966299

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VDIXXX10.1177/1040638720966299Immunomagnetic separation PCR assay for pathogenic LeptospiraTomckowiack et al.

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Journal of Veterinary Diagnostic Investigation

Analytical evaluation of an immunomagnetic 2021, Vol. 33(1) 52­–58

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DOI: 10.1177/1040638720966299
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Leptospira in cattle urine samples
obtained under field conditions

Camilo Tomckowiack, Claudio Henriquez, Alfredo Ramirez-Reveco,

Priscila Muñoz, Bernardita Collado, Daniel Herzberg, Hugo Folch,
Miguel Salgado1

Abstract. Clinical manifestations of leptospirosis are diverse and very similar to other febrile diseases, hence early and
accurate detection of subclinical infections is a key element in disease control. We evaluated immunomagnetic separation (IMS)
capture technology coupled with a standard quantitative PCR (qPCR) system for the detection of pathogenic Leptospira in urine
samples from 803 cows from dairy herds with a history of clinical cases of leptospirosis. The urine samples were first processed
in a purification step, then subdivided into 2 subsamples, one that continued to DNA extraction and direct qPCR, and one that
was pretreated by IMS before continuing to DNA extraction and qPCR. Overall, 133 of 803 (16.6%) samples were IMS-qPCR
positive, whereas only 92 of 803 (11.5%) were positive when using direct qPCR. Statistically significant differences were
observed between the mean estimated Leptospira load between the IMS-qPCR and the direct qPCR positive urine samples. The
IMS-qPCR technology revealed a larger number of positive results and higher bacterial loads than direct qPCR. This difference
is most likely the result of the high antigen-binding capacity and capture efficiency of the IMS system. The use of polyclonal
antibodies produced by the inoculation of 3 synthetic peptides, which make up the extracellular regions of the LipL32 protein,
provided a high detection capacity to the IMS-qPCR technique, resulting in performance superior to direct qPCR.

Key words: immunomagnetic separation qPCR; Leptospira; leptospirosis; LipL32.

Introduction Immunomagnetic separation (IMS) has been reported to

Leptospirosis, a bacterial zoonotic disease with a worldwide
distribution, is caused by spirochetes of the genus Leptospira
and encompasses a wide spectrum of clinical diseases in
humans, including multi-organ failure with a high mortality
rate.10 Farming activities were recognized as an important
risk factor before animal host species were identified.1
Rodents were first identified as a potential source of human
infection, followed by dogs.2,4 The role of livestock as reser-
voirs was not determined until several decades later.4 Fre-
quently, abortion or stillbirth is the only clinical sign detected
in adult cattle infected by pathogenic Leptospira.8 Infected
calves usually develop a severe, acute form of the disease
(with clinical signs such as fever, jaundice, and hematuria)
that is frequently fatal.2
Because of the diversity of clinical signs, the detection of
pathogenic Leptospira is difficult and depends upon a variety
of laboratory assays such as the detection of specific anti-
bodies by microscopic agglutination test, indirect hemag-
glutination assay, or ELISA. In addition, Leptospira or their
components may be detected in urine or tissues by culture,
dark field microscopy, immunostaining, or PCR.1,4,6
be used for Leptospira detection.5 This concentration method
can be coupled with any test for Leptospira detection regard-
less of whether it is based on antigens, genes, phage binding,
or growth in culture media. If effective, IMS can provide
cleaner samples (i.e., free of contaminating microbes or PCR
inhibitors) and a higher yield of Leptospira (i.e., improved
analytical sensitivity) via a one-step, low-cost procedure.5
The combination of IMS and PCR increases both test
specificity and sensitivity.13,17,18 There remain some hurdles
Instituto de Medicina Preventiva Veterinaria (Tomckowiack, Collado,
Salgado), Instituto de Farmacología y Morfofisiología (Henriquez),
Instituto de Ciencias Clínicas Veterinarias (Herzberg) and Instituto de
Ciencia Animal (Ramirez-Reveco), Facultad de Ciencias Veterinarias,
Universidad Austral de Chile, Valdivia, Chile; Instituto de Inmunología,
Facultad de Medicina, Universidad Austral de Chile, Valdivia, Chile
(Muñoz, Folch).
1
Corresponding author: Miguel Salgado, Instituto de Medicina
Preventiva Veterinaria–Facultad de Ciencias Veterinarias, Universidad
Austral de Chile, Saelzer Building 5° Floor, Campus Isla Teja, Valdivia,
50 90000, Chile. miguelsalgado@uach.cl
 Immunomagnetic separation PCR assay for pathogenic Leptospira
to widespread use of the IMS-PCR technology for Lepto-
spira detection in veterinary specimens. The analytical sensi-
tivity of the IMS method has been evaluated with only a
small number of Leptospira strains and under experimental
conditions.5 We describe herein the development, optimiza-
tion, and analytical evaluation of an IMS–quantitative PCR
(IMS-qPCR) protocol to facilitate the detection of patho-
genic Leptospira from cattle urine samples obtained under
field conditions.
Materials and methods
Study population and sample collection
Sampling was conducted among 15 smallholder and 23
medium-size dairy farms located in the Los Ríos and Los
Lagos regions of southern Chile, between October 2016 and
January 2017. Verbal consent was obtained from all farmers
who participated in the study. The smallholder farmers are
subsistence farmers who produce < 100,000 L of milk/y, and
their cattle graze outside year-round and are fed little or no
concentrate. The medium-size herds represent the typical
dairy farms of the area in terms of breed (Holstein), herd size
(200–500 animals), and management practices (graze in
rotational paddocks year-round, milked twice a day, 305-d
milk production, 220,000–4,500,000 L/y).
A targeted sampling strategy was used to maximize the
likelihood of testing Leptospira-infected cattle by selecting
herds with a history of clinical leptospirosis, mainly associ-
ated with Pomona and Hardjo serovars. We collected 803
urine samples from adult cattle from the 38 study herds. In
order to detect pathogenic Leptospira in each of the sam-
ples, urine samples (25 mL) were collected by direct stimu-
lation of the vulvar area. Urine samples were kept at room
temperature until they were transferred and processed, on
average within 4 h, at the Laboratorio de Enfermedades
Infecciosas, Instituto de Medicina Preventiva Veterinaria,
Facultad de Ciencias Veterinarias, Universidad Austral de
Chile.
Detection of pathogenic Leptospira in urine specimens
was conducted through a comparative approach based on
both direct qPCR and IMS-qPCR from each urine sample.
Each urine sample was centrifuged at 4,000 × g for 15 min;
the pellet was then resuspended in 1 mL of phosphate-
buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 4.3 mM
Na2HPO4, 1.4 mM KH2PO4 [pH 7]), transferred to a 1.5-mL
microcentrifuge tube, and re-centrifuged at 11,000 × g for
5 min. Finally, the supernatant was discarded, and the pellet
was resuspended in 1 mL of PBS. After this first cleaning and
purification step, the sample was subdivided, with a 100-μL
aliquot used for DNA extraction and direct qPCR and another
100-μL aliquot undergoing a pretreatment step of immune
separation before proceeding with DNA extraction and direct
qPCR.
53
Polyclonal antibody production
In order to produce polyclonal antibodies against LipL32 (an
outer membrane lipoprotein7), 3 peptide chains (EP3, EP4,
and EP6),9 each with a size of ~20 kDa, were chemically
synthesized (this mixture hereafter referred to as P2).
Sequences of these LipL32 epitopes have been described in
silico as the most immunogenic and are found outside the
leptospiral bacterial cell wall.9 A glutaraldehyde cross-link-
ing protocol with hemocyanin carrier protein (Imject Blue
carrier protein; Thermo Fisher Scientific) was performed on
the peptides, as described previously,11 with some modifica-
tions. Briefly, 10 mg of hemocyanin was incubated with 1 mg
of each peptide in the presence of 0.15% glutaraldehyde
(MilliporeSigma) at room temperature for 2 h. The reaction
was terminated by the addition of 1/10 (v/v) of 1 M glycine,
and the sample was dialyzed against 0.1 M borate buffer
(18.55 g of boric acid; 2,850 mL of H2O; pH 8.5) for 24 h at
4°C. From this protocol, an emulsion was made with 200 μg
of each of the peptides and Freund complete adjuvant
(MilliporeSigma), with 1 mL injected subcutaneously in 4
different locations on the back of a 2.5-kg New Zealand male
rabbit. The laboratory animal was provided by the Instituto
de Salud Pública (Chilean Government). This rabbit was
managed in strict accordance with the recommendations in
the Guide for Use of Animals for Research of the Universi-
dad Austral de Chile (https://www.uach.cl/organizacion/
vicerrectoria-investigacion-desarrollo-y-creacion-artistica/
utiles/subcomite-en-uso-de-animales-en-investigacion,
Spanish.). The animal was housed in an individual metal
cage with free access to food and water. Polyclonal antibod-
ies were obtained from the rabbit serum and stored at −20°C
until use. The serum was used directly to perform the IMS
without any purification step.
Polyclonal antibody validation
In order to evaluate the binding of the anti-LipL32 antibody
to the target protein in pathogenic Leptospira, one strain
(107 bacteria/mL) of each of 6 serovars (Table 1) was used.
The Leptospira strains were provided by the National Collab-
orating Centre for Reference and Research on Leptospirosis
(Amsterdam, The Netherlands). The cultures were homoge-
nized in the extraction buffer (50 mM Tris, pH 8.0; 1 mM
EDTA; 100 mM NaCl; 1 mM phenylmethylsulfonyl fluoride).
The Leptospira cells were disrupted by tip sonication at
70% in an ice bath with two 10-s rounds. Samples were
quantified (Bradford reagent; MilliporeSigma). Twenty µg
of whole protein extract was separated by SDS-PAGE and
electro-transferred to a 0.45-µm nitrocellulose membrane
(GE Healthcare). Membranes were blocked with 1% bovine
serum albumin (BSA) in 0.2% Tween-20 for 1 h at room
temperature. Primary antibody (1:1,000 v/w) incubation was
performed overnight, using continuous stirring at 4ºC. The
54
Tomckowiack et al.
Table 1.  Details of Leptospira strains used in evaluation of an immunomagnetic separation PCR assay to detect pathogenic Leptospira
in bovine urine samples.
Species Serogroup
L. borgpetersenii Tarassovi
L. interrogans Canicola
L. interrogans Icterohaemorrhagiae
L. interrogans Pomona
L. interrogans Sejroe
L. interrogans Australis
All reference strains were provided by the National Collaborating Centre for Reference and Research on Leptospirosis (Amsterdam, The Netherlands).
following day, membranes were washed in 0.2% Tween-20
solution in Tris-buffered saline (TBS; pH 7.4) and then incu-
bated with anti-rabbit IgG–horseradish peroxidase second-
ary antibody (1:2,000 v/w; Santa Cruz Biotechnology) for
1 h at room temperature. Finally, the immunoreactive signal
was revealed with a solution of luminol–hydrogen peroxide
(ECL; Thermo Scientific) and digitally displayed (LI-COR
Odyssey Fc system; Biosciences). Primary antibody speci-
ficity was evaluated in parallel by the use of primary anti-
body that had been pre-adsorbed with immunogenic peptides
(P2) overnight.
Indirect immunofluorescence
To demonstrate the binding of the anti-LipL32 antibody to
the membrane of pathogenic Leptospira, an immunocyto-
chemistry assay was performed using L. interrogans serovar
Icterohaemorrhagiae strain Icterohaemorrhagiae (Table 1).
Smears of this Leptospira strain were prepared on slides,
fixed with 2% paraformaldehyde in 2.5 mM MgCl2, and then
blocked with 5% BSA in 0.2% Tween-20 in TBS for 60 min
at room temperature. Samples were incubated overnight with
the primary antibodies (1:50 v/w) in a moist chamber at 4°C.
As a negative control, the primary antibody was not added.
In a second control, the primary antibody was pre-absorbed
with the immunogenic peptides (P2). Next, incubation with
the secondary antibody was performed, with antibodies
conjugated to fluorochrome Alexa 488, for 60 min at room
temperature. Bacterial DNA was counterstained with 2 µM
propidium iodide and washed twice in TBS. Finally, the
slides were mounted, and stained cells were visualized
and evaluated in an inverted epifluorescence microscope
(DMI3000 B; Leica) coupled to a digital camera (DFC 425
C; Leica). The images obtained were processed with Adobe
Photoshop 6.0.
Use of coated magnetic beads
Magnetic beads coated with anti-rabbit antibodies (M-280
sheep anti-rabbit IgG; Dynabeads) were used. The 100-µL
aliquot of the resuspended urine pellet was subjected to the
following pre-treatment. First, a 100-µL aliquot of the
polyclonal antibody produced against LipL32 (1:1,000)
Serovar Strain
Tarassovi Perepelitsin
Canicola Hond Utrecht IV
Icterohaemorrhagiae Icterohaemorrhagiae
Pomona Pomona
Hardjo Ar
Bratislava Jez Bratislava
was added and the mixture incubated for 30 min at 37°C.
The mixture was then washed with 1 mL of PBS, centri-
fuged twice (11,000 × g, 5 min), and finally 0.5 mL
(containing 5.0 × 105) of the coated beads was added.
Magnetic separation of pathogenic Leptospira from urine
samples and the subsequent washing steps were carried out
(Invitrogen BeadRetriever system; Life Technologies).
Leptospira were selectively concentrated from the 0.5 mL
of processed samples obtained from the coated magnetic
beads protocol, as described above. The final product of
the IMS process was suspended in 0.5 mL of PBS for DNA
extraction and purification followed by qPCR, as described
below.
DNA extraction and purification
The sample obtained from the IMS step or direct from the
re-suspended bacterial pellet was mixed with 500 μL of lysis
buffer (2 mM EDTA, 400 mM NaCl, 10 mM Tris-HCl [pH
8.0], and 0.6% SDS) and 5 μL of proteinase K (10 μg/μL;
MilliporeSigma) in a 1.5-mL microcentrifuge tube (Eppen-
dorf; MilliporeSigma). The mixture was incubated at 56°C
for 1 h, then 500 μL of 100% ethanol was added. The tubes
were left standing for 2 min at room temperature before being
vortexed for 5 s and centrifuged (11,000 × g, 5 min). The
supernatant was discarded, and the pellet was washed once
in 200 μL of 70% ethanol by re-suspension and centrifuged
as described above. Next, the pellet was re-suspended in
50 μL of sterile distilled water. The tubes were placed in a
dry heating block (Eppendorf) at 100°C for 5 min. The solu-
tion was centrifuged briefly (16,000 × g, 30 s) to remove any
contaminating material. Finally, a 25-μL aliquot of superna-
tant was placed into a new tube (Eppendorf) to be used as a
template for PCR.
qPCR from urine samples
The DNA templates obtained from either the IMS prepara-
tion or directly from the re-suspended pellet obtained from
the urine were analyzed (LightCycler 2.0; Roche), using a
TaqMan probe and targeting the LipL32 gene, which is spe-
cific for pathogenic Leptospira species.16 The amplification
mixture for each sample was as follows: 0.7 μM of primers,
 Immunomagnetic separation PCR assay for pathogenic Leptospira
0.15 μM of probe, 10 μL of TaqMan universal master mix
(Roche), and 5 μL of DNA template, in a total volume of
20 μL. Samples were amplified using the following para­
meters: initial denaturation at 95°C for 2 min, followed by 40
cycles of denaturation at 95°C for 5 s, and annealing/elonga-
tion at 58°C for 30 s. Negative and positive controls to con-
firm the validity of the reaction were used as well as negative
and positive controls of DNA extraction. The Roche system
reports crossing point (Cp) values reflecting the number of
pathogenic Leptospira in the sample, and a Cp > 40 was
regarded as a negative result.
In vitro evaluation of the IMS-qPCR Leptospira
detection capacity
The IMS-qPCR detection system was first subjected to an in
vitro preliminary evaluation. Five pure cultures of patho-
genic Leptospira (L. interrogans serovar Icterohaemor­
rhagiae strain Icterohaemorrhagiae; L. interrogans serovar
Bratislava strain Jez Bratislava; L. interrogans serovar Cani-
cola strain Hond Utrecht IV; L. interrogans serovar Pomona
strain Pomona; L. borgpetersenii serovar Tarassovi strain
Perepelitsin) were used. From these pure cultures, Lepto-
spira concentration was estimated using direct qPCR accord-
ing to the genome-equivalent estimation principle described
below. Four dilutions with a known concentration of Lepto-
spira (101, 102, 103, and 104 bacteria/mL) were then evalu-
ated by IMS-qPCR using the same quantification method.
Estimation of pathogenic Leptospira shedding
level
Pathogenic Leptospira cell numbers (genome equivalents)
were estimated according to a published protocol3 using the
molecular weight of the genome of L. interrogans serovar
Hardjo type Prajitno strain Hardjo-prajitno (GenBank acces-
sion EU357983.1) to establish a standard curve for estima-
tion of Leptospira numbers by Roche 2.0 real-time PCR,
according to the following equation:
DNA concentration ( ng / µL )
genomeequivalent =
(
× 6.022 × 1023 / mol−1 ) Table 2). More specifically, 41 samples positive by IMS-
( )
4.659 × 106 base pairs ×
(1×10 ng / g × 660g / mol )
9
( accesion EU357983.1)( base mass )
Statistical analysis
The pathogenic Leptospira detection results obtained by
direct PCR and IMS-qPCR were compared using the
McNemar test. Bacterial load differences detected by the 2
detection systems were assessed. Because both direct PCR
and IMS-qPCR results were not normally distributed, the
55
Wilcoxon matched-pairs signed-rank test was used. For all
statistical analyses, p ≤ 0.05 was considered significant.
Statistical analyses were done using Prism 6 software
(GraphPad).
Results
The SDS-PAGE Coomassie blue stain showed a similar
pattern for all 6 pathogenic Leptospira strains (Fig. 1A). The
specific immunoreactivity of a major band of the expected
molecular size (32 kDa) in all strains was confirmed by
western blot (Fig. 1B). Minor bands of different molecular
weights were detected, possibly derived from cleavage of
the LipL32 target protein. A specific band of low molecular
weight (< 9 kDa) was expected for P2 (Fig. 1B). A markedly
diminished immunoreaction was observed when antibody
was pre-absorbed with P2 immunogenic peptide for all bac-
terial protein samples (Fig 1C). In the sample containing the
P2 immunogenic peptide used to produce antibodies, a total
loss of immunoreactivity was observed with the pre-absorbed
antibody (Fig. 1C).
A negative indirect immunofluorescence result was
obtained when cell samples were incubated with only sec-
ondary antibodies (Fig. 1D). With the same technique, the
signal was compatible with the presence of LipL32, with a
positive and strong signal for LipL32 in all cells present in
the smears, including those cells with a polymorphic size
pattern (Fig. 1E). A weak signal was obtained when cell sam-
ples were incubated using the anti-Lip32 antibody that had
been pre-absorbed with P2 immunogenic peptides (Fig. 1F).
The IMS-qPCR technique with polyclonal antibodies was
optimal at 1:1,000 serum dilution (data not shown). In the in
vitro evaluation, statistically significant differences were
observed from dilutions 101–104 (Fig. 2). The quantitative
analysis of IMS-qPCR when used on pure cultures showed
that detection of Leptospira loads of < 102 bacteria/mL was
not possible.
Urine samples from the 38 cattle herds were positive for
leptospires by both direct qPCR and IMS-qPCR, with a
greater number detected by the latter. Overall, 133 of 803
(16.6%) of the urine samples were IMS-qPCR positive; only
92 of 803 (11.5%) were direct qPCR-positive (p = 0.0001;
qPCR within the range of 100–103 bacteria/mL gave negative
results for direct qPCR (Table 3). Statistically significant dif-
ferences in median load estimation values of the positive
samples were observed between the IMS-qPCR assay (4.69
log10 bacteria/mL for the 133 positive urine samples) and the
direct qPCR assay (1.49 log10 bacteria/mL for the 92 positive
urine samples; Fig. 3).
Discussion
Our results confirm specific binding of our anti-LipL32 anti-
body in the outer membrane region of pathogenic Leptospira,
56
Tomckowiack et al.

Figure 1.  Detection of LipL32 protein in pathogenic Leptospira. A–C. Each gel consists of the following: lanes 1, 9 = molecular
weight standards (SDS-PAGE proteins or immunoblotting proteins; lane 2 = lysates from L. interrogans serovar Icterohaemorrhagiae
strain Icterohaemorrhagiae; lane 3 = L. interrogans serovar Bratislava strain Jez Bratislava; lane 4 = L. interrogans serovar Canicola strain
Hond Utrecht IV; lane 5 = L. interrogans serovar Pomona strain Pomona; lane 6 = L. interrogans serovar Hardjo strain Ar; lane 7 = L.
borgpetersenii serovar Tarassovi strain Perepelitsin; lane 8 = saline buffer. A. Proteins stained with Coomassie stain. SDS-PAGE gel for total
protein lysate (20 µg) of 6 strains (lanes 2–7) and immunogenic peptide (lane 10). B. Western blot analysis for 20 µg of total protein using
serum anti-LipL32 (rabbit). Arrowhead indicates the strong immunodetection signal. C. Western blot analysis (negative control) of total
protein using serum antibodies (anti-LipL32) pre-absorbed overnight with immunogenic peptide. Arrowhead indicates immunodetection
signal for LipL32. For both B and C, arrow indicates peptide detection. These are representative images from 3 independent determinations.
D–F. Indirect immunofluorescent detection of LipL32 protein in L. interrogans serovar Icterohaemorrhagiae strain Icterohaemorrhagiae.
An absent (D), strong (E), and weak (F) in situ signal is shown when using only secondary antibodies, primary serum antibodies, and pre-
absorbed serum antibodies, respectively. Asterisks in D indicate DNA staining cells with propidium iodide (2 µg/mL); arrow and arrowhead
in E indicate elongated or curly cells; asterisks in F indicate the displacement of the primary antibody–antigen binding produced when using
pre-absorbed antibody. Bars = 20 µm. These are representative images from three independent determinations.

which is consistent with knowledge of the LipL32 protein as
an immunogenic target.1,7 This finding was a key element for
the implementation of the IMS proposed protocol to be used
in urine samples.
The IMS-qPCR technique, when used on pure cultures
with known concentrations of pathogenic Leptospira, had
limited recovery capacity, resulting in lower sensitivity than
qPCR. On the other hand, different results were observed
from pathogenic Leptospira obtained from clinical urine
samples, especially in bacterial load estimation values of
the positive samples. This better performance with clinical
samples may be explained by the fact that, for Leptospira
strains under laboratory experimental conditions, a significant
decrease of LipL32 protein in the outer membrane is
expected,14 possibly because of down-regulation of the
LipL32 gene when the pathogen is in a non-infectious envi-
ronment. In contrast, LipL32 protein is normally expressed in
pathogenic Leptospira from infected tissue, and the O-anti-
gen from Leptospira LPS is significantly reduced, leaving
LipL32 more exposed.12 Therefore, the natural Leptospira
infectious state may influence the difference in efficiency
of the IMS-qPCR protocol. The fact that LipL32 is over-
expressed and more exposed on the surface would improve
the binding capacity of our anti-LipL32 antibodies and
enhance the separation efficiency of IMS when used on
clinical samples. The IMS-qPCR technology that we used
 Immunomagnetic separation PCR assay for pathogenic Leptospira 57

Figure 3.  Comparison of the bacterial load detection (cells/mL)

for the 133 urine samples positive in the immunomagnetic separation
qPCR (IMS-qPCR) assay and the 92 urine samples positive in the
direct qPCR assay. *** = p < 0.05 significant difference on paired
comparison.
Figure 2. Analytical sensitivity estimation of the immuno-
magnetic separation qPCR (IMS-qPCR) compared with direct
There are several possible reasons for the dramatically
qPCR for pathogenic Leptospira in different dilutions of pure
cultures, expressed in bacteria/mL. Plots represent the mean (central higher sensitivity of IMS-PCR than direct PCR that we
bar) ±1 SD for different culture populations. Statistically significant found. Most of these explanations are focused on the effec-
differences were observed from dilutions 101–104 (p < 0.05). tiveness of an IMS system defined by its antigen-binding
capacity or capture efficiency.18 Furthermore, although direct
PCR loses sensitivity because of PCR inhibitors in urine
Table 2.  Cross-classification of direct qPCR and
immunomagnetic separation qPCR (IMS-qPCR) results on
samples,15 the IMS-qPCR can remove PCR inhibitors during
803 urine samples. the processing of clinical urine samples. Hence the IMS-
qPCR technique improves the analytical sensitivity over tra-
Result of direct qPCR ditional techniques. For direct qPCR assays, inhibitors may
Result of
IMS-qPCR Positive Negative Total be removed using specialized DNA extraction kits, but those
kits are expensive, making the IMS-qPCR even more advan-
Positive 86 47 133 tageous.
Negative 6 664 670 The development of the IMS-PCR technique with anti-
Total 92 711 803 bodies against only Leptospira borgpetersenii serovar Hardjo
has been reported.17 An improvement in diagnostic sensitiv-
ity in bovine urine samples has been reported for the same
Table 3.  Frequency distribution of positive direct qPCR
serovar.19 However, in both these reports, the application was
results for 133 urine samples with the indicated bacterial load as
determined by immunomagnetic separation (IMS) qPCR.
limited to one serogroup. Our results show a detection capac-
ity for multiple serogroups and serovars of Leptospira. A
IMS-qPCR result expressed No. of the same urine samples previous study5 produced monoclonal antibodies targeting
as estimated bacteria/mL positive in direct qPCR the LipL32 antigen using a recombinant protein instead of
100–103 (42) 1 peptides as we did. However, unlike our study, the difference
104–105 (49) 43 in bacterial concentration detection between PCR and IMS-
106–107 (42) 42 PCR reported in this prior study was only 1 log10 value.5
Our novel detection tool had a much higher analytical
n in parentheses.
sensitivity than direct urine qPCR. This high capacity to
detect Leptospira-infected animals that shed the pathogen in
on clinical samples provided results that yielded a signifi- low concentration will significantly strengthen leptospirosis
cantly higher number of positive results and with a higher control efforts in cattle herds.
estimated bacterial load (> 3 log10 differences) than direct
qPCR in cattle clinical urine samples obtained under field Acknowledgments
conditions. We thank all of the farmers for helping with sample collection.
58
Tomckowiack et al.
Declaration of conflicting interests
The authors declared no potential conflicts of interest with respect
to the research, authorship, and/or publication of this article.
Funding
This work was supported by Proyecto FIC 15-8 (código BIP
30421496), del Gobierno Regional de Los Ríos, Chile.
ORCID iDs
Camilo Tomckowiack https://orcid.org/0000-0002-1025-9265
Miguel Salgado https://orcid.org/0000-0002-2144-7982
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