Performance of a Recombinant LipL32
Based Rapid In-clinic ELISA (SNAP®
Lepto) for the Detection of Antibodies
Against Leptospira in Dogs
Curtis, KM1
Foster, PC1
Smith, PS1
Monn, MP
Stillman, BA1
Chandrashekar R1
Lappin, MR2
Goldstein RE3
IDEXX Laboratories Inc, Westbrook, ME

1

Colorado State University, Fort Collins, CO

2

Animal Medical Center, New York, NY.

3

KMC: kris-curtis@idexx.com
PCF: paulette-foster@idexx.com
PSS: phillip-smith@idexx.com
MPM: michael-monn@idexx.com
BAS: brett-stillman@idexx.com
RC: chandra@idexx.com
MRL: michael.lappin@colostate.edu
REG: Richard.Goldstein@amcny.org*Correspondence: K. Curtis, PhD. kris-curtis@idexx.com.

KEY WORDS: Leptospira, serology,
leptospirosis, ELISA, LipL32
ABSTRACTThe purpose of this study was to compare the performance of SNAP® Lepto, a
LipL32-based rapid enzyme-linked immunosorbent assay (ELISA) to the serologicalgold standard microscopic agglutination
test (MAT) for the detection of antibodies
to Leptospira spp in a broad population ofcanine samples. Four hundred and sixty
serum samples submitted for MAT testing at

182

IDEXX Reference Laboratories were tested
on SNAP Lepto. Positive percent agreement
between the two tests was 83.2% for samples with peak MAT titers ≥ 1:800, 64.9%
for samples with peak MAT titers ranging
from 1:100 to 1:400, and 79.2% for samples
with peak MAT titers ≥ 1:100. Negative
percent agreement between these tests was
82.1% in this population. One hundredand fifty MAT negative serum samples
from healthy dogs in Alaska were tested by
SNAP Lepto, and the percent agreement was
Vol. 13, No.3, 2015 • Intern J Appl Res Vet Med.

96.0%. Fifty-two serum samples positive
for anti-Borrelia burgdorferi antibodies
were tested. All 52 samples were negativeby MAT, and 51/52 samples were negativeon SNAP Lepto. Serial serum samples from28 dogs following Leptospira vaccination
were tested by MAT and SNAP Lepto. MAT
results were positive in 27/28 dogs at week
3 (highest peak titer of 1:3200), 28/28 dogs
at week 4 (highest peak titer of 1:6400),
3/21 dogs at week 52 (highest peak titer of
1:200), and 15/19 dogs at week 56 (highest
peak titer of 1:1600). In comparison, positive SNAP Lepto test results were observedin 15/28 dogs at week 3, 22/28 dogs at week
4, 5/21 dogs at week 52, and 16/19 dogs at
week 56. In this study, SNAP Lepto demonstrated similar performance to the MAT
for the detection of antibodies to Leptospira
spp.
INTRODUCTION
Canine leptospirosis is a zoonotic bacterial
infection with worldwide distribution that iscaused by spirochetes of the genus Leptospira.1-3 Infection in dogs most commonlyoccurs through direct contact with urine
or water containing Leptospira. Clinicalpresentation in dogs is often variable andcan range from subclinical infection to anacute, fatal disease that is characterized bymulti-organ dysfunction. Currently, laboratory testing is required for proper differential
diagnosis.
Because culture of the organism fromblood or urine is difficult and not sensitive,
serology is typically used for diagnosis of
leptospirosis.1,4-6 The microscopic agglutination test (MAT) for detection of antibodies is
the most common diagnostic test used today
to aid in the diagnosis of canine leptospirosis.3,4,6 The MAT is performed by mixing
serial dilutions of canine sera with cultured
Leptospira organisms of different serovars
representing different serogroups. The titeragainst a specific serogroup is defined as
the highest dilution of the sera that caused
50% or more agglutination of the organisms
representing that serogroup. The serogroupwith the highest titer is typically interpreted
Intern J Appl Res Vet Med • Vol. 13, No. 3, 2015.

as the infecting serogroup; however, due toa high degree of cross-reactivity across serogroups, that may not always be accurate.7
A peak MAT titer of > 1:3200 to a vaccinal
serovar and > 1:1600 to a non-vaccinal
serovar is suggestive of active infection.4
Standardization of the MAT across laboratories has been difficult, and variations in titers
reported may occur with multiple testing
events on the same sample and also between
different laboratories.8
LipL32 is the major outer membraneprotein of Leptospira, with the highest number of copies per organism than any othermembrane protein.9,10 LipL32 is expressed
only by pathogenic Leptospira, and is highly
conserved in these species.9-12 Previous
studies of the human immune response to
Leptospira indicate that LipL32 is the immunodominant protein antigen during infection,12,13 and its use as a diagnostic target
has looked promising in several studies withhuman and canine samples.12,14-19 A recombinant LipL32-based antibody detection
system is an attractive marker for use inserological tests for leptospirosis.
Because of the zoonotic potential of
leptospirosis and the possibility of infected
dogs serving as a reservoir for other dogs
and humans, achieving a rapid diagnosis isof high importance. The potentially fatal
consequences of inadequate therapy and thezoonotic risks related to handling dogs withleptospirosis create a need for a rapid, sensitive, and convenient diagnostic tool to be
used at the point-of-care to detect antibodies
against pathogenic Leptospira in dogs. The
purpose of this study was to compare the
LipL32-based SNAP Lepto to the MAT for
detection of anti-Leptospira spp antibodies in the following groups: serum samples
previously submitted for MAT testing fromdogs with an unknown infection status andvaccination history, MAT negative sera fromhealthy dogs living in Alaska, canine sera
positive for antibodies to Borrelia burgdorferi, and serial serum samples obtained fromhealthy dogs after Leptospira vaccination.
MATERIAL AND METHODS183

SamplesCanine serum samples (n=460) that were
submitted to IDEXX Reference Laboratories, Inc. (IRL) for MAT testing from locations throughout the United States were collected for this study. Of these, 201 samples
were MAT negative and 259 samples were
MAT positive. MAT positive samples had atiter of 1:100 or greater to one of the following 6 serogroups: Grippotyphosa, Canicola,
Pomona, Icterohaemorrhagiae, Bratislava,
and Autumnalis. Differentiation between
the serovars in positive samples was accomplished by identification of the serovar associated with the highest agglutinating titer(peak MAT titer). All samples were divided
into aliquots and stored at –20°C until use.
Serum samples (n-150) were obtained
from a veterinary clinic located in Alaska,
which is considered a Leptospira nonendemic region. These samples were drawn
from healthy dogs with no known history of
vaccination against leptospires. All samples
were divided into aliquots and stored at–20°C until use.
Serum samples (n=52) from dogs with
antibodies to B burgdorferi (Lyme Quant
C6®)20 were obtained from a veterinary
clinic located in Minnesota. The clinic does
not vaccinate its patients against leptospirosis. However, travel and prior vaccination
history was not available for these dogs.
All samples were divided into aliquots andstored at –20°C until use.
Serum samples from healthy, clientowned dogs (n=28) with no history of Leptospira vaccination for at least 1 year wereobtained from an IACUC approved study
at Colorado State University.21 Inclusion
criteria stipulated dogs be between 1 and8 years of age, >15 kg, healthy, and have aknown vaccination history with no Leptospira spp vaccination in the previous year.
Dogs were randomly assigned to be administered one of four commercially available
vaccinesb-e containing the Canicola, Grippotyphosa, Icterohaemorrhagiae, and Pomona serovars. All vaccinations were delivered
subcutaneously at the left shoulder. After184

the initial vaccine administration (week 0),
all dogs were administered the same vaccine
within 3 days of week 3. Approximately
52 weeks later, dogs were administered
the same vaccine as used at weeks 0 and 3.
Blood was collected by jugular or cephalicvenipuncture prior to vaccination on week
0 and then again approximately on weeks 3,
4, 7, 15, 29, 52, and 56. Sera samples fromweeks 0, 3, 4, 52, and 56 were tested in thisstudy. Sera were separated and stored at-20°C until tested.
Microscopic Agglutination Test
MAT was performed either at the IRL(percent agreement study, Borrelia burgdorferi antibody cross-reactivity study) or the
Colorado State University Veterinary Diagnostic Laboratory (vaccine study population;www.dlab.colostate.edu) in accordance with
their standard operating procedures. IRL
testing utilized the following six serovars:
Pomona, Canicola, Icterohaemorrhagiae,
Grippotyphosa, Bratislava, and Autumnalis;
Colorado State University testing utilized
five of these serovars, substituting serovar
Hardjo in place of Autumnalis. MAT was
performed by mixing dilutions of canine
sera (starting at 1:100) with live Leptospiraorganisms of each serovar. The highest dilution of serum in which >50% agglutinationof organisms occured was reported as the titer for a given serovar. Samples that demonstrated agglutination to any of the serovars
at any serum dilution were characterized as
positive. Differentiation between the serovars in positive samples was accomplishedby identification of the serovar associatedwith the highest agglutinating titer. MAT
positive samples were characterized further
by serovar and peak titer value. Samples
that did not demonstrate agglutination withany of the serovars at a 1:100 serum dilution
were characterized as negative.
SNAP Lepto
A rapid ELISA was used to detect antibodies
against Leptospira in canine serum samples.
This SNAP assay makes use of reversible
chromatographic flow of sample and automatic, sequential flow of wash solution andVol. 13, No.3, 2015 • Intern J Appl Res Vet Med.

Table 1. SNAP Lepto performance with MAT positive samples by peak titer.
Peak MAT Titer# Samples# Rapid ELISA Positive% ELISA Positive

200

20

11

55.0%

800

53

100

400

8

5

29

21

1600

34

25

6400

19

3200
12800
25600

51200

>102400
Total

13
32
14

18

19

259

37
10
16
29

62.5%

72.4%
69.8%
73.5%
76.9%
84.2%
90.6%

14

100.0%

19

100.0%

18

205

100.0%
79.2%

enzyme substrate. Color development in
spots on the flow matrix is used to provide
the assay results, which are read visually.
The assay detects antibodies against
Leptospira recombinant LipL32 protein.11
Recombinant LipL32 was expressed andpurified using conventional molecular techniques.22 Polystyrene latex particles werecovalently coupled to recombinant LipL32
and deposited on the flow matrix (sample
spot). Additionally, recombinant LipL32
was conjugated with horseradish peroxidase(HRP). The assay also incorporates a positive control spot, which consists of antibodies to horseradish HRP and captures a small
portion of the conjugate during the assay andturns blue upon addition of the substrate.
Briefly, 3 drops of test sample weremixed with 4 drops of the LipL32-HRP
conjugate and deposited in the sample well.
Leptospira-specific antibodies, if present in
the sample, would bind to the LipL32–HRP
in the conjugate. Immune complexes thatformed would bind to the LipL32 coatedparticles on the solid phase (sample spot) of
the ELISA. The ELISA was then exposed towash solution and substrate reagents.
The presence or absence of antibody wasdetermined by visual interpretation, comparing color intensities of the sample spot withthe background color intensity of the flow
matrix in the result window of the assay.
Color in the sample spot that was greaterthan color of the background indicated that asample was positive for antibody to Leptospira. A colorless sample spot indicated thata sample was negative for antibody to Leptospira. The positive control spot must haveturned blue for the assay to be validated.
Statistical Methods
Statistical analysis was performed using standard formulas to calculate percent
agreement between the MAT and SNAP
Lepto (SAS® 9.3). All confidence intervals(Clopper-Pearson) are two-sided and calculated as 95% confidence intervals.

Intern J Appl Res Vet Med • Vol. 13, No. 3, 2015.

185

RESULTS
A population of 460 canine serum samples
that had been submitted to the IRL by aveterinary clinic or hospital for MAT testing
was obtained and tested with SNAP Lepto.
Two hundred and fifty-nine samples were
MAT positive with a titer >1:100 to one ofsix serogroups tested. Overall, SNAP Lepto
yielded positive results for 205/259 samples(79.2% agreement; 95% confidence limits
73.7 - 83.9%). Percent agreement between
SNAP Lepto and MAT increased withincreasing peak MAT titer (Table 1). With

Table 2. SNAP Lepto performance with MAT positive samples by peak titer.
SerovarAutumnalis
Bratislava
Canicola# Samples
17

12

36

Grippotyphosa

118

Pomona60

Icterohaemorrhaigiae
Total

16

259

samples that had a peak titer suggestive ofactive infection, >1:3200,4 the percent agreement between SNAP Lepto and MAT was
92.2% (106 of 115 samples; 95% confidence
limits 85.7 – 96.4%). SNAP Lepto detected
samples with peak MAT titers to each of
the 6 serogroups tested, and performance
was similar across each of these serogroups(Table 2).
Two hundred and one of the 460 canine
serum samples obtained from IRL tested
MAT negative and were also tested with
SNAP Lepto. The percent agreement intesting this sample population was 82.1%(165 of 201 samples; 95% confidence limits
76.1 – 87.1%). In order to better understandthe performance of the SNAP Lepto with
MAT negative samples, 150 MAT negative
samples from healthy dogs in a non-endemic
area (Alaska) were also tested. The percent
agreement in this population was 96.0%(144 of 150 samples; 95% confidence limits
91.5 – 98.5%).
Fifty-two canine serum samples that had
positive results for B burgdorferi antibodies (Lyme Quant C6®<, IRL)20 were testedon the IRL MAT and SNAP Lepto to assesscross-reactivity in dogs with another spirochaetal disease. This population spanned
the range of Lyme Quant C6® values, fromweak to strong. Ten of the 52 Lyme positive
samples exceeded the reportable range of
the Quant C6® assay. The range of Quant
C6® values for the remaining 42 samples
was 42-297 U/mL and the median value was
125 U/mL. The IRL MAT results for all 52

186# Rapid ELISA Positive
13
11

27

95

12

47

205

% ELISA Positive
76.5%

91.7%

75.0%

80.5%

75.0%

78.3%

79.2%

samples were negative for all six serogroups
tested. Fifty-one of the 52 Lyme positive
samples (98.1%) tested negative on SNAP
Lepto. The one sample that tested positive
on SNAP Lepto had a weak Quant C6®
value of 44.
In order to evaluate whether SNAP
Lepto detects antibodies resulting from
Leptospira vaccination, samples from astudy21 at Colorado State University withhealthy client-owned dogs were tested.
Pre-vaccination samples (day 0) were
tested by SNAP Lepto and MAT, and only
samples with negative day 0 results on bothtests were considered for further evaluation(n=28). Following initial vaccination on
day 0, 27 of 28 dogs had a positive MAT
titer at week 3, and 7 of these had peak titersof 1:3200.21 Fifteen of the 28 dogs tested
positive on SNAP Lepto, and all were also
MAT positive. The number of SNAP Lepto
positive dogs after the second vaccination
at week 3 increased to 22 of the 28 dogs
(tested at week 4). All 28 dogs were MAT
positive at week 4, and the highest observed
MAT titer increased to 1:6400 (n=7 dogs).21
The number of antibody positive dogs
decreased over time in this one year study.
Samples from 21 of the 28 dogs were available one year post-vaccination. Three of 21
dogs had positive MAT titers, with 1:200
being the highest titer observed.21 Five of
21 dogs tested positive on SNAP Lepto,
and one of these was also MAT positive. A
total of 19 dogs were administered one doseof the same vaccine approximately 1 year
Vol. 13, No.3, 2015 • Intern J Appl Res Vet Med.

following the initial vaccination. Fifteen ofthese 19 dogs had positive MAT titers 4-6
weeks post-vaccination (weeks 56-60), with
1:1600 being the highest MAT titer observed(n=5 dogs).21 Sixteen dogs were positive
on SNAP Lepto, and 14 of these were also
positive by MAT.
DISCUSSION
Definitive diagnosis of canine leptospirosis currently requires laboratory testing todetect the organism in clinical samples or
use of the MAT.4 Because of the zoonotic
potential of leptospirosis and the potentiallyfatal consequences of inadequate therapy,
there is great need for a rapid, sensitive, andconvenient diagnostic tool to be used at the
point-of-care. The current study compared
the percent agreement of the LipL32-based
SNAP Lepto to the MAT for detection of
antibodies to canine pathogenic Leptospira
spp. Agreement of SNAP Lepto in a population of MAT positive samples increased
with increasing peak MAT titers, reaching
90.6% with peak titers >12,800. Agreementof SNAP Lepto with MAT negative samples
was 82.1% in samples submitted to IRL for
MAT testing and was highest with samples
from healthy dogs (96.0%). Both SNAP
Lepto and the MAT detected antibodies
induced by Leptospira vaccination.
The canine serum samples submitted to
the IRL for MAT testing were presumably
obtained from sick dogs suspected of having
leptospirosis. However, because of the lackof patient histories, the lack of access toconvalescent MAT results, and the lack ofvaccination history, the true clinical status of
the patients cannot be determined. Althougha single positive titer can serve to increase
suspicion for leptospirosis, even a hightiter does not confirm a diagnosis.3 In dogs
with a known history of vaccination, thisis particularly important because although
postvaccinal titers tend to be low, titers >1:1600 have the potential to persist after
vaccination.23 For these reasons, the current
study only compared performance of thepoint-of-care ELISA to MAT status and peak
MAT titer without any attempt to interpret
Intern J Appl Res Vet Med • Vol. 13, No. 3, 2015.

clinical status of each dog.
In testing the MAT positive canine
serum samples from the IRL, 21.8% tested
negative on SNAP Lepto. These discrepant
results may represent false negative SNAP
Lepto results, whereby these discrepant
samples may contain agglutinating antibodies in the absence of detectable anti-LipL32
antibodies on SNAP Lepto. In the earlystages of infection, dogs may frequently
have negative serology results.3,4 It is forthis reason that the current recommendation for MAT testing is to submit acute andconvalescent samples, looking for a 4-foldchange in titers in order to make a definitive
diagnosis.3 It is unknown whether repeatedtesting of samples from dogs with these
discrepant results would later yield positive
SNAP Lepto results. In suspected casesof canine leptospirosis where the SNAP
Lepto result is negative, yet the level ofsuspicion remains high due to clinic signs,
use of the MAT and/or polymerase chain
reaction (PCR) assays are valuable adjuncts
to the diagnosis of leptospirosis. Further
clinical studies that include cases definedby acute and convalescent MAT titers, aswell as PCR, are required to understand thisdiscrepancy.
In testing of MAT negative canine serum
samples from the IRL, 17.9% were positive
on SNAP Lepto. These samples were originally submitted for MAT testing, and presumably obtained from sick dogs suspectedof having leptospirosis. These discordantresults may represent false negative MAT
results. In contrast, samples from healthy,
non-vaccinated dogs in Alaska produced asignificantly lower positive rate (4.0%) on
SNAP Lepto. These data suggest that MAT
negative, SNAP Lepto positive results are
more common in dogs suspected of having leptospirosis, and that these discordantresults may be the result of false-negative
MAT results. MAT titers typically become
positive after approximately 1 week andpeak after 3-4 weeks.4 Because initial positive MAT results can take a week or more todevelop, negative initial MAT results are not187uncommon. It is for this reason that acuteand convalescent testing is the recommended protocol for MAT testing.3 Unfortunately, paired acute and convalescent samples
were not available from these dogs to allow
for subsequent testing. Another possible
explanation for the discordant results is thatthese samples may have been obtained fromdogs later in the disease, when the level ofcirculating agglutinating antibodies tends todecrease.23
An alternative explanation for these
MAT negative, SNAP Lepto positive discordant results is that these samples containanti-LipL32 antibodies but lack agglutinating antibodies detectable by MAT. These
results could occur as a result of exposure to
Leptospira organisms in the environment or
prior Leptospira vaccination. The present
study demonstrates that some currently vaccinated dogs tested positive on SNAP Lepto
for a varying amount of time post-vaccination. Complete vaccine histories were notavailable for patients included in this study,
and it is possible that Leptospira vaccination
produced positive results on SNAP Lepto inthis study. Detection of antibodies inducedby vaccination is a limitation to interpretation of all Leptospira serological tests,
including the MAT, particularly when only
an acute titer is obtained. Paired MAT titers
may facilitate the interpretation of the results
if vaccinal antibodies are present. 21,23,24
Finally, these MAT negative, SNAP Lepto
positive discordant results could represent
false-positive SNAP Lepto results. Falsepositive SNAP Lepto results could occur
from cross-reactivity with homologous
antigens or infection with other pathogens.
LipL32 is expressed only by pathogenic
Leptospira,9,11,12,25 and the only ortholog
identified to date is in the unrelated marine
bacterium Pseudoalteromonas tunicata.26
However, the existence of undiscovered
homologous antigens cannot be ruled out.
Furthermore, data obtained in testing the
MAT negative, Lyme positive samples
supports the lack of cross-reactivity with antibodies against another common pathogenic

188

spirochete, B burgdorferi.
Leptospirosis can be a challenging
disease to diagnose. The microscopic
agglutination test (MAT) for detection of
antibodies is the most common diagnostic
test used today to aid in the diagnosis of
canine leptospirosis.3,4,6 The MAT has some
limitations due to the inability for it to be
run as point-of-care assay. The primaryadvantage of a rapid ELISA, such as SNAP
Lepto, is the convenience of performing thetest in a hospital or reference laboratory setting without the need for expensive culturesand equipment. This makes a serologic test
for leptospirosis more available to practitioners in a hospital setting, thereby ensuring
adequate precautions are taken to reduce therisk of handling dogs with a zoonotic disease, as well as address the need to administer adequate therapy in a timely manner.
SNAP Lepto also provides the ability to runconsecutive tests over a matter of days inorder to detect seroconversion in a patient,often prior to receiving results of an initialsample submission for MAT testing. However, future study with clinically defined
canine leptospirosis cases is required toevaluate the potential utility of SNAP Lepto
in repeated convalescent sample testing.
In summary, the current study describes theperformance of the LipL32-based SNAP
Lepto for detection of anti-LipL32 antibodies. This test does not distinguish between
serovars, including vaccinal versus nonvaccinal serovars, and does not provide atiter magnitude, and these factors must be
taken into consideration when interpretingthe test results. However, SNAP Lepto provides a convenient tool to assess Leptospiraantibody status in dogs, thereby providing
potentially valuable information to this complex diagnostic work up.
FOOTNOTES
a Lyme Quant C6®, IDEXX Laboratories,
Inc., Westbrook, ME.
b LeptoVax 4, Boehringer-Ingelheim, St.
Joseph, MO
c Nobivac Lepto4, Merck Animal Health,Vol. 13, No.3, 2015 • Intern J Appl Res Vet Med.

Whitehouse Station, NJ
d RECOMBITEK 4 Lepto, Merial, Duluth,
GAe Vanguard L4, Pfizer Animal Health, New
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