Veterinary Parasitology 104 (2002) 275–285
Evaluation of the Leishmania recombinant K39
antigen as a diagnostic marker for canine
leishmaniasis and validation of a standardized
enzyme-linked immunosorbent assay
A. Scalone a , R. De Luna b , G. Oliva b , L. Baldi c , G. Satta d ,
G. Vesco e , W. Mignone f , C. Turilli g , R.R. Mondesire h ,
D. Simpson h , A.R. Donoghue h , G.R. Frank h , L. Gradoni a,∗
a
Laboratorio di Parassitologia, Istituto Superiore di Sanità, Rome, Italy
Istituto di Clinica Medica Veterinaria, Università “Federico II”, Naples, Italy
c Istituto Zooprofilattico Sperimentale del Mezzogiorno, Portici, Naples, Italy
d Istituto Zooprofilattico Sperimentale della Sardegna, Sassari, Italy
e Istituto Zooprofilattico Sperimentale della Sicilia, Palermo, Italy
Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, Imperia, Italy
g Istituto Zooprofilattico Sperimentale delle Venezie, Padua, Italy
h Heska Corporation, Fort Collins, CO, USA
b
f
Received 25 June 2001; accepted 3 December 2001
Abstract
Canine infections with Leishmania infantum are important as a cause of serious disease in the dog
and as a reservoir for human visceral leishmaniasis (VL). Accurate diagnosis of canine infections
is essential to the veterinary community and for VL surveillance programs. A standardized ELISA
using a purified recombinant antigen (rK39) specific to VL was compared to the immunofluorescent
antibody test (IFAT) as the standard. The ELISA was developed, optimized and evaluated using sera
from 6368 dogs. The standardized ELISA and IFAT results were highly concordant. The timing and
pattern of ELISA and IFAT seroconversion in dogs followed prospectively after natural infections
were very similar. Antibodies reacting with rK39 were more common in asymptomatic canine
infections than reported for subclinical human VL. The rK39 ELISA is a relatively simple and
rapid assay for assessing the infection status of dogs, and is an alternative to IFAT, especially when
screening large numbers of samples. © 2002 Elsevier Science B.V. All rights reserved.
Keywords: Leishmania infantum; Recombinant antigen; Dog; Diagnosis; IFAT; ELISA
∗ Corresponding author. Tel.: +39-6-4990-2309; fax: +39-6-4938-7065.
E-mail address: [email protected] (L. Gradoni).
0304-4017/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 0 1 7 ( 0 1 ) 0 0 6 4 3 - 4
276
A. Scalone et al. / Veterinary Parasitology 104 (2002) 275–285
1. Introduction
Dogs are the domestic reservoir for Leishmania infantum (synonym: L. chagasi) (Maurı́cio
et al., 2000), the parasite causing zoonotic visceral leishmaniasis (VL) in both the Old and
New Worlds (World Health Organization, 1990). Clinical forms of canine leishmaniasis,
characterized by chronic evolution of viscerocutaneous signs, occur in less than 50% of infected dogs (Lanotte et al., 1979). On the other hand, both symptomatic and asymptomatic
animals are infective to sandfly vectors (Gradoni et al., 1987; Molina et al., 1994). Furthermore, routine canine leishmaniasis diagnosis is important for monitoring L. infantum
transmission in a given territory, as the dog may serve as a sentinel host for the assessment
of leishmaniasis risk to human susceptible populations, such as HIV-infected individuals
(Gradoni et al., 1996).
Given the frequent lack of signs in dogs and the difficulty of direct detection of the organism, rapid and accurate indirect diagnosis of canine infection represents an essential tool in
VL surveillance programs. The principal serodiagnostic tests include immunofluorescent
antibody test (IFAT), direct agglutination, enzyme-linked immunosorbent assay (ELISA),
dot-ELISA, immunochromatography and immunoblot. Among them, IFAT appears to be
recognized as the gold standard, being the most sensitive and specific test (see Gradoni
(1999) for a review). These conventional tests employ crude antigen preparations, either as
whole promastigotes or their soluble extracts, which limit standardization of the assays and
reproducibility of results.
A recombinant (r) antigen suitable for VL diagnosis is K39, a repetitive immunodominant
epitope in a kinesin-related protein that is highly conserved among viscerotropic Leishmania
species (Burns et al., 1993). It has been demonstrated in a number of endemic sites that
rK39 ELISA is sensitive and specific for serodiagnosis and prognosis of human VL (Qu
et al., 1994; Singh et al., 1995; Badaro et al., 1996; Houghton et al., 1998; Zijlstra et al.,
1998). Preliminary studies have shown that this test was suitable also for the serodiagnosis
of canine leishmaniasis (Ozensoy et al., 1998; Zerpa et al., 2000). Here, we report on the
development and standardization of a rK39 ELISA, which was validated with a large number
of canine sera through a multi-center study involving five regions of Italy.
2. Materials and methods
2.1. Study design and dogs
Preliminary antigen evaluation and determination of test conditions using a prototype
rK39 ELISA was performed with serum samples from the following dog groups: (i) 209
dogs from an endemic area in southern Italy, in which a well-established natural L. infantum
infection was demonstrated by standard parasitological techniques (all were part of a population screened for drug clinical trials (Oliva et al., 1995, 1998) and displayed clinical signs
of different degrees of severity); (ii) 81 clinically healthy dogs from the same endemic
area and proven to be negative by standard parasitological techniques and IFAT; (iii) 90
normal dogs from the USA; (iv) 62 Leishmania-free dogs known to be infected with other
parasites.
A. Scalone et al. / Veterinary Parasitology 104 (2002) 275–285
277
For standardization of test conditions using field samples, 415 sera were collected from
a dog population of northern Sardinia as part of the local VL surveillance program.
To monitor the appearance and trend of anti-rK39 antibodies, four naive beagles (Stefano
Morini Co., Reggio Emilia, Italy) were housed in a kennel for stray dogs in a highly endemic focus of disease and exposed to natural Leishmania transmission during one sand fly
season (from June to September 1997). Serum samples were collected periodically over 13
months.
A standardized laboratory kit ELISA using rK39 (Heska Corporation, Fort Collins, CO;
distributed in Italy by A.T.I. s.r.l., Ozzano Emilia, Bologna) was validated in a multi-center
study using 5507 serum samples collected as part of the routine sampling of both owned and
stray dogs. The samples were collected by veterinary practitioners of five Italian regions
endemic for human and canine leishmaniasis caused by L. infantum, and sent for diagnosis to
the local Institutes for Zooprophylaxis (IZs). The IZs are public institutes for animal health
that act as diagnostic reference centers for canine leishmaniasis. Diagnostic evaluation was
requested due to suspicion of clinical disease or as part of epidemiological surveys in the
VL surveillance programs. Thus, this group of dogs included both clinically ill and healthy
animals.
2.2. Standard Leishmania serology and parasitology
IFAT was used as reference serological test following standard protocols (Gradoni and
Gramiccia, 2000). The antigen was prepared from promastigotes of the WHO reference
strain of L. infantum (MHOM/TN/80/IPT1). A titer of 40 or greater was considered as
positive at the conditions developed at the Department of Parasitology of Istituto Superiore
di Sanità (Pozio et al., 1981). Material from bone marrow and popliteal or pre-scapular
lymph node aspirates was smeared on slides and stained with Giemsa’s stain and/or cultured
in Evan’s modified Tobie’s medium.
2.3. K39 antigen
The prototype ELISA was developed using a histidine tagged antigen (rK39His ). The
pET-17b plasmid (Novagen Inc., Madison, WI) containing the insert for rK39His was
obtained from Corixa Corporation, Seattle, Washington. The plasmid was transformed into
BL21(DE3) Escherichia coli via standard methods (Sambrook et al., 1989). The protein
rK39His consisted of amino acids 658–955 from GenBank accession L07879, with the addition of seven amino acids at the amino-terminus (MHHHHNH) and 14 amino acids at the
carboxy-terminus (RNSAEFAQKPRHGD). Purification from the cell lysate was performed
by nickel-chelate chromatography using a Ni-NTA matrix (Qiagen Inc., Valencia, CA) and
EDTA elution.
The laboratory kit ELISA was developed using a slightly different antigen (rK39␭cro ),
which was identical to that described for the prototype ELISA except that amino acids 2–7
were removed (HHHHNH). The insert encoding rK39␭cro was subcloned into the plasmid
␭PRcro/T2ori/RSET-B (PCT Publication no. WO 95/24198, 14 September 1995). The
plasmid was transformed into HB101 E. coli via standard methods (Sambrook et al., 1989).
Purification from the cell lysate was performed by anion-exchange chromatography using
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A. Scalone et al. / Veterinary Parasitology 104 (2002) 275–285
a Q-Sepharose high performance column (Amersham Pharmacia Biotech Inc., Piscataway,
NJ) and NaCl gradient elution.
2.4. ELISA (prototype and laboratory kit formats)
2.4.1. Prototype ELISA
Nunc PolySorp® 96-microwell plates (Nalgene Nunc International, Rochester, NY) were
coated overnight at 4 ◦ C with 25 ng per well of rK39His in 100 ␮l per well of 50 mM carbonate buffer, pH 9.6. The microwells were blocked with StabilCoat (Surmodics, Eden Prairie,
MN) and dried. Dog serum was added to the microwells at a final concentration of 1/50 in
4% (v/v) fetal bovine serum/phosphate-buffered saline containing 0.05% (v/v) Tween-20
(FBS/PBS-T) and the plates were incubated 30 min at room temperature. The microwells were washed four times with PBS-T, followed by the addition of peroxidase labeled
goat–anti-dog IgG (H+L) (KPL, Gaithersburg, MD) diluted to 100 ng/ml in FBS/PBS-T and
incubated 30 min at room temperature. The microwells were washed four times as before,
and the bound enzyme detected by incubating 10 min with the TMB Microwell Peroxidase
Substrate System (KPL). The reaction was stopped with 2.5N H2 SO4 and absorbance was
read at 450 nm.
2.4.2. Laboratory kit ELISA
The protocol was identical to the prototype ELISA except the microwells were coated
with 2.2 ng rK39␭cro per well and the peroxidase labeled goat–anti-dog IgG (H + L) was
diluted to 22 ng/ml.
2.5. Statistical analysis
To compare ELISA and IFAT results, concordance between the tests was further analyzed
using kappa statistics. Kappa is an index which indicates how much greater test concordance
is than would be expected by chance (Fleiss, 1986). The kappa values range from 0 (chance
agreement only) to 1.0 (perfect agreement). Values above 0.4 represent good concordance,
and those above 0.75 represent excellent concordance.
3. Results
3.1. Sensitivity and specificity of the prototype ELISA in established L. infantum infection
The prototype ELISA was used to test sera from 209 dogs with parasitological evidence
of leishmaniasis. In these animals, amastigote density in bone marrow and lymph nodes
ranged from one parasite per 1000 microscopic fields to one parasite per microscopic field.
IFAT was positive in all these dogs, with titers ranging from 80 to 5120. Sera from 81
uninfected Italian dogs and 90 normal US dogs were used as negative controls.
The mean absorbance plus 3 standard deviations for all of the negative dogs was A450 =
0.220. However, if only the Italian negative dogs were used, the value was A450 = 0.330.
Of the confirmed infected dogs, 203/209 (97.1%) were positive at absorbance greater
than 0.220. For the normal dogs, 1/81 (1.2%) of the Italian dogs and 0/90 of the US dogs
A. Scalone et al. / Veterinary Parasitology 104 (2002) 275–285
279
were positive at the absorbance greater than 0.220. Thus, the sensitivity of the assay was
97.1% while the specificity was 98.8%, if only the Italian dogs were included, or 99.4%
if the US dogs were also included. There was no correlation between absorbance values
recorded with the ELISA and either the IFAT titer or the severity of disease (data not
shown).
Sera from 62 Leishmania-free dogs infected with Erlichia canis (13), Babesia gibsoni
(9), Trypanosoma cruzi (2), Dirofilaria repens (4), Dirofilaria immitis (7), Dipetalonema
reconditum (3), Dipylidium caninum (5), Trichuris vulpis (6) or Ancylostoma caninum
(13) were also examined and found to have absorbance values less than 0.220 (data not
shown).
3.2. Sensitivity and specificity of the prototype ELISA in field samples
Based on the absorbance mean and standard deviation for Italian negative dogs above,
the cut-off value was set at A450 = 0.400. Using these test conditions, the prototype ELISA
was further evaluated on a sample of 415 sera with known IFAT values collected from the
northern Sardinia area during an epidemiologic survey. Parasitological and clinical features
of the animals were not available. Of the 157 IFAT-positive sera with a titer of 40 or
greater, 156 (99.4%) were also positive in the prototype ELISA. The single ELISA negative
sample had a corresponding IFAT titer of 40. Among the 258 IFAT-negative sera, 1 (0.4%)
was positive in the prototype ELISA (Fig. 1). Again, there was no correlation between
absorbance values and IFAT titers (data not shown). The test sensitivity and specificity as
compared to IFAT were 99.4 and 99.6%, respectively. The observed concordance between
the two tests was 0.99 and the kappa value 0.89.
Fig. 1. Distribution of absorbance (A450 ) values obtained using the prototype rK39 ELISA with 415 canine serum
samples collected in northern Sardinia and examined by IFAT at the threshold titer of 40. Values lower and greater
than 0.400 are shown at different scales on the x-axis.
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A. Scalone et al. / Veterinary Parasitology 104 (2002) 275–285
281
3.3. Appearance and trend of anti-rK39 antibodies using the standardized laboratory
kit ELISA
Four naive beagle dogs were housed in a kennel from June 1997 through March 1998. By
the end of March 1998 all animals showed evidence of an established Leishmania infection
as demonstrated by positive microscopy and culture. Only one dog (dog 1, Fig. 2) developed
clinical disease during the observation period. Serological assessments were made in parallel
using the laboratory kit ELISA and IFAT. As shown in Fig. 2, both the appearance and trend
of anti-leishmanial antibodies showed high concordance with the two tests (0.88; kappa:
0.85). In dog 1, high levels of antibodies were detected very early after Leishmania exposure,
with an increasing trend up to the animal’s euthanasia. A similar trend, but with temporary
seroconversion to negative revealed by both assays, was observed in dog 2. Both dogs 3
and 4 developed a late positive serologic response, about 10 months after placement at the
endemic site. Interestingly, IFAT titers and ELISA absorbance values recorded prospectively
in each dog demonstrated similar patterns and appeared to be correlated, while this was not
evident in the cross-sectional determinations reported above.
3.4. Multi-center study for the validation of the standardized laboratory kit ELISA
This study was designed to evaluate the performance of the standardized laboratory kit
ELISA at IZ departments that routinely run a high number of IFAT for canine leishmaniasis.
The sera were examined by both methods for 1–2 months. To maximize the standardization
of IFAT procedures, the collaborating IZs were provided with the same L. infantum strain
for antigen preparation and batch of FITC-conjugated anti-dog immunoglobulins, as well
as with a common protocol for the assay procedure. To facilitate the evaluation, only two
serum dilutions (1:40 and 1:80) were examined by IFAT. If a sample had discordant results
with the two tests, it was sent to the Department of Parasitology of Istituto Superiore di
Sanità for blind re-evaluation.
Five IZs, hereafter referred to as Palermo, Sassari, Naples, Imperia and Padua, examined
a total of 5507 sera. Of these sera, 275 (5.0%) had discordant results between the two tests,
145 of which were re-evaluated. In Table 1, which shows the laboratory kit ELISA results at
the IFAT threshold titer of 40, data are presented after re-evaluation of discordant samples.
The observed test concordance varied from 0.91 to 0.99 between the IZs, whereas the kappa
value varied from 0.76 to 0.98.
Because several sera giving discordant results had an IFAT titer of only 40, and because the
percentage of the positives represented by these sera varied greatly between the IZs (4.1%
Sassari, 11.7% Palermo, 11.8% Padua, 18.5% Naples, 42.6% Imperia), the laboratory kit
ELISA results were also compared with the IFAT threshold titer of 80 (Table 2). Using this
criteria, both the observed test concordance (0.95–0.98) and the kappa values (0.84–0.95)
were much more consistent between the IZs.
Information on the clinical condition was available in a subset of 1028 dogs. Of these,
628 dogs showed one or more signs referable to a leishmanial infection, including aspecific
signs such as slight lymph adenopathy and/or loss of weight. In this group of dogs, 265
were IFAT-positive at the threshold titer of 80, and 259 were positive in the laboratory kit
ELISA. Sensitivity and specificity of the ELISA as compared to IFAT for this group of dogs
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Table 1
Laboratory kit ELISA results compared to an IFAT threshold titer of 40 with 5507 canine sera examined by five
Italian Institutes for Zooprophylaxis (IZs)
Result
Positive
Negative
FPa
FNb
OCc
Kappa
IZ Palermo
(N = 1839)
IZ Sassari
(N = 1119)
IZ Naples
(N = 1675)
IZ Imperia
(N = 467)
IZ Padua
(N = 407)
IFAT
IFAT
IFAT
IFAT
IFAT
ELISA
733
743
1106
1096
18
8
0.99
0.98
ELISA
344
330
775
789
3
17
0.98
0.95
ELISA
365
331
1310
1344
19
53
0.96
0.88
ELISA
134
94
333
373
0
40
0.91
0.76
ELISA
66
71
341
336
5
0
0.99
0.96
a
rK39 ELISA false positive compared to IFAT.
rK39 ELISA false negative compared to IFAT.
c Observed concordance between rK39 ELISA and IFAT.
b
Table 2
Laboratory kit ELISA results reported in Table 1 compared to an IFAT threshold titer of 80
Result
Positive
Negative
FPa
FNb
OCc
Kappa
IZ Palermo
IZ Sassari
IZ Naples
IZ Imperia
IZ Padua
IFAT
IFAT
IFAT
IFAT
IFAT
ELISA
647
743
1192
1096
98
2
0.95
0.89
ELISA
330
330
789
789
13
13
0.98
0.95
ELISA
308
331
1367
1344
49
26
0.95
0.84
ELISA
94
94
373
373
8
8
0.97
0.91
ELISA
59
71
348
336
12
0
0.97
0.89
a
rK39 ELISA false positive compared to IFAT.
rK39 ELISA false negative compared to IFAT.
c Observed concordance between rK39 ELISA and IFAT.
b
were 96.6 and 99.2%, respectively. Four hundred dogs were apparently in good health. In
this group of asymptomatic animals, 53 were IFAT-positive at the threshold titer of 80, and
52 were positive at the laboratory kit ELISA. Sensitivity and specificity of the ELISA as
compared to IFAT for this group of dogs were 92.4 and 99.1%, respectively.
4. Discussion
Among 380 dogs examined in the first part of the study, IFAT was positive at titers of 80 or
greater in 100% of animals with parasitological evidence of leishmaniasis, whereas the assay
was negative at the threshold titer of 40 in 100% of clinically healthy, Leishmania-free dogs.
Therefore, the first set of experiments have confirmed the value of IFAT for the evaluation of
the new serodiagnostic method, and justified its use as the sole reference test in subsequent
investigations involving larger dog samples.
A. Scalone et al. / Veterinary Parasitology 104 (2002) 275–285
283
When used with sera from two large group of dogs, both the sensitivity and the specificity of the prototype ELISA were remarkably high as compared to IFAT (97.1–99.4 and
99.4–99.6%, respectively). Sensitivity was similar to, and specificity higher than those
reported for conventional ELISA (Ashford et al., 1993; Mancianti et al., 1995; Sideris et al.,
1996) or dot-ELISA (Mancianti et al., 1996) using parasite derived antigen. Furthermore, the
distribution of absorbance values revealed a clear difference between negative and positive
sera (see Fig. 1), while this is not usually seen with the conventional Leishmania ELISA. It
is noteworthy, however, that in early canine infections both IFAT and rK39 ELISA failed to
reveal specific antibodies for a variable period of time, as shown in three out four naive
beagles exposed to natural Leishmania transmission. Due to serological latency, which appears to be common in dogs and may include both incubation and transient seroconversionto-negative periods (Acedo-Sánchez et al., 1998), Dye et al. (1993) have estimated that even
the most efficient serological method will probably miss more than 20% of newly established
infections. As a consequence, serological techniques should be complemented by other
methods currently in use when an accurate diagnosis is necessary (Campino et al., 2000),
or suspected cases that are seronegative should be re-tested after a suitable period of time.
The laboratory kit ELISA detected anti-leishmanial antibodies in a large number of
asymptomatic dogs. The high sensitivity of the assay in this group of dogs (92.4%), which
was only slightly lower than in dogs showing signs referable to leishmanial infection
(96.6%), indicates that antibodies reacting with rK39 are more common in asymptomatic canine infections than in subclinical human VL. Only 4% of sera from 130 subjects with early,
stationary or self-healing subclinical VL (as detected by serology using crude Leishmania
antigen) reacted with rK39, whereas 87% of sera from 15 subclinical cases progressing to
clinical VL reacted with the antigen (Badaro et al., 1996). These different patterns suggest
that the asymptomatic condition should be treated as a markedly different biological feature
between canine and human subjects infected with L. infantum.
Data from the multi-center study on the laboratory kit ELISA validation showed some
variation between collaborating institutes in performance of the IFAT, the rK39 ELISA, or
both. It is unlikely that the dog populations examined by the IZs differed somehow in the
biological response to Leishmania, however, it should be noted that the dogs tested at each
IZ were different, and each IZ received these samples based on the different criteria for that
area. The great variation in frequency of sera showing the low IFAT titer of 40 (from 4.1 to
42.6%) strongly suggests the greatest variation was due to IFAT interpretation. To overcome
this, the IFAT threshold value was increased to 80 for all IZs, which may have resulted in
the removal of some true positives from some locations and, therefore, a slight decrease of
specificity. The “gray zone” for a positive interpretation appears to be a common problem
for IFAT in many laboratories, which report threshold titers ranging from 20 to 160 (Jambou
et al., 1986; Abranches et al., 1991; Dye et al., 1993; Berrahal et al., 1996; Sideris et al.,
1996; Acedo-Sánchez et al., 1996, 1998).
In conclusion, the laboratory kit ELISA is a relatively simple and rapid assay for assessing the infection status of dogs. The use of a specific purified recombinant protein as the
K39 antigen allows better standardization than culture derived parasite material, and the
quantitative assessment removes the operator subjectivity that does exist with IFAT. The
laboratory kit ELISA can be used as an alternative to IFAT, especially when screening large
numbers of samples.
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Acknowledgements
We wish to acknowledge Corixa Corporation for their technical advise and supplying
the original plasmid encoding the K39 antigen. These studies were supported in part by
funding from Heska Corporation.
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