Evaluation of SNAP® Lepto in the
Diagnosis of Leptospirosis Infections in
dogs: Twenty two Clinical Cases
Winzelberg, S1
Tasse, SM2
Goldstein, RE1
Chapman, PS3
Benedict, AG4
Mason, GD5
Noble, SJ5
Curtis, KM2
Chandrashekar, R2
Animal Medical Center, New York, NY 10065(Winzelberg’s present address is VERG North Internal Medicine Brooklyn, NY)

1

2

IDEXX Laboratories Inc,Westbrook, ME

3

Veterinary Specialty and Emergency Center, Levittown, PA

Port City Veterinary Referral Hospital, Portsmouth, NH(Benedict’s present address is VCA Shoreline Veterinary Referral andEmergency Center, Shelton, CT)
45Portland Veterinary Specialists, Portland, ME

SW: Swinzelbergvmd@gmail.com
SMT: sarah-tasse@idexx.com
REG: Richard.Goldstein@amcny.org
PSC: PChapman@vsecvet.com
AGD: aghadden04@hotmail.com
GDM: gaildmason@gmail.com
SJN: nsvet@aol.com
KMC: kris-curtis@idexx.com
RC: chandra@idexx.com*Correspondence: Tasse S, DVM. sarah-tasse@idexx.com

KEY WORDS: Leptospira, leptospirosis,
SNAP® Lepto, MAT
ABSTRACT
Leptospirosis affects many mammalian
species, including both humans and dogs.
It is caused by spirochetes of the species
Leptospira interrogans sensu lato and is a
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common zoonotic bacterial infection withworldwide significance.1-3 The purposeof this study was to evaluate the correlation between Leptospira species antibody
detection using the SNAP® Lepto test,
an enzyme-linked immunosorbent assay(ELISA), and the microscopic agglutination
test (MAT). A total of 162 serum samples

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from dogs with a differential diagnosis of
leptospirosis were evaluated with the MAT
and the SNAP® Lepto to detect antibodies
to Leptospira. A subset of whole blood andurine samples (n=59) were also tested on
Leptospira real-time polymerase chain reaction (PCR) assay. On initial presentation,
the SNAP® Lepto was positive in 15/22 confirmed leptospirosis cases, 4/9 suspected leptospirosis cases and 20/131 dogs with otherillnesses. The MAT was positive (1:100 titeror greater) in 18/22 confirmed leptospirosis
cases, 6/9 suspected leptospirosis cases and29/131 dogs with other illnesses. The PCR
was positive in 5/22 confirmed leptospirosis cases. The overall agreement with theclinical diagnosis of leptospirosis was 80%
for the SNAP® Lepto and 78% for MAT.
The results of this study support the utilityof the SNAP® Lepto test in assisting in the
diagnosis of leptospirosis in dogs with clinical signs consistent with infection.
INTRODUCTION
Early diagnosis of leptospirosis and prompt
administration of appropriate antimicrobial
treatment is critical due to the zoonotic andprogressive nature of the disease. Dogs suspected of leptospirosis have variable clinicalpresentations, and the course of the disease
can range from subclinical infection to anacute, fatal disease that is characterized bymulti-organ dysfunction.3-5 Current methods
for diagnosis of leptospirosis include MAT,
ELISA(SNAP® Lepto, IDEXX Laboratories,
Inc., Westbrook, ME), PCR and histopathology.
Due to the difficulty in culturing Leptospira from blood or urine, it is not readily
performed in clinical veterinary medicine.4
The most common diagnostic test used
today to aid in the diagnosis of canine
leptospirosis is the MAT, which detects
antibodies capable of agglutinating cultured
organisms.5 Due to the complexity in performing the MAT, this test is only performedin commercial diagnostic laboratories.
A second serologic test is available, theSNAP® Lepto, an ELISA test that detects antibodies to a major outer membrane protein,

194

LipL32.6 There are inherent limitationsin the performance of serologic tests forthe diagnosis of leptospirosis. In the earlystages of infection, dogs may frequently
have negative serology results.3,5,7 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
In addition, serologic tests
for leptospirosis, including both MAT and
SNAP® Lepto, may not reliably differentiate
infection from vaccination.6 Real-Time PCR
testing is also available to aid in the diagnosis of leptospirosis in dogs. The detectionof leptospiral DNA in either blood, urine, or
tissue samples is most useful in acute phasesof infection when assays for antibody detection may be negative.3 A PCR positive resultin any of these sample types is considereddiagnostic for a Leptospira infection inpatients presenting with clinical signs consistent with leptospirosis. Although positive
PCR results indicate the presence of DNA
and will confirm infection, a negative result
does not necessarily rule out infection due tothe limitations of PCR testing and the variable course of Leptospira infections. Due tothe limitations of both serology and PCR,
the variable nature of the infection and thepotential for acute onset of clinical signs, serology, and PCR should be used in combination to test dogs suspected of leptospirosis.
The purpose of this study was to evaluate
the SNAP® Lepto test in the diagnosis of
canine leptospirosis in 22 confirmed, clinicalcases.
MATERIAL AND METHODS
Sample Populations
Serum, whole blood, and urine (where possible) were obtained as part of the routine
standard of care from 162 dogs presenting toveterinary referral hospitalsb-e with a clinicalsuspicion of leptospirosis. Any remaining samples were donated to the study withconsent from the owner.
Microscopic Agglutination Test
The MAT was performed at a diagnostic
laboratory.a All samples had MAT performed
Vol. 13, No.3, 2015 • Intern J Appl Res Vet Med.

Table 1. Criteria used to classify the clinical canine population
having a differential diagnosis of leptospirosis.4,5Category

Confirmed Leptospirosis

Suspected Leptospirosis

Other Illness

Description• PCR positive for Leptospira inblood or urine on initial testing OR• MAT titer of 1:800 or greater on
initial testing with no history of
Leptospira vaccination OR• MAT titer of ≥1:3200 on initial
testing with a previous history of
Leptospira vaccination or an unknown vaccine history OR• A 4-fold increase in MAT titer
between acute and convalescentsamples

None of the above categories could
be satisfied, an alternative cause ofillness could not be identified, thepatient demonstrated an appropriate response to treatment, and theattending veterinarian’s clinicaldiagnosis was leptospirosis
Patient was diagnosed with adisease that was not leptospirosis

Leptospira PCR
Leptospira real time
PCR was performed
by a national referencelaboratorya on paired
whole blood and urine
samples for a subset(n=59) of clinicallyill dogs presenting
to veterinary referral
hospitals with a differential diagnosis of
leptospirosis. A PCR
positive result in either
sample type was considered diagnostic fora Leptospira infection
in patients presenting
with clinical signs consistent with leptospirosis.
Diagnosis
All patients wereclassified into one ofthree categories by theattending veterinarian:
1. Confirmed leptospi-

for the following serovars: Pomona, Canicola, Icterohaemorrhagiae, Grippotyphosa,
Bratislava, and Autumnalis. Samples wereconsidered positive if the serum agglutinated at 1:100 dilution. Samples that did
not demonstrate agglutination with any of
the serovars tested at a 1:100 serum dilution
were characterized as negative.
SNAP® Lepto
The SNAP® Lepto test is an in-clinic ELISA
licensed by the USDA for the detection of
Leptospira spp antibodies. The presence orabsence of antibody was determined by visual interpretation, comparing color intensities of the sample spot with the backgroundcolor intensity of the flow matrix in the
result window of the assay. The presenceof color in the sample spot indicated that asample was positive for antibody to Leptospira. A colorless sample spot indicated thata sample did not have detectable levels ofantibody to Leptospira. The positive control
spot must be blue for the ELISA to be valid.

RESULTS AND DISCUSSION
The presenting signs of leptospirosis in dogs
can vary depending on severity of illness, infecting strain, and patient’s immune status.3
Veterinarians often suspect leptospirosis indogs when signs are consistent with renalor hepatic failure.3 In this study, the mostcommon presenting signs, physical exam

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195

rosis
2. Suspected leptospirosis, or
3. Other illness (no leptospirosis).
Criteria for classification are shown in Table
1.4,5
Statistical Methods
The positive and negative percent agreement
values obtained with the LipL32 ELISA in
relation to the MAT results performed at anational reference laboratorya were calculated using SAS® 9.4. All confidence intervals(Clopper-Pearson) are two-sided and calculated as 95% confidence intervals.

Table 2. Most common clinical signs, physical exam findings
and laboratory abnormalities of patients having a differential
diagnosis of leptospirosis.

other illnesses.
On initial presentation,
the SNAP® Lepto was positive for 15/22 confirmed
Clinical Signs
All Patients
Confirmed
leptospirosis cases, 4/9 sus(n=162)
Leptospirosis
pected leptospirosis cases,(n=22)
and 20/131 dogs with other
Lethargy
131
21
illnesses. The MAT was
positive (1:100 titer orAnorexia
129
21
greater) in 18/22 confirmed
Vomiting
114
21
leptospirosis cases, 6/9 susPolyuria/Polydipsia
61
9pected leptospirosis cases,
Diarrhea
58
11
and 29/131 dogs with otherillnesses. The MAT titers
in the dogs with other illPhysical Examnesses ranged from 1:100
Dehydration
85
8
to 1:800 with a mediantiter of 1:200. These lower
Icterus49
10
MAT values are in contrast
Fever
30
3
to those seen in dogs in
CRT >2 seconds
28
6
the confirmed leptospirosis category with single
Dyspnea/Tachypnea
172
MAT values ranging from
1:12,800 to 1:102,400, with
Laboratory Abnormalities
the exception of one dog at
Increased liver enzymes
104
12
1:1600. Nine of the confirmed leptospirosis cases
Increased BUN
91
21
had convalescent samples
Increased creatinine
86
21
collected; four had previAbnormal urinalysis
78
12
ously tested positive on
SNAP® Lepto and repeatedLeukocytosis
50
12
as positive, 2 remained
Thrombocytopenia
47
10
negative, and 3 seroconverted from negative tofindings, and laboratory abnormalities of
positive within 11 days.
the canine patients selected based on having
The population of clinically ill dogs
a differential diagnosis of leptospirosis arecontained 31 that had previously received
summarized in Table 2.
Leptospira vaccination, 78 with an unknown
The population of dogs consisted of 88
history of vaccination, and 53 that had not
females (12 intact) and 74 males (12 intact)previously been vaccinated. The SNAP®
with an age range from 3 months to 16 years
Lepto was positive for 14/31 dogs that had(median age 7 years). All patients (n=162)
been vaccinated (1 confirmed and 1 suswere evaluated by both MAT and thepected of leptospirosis), 14/78 dogs that had
SNAP® Lepto test at the time of initial exan unknown vaccine history ( confirmed andamination. For a subset of patients (n=59),
1suspected of leptospirosis), and 11/53 dogs
Leptospira PCR was also performed.
that had not previously been vaccinated (10Patients were categorized according to theconfirmed and 1 suspected of leptospirosis).
established criteria (Table 2), resulting in 22
The MAT test was positive for 17/31
cases of confirmed leptospirosis, 9 cases ofdogs that had been vaccinated (2 suspectedsuspected leptospirosis and 131 dogs with

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

of leptospirosis), 19/78 dogs that had unknown vaccine history (6 confirmed and 2
suspected of leptospirosis), and 17/53 dogs
that had not previously been vaccinated (12confirmed and 2 suspected of leptospirosis). It is important to note that all dogs in
the diagnosis category of other illness thatwere not vaccinated tested negative by both
SNAP® Lepto and MAT. As discussed earlier, serologic tests for leptospirosis, including both MAT and SNAP® Lepto may notreliably differentiate infection from vaccination.6 In this study, performance of MAT
and the SNAP® Lepto test were comparable
across these subsets of patients.
The 22 confirmed leptospirosis cases
were broadly distributed among the four
criteria used for classification. Four out of
the 22 confirmed dogs were PCR positive
for Leptospira in blood or urine on initial
testing. All had corresponding MAT negative results. In this group, only one dog
was positive on the SNAP® Lepto, and thisparticular dog had a history of Leptospiravaccination one year prior to clinical presentation. The dogs in this category were likely
in the acute phases of the infection, andthese results highlight the utility of the PCR
during this stage.
In the next confirmed leptospirosis
category, 8/22 dogs had a MAT titer of
1:800 or greater on initial testing, with nohistory of Leptospira vaccination and 7/22 of
those dogs were also positive on the SNAP®
Lepto, with MAT values ranging from
1:1600 to >1:102,400. In the four dogs that
had MAT titer of ≥1:3200 on initial testingwith either a previous history of Leptospiravaccination or an unknown vaccine history,
4/22 were positive on SNAP® Lepto. The
MAT values in this group of dogs ranged
between 1:12,800 and 1:102,400. In the last
category of confirmed leptospirosis cases,
6/22 dogs had a 4-fold increase in MAT titer
between acute and convalescent samples.
Of these dogs, 2/6 were SNAP® Lepto
negative on initial presentation and all six
were positive on convalescent testing. These
results highlight the importance of convales-

cent serologic testing in a patient suspectedof leptospirosis. The overall agreement witha clinical diagnosis of leptospirosis was 80%
for the SNAP® Lepto and 78% for MAT.
The utilization of ELISA technology indeveloping a diagnostic tool for leptospirosis presents an opportunity for a more rapid,
sensitive, and convenient test. The results ofthis study indicate that the SNAP® Lepto test
provides a tool for rapid assessment of Leptospira antibody status, providing information that may increase the index of suspicion
for leptospirosis in an unvaccinated dog
or a dog with an unknown vaccine history.
Providing a serologic test for leptospirosisthat is easily accessible to practitioners at
the point-of-care facilitates the diagnosis ofa zoonotic and potentially fatal disease toensure that appropriate therapeutic interventions are initiated and that adequate
precautions are taken to reduce the risk oftransmission. The results of this study also
highlight the importance of using both serology and PCR in testing samples from dogs
suspected of leptospirosis and that as withother serologic assays, results of the SNAP®
Lepto test should be interpreted in the context of clinical findings, vaccination history,
and other diagnostic test results.8

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FOOTNOTESa IDEXX Reference Laboratories, Inc., 1
IDEXX Drive, Westbrook, ME 04092
bVeterinary Specialty and Emergency Center, Levittown, PA
cPort City Veterinary Referral Hospital,
Portsmouth, NH
dPortland Veterinary Specialists, Portland,ME
eAnimal Medical Center, New York, NY
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