Seroepidemiology of canine leptospirosis by iELISA and MAT

Aim: The present study was conducted to determine the prevalence of antibodies against leptospira using a genus specific and serovar specific antigen by indirect ELISA (iELISA) and Microscopic Agglutination test (MAT), respectively. Materials and Methods: 300 sera samples were collected from dogs, from Pondicherry, suspected to be suffering from Canine leptospirosis and with no history of vaccination. Using the outer membrane proteins (OMP's) of Leptospira interrogans serovar canicola as antigen the leptospiral antibodies were detected by iELISA and compared with the MAT using five commonly infecting serovars (canicola, icterohaemorrhaegiae, grippotyphosa, pomona, australis) as antigens. Statistical analysis of the data was carried out using chi-square test. Result: Out of 300 sera samples, 159 (53 %) and 99 (33 %) sera samples were found to be positive for leptospiral antibodies by iELISA and MAT, respectively. The iELISA employed in this study revealed a very high sensitivity of 94.94 % in comparison with the MAT (59.11 %). However, iELISA showed less specificity (67.66 %) when compared to MAT (96.45 %). By chisquare test, a significant difference (P<0.001) was obtained between the results from iELISA and MAT. Conclusion: The present study revealed that the iELISA was found to be highly sensitive, rapid and easy to perform in comparison with MAT for the detection of canine anti-leptospiral antibodies.


Introduction
Leptospira by inexperienced workers [2].Hence microscopic examination is used as a parallel test along Leptospirosis is a widespread zoonotic disease with the culturing of these organisms (gold standard and is a real public health concern around the world.test) which is very laborious, time consuming and does The disease is caused by spirochetes of the genus not aid in the early diagnosis of the disease [3].Leptospira which comprises of more than 260 serovars Serology appears to be the most reliable tool for classified under 24 serogroups based on agglutinating the diagnosis of leptospiral infections carried out with antigens and is classified into multiple genomospecies Microscopic Agglutination Test (MAT) which is based on DNA studies [1].These highly invasive recognized as the international standard technique [4].spirochaetal pathogens are capable of infecting a wide The MAT is a specific test, which requires maintenance range of mammalians either by direct contact or by of live organisms that may cause problem in handling, indirect contact with soil/water contaminated with the standardization and its interpretation is relatively urine of the carrier animals.
subjective [5].Leptospiral outer membrane proteins Leptospira are obligate aerobes with an optimum (OMP) contain both transmembrane proteins, porin growth at 28-30°C in the EMJH medium, supple-(OMP L1) and lipoproteins, such as LipL41 and mented with Tween-80 and bovine serum albumin LipL36.Most research on leptospiral antigens is being fraction V [2].The diagnosis of this disease is done focused on Lipopolysaccharide (LPS) and their variations mainly by direct microscopic examination and by in the carbohydrate side chains which are responsible isolation of these microorganisms.The microscopic for the antigenic diversity among leptospiral serovars.examination is done using dark-field microscopy and Because of this antigenic variability, the use of aids in the early diagnosis of the disease.However it leptospiral LPS has limitations for serodiagnosis.Ideally, has two major drawbacks; i) too low concentration of an antigen of choice for the purpose of developing a leptospires (less than 104 cells / ml) which may not be diagnostic test which can be applied for the variety of detected and ii) artifacts such as fibrin and extrusions epidemiological situations associated with human and from cellular elements can be easily mistaken for animal leptospirosis is to be highly conserved among the diverse pathogenic leptospiral serovars.In this context, OMPs is considered to be a potential antigen as they are conserved within the various pathogenic

Seroepidemiology of canine leptospirosis by iELISA and MAT
serovars and such proteins and their associated bicarbonate buffer (pH-9.6) to provide dilutions molecules, selectively solubilised using Triton-X 114 ranging from 3.66 µg/well to 25ng/well across the columns.detergent, would be of immense diagnostic value [6].
The ELISA has been used for the serodiagnosis of Indirect ELISA: Antigen in carbonate -bicarbonate a wide range of infectious diseases and also to detect buffer was coated on to each well by incubating at 4° C leptospira -specific antibodies in humans and animals overnight.On the next day, the plates were washed five [7,8].This immunosorbent assay is reported to be times with PBS with 0.05% Tween 20. 300 micro litre simple, safe, specific, sensitive, easily automated and of 5% skim milk powder was added to block the suitable for the examination of a large number of sera uncoated sites and incubated at 37° C for one hour.To assess the protein was extracted as per the method described by antibody titres of canine sera samples against Haake [6].Briefly, leptospires were washed in Leptospira, MAT [10] was performed in microtitre phosphate-saline-5 mM MgCl and then extracted in 2 plates using 5 Leptospiral interrogans serovars viz., the presence of 1% protein-grade Triton X-114 -150 canicola, icterohaemorrhagiae, pomona, grippotyphosa mM NaCl-10 mM Tris (pH 8)-1mM EDTA at 4°C.The and australis.A serum dilution of 1:50 was first used to insoluble material was removed by centrifugation at screen the samples for leptospirosis.The positive 17,000 g for 10 min.After centrifugation, 20 mM CaCl 2 samples were identified and titrated using serial two was added to the supernatant.Phase separation was fold dilutions from 1:50 to 1:3200 in the following performed by heating the supernatant to 37°C followed manner.100 micro litre of PBS was added to wells 2 to by centrifugation at 1000 g for 10min.The detergent 8 in 96 well flat bottomed microtitre plate.100micro and aqueous phases were then separated and litre of pre-diluted serum (1:25) was added to wells 1 precipitated with acetone.and 2 including positive controls corresponding to Serum samples: 300 sera samples were collected from those serovars.Doubling dilution of sera was made dogs suspected to be suffering from Canine from well 2 to 8. 100micro litre of antigen (5-7 day old leptospirosis and those with no history of vaccination live culture) was added to wells 1 to 8 so that the final from Pondicherry for the detection of leptospiral dilution was 1:50 to 1:6400.PP values were calculated using the average OD values with MAT was 94.94 % and 67.66 % respectively obtained from the negative control serum samples.
(Table -3).The sensitivity and specificity of MAT in Based on these OD values, the mean PP value of the comparison to iELISA was 59.11 % and 96.45% negative control group was calculated as 14.51.
respectively.Surujballi [14] showed a higher sensitivity Therefore, any serum sample showing a PP value of and specificity 95.3 % and 100 %, respectively.The 29.02 (twice the mean negative PP value) or more was iELISA developed by Ribotta [13], showed a higher considered as positive for leptospirosis (Table 2).Out sensitivity (100%) and higher specificity (95.6 %) than of these 300 sera samples, 159 (53 %) were found to be our study.The relative sensitivity of iELISA obtained positive for leptospirosis by iELISA.
in our study was 94.94 % suggesting that it was as efficient as MAT in detecting true positive animals.MAT: Out of the 300 dog serum samples tested against However, the specificity was only 67.66 % signifying 5 serovars of leptospira, 99 (33 %) serum samples were the inability of the iELISA to detect all the MAT found to be positive for at least one serovar tested.In negative animals as iELISA was negative too.This that 60 (60.60 %) serum samples were found to be could be explained on the basis that all those sera positive for serovar canicola, 11 (11.11%) serum turned out to be MAT negative may not actually be samples for serovar icterohaemorrhagiae, 11 (11.11negative as only 5 leptospira serovars were used as %) serum samples for serovar grippotyphosa, 8 (8.08 antigens for antibody detection in our MAT technique.%) serum samples for serovar Pomona and 9 (9.09%) However, the findings supported the prevailing view sera samples were found to be positive for serovar that the iELISA is more sensitive than MAT [18].australis (Figure -3).It was observed that 40 serum samples cross reacted with various serovars, A total of Statistical analysis: Chi-square analysis of the data 11 sera samples cross reacted with canicola and revealed that there is a significant difference between icterohaemorrhagiae; 9 sera with canicola and iELISA and MAT employed in this study, where the pomona, 8 sera with canicola and australis; 5 sera with chi-square value is 104.4 with a Probability value of grippotyphosa and canicola, 2 with australis and 0.0001 i.e., <0.01.So, it is highly significant at 1 % pomona, and 5 sera with icterohaemorrhagiae and level.pomona.These cross reacted sera were verified in Conclusion different dilutions to find out the serovar.
Among many serovars of leptospira interrogans, The present study revealed that the iELISA was we have selected only five serovars for this study to found to be highly sensitive, rapid and easy to perform test the antibodies in the dog sera.Most clinical cases in comparison with MAT for the detection of canine of Leptospirosis in dogs are caused by serovars anti-leptospiral antibodies.The immunosorbent assay canicola, icterohaemorrhagiae, grippotyphosa and employed a non-hazardous, highly purified, reproducible pomona [15].In addition, other serovars namely antigenic preparation which can be prepared in large australis, javanica, ballum, hardjo, autumnalis and quantities and stored for long periods.Moreover, they pyrogenes were recorded by Senthilkumar [16].
do not require the maintenance of a constant supply of Immunity to leptospirosis is largely humoral and is live leptospiral cultures of different serovars as in the relatively serovar specific [7].MAT is the most case of MAT.Therefore iELISA can be recommended commonly used serological diagnostic method, as a valuable test in routine diagnostic laboratories.although it also detects antibodies produced by The most prevalent serovar was found to be Leptospira vaccination [17].According to our study the frequency interrogans serovar canicola by serovar specific MAT of leptospiral infection with canicola serogroup is test.Many of the samples subjected in the present more common in canines of Pondicherry.
study were from animals either convalescing or Sensitivity and specificity of iELISA and MAT: The suffering with subclinical leptospirosis.Data showed sensitivity and specificity of the iELISA in comparison that 53% of these animals were at least one time or coat the nunc maxisorb ELISA plates for screening of test sera samples.Antigen extraction and Protein estimation: A seven day old Leptospira interrogans serovar canicola with a Determination of the cut-off value: Ribotta [13] used 7 concentration of 4.0 x 10 organisms per ml was used to at a much higher concentration of antigen, 2.2 µg per obtain OMPs by Triton X-114 extraction method.The well and a serum dilution of 1:200.Surujballi [14] used concentration of OMP extracted was 3.66 µg/µl by comparatively lower concentration of antigen, 0.1 µg Lowry's method.The advantages of using Triton X-114 per well which gave an OD of one with strong positive extracted OMP antigen in iELISA is that the bulk serum, 0.6 with moderate and 0.05 with negative amount of the extracted antigen can be obtained serum at a dilution of 1:200.But in the present study without handling the live Leptospira organisms.It is we used a very minimal concentration of antigen and safer than MAT because it eliminates the risk of this is likely due to the quality and purity of antigen handling live leptospires.Another advantage with this extracted.A concentration of 0.4575 µg per well of antigen is that it can detect genus-specific antibodies antigen, average OD values of 1.2, 0.6 and 0.18 were which MAT does not.obtained for strong positive, moderate and healthy serum, respectively.The conversion of raw OD values Standardization of indirect ELISA (iELISA): iELISA into percent positivity (PP) values offers advantages, was standardized by optimising antigen dilution at as it obviates the variations arising out of OD values of varying dilutions of hyperimmune sera ranging from test samples in comparison with control values of each 1:10 to 1:100 and healthy serum collected from dogs plate on a day to day basis.Thus results interpreted in before immunisation that served as a negative control the present study based on PP values offer a means to and rabbit anti-dog IgG-HRPO conjugate at a constant distinctly separate a population of animals which are dilution of 1:1000.The OD values were obtained with either positive or negative.The mean OD value of respect to varying dilutions of OMP antigen against a control negative serum samples at 1:50 dilution was constant dilution of hyperimmune serum in each 0.180 and that of control positive serum samples was column (Figure-2).It was observed that the antigen 1.500 using 0.4575 µg/well of OMP antigen.Based on concentration of 0.4575 µg/well at 1:50 of these OD values, twice the mean OD values of the hyperimmune sera and 1:1000 dillution of rabbit antinegative control serum was considered as the cut-off dog HRPO was found to be satisfactory to screen the OD value to screen the test serum samples.Therefore test serum samples (Table-1).At this concentration of any serum sample showing the OD of 0.36 and above antigen, the OD value of strong positive at 492 nm was was considered as positive for leptospirosis.1.0417.So, the antigen concentration of 0.4575 Calculation of the percent positivity (PP) values: The µg/well was taken as optimal antigen concentration to

Figure- 2 .Figure- 3 .
Figure-2.Determination of optimum concentration of OMP antigen for iELISA Eight standard serovars of Leptospira final washed and 100 microlitre of freshly prepared viz; Leptospira interrogans serovars canicola, chromogen -substrate solution containing OPD and icterohaemorrhagiae, pomona, grippotyphosa, urea H O as substrate was added to each well and the 2 2 javanica, australis, autumnalis, hebdomadis were O plate was kept at room temperature (25 C) for 10 min at obtained from the National Leptospira Laboratory, dark.The enzyme substrate reaction was stopped by Portblair, Andaman and Nicobar Islands and are adding 50 µl of 1M H SO .The optical density of each 2 4 maintained at the Department of Microbiology, of the samples was recorded at 492 nm by ELISA RAGACOVAS, Pondicherry.reader (Biorad).Triton X-114 extraction: L. interrogans outer membrane Microscopic agglutination test (MAT): Relative sensitivity and specificity interrogans serovar canicola OMP antigen to be used of iELISA for the detection of leptospiral antibodies in to coat the ELISA plates.It was performed by keeping dog sera were determined in comparison with MAT, as the serum dilution constant 1:100 against serial two described by Zweing and Robertson [12].Comparative fold dilutions of Leptospira canicola antigen.A serial evaluations of both serological tests were carried out two fold dilutions of Leptospira interrogans serovar by chi-square test (SPSS 17.0 software).canicola OMP antigen was prepared in carbonate - OResults and Discussion

Table - 1
. End point titration to find out the optimum conentration of antigen for iELISA

Table - 2
. Distribution of PP values obtained from dog serum samples

Table - 3
. Relative sensitivity and specificity of MAT compared with iELISA for the detection of anti-leptospiral antibodies