml, 100, and 50 mg/ml) and the intensities of colour development to determine the optimal concentration of the capture antigen (Table 2). Various dilutions of serum samples and the intensities of colour development were also checked (Data not shown). The assay was optimized using 2 mg/ml concentrations of capture antigen (rLipL41) and 1:10 dilution of the serum samples to be tested (Fig. 2).
Table 1 Comparison of IgG-ELISA and MAT for serodiagnosis of canine leptospirosis. MAT
d 100 bþd
where ‘d’ is the number of serum samples negative by test and MAT and ‘b’ is the number of serum samples negative by MAT but positive by test: aþd concordance ¼ 100 aþbþcþd
+
Total
IgG-ELISA +
123 (a) 40 (c)
11 (b) 153 (d)
134 193
Total
163
164
327
2
x = 159.77**; K = 0.69. Sensitivity: 75.46%; specificity: 93.29%; concordance: 84.40%. **Highly significant P 0. 01.
M. Subathra et al. / Comparative Immunology, Microbiology and Infectious Diseases 34 (2011) 17–22
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Table 2 Determination of antigen concentration for flow-through-based dot-immunoassay. Concentration of rLipL41
10 mg/ml 5 mg/ml 2 mg/ml 1 mg/ml 100 mg/ml 50 mg/ml
Canine serum samples with reciprocal MAT titres in brackets 1 (<100)
2 (100)
3a (100)
4 (200)
5 (400)
6 (800)
+ +
+++ +++ +++ +
+++ +++ +++ +++ ++
+++ +++ +++ ++
+++ +++ +++ ++ +
+++ +++ +++ ++ +
+++ High intensity of the colour; ++ moderate intensity of the colour; + poor intensity of the colour; negative. a Slightly haemolysed serum sample.
Fig. 2. Flow-through-based dot-immunobinding assay. Modules 1 and 3, positive reactions; modules 2 and 4, negative reactions; C, control reactions; T, test reactions.
Table 3 Comparison of flow-through-based dot-immunoassay and MAT for serodiagnosis of canine leptospirosis. MAT
Among 327 canine serum samples, 160 samples were found positive. The sensitivity, specificity and concordance of the assay in comparison with standard MAT are shown in Table 3. All the five negative samples were also negative by MAT, IgG-ELISA and flow-through-based dot-immunoassay.
+
Total
Flow-through +
143 (a) 20 (c)
17 (b) 147 (d)
160 167
4. Discussion
Total
163
164
327
Highly conserved OMPs are of special significance in serodiagnosis and vaccine development for leptospirosis. The leptospiral OMPs expressed during mammalian
2
x = 195.80**; K = 0.77. Sensitivity: 87.73%; Specificity: 89.63%; Concordance: 88. 69%. **Highly significant P 0. 01.
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infection may have potential immunoprotective capabilities [19,17]. However, the lack of an effective, widely available laboratory tool remains a major problem [20] and standard serological tests for case confirmation need to be optimized [21]. Currently available methods are not easy to perform; all require special and expensive equipments and can be used only by trained personnel. There is a need to develop a method that is rapid, simple with high sensitivity and specificity. Recombinant protein-based serologic tests may achieve high sensitivity and specificity because of high concentration of immunoreactive antigens, which could be used in the assays, and also lack of non-specific moieties present in whole cell preparations [20]. In the present study, one of the outer membrane proteins, that are highly conserved among pathogenic leptospires [17], LipL41 was expressed as recombinant fusion protein and utilized for developing a rapid flow-through-based dot-immunoassay. Most of the rapid assays were developed and evaluated for serodiagnosis of human leptospirosis. The disease being considered mainly as occupational, and of zoonotic in nature, the early diagnosis and treatment of animals are essential for prevention of human leptospirosis. ELISA-based assays were developed by many workers and evaluated as an alternative to MAT for diagnosis of leptospirosis [22–24]. In this study, IgG-ELISA and dot-immunobinding assay were developed using recombinant antigen (LipL41), and their results were analysed with standard MAT. The MAT result indicated that commonly occurring L. interrogans serovars in canine species were Canicola, Australis, Javanica and Icterohaemorrhagiae. IgG-ELISA with the recombinant LipL41 antigen developed in this study had higher sensitivity and specificity than that reported by Flannery et al. [20] who evaluated the same recombinant antigen for the diagnosis of human leptospirosis. They evaluated sera from 50 healthy residents and paired sera from 50 cases of leptospirosis confirmed by the MAT. They had the sensitivity and specificity of 24% and 44%, respectively. Natarajaseenivasan et al. [25] evaluated the IgM based recombinant LipL41 ELISA which showed 89.3% sensitivity and 89.2% specificity, because IgM antibody level increases in the first week but becomes negative within 3–5 weeks, whereas IgG antibodies are detected at about the same time as IgM but persists for a much longer period [26]. Terpstra et al. [27] who reported high IgM titres between 10 and 60 days of illness while the combination of moderately increase IgM titres and low IgG titres was most often observed in the first 10 days of the disease. Here the developed assay was based on IgG response, so the sensitivity rate is 75.46% when compared to IgM based MAT. In future, we can increase the sensitivity rate by IgM based ELISA and Mariya et al. [28] 2006 evaluated the recombinant leptospiral lipoprotein LipL41 as an antigen for conducting ELISA for diagnosis of bovine leptospirosis showed 100% sensitivity and 85.3% specificity. However, the specificity was 85.3% which indicates that only limited Leptospira serovars were used as antigens for antibody detection in MAT. The flow-through-based dot-immunoassay developed in this study combines the ease of flow-through technology and dot-ELISA principle for rapid, sensitive serodiag-
21
nosis of canine leptospirosis. The sensitivity and specificity obtained by screening of 327 dog serum samples proved that this test could be a valid screening test for canine leptospirosis. The results could be read within 5–10 min whereas the conventional dot-ELISA techniques required 1–3 h. Moreover, the development of clear dots avoided the ambiguity about the results. Eliades et al. [29] developed a dot-immunobinding assay for the serodiagnosis of human hydatidosis and concluded that the detection of anti-HA antibodies had agreed closely with the data from ELISA. In this study, the sensitivity of the flow-through-based dot-immunoassay was found to be higher than that of IgG-ELISA and the specificity found to be reduced. This could be due to the quality of the serum samples used in the assays. Sometimes haemolysed serum samples showed mild positivity in the flow-through-based dot-immunoassay; because of pale red in colour of serum react with membrane bound antigen, though they were MAT negative. Hence, the haemolysed serum samples should be avoided in this assay. The developed assay gave a reliable result and interesting alternative tests for serodiagnosis of canine leptospirosis especially at peripheral veterinary health care units in developing countries where sophisticated equipments are not available. Acknowledgments The study is supported by Grants from Department of Biotechnology, Ministry of Science and Technology, Government of India to T.M.A.S. References [1] Faine S. Leptospira and leptospirosis. Boca Raton, USA: CRC Press; 1994. [2] Hartskeerl RA, Terpstra WJ. Leptospirosis in wild animals. Vet Quart 1996;18:149–50. [3] Communicable disease surveillance kit. Geneva, Switzerland: World Health Organization; 1998. [4] Petchclai B, Kunakorn M, Hiranras S, Potha U, Liemsuwan C. Enzymelinked immunosorbent assay for leptospirosis immunoglobulin M specific antibody using surface antigen from a pathogenic Leptospira: a comparison with indirect hemagglutination and microagglutination tests. J Med Assoc Thai 1992;75:203–8. [5] Cumberland P, Everard COR, Levett PN. Assessment of the efficacy of an IgM-ELISA and microscopic agglutination test (MAT) in the diagnosis of acute leptospirosis. Am J Trop Med Hyg 1999;61: 731–4. [6] Gussenhoven GC, van der Hoorn MAWG, Goris MGA, Terpstra WJ, Hartskeerl RA, Mol BW, et al. LEPTO dipstick, a dipstick assay for detection of Leptospira-specific immunoglobulin M antibodies in human sera. J Clin Microbiol 1997;35:92–7. [7] Pradutkanchana S, Pradutkanchana J, Khuntikij P. Detection of IgM specific antibody using indirect immunofluorescent assay for diagnosis of acute leptospirosis. J Med Assoc Thai 2003;86:641–6. [8] Ko AI, Galvao Reis M, Ribeiro Dourado CM, Johnson WDJR, Riley LW. Urban epidemic of severe leptospirosis in Brazil. Salvador Leptospirosis Study Group. Lancet 1999;354:820–5. [9] Russell KL, Gonzalez MAM, Watts DM, Lagos-Figueroa RC, Chauca G, Ore M, et al. An outbreak of leptospirosis among Peruvian military recruits. Am J Trop Med Hyg 2003;69:53–7. [10] Vijayachari P, Sehgal SC, Goris MGA, Terpstra WJ, Hartskeerl RA. Leptospira interrogans serovar Valbuzzi: a cause of severe pulmonary haemorrhages in the Andaman Islands. J Med Microbiol 2003;51:1–6. [11] Meeyam T, Tablerk P, Petchanok B, Pichpol D, Padungtod P. Seroprevalence and risk factors associated with leptospirosis in dogs. Southeast Asian J Trop Med Pub Health 2006;37:148–53. [12] Goldstein RE, Lin RC, Langston CE, Scrivani PV, Erb HN, Barr SC. Influence of infecting serogroup on clinical features of leptospirosis in dogs. J Vet Int Med 2006;20:489–94.
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[13] Nunes-Edwards PL, Thiermann AB, Bassford Jr PJ, Stamm LV. Identification and characterization of the protein antigens of Leptospira interrogans serovar Hardjo. Infect Immun 1985;48:492–7. [14] Brown JA, LeFebvre RB, Pan MJ. Protein and antigen profiles of prevalent serovars of Leptospira interrogans. Infect Immun 1991;59: 1771–7. [15] Paepens C, De Saeger S, Sibanda L, Barna-Vetro I, Leglise I, Van Hove F, et al. A flow-through enzyme immunoassay for the screening of fumonisins in maize. Anal Chim Acta 2004;523:229–35. [16] Cole JR, Sulzer CR, Pursell AR. Improved microtechnique for the leptospiral microscopic agglutination test. Appl Microbiol 1973;25: 976–80. [17] Haake DA, Mazel MK, Mccoy AM, Milward F, Chao G, Mastuanga J, et al. Leptospiral outer membrane proteins OmpL1 and LipL41 exhibit synergistic immunoprotection. Infect Immun 1999;67: 6572–82. [18] Dey S, Madhan Mohan C, Senthilkumar TMA, Ramadass P, Mahalinga Nainar A, Nachimuthu K. Recombinant LipL32 antigen-based single serum dilution ELISA for detection of canine leptospirosis. Vet Microbiol 2004;103:99–106. [19] Barnett JK, Barnett D, Bolin CA, Summers TA, Wager EA, Cheville NF, et al. Expression and distribution of leptospiral outer membrane components during renal infection of hamsters. Infect Immun 1999;67:853–61. [20] Flannery B, Coasta D, Carvalho FP, Guerreiro H, Mastuanga J, Silva ED, et al. Evaluation of recombinant Leptospira antigen-based enzyme– linked immunosorbent assays for the serodiagnosis of Leptospirosis. J Clin Microbiol 2001;39:3303–10. [21] Bajani MD, Ashford DA, Bragg SL, Woods CW, Aye T, Spiegel RA, et al. Evaluation of four commercially available rapid serologic tests for diagnosis of leptospirosis. J Clin Microbiol 2003;41:803–9.
[22] Adler B, Faine S, Gordon LM. The enzyme-linked immunosorbent assay (ELISA) as a serological test for detecting antibodies against Leptospira interrogans serovar Hardjo in sheep. Aust Vet J 1981;57: 414–7. [23] Biancifiori F, Cardaras P. Enzyme-linked immunosorbent assay in the diagnosis of leptospirosis in domestic animals using peroxidaseconjugated protein A. Comp Immun Microbiol Infect Dis 1983;55: 57–65. [24] Ribotta MJ, Higgins R, Gottschalk M, Lallier R. Development of an indirect enzyme-linked immunosorbent assay for the detection of leptospiral antibodies in dogs. Can J Vet Res 2000;64:32–7. [25] Natarajaseenivasan K, Vijayachari P, Sharma S, Sugunan AP, Selvin J, Sehgal SC. Serodiagnosis of severe leptospirosis: evaluation of ELISA based on the recombinant OmpL1 or LipL41 antigens of Leptospira interrogans serovar autumnalis. Ann Trop Med Parasitol 2008;102 (8):699–708. [26] Cousins DV, Robertson GM, Hustas L. The use of the enzyme-linked immunosorbent assay (ELISA) to detect the IgM and IgG antibody response to Leptospira interrogans serovars hardjo, Pomona and tarassovi in cattle. Vet Microbiol 1985;10:439–50. [27] Terpstra WJ, Ligthart GS, Schoone GJ. ELISA for the detection of specific IgM and IgG in human leptospirosis. J Gen Microbiol 1985;131:377–85. [28] Mariya R, Chaudhary P, Kumar AA, Thangapandian E, Amutha R, Srivastava SK. Evaluation of a recombinant LipL41 antigen of Leptospira interrogans serovar Canicola in ELISA for serodiagnosis of bovine leptospirosis. Comp Immunol Microbiol Infect Dis 2006;29: 269–77. [29] Eliades P, Karagouni E, Stergiatou I, Miras K. A simple method for the serodiagnosis of human hydatid disease based on a protein A/ colloidal dye conjugate. J Immun Methods 1998;218:123–32.
Contents lists available at ScienceDirect
Comparative Immunology, Microbiology and Infectious Diseases journal homepage: www.elsevier.com/locate/cimid
Development of rapid flow-through-based dot-immunoassay for serodiagnosis of leptospirosis in dogs M. Subathra, T.M.A. Senthilkumar *, P. Ramadass, G. Dhinakar Raj Department of Animal Biotechnology, Madras Veterinary College, TANUVAS, Chennai 600007, India
A R T I C L E I N F O
A B S T R A C T
Article history: Accepted 5 October 2009
An IgG-ELISA used recombinant antigen and a rapid flow-through enzyme immunoassay were developed for rapid screening of leptospiral antibodies in dogs using recombinant LipL41, which is one of the conserved outer membrane proteins in pathogenic leptospires as the coating antigen. Results from this study were compared with the standard microscopic agglutination test and found that the sensitivity and specificity of the enzyme-linked immunosorbent assay were 75.46% and 93.29% and whereas that of flowthrough-based dot-immunobinding assay were 87.73% and 89.63%, respectively. Relative merits of these tests were also assessed. The flow-through-based dot-immunobinding assay was thus proved to be a valid screening test for canine leptospirosis. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: Canine leptospirosis Enzyme-linked immunosorbent assay Flow-through-based dot-immunobinding assay Microscopic agglutination test Recombinant LipL41
1. Introduction Leptospirosis is an anthropozoonosis of ubiquitous distribution, caused by spirochaetes of the pathogenic Leptospira species. Leptospirosis affects a wide range of hosts including humans, domestic and wild animal species [1,2]. Laboratory confirmation of leptospirosis is obtained when either the pathogen is isolated or a positive serological result is obtained. The microscopic agglutination test (MAT) is considered as the reference test for leptospirosis. The result of MAT is considered consistent with leptospirosis when either a four-fold increase in titre is observed between paired serum samples or a significantly increased titre for a single serum sample [3]. Both culture and the MAT seem to be laborious as well as expensive. Several methods for detecting specific IgM antibody including, enzyme-linked immunosorbent assay (ELISA) [4,5], Dipstick ELISA [6], and indirect immunofluorescent assay [7] are widely used and some of them are available commercially. As all the existing tests have advantages as well as limitations, the development of new diagnostics assumes
* Corresponding author. Tel.: +91 44 2536 9301; fax: +91 44 2536 9301. E-mail address: [email protected] (T.M.A. Senthilkumar). 0147-9571/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.cimid.2009.10.003
greater significance since leptospirosis has become an important public health problem in most countries of the world with many outbreaks reported in the recent past [8– 12]. Proteins located on the leptospiral outer membrane are of the greatest interest, as outer membrane proteins (OMPs) are potentially exposed to the host immune system. Antigenic conservation of leptospiral proteins was demonstrated in immunoblot studies that showed that sera from rabbits hyperimmunized with one strain recognized numerous proteins of a wide variety of serovars [13,14]. Several of these protein antigens have been identified, including the outer membrane lipoprotein LipL41. In the present study, a simple dot-immunobinding assay was developed based on the flow-through principle [15] utilizing the recombinant LipL41 (rLipL41) protein expressed in Escherichia coli as capture antigen. 2. Materials and methods 2.1. Bacterial strains and media The panel of eight Leptospira interrogans serovars included in MAT was Australis, Autumnalis, Canicola, Javanica, Pomona, Icterohaemorrhagiae, Grippotyphosa,
18
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and Pyrogenes. They were grown and maintained in EMJH liquid and semi-solid medium (Difco, USA) at 29–30 8C. The concentration of organisms was assessed regularly by dark field microscopy. These reference leptospiral strains were obtained from Koninklijk Institutut voor de Tropen (KIT), Amsterdam, The Netherlands, and National Leptospirosis Reference Center, Port Blair, Andaman and Nicobar Islands, India. E. coli DH5a cells (F f80DlacZDM15D (lacZYA-argF) U169 recA1 endA1 hsdR17 (rk, mk+) phoA supE44 l-thi-1 gyrA96 relA1) (Invitrogen, USA) were used as host cells for the expression of recombinant antigen and were grown and maintained in LB broth and LB agar.
tion. The polyhistidine tag (6X-His) of the fusion protein was utilized for purification of fusion protein by Ni2+-NTA affinity chromatography (Qiagen). The purified protein was analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The concentration of the protein was determined by Lowry’s method and stored in aliquots at 80 8C until used. 2.5. IgG-enzyme-linked immunosorbent assay (IgG-ELISA)
The gold standard serodiagnostic test for leptospirosis is MAT was performed as per the method of Cole et al. [16]. Briefly, the sera from the patients were serially diluted from 1:50 to 1:3, 200 in phosphate buffered saline (PBS), pH 7.2 and reacted with live antigen suspensions of the panel of eight reference leptospiral serovars. After 2 h incubation at 37 8C, the serum–antigen mixtures were examined by dark field microscope for the presence of agglutination/clearance and the titres were determined. Reciprocal agglutination titres of greater than or equal to 100 were considered as positive reactions.
IgG-ELISA was carried out initially to evaluate the purified rLipL41 protein as antigen for serodiagnosis of leptospirosis. The optimum concentration of the antigen was determined by the checkerboard titration [18]. Flatbottom polystyrene microtitre plates (MaxiSorp, Nunc) were coated with purified antigen diluted in 0.06 M carbonate–bicarbonate buffer (Na2CO3 1.59 g and NaHCO3 2.93 g in 1 l of distilled water, pH 9.6) overnight at 4 8C. The plates were washed thrice with phosphate buffered saline, pH 7.2 with 0.05% Tween 20 (PBST) and blocked with 2% bovine serum albumin (BSA) in PBST for 1 h at 37 8C. The plates were washed thrice with PBST and incubated at 37 8C with 100 ml of two-fold serially diluted serum samples to be tested in PBST from 1:100 to 1:3, 200 dilutions for 1 h. After three washes with PBST, wells were added with 1:15,000 dilution of rabbit anti-dog IgGperoxidase (HRP) conjugate (Bangalore Genei, India) and incubated for 1 h at 37 8C. The substrate solution ABTS (2,20 -Azino di-ethyl benz-thiozoline-6-sulphonic acid) (Sigma, USA) was prepared and added to the wells at 100 ml volume and allowed to react for 10 min. When the colour developed, the absorbance at 405 nm was measured in an ELISA reader (Biotek instruments Inc., USA). The mean absorbance of three negative control wells was calculated. Double the values of mean negative control optical densities (ODs) were considered as positive OD, and the reciprocal dilution of the sample showing positive OD was observed as the titre value of the sample.
2.4. Recombinant leptospiral antigen LipL41
2.6. Flow-through-based dot-immunoassay
The outer membrane protein (OMP) gene LipL41 was partially amplified from the reference L. interrogans serovar Icterohaemorrhagiae (strain RGA) by polymerase chain reaction (PCR). The primers with built in restriction enzyme sites were selected based on the published LipL41 primer sequence of L. krischneri species [17]. The PCR amplicon size of 1077 bp (GenBank accession no. DQ132992) was cloned into an expression vector pPROEXHT‘b’ (Invitrogen, USA). The expression of recombinant fusion protein (rLipL41) was achieved by adding isopropyl b-D thiogalactoside (IPTG) at 1 mM final concentration. Purification of the expressed protein was carried out by sequential addition of buffer A (50 mM Tris–HCl, pH 8.0, 7.5% glycerol, 0.1 mM EDTA and 50 mM NaCl), buffer B (50 mM EDTA, 10% Triton X-100, 2.5 mg/ml lysozyme and 2 mM PMSF or b-Mercaptoethanol) and 5 mM MgCl2 to the cell pellet and incubation in ice for 1 h. Then the cells were sonicated at constant pulse twice for 30 s. The lysate was clarified at 17,950 g for 30 min at 4 8C. The supernatant was subjected for affinity purifica-
The flow-through assay was developed as per the method of Paepens et al. [15] with minor modifications. Briefly, NCMs (0.45 mm, Advanced Microdevices Pvt. Ltd., Ambala Cantt, India) were cut into small squares (2 cm 2 cm). The test was performed in an immunofiltration device, a small rectangular plastic module with test window holding water absorbing material and a nitrocellulose membrane (NCM). The NCM was pressed tightly over the water absorbing material in the test window. One microlitre of the antigen (rLipL41) diluted in PBS, pH 7.2 was dotted on the NCM in the ‘T’ side of the test window and one ml of 1:100 diluted healthy dog serum (Bangalore Genei, India) in PBS, pH 7.2 was dotted in the ‘C’ side of the test window to serve as reagent control. The modules were incubated at 37 8C for 1 h and stored in a sealed plastic bag at room temperature. The assay was performed at room temperature. Hundred microlitres of wash buffer (1 M Na2HPO4, 1 M NaH2PO4, pH 6.0, 0.9% NaCl, 0.5% Triton X-100, and 1% BSA) was added twice to the test window to wet and 2%
2.2. Canine serum samples Three hundred and twenty-two blood samples were collected from the dogs suspected for leptospirosis with pyrexia, jaundice and other malaise, irrespective of their age, breed and sex in the Small Animal Clinics, Madras Veterinary College Hospital, Chennai, India. Blood samples were also collected from five apparently healthy dogs that were brought for general health check up with vaccination details to serve as negative controls. Sera were separated on the same day and stored at 20 8C until used. 2.3. Microscopic agglutination test (MAT)
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Fig. 1. Evaluation and standardization of the rLipL41 concentration (5, 10, 25, 50, 100, 200, 400 and 800 ng) at a serum dilution of 1:100. P 0.01.
BSA in PBS, pH 7.2 was added to block the NCM. The antigen concentration for dotting the NCM was assessed using checkerboard titration by comparing the intensities of colour developed with different antigen concentrations and serum dilutions. The suspected dog serum samples were diluted to 1:10 with PBS, pH 7.2 and 100 ml was added to the test window. The unbound antibodies were washed by adding 100 ml of wash buffer twice. The goat anti-dog IgG-peroxidase conjugate (Bangalore Genei, India), 100 ml was added in the dilution of 1:500 in PBS, pH 7.2. After washing with wash buffer twice, the substrate Diamino benzidine (DAB) (Sigma, USA) was added and allowed for absorption. Immediately the NCM was rinsed with 100 ml of distilled water twice. The appearance of two brown dots in the ‘T’ and ‘C’ sides of test window indicated a positive reaction, and the absence of dot in the ‘T’ side of test window indicated a negative reaction. If there were no dots in the test window, the test was considered invalid. The entire protocol could be completed within 5–10 min. 2.7. Relative sensitivity, specificity and concordance The relative sensitivity, specificity and concordance (in percent) of the IgG-ELISA and flow-through-based dotimmunoassay for the detection of leptospiral antibodies in dog serum samples were determined in comparison to the MAT as described below: a sensitivity ¼ 100 aþc where ‘a’ is the number of serum samples positive by the test and MAT and ‘c’ is the number of serum samples positive by MAT but negative by test: specificity ¼
3. Results 3.1. IgG-enzyme-linked immunosorbent assay The optimum concentration of the purified rLipL41 antigen, which showed maximum difference in optical density (OD) values, between the positive and negative serum samples was determined to be 10 ng/well (Fig. 1). Though all the concentrations of the antigen are significant (P 0.01), the concentration at which three-fold difference between the positive OD and negative OD was taken as the coating antigen concentration of LipL41. Among 327 canine serum samples, 134 samples were found positive. The sensitivity, specificity and concordance of the assay in relation to the standard MAT are shown in Table 1. 3.2. Flow-through-based dot-immunoassay A series of experiments were performed to determine the optimal conditions and to adjust the test sensitivity to an appropriate level. Checkerboard titrations were carried out with various concentrations of antigen (10, 5, 2 mg/ml, 100, and 50 mg/ml) and the intensities of colour development to determine the optimal concentration of the capture antigen (Table 2). Various dilutions of serum samples and the intensities of colour development were also checked (Data not shown). The assay was optimized using 2 mg/ml concentrations of capture antigen (rLipL41) and 1:10 dilution of the serum samples to be tested (Fig. 2).
Table 1 Comparison of IgG-ELISA and MAT for serodiagnosis of canine leptospirosis. MAT
d 100 bþd
where ‘d’ is the number of serum samples negative by test and MAT and ‘b’ is the number of serum samples negative by MAT but positive by test: aþd concordance ¼ 100 aþbþcþd
+
Total
IgG-ELISA +
123 (a) 40 (c)
11 (b) 153 (d)
134 193
Total
163
164
327
2
x = 159.77**; K = 0.69. Sensitivity: 75.46%; specificity: 93.29%; concordance: 84.40%. **Highly significant P 0. 01.
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Table 2 Determination of antigen concentration for flow-through-based dot-immunoassay. Concentration of rLipL41
10 mg/ml 5 mg/ml 2 mg/ml 1 mg/ml 100 mg/ml 50 mg/ml
Canine serum samples with reciprocal MAT titres in brackets 1 (<100)
2 (100)
3a (100)
4 (200)
5 (400)
6 (800)
+ +
+++ +++ +++ +
+++ +++ +++ +++ ++
+++ +++ +++ ++
+++ +++ +++ ++ +
+++ +++ +++ ++ +
+++ High intensity of the colour; ++ moderate intensity of the colour; + poor intensity of the colour; negative. a Slightly haemolysed serum sample.
Fig. 2. Flow-through-based dot-immunobinding assay. Modules 1 and 3, positive reactions; modules 2 and 4, negative reactions; C, control reactions; T, test reactions.
Table 3 Comparison of flow-through-based dot-immunoassay and MAT for serodiagnosis of canine leptospirosis. MAT
Among 327 canine serum samples, 160 samples were found positive. The sensitivity, specificity and concordance of the assay in comparison with standard MAT are shown in Table 3. All the five negative samples were also negative by MAT, IgG-ELISA and flow-through-based dot-immunoassay.
+
Total
Flow-through +
143 (a) 20 (c)
17 (b) 147 (d)
160 167
4. Discussion
Total
163
164
327
Highly conserved OMPs are of special significance in serodiagnosis and vaccine development for leptospirosis. The leptospiral OMPs expressed during mammalian
2
x = 195.80**; K = 0.77. Sensitivity: 87.73%; Specificity: 89.63%; Concordance: 88. 69%. **Highly significant P 0. 01.
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infection may have potential immunoprotective capabilities [19,17]. However, the lack of an effective, widely available laboratory tool remains a major problem [20] and standard serological tests for case confirmation need to be optimized [21]. Currently available methods are not easy to perform; all require special and expensive equipments and can be used only by trained personnel. There is a need to develop a method that is rapid, simple with high sensitivity and specificity. Recombinant protein-based serologic tests may achieve high sensitivity and specificity because of high concentration of immunoreactive antigens, which could be used in the assays, and also lack of non-specific moieties present in whole cell preparations [20]. In the present study, one of the outer membrane proteins, that are highly conserved among pathogenic leptospires [17], LipL41 was expressed as recombinant fusion protein and utilized for developing a rapid flow-through-based dot-immunoassay. Most of the rapid assays were developed and evaluated for serodiagnosis of human leptospirosis. The disease being considered mainly as occupational, and of zoonotic in nature, the early diagnosis and treatment of animals are essential for prevention of human leptospirosis. ELISA-based assays were developed by many workers and evaluated as an alternative to MAT for diagnosis of leptospirosis [22–24]. In this study, IgG-ELISA and dot-immunobinding assay were developed using recombinant antigen (LipL41), and their results were analysed with standard MAT. The MAT result indicated that commonly occurring L. interrogans serovars in canine species were Canicola, Australis, Javanica and Icterohaemorrhagiae. IgG-ELISA with the recombinant LipL41 antigen developed in this study had higher sensitivity and specificity than that reported by Flannery et al. [20] who evaluated the same recombinant antigen for the diagnosis of human leptospirosis. They evaluated sera from 50 healthy residents and paired sera from 50 cases of leptospirosis confirmed by the MAT. They had the sensitivity and specificity of 24% and 44%, respectively. Natarajaseenivasan et al. [25] evaluated the IgM based recombinant LipL41 ELISA which showed 89.3% sensitivity and 89.2% specificity, because IgM antibody level increases in the first week but becomes negative within 3–5 weeks, whereas IgG antibodies are detected at about the same time as IgM but persists for a much longer period [26]. Terpstra et al. [27] who reported high IgM titres between 10 and 60 days of illness while the combination of moderately increase IgM titres and low IgG titres was most often observed in the first 10 days of the disease. Here the developed assay was based on IgG response, so the sensitivity rate is 75.46% when compared to IgM based MAT. In future, we can increase the sensitivity rate by IgM based ELISA and Mariya et al. [28] 2006 evaluated the recombinant leptospiral lipoprotein LipL41 as an antigen for conducting ELISA for diagnosis of bovine leptospirosis showed 100% sensitivity and 85.3% specificity. However, the specificity was 85.3% which indicates that only limited Leptospira serovars were used as antigens for antibody detection in MAT. The flow-through-based dot-immunoassay developed in this study combines the ease of flow-through technology and dot-ELISA principle for rapid, sensitive serodiag-
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nosis of canine leptospirosis. The sensitivity and specificity obtained by screening of 327 dog serum samples proved that this test could be a valid screening test for canine leptospirosis. The results could be read within 5–10 min whereas the conventional dot-ELISA techniques required 1–3 h. Moreover, the development of clear dots avoided the ambiguity about the results. Eliades et al. [29] developed a dot-immunobinding assay for the serodiagnosis of human hydatidosis and concluded that the detection of anti-HA antibodies had agreed closely with the data from ELISA. In this study, the sensitivity of the flow-through-based dot-immunoassay was found to be higher than that of IgG-ELISA and the specificity found to be reduced. This could be due to the quality of the serum samples used in the assays. Sometimes haemolysed serum samples showed mild positivity in the flow-through-based dot-immunoassay; because of pale red in colour of serum react with membrane bound antigen, though they were MAT negative. Hence, the haemolysed serum samples should be avoided in this assay. The developed assay gave a reliable result and interesting alternative tests for serodiagnosis of canine leptospirosis especially at peripheral veterinary health care units in developing countries where sophisticated equipments are not available. Acknowledgments The study is supported by Grants from Department of Biotechnology, Ministry of Science and Technology, Government of India to T.M.A.S. References [1] Faine S. Leptospira and leptospirosis. Boca Raton, USA: CRC Press; 1994. [2] Hartskeerl RA, Terpstra WJ. Leptospirosis in wild animals. Vet Quart 1996;18:149–50. [3] Communicable disease surveillance kit. Geneva, Switzerland: World Health Organization; 1998. [4] Petchclai B, Kunakorn M, Hiranras S, Potha U, Liemsuwan C. Enzymelinked immunosorbent assay for leptospirosis immunoglobulin M specific antibody using surface antigen from a pathogenic Leptospira: a comparison with indirect hemagglutination and microagglutination tests. J Med Assoc Thai 1992;75:203–8. [5] Cumberland P, Everard COR, Levett PN. Assessment of the efficacy of an IgM-ELISA and microscopic agglutination test (MAT) in the diagnosis of acute leptospirosis. Am J Trop Med Hyg 1999;61: 731–4. [6] Gussenhoven GC, van der Hoorn MAWG, Goris MGA, Terpstra WJ, Hartskeerl RA, Mol BW, et al. LEPTO dipstick, a dipstick assay for detection of Leptospira-specific immunoglobulin M antibodies in human sera. J Clin Microbiol 1997;35:92–7. [7] Pradutkanchana S, Pradutkanchana J, Khuntikij P. Detection of IgM specific antibody using indirect immunofluorescent assay for diagnosis of acute leptospirosis. J Med Assoc Thai 2003;86:641–6. [8] Ko AI, Galvao Reis M, Ribeiro Dourado CM, Johnson WDJR, Riley LW. Urban epidemic of severe leptospirosis in Brazil. Salvador Leptospirosis Study Group. Lancet 1999;354:820–5. [9] Russell KL, Gonzalez MAM, Watts DM, Lagos-Figueroa RC, Chauca G, Ore M, et al. An outbreak of leptospirosis among Peruvian military recruits. Am J Trop Med Hyg 2003;69:53–7. [10] Vijayachari P, Sehgal SC, Goris MGA, Terpstra WJ, Hartskeerl RA. Leptospira interrogans serovar Valbuzzi: a cause of severe pulmonary haemorrhages in the Andaman Islands. J Med Microbiol 2003;51:1–6. [11] Meeyam T, Tablerk P, Petchanok B, Pichpol D, Padungtod P. Seroprevalence and risk factors associated with leptospirosis in dogs. Southeast Asian J Trop Med Pub Health 2006;37:148–53. [12] Goldstein RE, Lin RC, Langston CE, Scrivani PV, Erb HN, Barr SC. Influence of infecting serogroup on clinical features of leptospirosis in dogs. J Vet Int Med 2006;20:489–94.
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