Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci.

DOI 10.1007/s40011-014-0421-9

RESEARCH ARTICLE

Recombinant LipL32 Based Enzyme Linked Immunosorbent

Assay for Seroprevalence Studies of Leptospirosis in Livestock
Farmers
Sujit K. Behera • Sabrinath Thankpan • Sasmita Khatua • Deepak Pal •
Suresh C. Das • Gitanjali Sarangi • Pallab Chaudhary • Ashok Kumar

Received: 9 November 2013 / Revised: 16 July 2014 / Accepted: 14 August 2014

Ó The National Academy of Sciences, India 2014

Abstract Leptospirosis is one of the most widespread to MAT. The presence of Leptospira antibody in human
zoonosis, where humans are the accidental hosts and get subjects associated with animal rearing is an important
infected by urine of rodents and carrier mammals. In concern to public health personnel and sero-epidemiolog-
general, synchronized study on leptospirosis is scanty ical survey is an important tool to lower the number of
among occupational exposed groups in eastern India. outbreaks.
Therefore, the present investigation was undertaken to
study seroprevalence of leptospirosis among livestock Keywords Leptospirosis  Recombinant LipL32 
handlers using recombinant LipL32 enzyme linked Animal handlers  Eastern India
immunosorbent assay (rLipL32 IgG ELISA). A total of 155
sera sample were collected randomly from people having
history of animal rearing and subjected to microscopic Introduction
agglutination test (MAT) and rLipL32 IgG ELISA. Of the
sera sample tested, 22 (14 %) and 25 (16 %) were found Leptospirosis is a zoonotic disease of worldwide occur-
positive by both MAT and rLipL32 with Grippotyphosa as rence, caused by spirocheate of genus Leptospira. The
the predominant serogroup (10 %), followed by Ict- organisms are maintained in the nature by chronic renal
erohaemorrhagiae (6.5 %), Australis (3 %), and Hardjo infection of carrier mammals, which excrete the organisms
(1 %). Sensitivity and specificity of rLipL32 IgG ELISA in their urine [1–3]. Leptospire can be contacted through
was found to be 100 and 97 % respectively in comparison several routes; however, exposure of abraded or shoddy
skin and mucous membrane to contaminated water and soil
containing urine of infected rats and animals is of prime
S. K. Behera (&)  A. Kumar
importance [1–3]. Workers like those in sewer and abattoir
Veterinary Public Health Division, Indian Veterinary Research
Institute, Izatnagar, Bareilly 243 122, Uttar Pradesh, India services, farmers, veterinarians are at higher risk of lepto-
e-mail: [email protected] spirosis by occupational exposure [1–3]. Leptospires are
classified into at least 12 pathogenic and 4 saprophytic
S. Thankpan  P. Chaudhary
species with more than 250 pathogenic serovars [1].
B&M Division, Indian Veterinary Research Institute, Izatnagar,
Bareilly, UP, India Leptospira flares up both in rural and urban areas where
the environmental factors (weather, hydrographic distri-
S. Khatua  G. Sarangi bution, urban infrastructure) are combined together with
Department of Microbiology, S.C.B. Medical College &
life styles (close contacts with domestic animals, harmful
Hospital, Cuttack, Odisha, India
fauna and recreational activities) promoting its develop-
D. Pal ment [4]. It is characterized by a wide spectrum of clinical
S.S.K.M. P.G. Hospital, Kolkata, West Bengal, India conditions in humans including multi-organ failure with
high mortality [5]. The clinical symptoms of leptospirosis
S. C. Das
VPH Section, Indian Veterinary Research Institute (ERS), mimic that of several other diseases, so timely diagnosis is
Kolkata, West Bengal, India a pre-requisite to ensure a favourable clinical outcome [6].

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 S. K. Behera et al.

Laboratory diagnosis of leptospirosis is primarily based et al. [12] with dilution of sera serially done from 1:50 to
on either isolation of Leptospira spp. from the samples 1:3,200 in phosphate buffered saline (PBS, pH 7.2). Sera
collected during clinical examination or necropsy proce- were incubated at 30 °C for 3 h with suspension of live
dure or demonstration of the antibodies in the serum [7]. Leptospira serovars having concentration 1–2 9 108. The
However, cultural isolation method has poor sensitivity and positive reaction was characterized by agglutination of at
is also affected by the slow growth rate of leptospires least 50 % of Leptospira cells.
requiring 4–6 month of incubation and there is high risk of
culture contamination [3]. Recombinant LipL32 Enzyme Linked Immunosorbent
Serological assay like microscopic agglutination test Assay
(MAT) wherein the live leptospires are used as antigen was
accepted as gold standard for identifying the serovars from Recombinant LipL32 protein was expressed, purified and
human beings and animals. However, MAT has several IgG based enzyme linked immunosorbent assay (ELISA)
drawbacks as it is slow, tedious, potentially bio-hazardous was performed as per the method described earlier [6, 8,
and pain staking [2, 3]. In recent years, outer membrane 9]. The recombinant plasmid pPROHTb containing
proteins (OMPs) are the target antigens for carrying out LipL32 gene available with genetic engineering lab in
immunodiagnosis. Recombinant protein antigens such as Bacteriology and Mycology Division, IVRI, was used to
LipL32, flab, LipL41, Hsp etc., when used as diagnostic transform competent DH5a strains of Escherichia coli
antigens, have been found to give promising result both in cells treated with 0.1 M CaCl2. The transformants which
respect of the sensitivity as well as specificity as compared appeared on LB Ampicillin plate were grown in bulk in
to MAT [6, 8, 9]. In this regard higher efficacy of rLipL32 LB broth containing Ampicillin and then induced with
protein in ELISA in comparison to MAT for serodiagnosis 1 mM IPTG during early log phase of growth and after
of leptospirosis is observed. In India, serosurveillance of overnight incubation it was centrifuged. The cell pellet
leptospirosis has been reported at different times with obtained was treated with Lysis buffer (pH 8.0) and then
difference in distribution of serovars among human beings centrifuged twice. The supernatant obtained was passed
[10]. In eastern India, epidemiological studies about lep- through a Nickel-Nitrilotriacetic acid affinity chroma-
tospirosis are very fragmentary though earlier reports of tography column. The column was later washed with
outbreaks are there in late nineties [6] and early twenties wash buffer (pH 6.3) to remove any unbound protein in
[4, 11]. However there was no report about Leptospira sero the column. Only 69 His tagged LipL32 protein
prevalence among occupational exposed groups in eastern remained in the column which is eluted on addition of
India. So, the present study was undertaken to give an Elution buffer at pH 4.5. The eluted proteins were then
overview about the presence of leptospiral antibodies dialysed with 19 PBS to remove the urea. The dialysed
among livestock farmers in eastern India. LipL32 protein was then buffered with glycine buffered
saline (pH 8.2) and this protein was then used for per-
forming ELISA. The optimum concentration of antigen
Material and Methods (100 ng/well) for coating the plates as well as dilution of
serum (1:100) was attained by the method of checker-
One hundred fifty five (155) sera samples from human board titration [6, 8, 9].
volunteers with history of animal rearing were collected
from different parts of Odisha and West Bengal with prior Blood Culture
consent. Ten blood samples were collected from malaria
and dengue patients to serve as negative control. All the Of the 155 human beings, blood from few (7) of them were
sera sample are stored at -20 °C until use. cultured in semi-solid EMJH-enrichment medium (5 ml)
with 5-fluorouracil (100 lM). A drop of each culture was
Microscopic Agglutination Test (MAT) examined by dark field microscopy after a week of incu-
bation till 11 weeks of incubation.
The sera samples collected from human beings were tested
by MAT for detecting antibodies using 12 leptospiral ser- Statistical Analysis
ovars viz. Australis, Autumnalis, Ballum, Canicola, Grip-
potyphosa, Hardjo, Hebdomadis, Icterohaemorrhagiae, The relative sensitivity, specificity, ?ve, -ve predictive
Pomona, Pyrogenes, Javanica and Tarrasovi grown in El- value and concordance value of the recombinant LipL32
linghausen, McCullough, Johnson and Harris (EMJH) antigen based ELISA for the detection of anti-leptospiral
liquid media (Difco laboratories, USA) at 30 °C. MAT was antibodies in human sera were compared with MAT as
performed as per the standard protocol described by Cole described below [13].

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Leptospira Seroprevalence Among Livestock Farmers Using rLipL32 Protein

Sensitivity ð%Þ ¼ ½a=ða þ cÞ  100; [50 years, 12 (21 %) and 14 (25 %) were found positive
by MAT and rELISA, respectively (Table 3). On the basis
where ‘‘a’’ is the number of serum samples positive by the of sex, seroprevalence was higher in case of males with
ELISA and MAT, ‘‘c’’ is the number of serum samples 21.4 and 23 % by both MAT and rELISA, respectively
positive by MAT but negative by the ELISA test. (Table 4).
Specificity ð%Þ ¼ ½d=ðb þ dÞ  100 No culture was found to be positive. It may be attributed
to the factor that the infection was not in acute condition
where ‘‘d’’ is the number of serum samples negative by the
and not attained the bacteraemia state.
ELISA and MAT, ‘‘b’’ is the number of serum samples
In the present study, out of 155 human serum samples
negative by MAT but positive by the ELISA
screened by MAT, 14.2 % were found to be positive, with
þve predictive value ¼ ½a=ða þ bÞ  100 Grippotyphosa as the predominant serovar which is in
where ‘‘a’’ is the number of serum samples positive by the partial agreement with sero-surveillance study conducted
ELISA and MAT, ‘‘b’’ is the number of serum samples in Odisha state following super cyclone, where 14 %
negative by MAT but positive by the ELISA seropositivity was detected with Canicola as the predomi-
nant serovar followed by low titre levels for serovars Ict-
ve predictive value ¼ ½d=ðc þ dÞ  100 erohaemorrhagiae, Grippotyphosa and Australis [14]; but
where ‘‘d’’ is the number of serum samples negative by the the present study is not in array with another study con-
ELISA and MAT, ‘‘c’’ is the number of serum samples ducted in West Bengal state in 2005 in which Pomona was
positive by MAT but negative by the test ELISA. the predominant serovar followed by Grippotyphosa [11].
The present study is in array with earlier reported sero-
Concordance ¼ ½ða þ dÞ=ða þ b þ c þ dÞ  100
prevalence (18 %) among the patients of pyrexia of
unknown origin [10]. In view of serovar positivity the
Results and Discussion present study is in agreement with the studies conducted in
Andaman archipelago and in Kerala, where Grippotyphosa
Leptospirosis is a major globally important public health was detected as the major serovar [15–17]. The variation in
threat, both in developing and industrialized countries. occurrence of serovar in the same place in different time
Incidence of leptospirosis is remarkably underestimated periods may partially be supplemented by the idea that with
and the disease is mostly under-diagnosed in the develop- the passage of time, interaction of pathogen in variable
ing world. Leptospirosis as a cause of several ailments environments might have allowed the emergence and
remains obscure till date in India. Although MAT is the test dominance of new serovar in a population that might out-
of choice, however, it is studded with several draw backs as number the existing frequency of prevalent serovar.
it is hazardous and time consuming, requiring trained The age and sex distribution of human beings in the
personnel. Recombinant antigen-based serologic tests may present study indicates that leptospirosis is the disease
have higher sensitivity and specificity as the target antigen amongst young to adults with the dominance in male
is immunodominant and highly conserved [6, 8]. The population. This finding was in congruence with earlier
recombinant leptospiral antigens utilized in the diagnostic study in West Bengal and Kerala where 63.6 and 38.1 %
assay in the present study are constitutively expressed by seropositivity in older age group patients were recorded in
all pathogenic serovars of Leptospira, hence, are of the high risk people [17, 18]. In the present study maxi-
importance for the diagnosis of leptospirosis. Out of the mum seroprevalence was seen in the age group [50 years.
total 155 human sera samples, 22 (14 %) and 25 (16 %) It may be attributed to lowering of immune status in older
sera were found to be positive for leptospiral antibody by age group people and precipitation of disease. In the
MAT and rELISA, respectively. Most predominant serovar present study, comparatively higher seropositivity was
reported in the present study include 16 Grippotyphosa detected in male than female population as described ear-
(10 %), followed by 10 Icterohaemorrhagiae (6.5 %), 5 lier [10, 19]. This higher prevalence may partially be due to
Australis (3 %), and 2 Hardjo (1.3 %) (Table 1). Sera more exposure of male population in outdoor professional
sample with titre 1 C 100 were taken as positive. Sero- activities.
positivity was found to be in the range of 1:100 to 1:400 This finding corroborated with earlier studies from
(Table 2). Most of the sera (n = 17) were positive for Yucatan State, Mexico situated in the inter-tropical belt,
multiple serovars with 1:100 titres. where 14.25 % (57/400) sero-positivity was reported from
Sera sample collected from human beings were divided a randomly selected human population [20]. Sero-epide-
into different age groups viz. \20, 20–30 and 30–50 and miological studies from north eastern Alpine regions of
[50 years. Out of the 45 sera sample in the age group Italy detected 10–12 % sero-prevalence of leptospirosis

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 S. K. Behera et al.

Table 1 Serovars reported in human beings in two states of eastern Table 5 Comparison of recombinant LipL32 IgG ELISA with
India Microscopic agglutination test (MAT)
Serovars reported Number (% seropositivity) MAT Total

Grippotyphosa 16 (10) Positive Negative

Icterohaemorrhagiae 10 (7) rLipL32IgGELISA
Australis 05 (3) Positive 22 (a) 04 (b) 26
Hardjo 02(1) Negative 00 (c) 129 (d) 129
Total 22 133 155
Sensitivity = 100 %; Specificity = 97 %; Concordance = 97 %;
Table 2 Distribution of antibody titres among the different serovars
K-value = 96 %
MAT Titre Grippotyphosa Icterohaemorrhagiae Australis Hardjo
kappa value of 96 % (Table 5) necessitates that in place of
1:100 10 04 03 00 MAT, rELISA may be efficiently used as the screening test
1:200 02 04 00 02 for serodiagnosis of leptospirosis in human beings; how-
1:400 04 02 01 00 ever, MAT would be the suitable test for detection of
1:800 00 00 00 00 serovar present in the sample.
1:1,600 00 00 00 00 Further, it was found that serum samples from malaria
1:3,200 00 00 00 00 and dengue patients were negative in both MAT and rELISA
which is similar to earlier findings [6, 8]. In the present
study, significant number of people were found positive for
Table 3 Age-wise sero-prevalence of leptospirosis in human Leptospira serovar Grippotyphosa which is also a common
Age (years) No. Microscopic Recombinant
serovar of cattle [3]. High seroprevalence may be possible
agglutination test (%) LipL32 IgGELISA (%) due to close proximity of human beings with their livestock
in context to their day to day professional attachment and
\20 04 00 00 need for livelihood as explained earlier [13, 24] and inclu-
20–30 09 01 (11) 01 (11) sion of less number of sera sample for study.
30–40 37 02 (5) 03 (8)
40–50 48 07 (9) 07 (9)
[50 57 12 (21) 14 (25) Conclusion
Total 155 22 (14) 25(16)
Leptospira interrogans Grippotyphosa is the most prevalent
serovar reported in the present study. However, the role of
Table 4 Sex-wise sero-prevalence of leptospirosis in human beings several risk factors associated with human leptospirosis
need to be elucidated. It may be apprehended that further
Sex No. Microscopic Recombinant LipL32
agglutination test (%) IgGELISA (%) comprehensive study including wide range of samples and
suitable molecular epidemiological methods would support
Male 84 18 (21) 19 (23) to establish the zoonotic transmission of Leptospirosis.
Female 71 04 (6) 06 (8)
Total 155 22 (14) 25(16) Acknowledgments The authors are thankful to Director, Indian
Veterinary Research Institute (IVRI), Izzatnagar for providing facil-
ities and financial support through Outreach of Zoonosis Project to
carry out the study.
among farmers and forestry workers [3]. In eastern Bra-
zilian Amazon, serologic evidence of leptospirosis of sera
collected from human beings associated with cattle was
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