Evaluation of recombinant LipL 32 based latex agglutination test for serodiagnosis of porcine leptospirosis

Aim: To evaluate the efficacy of recombinant LipL32 based Latex Agglutination Test (LAT) in serodiagnosis of porcine leptospirosis. Materials and Methods: Recombinant protein was produced, purified and they are coated with latex bead for performing Latex Agglutination Test. A total of 85 sera samples of pigs were collected and screened for the presence of antileptospiral antibodies using Microscopic agglutination test (MAT) and rLipL32LAT. Results: Seroprevalence rate of 18.22% was reported with Tarassovi as the predominant serovar. In the present study, the rLipL32-based LAT is having 81.25 % sensitivity, 98.55% specificity, 95.29% accuracy and k-value of 98.43%. Conclusion: The result revealed good sensitivity, specificity and accuracy of the serodiagnostic test of porcine leptospirosis. It can be useful as a pen side diagnostic test for swine leptospirosis in field condition.


Introduction
diagnostics such as LeptoDipstick, Lepto dri-dot, Lepto lateral-flow, Latex Agglutination test , which Leptospirosis is a multispecies infectious disease take the advantage of presence of genus specific outer caused by pathogenic spirochete Leptospira interrogans.
membrane proteins(OMP) of pathogenic leptospiral There are about 250 serovars, arranged into 25 organism [11][12].In the recent years, several Leptospira serogroups have been described under the species L.
surface proteins have been identified and characterized interrogans [1].The major losses to swine industry for their usage in diagnostic or vaccine candidate [13associated with leptospirosis are caused by abortion , 14].The usefulness of lipoprotein LipL32, which is still birth, infertility and birth of live, weak piglets [2highly conserved among pathogenic leptospires has 4].The disease has been reported in many countries [4already been investigated in human and animal sera [4,7].A report of swine leptospirosis is scanty in our 10,15,16].country except one report during mid nineties [8].
So, in the current study it was planned to evaluate Diagnosing leptospirosis in swine is a challenge.The the LipL32 based latex agglutination test (LAT) for culture of leptospires is difficult, time-consuming, and serodiagnosis of porcine leptospirosis.requires specialized culture medium and technical expertise; culture is usually only available at reference

laboratories [3]. Microscopic agglutination test (MAT),
A total of 85 porcine sera samples (24 sera from is the most commonly used serological test as it is Nekpur, Bareilly; 11 sera from Pig Farm, Indian inexpensive, widely available, and reasonably sensitive Veterinary Research Institute, 25 sera samples each [9].However, MAT requires the use of several from Tamil Nadu and Gorakhpur, Eastern Uttar leptospiral serovars in their active growth phase whose Pradesh) were collected.All the porcine sera samples maintenance is difficult, expensive, tedious and time were initially tested by MAT as described by Cole et al. consuming [1,4].Hence, numerous approaches have [17] using the whole cell live antigens of the following been investigated to improve diagnosis of leptospirosis serovars: Australis, Autumnalis, Ballum, Canicola, in swine such as enzyme immunoassays using whole Grippotyphosa, Hardjoprajitno, Hebdomadis, Icterocell of leptospire and recombinant LipL32 based haemorrhagiae, Javanica, Pomona, Pyrogenes and ELISA [3,4,10].These newer procedures are however Tarassovi.1: 100 or more MAT titre was taken as positive only available in a few laboratories.Therefore, more in this study.All the porcine sera were then tested by widely acceptable approaches of serodiagnosis involve rLipL32 based LAT and the test results of both the tests diagnosing the disease at field level include penside are compared.Thirteen out of the 16 sera samples, which reagent as the procedure described earlier [18,19].The tested positive by MAT, were tested positive by LipL32 sensitised Latex beads were then used to screen rLipL32-based LAT also.The three sera, which tested sera samples suspected for Leptospirosis.Ten micronegative by rLipL32 based LAT, were in fact liters of sensitised Latex beads in a clean glass slide is borderline sera samples with titre values of 1:100 by mixed with 10 µl of field sera and result noted within 2 MAT.It was also observed that a quantitative relation minutes.If agglutination is present, a halo rim of exist between both the tests.Strongly reacting field agglutinin is formed with a cleared centre (Figure -1).
sera showing titre of 1:400 by MAT invariably gave If agglutination is absent, a homogenous suspension +++/ ++++ test result with rLipL32 based LAT while will only be present.The remaining sensitized latex weakly and moderately reactive field sera showing titre beads will be stored at 4°C and used for performing of 1:100 and 1:400 respectively by MAT gave + and Latex Agglutination Test against porcine field sera +++ test result with rLipL32 based LAT (Figure -1).suspected for leptospirosis by detection of anti-LipL32 Table -1 provide the information regarding the antibodies.
sensitivity, specificity and Accuracy of rLipL32 based Evaluation of MAT and rLipL32 based LAT: The relative LAT in comparison with MAT.sensitivity, specificity and accuracy of rLipL32 based

LAT for serodiagnosis of leptospirosis were evaluated
Leptospirosis is often confused with other similar in comparison to Microscopic Agglutination test as diseases or ailments mimicking its symptoms.described below [19].
Diagnosis of this important disease of public health Sensitivity= a/(a + c) × 100, where 'a' is the number of significance is often cumbersome and challenging [1, sera positive by rLipL32 based LAT and MAT while 'c ' 20].Hence, Latex Agglutination Test employing is the number of sera positive by MAT but negative by recombinant LipL32 protein would serve as a simple, rLipL32 based LAT sensitive pen side diagnostic test, which can give rapid Specificity= d/(b + d) × 100, where 'd' is the number of and reliable diagnosis of leptospirosis under field sera negative by rLipL32 based LAT and MAT while 'b' conditions.This diagnostic test would not require the is the number of sera negative by MAT but positive by use of highly skilled labour and the test results would rLipL32 based LAT be very easy to interpret.Thus, LAT can be Accuracy= (a + d)/ (a + b +c +d) × 100 recommended for field studies in which speed and

Conclusion
simplicity are crucial [19].When the field sera is mixed with rLipL32 coated latex beads, anti-LipL32 In conclusion, the rLipL32 based LAT having antibodies present in the sera of pigs naturally infected good sensitivity, specificity and accuracy can be served by leptospirosis interact with the antigen to form fine as a pen side diagnostic test in well established and clearly visible granular agglutination.The strength Veterinary hospitals as well as in mobile Veterinary of the agglutination depends on concentration of the clinics operating in remote, resource-poor settings, but antibodies in a sera sample.Clearly visible granular validity of test can be established after testing more agglutination indicates the existence of specific sera in different geographical regions.Leptospirosis is antibodies to leptospires.In stronger reactions due to a health problem in swine, which causes major losses to sera of high antibody titre, fine granular clumps tend to pork industry.Infected swines are carrier for the settle at the edge of the circle and the reaction time to leptospira, which can transmit infection to people, is of form fine granular clumps is lesser than 60 seconds.
major public health concern.Hence, continuous Agglutination that occurs beyond 2-3 minutes may be seromonitoring in field condition should be done by due to evaporation and should be treated as doubtful using different pen side diagnostic tests to ensure cases.When Latex beads are sensitised with rLipL32 effective control and management of this disease.protein which is cross-reactive lipoprotein found on the Authors' contributions outer membrane of most pathogenic leptospiral serovars, this test would be able to detect infection SKB carried out the sample collection, analysis and caused by any of them.Unlike MAT, which employs manuscript preparation.TS and PC helped in scientific, live whole leptospiral antigens could pose danger to the intellectual and technical suggestions, AK designed the lab technician performing the test, whereas, LAT work, SCD and RKA helped in manuscript preparation employing rLipL32 protein is completely safe and nonand correction.All authors read and approved the final hazardous.The coated latex particles are stable for long manuscript.periods at 4ºC and this long shelf life and cost congruence with earlier report of this test in bovine and leptospirosis in animals and human beings in various region human leptospirosis [18,19], but opposite to canine of the country.Indian Journal of Compendium Microbiology leptospirosis [18].
Result division, IVRI was used to transform competent DH5α strains of E coli cells treated with 0.1M CaCl .The 2 Screening of 85 sera samples resulted in 16 transformants which appeared on LB Ampicillin plate (18.22%) sera showing positive for various serovars of were grown in bulk in LB broth containing Ampicillin Leptospira.It is also noteworthy that all the sera, which & then induced with 1M IPTG (Isopropyl β-D-1tested positive, were reacted against multiple serovars thiogalactopyranoside) during early log phase of of Leptospira viz.Icterohaemorrhagiae, Tarassovi, growth & after overnight incubation were centrifuged.Australis and Javanica.Twelve out of the 16 positive The cell pellet obtained was treated with Lysis buffer sera showed a titre value of 1:200 and remaining four (pH 8.0) and then centrifuged twice.The supernatant sera showed baseline titre of 1:100 for Icterohaemorrobtained was passed through a Nickel-Nitrilotriacetic hagiae.All the sera showed reactivity to serovar acid affinity chromatography column.The column was Tarassovi with titre value of 1:100 (n=12) and 1:400 later washed with Wash buffer (pH 6.3) to remove any (n=4).Serovars Australis and Javanica reacted positively unbound protein in the column.Only 6X tagged with 12 porcine sera with Australis reacting at baseline LipL32 protein remain in the column which is eluted on titre of 1:100 with seropositive porcine sera samples.addition of Elution buffer pH 4.5.The eluted protein However, two porcine sera each tested positive at titre were then dialysed with 1X PBS to remove urea.The value of 1:200 with the serovar Javanica and rest of the dialysed LipL32 protein was then buffered with sera at 1:100 titres.Glycine buffered saline pH 8.2 & this protein was then Comparison of rLipL32 based LAT and MAT test used to coat Latex beads buffered with the same results:

Table - 1
. Evaluation of diagnostic values of rLipL32 based LAT in comparison to MAT