Comparative Immunology, Microbiology and Infectious Diseases 73 (2020) 101542

Contents lists available at ScienceDirect

Comparative Immunology, Microbiology and

Infectious Diseases
journal homepage: www.elsevier.com/locate/cimid

Detection of Leishmania infantum DNA by real-time PCR in saliva of dogs

Ana Cantos-Barreda a, Damián Escribano b, c, *, Padet Siriyasatien d, José J. Cerón b,
M. Carmen Thomas e, Raquel N. Afonso-Lehmann e, Manuel C. López e, Luis J. Bernal b,
Atchara Phumee d, George Lubas f, Silvia Martínez-Subiela b
a
Department of Animal Health, Faculty of Veterinary Science, Regional Campus of International Excellence ‘Campus Mare Nostrum’, University of Murcia, 30100,
Espinardo, Murcia, Spain
b
Interdisciplinary Laboratory of Clinical Analysis, Interlab-UMU, Regional Campus of International Excellence ‘Campus Mare Nostrum’, University of Murcia, 30100,
Espinardo, Murcia, Spain
c
Department of Animal Production, Faculty of Veterinary Science, Regional Campus of International Excellence ‘Campus Mare Nostrum’, University of Murcia, 30100,
Espinardo, Murcia, Spain
d
Vector Biology and Vector Borne Disease Research Unit, Department of Parasitology, Faculty of Medicine, Chulalongkorn University, 10330, Bangkok, Thailand
e
Molecular Biology Department, Instituto de Parasitología y Biomedicina ‘López Neyra’, Consejo Superior de Investigaciones Científicas, 18016, Granada, Spain
f
Department of Veterinary Science, University of Pisa, 56122, San Piero a Grado, Pisa, Italy

A R T I C L E I N F O A B S T R A C T

Keywords: This study developed a real-time quantitative PCR (qPCR) assay to detect L. infantum kinetoplast DNA (kDNA) in
Dog canine saliva. The qPCR showed an efficiency of 93.8%, a coefficient of correlation of 0.996 and a detection limit
Experimental infection of 0.5 fg/reaction (0.005 parasites), although it detected until 0.25 fg/reaction (0.0025 parasites). When samples
Leishmania infantum
from 12 dogs experimentally infected with L. infantum were collected, L. infantum kDNA was detected at 16-
Real-time PCR
Saliva
weeks post-infection (wpi) in 41.7% and 91.7% of saliva and bone marrow samples, respectively, and at 47-
wpi in 75% of both samples. L. infantum kDNA can be detected by qPCR in canine saliva, with lower sensi­
tivity in the early stages of infection and a lower parasite load estimation compared to bone marrow. However,
saliva had similar sensitivities to bone marrow in the later stages of the infection and could be used to detect
L. infantum kDNA being aware of its limitations.

1. Introduction real-time quantitative PCR (qPCR) provides reproducible results with

good accuracy and sensitivity, depending on the type of target gene, the
Leishmania infantum is the main causative agent of zoonotic visceral samples selected, and the optimization of the reagent concentrations.
leishmaniosis in the Mediterranean basin [1]. Dogs acquire the parasite Several primers for use in qPCR assays for the diagnosis of Leishmania
through the bite of infected phlebotomine sandflies and are considered infection have been published [4–7]. The use of kinetoplast DNA
as the main reservoirs of infection in the domestic transmission cycle of (kDNA) minicircle qPCR has been demonstrated to be more sensitive
L. infantum, affecting humans and other mammals [2]. Canine leish­ than conventional PCR. In addition, the kDNA is the main target used for
maniosis (CanL) displays a wide range of clinical signs, usually affecting diagnostic screening due to its high specificity and sensitivity based on
the skin (dermatitis, alopecia, onychogryphosis), lymphoid organs the high number of copies (~10,000) per parasite [6].
(splenomegaly, adenomegaly), and kidneys (renal disease) [3]. How­ In previous studies where specific antibodies against Leishmania spp.
ever, a fraction of the canine population in Leishmania-endemic areas were quantified by immunoassay in dogs from Leishmania-endemic
remains asymptomatic but infectious to sandflies. In these cases, an areas, the reported prevalence for CanL was less than 30% [8–10].
accurate diagnosis of CanL is an important challenge [2]. However, a higher prevalence was found in studies that used conven­
Diagnosis of CanL can be performed by direct identification of the tional PCR techniques in the conjunctiva (32%) or skin (51%) [11], even
parasite, molecular detection of its DNA, or detection of Leishmania- reaching the 67% of prevalence by qPCR in a canine population sero­
specific antibodies. In the diagnosis by molecular methods, the use of positive and/or positive by PCR in lymphoid tissue samples [9]. As

* Corresponding author at: Interdisciplinary Laboratory of Clinical Analysis, Interlab-UMU, Regional Campus of International Excellence ‘Campus Mare Nostrum’,
University of Murcia, 30100, Espinardo, Murcia, Spain.
E-mail address: [email protected] (D. Escribano).

https://doi.org/10.1016/j.cimid.2020.101542
Received 8 May 2020; Received in revised form 3 August 2020; Accepted 28 August 2020
Available online 3 September 2020
0147-9571/© 2020 Elsevier Ltd. All rights reserved.
A. Cantos-Barreda et al. Comparative Immunology, Microbiology and Infectious Diseases 73 (2020) 101542

epidemiological studies show that the prevalence of infection is higher amplification was observed, as reported in a previous work which used
than the seroprevalence, some authors recommend investigating Leish­ the same experimentally infected dogs than the present study [10]. Dogs
mania infection using a combination of serological and molecular were infected by intravenous (i.v.) inoculation of 1 × 106
methods if serological titers are inconclusive [11,12]. stationary-phase promastigotes of L. infantum MCAN/BR/00/BA262. To
Several studies have detected and quantified by qPCR the presence of obtain those parasites, amastigotes were isolated from the spleen of an
L. infantum DNA in canine samples obtained by non-invasive procedures, experimentally infected hamster and cultured in Schneider’s Drosophila
including oral and conjunctival swabs [4,7,13,14]. In human medicine, medium (Biowest, Nuaillé, France) supplemented with 10%
DNA from different species of Leishmania has been detected in saliva by heat-inactivated fetal bovine serum (iFBS) and gentamicin (50 μg/mL)
PCR assays, even prior to being detected in blood or buffy coat [15–19]. at 26 ◦ C. The promastigotes transformed from spleen-derived amasti­
Saliva can contain cells from the oral mucosa and can be collected with gotes were grown in modified RPMI-1640 medium supplemented with
both cotton swabs and sponges, with differences in the sample volume 20% iFBS and gentamicin (50 μg/mL) at 26 ◦ C. The success of the
obtained and the number of cells. Saliva is a non-invasive and easy to infection was confirmed in the totality of the 12 dogs by qPCR using
collect sample – dog owners can do it themselves – that has been DNA from bone marrow aspirates [10]. Dogs were not vaccinated
demonstrated to be a suitable tool for quantification of Leishmania-s­ against Leishmania or receive any treatment that could interfere in the
pecific antibodies in dogs with CanL [20]. Changes in the salivary pro­ results and were free of other diseases.
teome of dogs after experimental infection with L. infantum in
comparison with the pre-infection (pre-i) time have been also evidenced 2.3. Sampling and experimental design
[21]. Furthermore, the use of this sample could have a large impact on
screening programs, especially in areas where facilities for sample Saliva, serum, and bone marrow aspirates were collected from each
storage and veterinary practice are poor or unavailable [22]. dog. Saliva and serum were used for the quantification of anti-Leish­
The main aim of the present study was to investigate the possible mania IgG2 levels, and saliva and bone marrow aspirate were used for
presence of L. infantum DNA in saliva samples of dogs obtained with a quantification of the L. infantum parasite load. Dogs were sampled three
sponge. For this purpose, a qPCR was validated from an analytical point times: at the pre-i time and at 16- and 47-weeks post-infection (wpi),
of view and applied to saliva samples of dogs before and after experi­ according to a previous report evaluating the parasite load in oral swab
mental infection with L. infantum. of dogs over one-year post-infection [23].
Unstimulated saliva was collected using Salivette® saliva collection
2. Materials and methods devices (Sarstedt, Nümbrecht, Germany) taking advantage of sponges to
maximize the volume of saliva obtained. The sponge clipped with
2.1. Ethics statement tweezers was rubbed against the buccal mucosa on each side of the
mouth, above and below the tongue, and through every nook of the oral
The research protocol was reviewed and approved by the Regional cavity for 1 min. Then, sponges were introduced into the Salivette®
Government of Murcia under the identification code number devices and centrifuged at 3,700 rpm for 10 min at 4 ◦ C. The recovered
A13170401. All procedures were conducted according to the legal re­ saliva was preserved in 1.5 mL vials at -80 ◦ C until analyses.
quirements of the European Community (Directive 2010/63/EU) and Blood samples were taken by venipuncture of the jugular vein, drain
the Spanish legislation (RD 53/2013) on animal protection. into 1 mL tubes without anticoagulant, and centrifuged at 3,500 rpm for
5 min at 4 ◦ C. The obtained serum was stored in 1.5 mL vials at -80 ◦ C
2.2. Animals until the time of analysis.
In order to obtain bone marrow aspirates, dogs were subjected to
A total of 12 healthy Beagle dogs purchased from an accredited sedation by i.v. inoculation of a combination of medetomidine (0.02
breeder (Isoquimen SL., Barcelona, Spain) were enrolled in this study. At mg/kg) (Domtor®; Ecuphar Veterinaria S.L.U.) and butorphanol (0.1
the time of infection, the age of the dogs varied between 9 and 36 mg/kg) (Torbugesic®; Zoetis Spain S.L.U., Madrid, Spain). Bone marrow
months, with a median of 36 months of age, and did not show clinical aspirate (approximately 450 μL) was collected from the costochondral
signs compatible with CanL. Dogs were allocated in open-air, partially junction of the rib by 18-gauge needle aspiration and mixed with the
covered kennels, in a research colony of the Animal Resources Center of same volume of NET (10 mM Tris-HCl, 10 mM NaCl, 10 mM EDTA, pH =
the University of Murcia located in the peri-urban area of Murcia city, 8; 0.1 mg/mL of proteinase K and 1% of SDS) for DNA extraction. Dogs
southeast of Spain. A number between 1 and 12 was arbitrarily assigned were reverted of sedation with atipamezole (0.1 mg/kg i.m.) (Anti­
to each dog’s microchip number in order to simplify their identification sedan®; Ecuphar Veterinaria S.L.U.).
for the study. Balanced chicken-based diet (LIBRA®; Affinity Petcare S.
A., Barcelona, Spain) and water were provided to dogs for ad libitum 2.4. DNA extraction
consumption. An external anti-sandfly activity insecticide for 10-25 kg
of body weight containing 500 mg/mL of permethrin and 100 mg/mL of DNA extraction from saliva and bone marrow samples was per­
imidacloprid (Advantix® Spot-on; Bayer Hispania S.L., Barcelona, formed using the phenol/chloroform method. Thus, 200 μL of saliva and
Spain) was applied every three weeks, following manufacturer’s in­ approximately 450 μL of bone marrow from each dog were mixed with
structions. Also, an internal deworming tablets containing 50 mg of NET buffer and incubated overnight at 56 ◦ C. Following phenol/chlo­
pyrantel, 50 mg of praziquantel, and 150 mg of febantel (Prazitel® Plus roform extraction, gDNA was ethanol precipitated and resuspended in
Worming tablets; Ecuphar Veterinaria S.L.U., Barcelona, Spain) were sterile MilliQ water up to 30 μL (saliva) or 100 μL (bone marrow). DNA
administered every three months based on the body weight, according to was spectrophotometrically quantified using the NanoDrop® 2000
manufacturer’s instructions. Dogs were vaccinated against distemper (Thermo Scientific, Wilmington, DE). High-quality template DNA (A260/
virus, canine adenovirus type 2, canine parvovirus, canine parainfluenza A280 ratio ≥ 1.7) was obtained in all samples.
virus type 2, Leptospira interrogans serogroups Canicola and Icter­
ohaemorrhagiae, and rabies virus (Eurican® DAPPi-LR; Boehringer 2.5. Amplification of Leishmania kDNA and quantification of parasite
Ingelheim Animal Health España S.A.U., Barcelona, Spain), and against load
Bordetella bronchiseptica (Eurican® Pneumo Bb/Pl2, Boehringer Ingel­
heim Animal Health España S.A.U.). Primers and probe used in this study were earlier described by
Prior to the experimental infection, dogs were tested for Leishmania Francino et al. [6]. The amplification and detection of DNA by qPCR was
infection by qPCR using DNA from bone marrow aspirates and no performed in the QuantStudio 5 Real-Time PCR System (Applied

2
A. Cantos-Barreda et al. Comparative Immunology, Microbiology and Infectious Diseases 73 (2020) 101542

Biosystems, Foster City, CA, U.S.A.) using iTaq Universal Probes of concentrations, respectively [29]. The intra-assay variance was
Supermix (Bio-Rad, Hercules, CA, U.S.A). An internal control consistent calculated by analyzing fifteen replicates of L. infantum DNA at LOD in
with the TaqMan Exogenous Internal Positive Control Reagents (VIC the same PCR run, while the inter-assay variance was calculated by
Probe) (Applied Biosystems, Foster City, CA, U.S.A.) was used with the analyzing these replicates on two different runs [28,30].
purpose of detecting possible PCR inhibition. The qPCR was performed
in a final reaction volume of 20 μL, which includes 17.5 μL of mix and 2.7. Clinical signs, serological status, and acute phase proteins (APPs)
2.5 μL of DNA at a final concentration of 1× iTaq Universal Probes concentrations of the experimentally infected dogs
Supermix, 900 nM of each primer amplifying a 120-base-pair fragment
of the conserved region in the Leishmania kDNA minicircle A clinical examination of the dogs was performed to assess the
(LEISH1 5′ -AACTTTTCTGGTCCTCCGGGTAG-3′ , LEISH2 5′ -ACC presence of clinical signs consistent with CanL. The serological status of
CCCAGTTTCCCGCC-3′ ) and 200 nM of TaqMan probe (FAM- the dogs for Leishmania spp. infection was evaluated by measurement of
5′ -AAAAATGGGTGCAGAAAT-3’-non-fluorescent quencher-MGB), 1× anti-Leishmania IgG2 levels in serum and saliva samples by time-
Exo IPC Mix, 1× Exo IPC DNA, and 125 or 250 ng of DNA from each resolved immunofluorometric assay (TR-IFMA), as previously reported
sample in triplicate. The thermal cycle conditions consisted of an initial [20,31]. The cut-offs (mean + 4 S.D. of 12 dogs Leishmania-negative by
incubation step at 50 ◦ C for 30 sec, followed by denaturation at 95 ◦ C for qPCR after their acquisition from Isoquimen S.L. (Barcelona, Spain))
10 min, and 45 cycles at 94 ◦ C for 30 sec and 55 ◦ C for 1 min each. Each were set at 22 UFL and 32 UFL for anti-Leishmania IgG2 in serum and
plate included in duplicate a positive control (0.5 ng of L. infantum DNA) saliva, respectively.
and negative controls (bone marrow from a Leishmania-negative dog, APPs including C-reactive protein (CRP), ferritin, and albumin, and
sterile water as non-template control (NTC), and 1× Exo IPC Block). also total proteins and globulins were analyzed in serum as previously
Amplification was performed in 96-well plates (Applied Biosystems, described in dogs [32].
Foster City, CA, U.S.A.) sealed with an adhesive film (Applied Bio­
systems, Foster City, CA, U.S.A.) to prevent evaporation of samples. To
2.8. Statistical analysis
assess the effectiveness of extraction of DNA, amplification of the
constitutive canine β-actin gene was performed.
The qPCR performance results were analyzed using the Quant­
To quantify the parasite load of each sample, the absolute quantifi­
StudioTM Design & Analysis Software v1.4 (Applied Biosystems). The
cation method was used, as it was previously reported for L. infantum
statistical analyses were carried out using GraphPad Prism 6 (GraphPad
DNA [24]. This method allows to obtain, by linear regression analysis,
Software Inc., La Jolla, CA) and GraphPad QuickCalcs (https://www.gr
the absolute number of parasites in the samples by plotting the Ct values
aphpad.com/quickcalcs/kappa2/). Pearson’s correlation between sero­
of the standards with a known amount of L. infantum DNA against the log
logical results of saliva and serum samples was calculated. Spearman’s
of the template DNA concentration from unknown samples tested in
correlations between saliva and bone marrow qPCR results, and between
triplicate. For this, a standard curve was constructed with eight 10-fold
serological and parasitological results were calculated. P < 0.05 was
serial dilutions of DNA in triplicate, ranging from 0.005 to 50,000 par­
considered statistically significant. Agreement between saliva and bone
asites of the DNA extracted from 4 × 106 parasites of L. infantum
marrow qPCR results was assessed using Cohen’s Kappa. Kappa (κ)
MCAN/BR/00/BA262. The Ct value was calculated for each sample by
values were scored according to the following guidelines: κ = 0, no
determining the point of the fluorescence value exceeding the threshold
better than chance; κ < 0.20, poor agreement; κ = 0.21–0.40, fair
limit [7]. The cycle cut-off point was selected as the cycle of quantifi­
agreement; κ = 0.41–0.60, moderate agreement; κ = 0.61–0.80, good
cation (Cq) value that corresponds to the limit of detection (LOD) of the
agreement; κ = 0.81–1.00, very good agreement [33]. Because of the
test. Samples were considered positive when the parasite load was above
small sample size of the groups of dogs, data regarding anti-Leishmania
the detection limit [24]. Results were expressed as parasites/ng of DNA
IgG2 levels, qPCR results, and APPs concentrations between sampling
[25], considering that each L. infantum parasite has 100 femtograms (fg)
times and groups were assessed by the non-parametric tests including
of genomic DNA [26].
Spearman’s correlations and Kruskal-Wallis test followed by Dunn’s
multiple comparisons test.
2.6. Performance characteristics and analytical validation of the TaqMan
assay
3. Results

The characteristics of qPCR have been determined by a few param­

3.1. Analytical validation of the Leishmania TaqMan assay
eters. The slope (m) was calculated by linear regression and is defined as
the regression coefficient of the regression line in the standard curve.
Performance characteristics were summarized in Table 1 and
Slopes between -3.6 and -3.1 are considered adequate [27]. The effi­
revealed a m of -3.6, a Y-intercept of 13.971, an E between 84.6%-
ciency (E) was calculated from the slope of the regression line of the
93.8%, and a r2 between 0.994 and 0.996. The LOD at the 95% of
standard curve. An E of 1 means that the quantity of DNA amplicons is
confidence interval was 0.5 fg/reaction, which corresponds to 0.005
doubled with each cycle [28] and E between 0.9 and 1.1 provide satis­
parasites based on an averaged genome size of 34 Mb [34,35]. However,
factory PCR performances [27]. The coefficient of correlation (r2) in­
this TaqMan assay was able to detect until 0.25 fg/reaction (0.0025
dicates the closeness of the relationship between two DNA concentration
points, and an acceptable r2 was set between 0.99 and 0.999 [27]. The
Table 1
linear dynamic range can be considered as the highest to the lowest
Performance characteristics of the qPCR assay performed.
quantifiable copy number established through a calibration curve over
which a reaction is linear. An acceptable dynamic range extends to 5 or 6 Performance characteristics Average

log10 concentrations [29]. The LOD, which is described as the lowest m − 3.6
copy number at which 95% of positive samples are detected, is a mea­ Y-intercept 13.971
E 84.6% - 93.8%
sure of the analytical sensitivity of the assay. The LOD was calculated
r2 0.994 - 0.996
using twenty replicates of the three lowest 10-fold serial dilutions of the LOD (at 95% confidence interval) 0.5 fg/reaction (0.005 parasites)
standard curve (2-0.02 fg/μL) [24]. Dynamic range 8 logs
The assay precision was evaluated by means of the intra-assay Intra-assay 0.36 S.D.
(repeatability) and inter-assay (reproducibility) variations. Intra- and Inter-assay 1.46 S.D.
Ct value for positive results ≤ 38
inter-assay variations were revealed as S.D. for the Cq variance and S.D.

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A. Cantos-Barreda et al. Comparative Immunology, Microbiology and Infectious Diseases 73 (2020) 101542

parasites). The dynamic range of detection of Leishmania DNA was DNA (dog #1). The differences in the crude data of parasitic load found
established in 8 logs. Samples were considered positive when their Ct between the bone marrow and the saliva of the same animal vary from
value was ≤ 38. There was no detectable Ct value in the non-template 4.66-fold (dog #3) up to 2,690-fold higher (dog #4). A significant cor­
control associated with non-specific reactions. Intra- and inter-assay relation between the parasite load found in paired saliva and bone
values among the replicates at LOD were 0.36 and 1.46 S.D., marrow samples at 47-wpi was observed (r = 0.695; p < 0.05) (Fig. 1A).
respectively. Dogs were classified in two groups based on the qPCR results and
according to a previous publication [3]: “control of infection” (CI) (n =
3.2. Application of the Leishmania TaqMan assay to the experimental 3) - dogs with negative results in saliva and a very low parasite load in
infection samples bone marrow at 16-wpi (Ct > 34, < 0.12 parasites/ng of DNA × 103) and
negative results in both samples at 47-wpi - “dissemination of infection”
The ability of the qPCR to detect Leishmania parasites using saliva (DI) (n = 9) - dogs for which the parasite load increases at 47-wpi in
samples of infected dogs was calculated taking into account the qPCR saliva and/or bone marrow with respect to 16-wpi. No correlation was
bone marrow results as the “gold standard”. Paired bone marrow and observed between qPCR results at 16- and 47-wpi of the CI and DI
saliva samples from 12 experimentally infected dogs before infection groups. Dogs #7, #8, and #11 were included in the CI group; while the
and at 16- and 47- wpi were analyzed by the Leishmania TaqMan assay to rest of dogs were included in the DI group.
compare the parasite load in both types of samples. All dogs were
Leishmania-negative by qPCR at the pre-i time in both saliva and bone 3.3. Clinical signs, serological status, and APPs concentrations of the
marrow samples. At 16-wpi, Leishmania DNA was detected in 5 out of 12 experimentally infected dogs
(41.7%) saliva samples and in 11 out of 12 (91.7%) bone marrow as­
pirates. At 47-wpi, Leishmania DNA was detected in 9 out of 12 (75%) Clinical signs, antibody titers, and APPs concentrations were evalu­
saliva and bone marrow samples. The assay was 100% specific, as no ated in order to have data about the acquired immunological response
amplification was observed in the negative control sample from an by mean of the clinical signs and the antibodies, and about the response
endemic area for CanL and neither in the NTC included in all qPCR runs. of the disease by the APPs.
The level of agreement at 16-wpi between the qPCR in saliva and bone No dogs belonging to the CI group presented clinical signs compat­
marrow was found poor (κ = 0.12; 95% CI: -0.116 – 0.360). At 47-wpi, ible with CanL neither at the pre-i time nor at 16- and 47-wpi (Tables 2
the level of agreement between the qPCR in saliva and bone marrow was and 3). At 47-wpi, two dogs (#7 and #8) of the CI group showed anti-
found to be very good (κ = 1; 95% CI: 1.000 – 1.000). Leishmania IgG2 levels under the cut-off value in both saliva and serum
Parasite load in both saliva and bone marrow samples of each dog at samples. The third dog of this group (dog #11) showed anti-Leishmania
16-wpi is summarized in Table 2. At 16-wpi, the assay detected a range IgG2 levels above the established cut off for saliva and serum. However,
of parasites from 0.02 × 10-3 parasites/ng of DNA (dog #10) up to 0.19 these values were lower than those in serum and most saliva samples
× 10-3 parasites/ng of DNA (dog #3) in saliva samples. DNA quantities from the DI group (Table 3). Median levels of anti-Leishmania IgG2 of the
in bone marrow samples were higher than in saliva, ranging from 0.02 × CI group at 47-wpi were 4.84 UFL (25th-75th percentiles: 4.84 - 427.3
10-3 parasites/ng of DNA (dogs #7 and #8) up to 45.11 × 10-3 parasites/ UFL) and 17.22 UFL (25th-75th percentiles: 16.7-96.6 UFL) in serum
ng of DNA (dog #10). When amplification was observed in saliva and and saliva, respectively. Concentrations of APPs are represented in
bone marrow samples, the differences in parasitic load found between Fig. 2. For this group, median concentrations of APPs above the refer­
both samples of the same animal vary from 59.7-fold (dog #12) up to ence range were observed in CRP at 47-wpi (24.7 μg/mL; 25th-75th
2,256-fold higher (dog #10). A significant but low correlation between percentiles: 2.5-93.3 μg/mL) in two dogs; and in ferritin at 16-wpi
the parasite load found in paired saliva and bone marrow samples at 16- (171.55 μg/L; 25th-75th percentiles: 134.8-178.4 μg/L), with 2 dogs
wpi was found (r = 0.285; p < 0.05). above the reference value at 16-wpi and one dog at 47-wpi.
Parasite load in both saliva and bone marrow samples of each dog at For the DI group (n = 9), all dogs were asymptomatic at the pre-i time
47-wpi is summarized in Table 3. At 47-wpi, the assay detected a range and at 16-wpi (Table 2). However, three dogs presented mild clinical
of parasites from 0.05 × 10-3 parasites/ng of DNA (dog #4) up to 61.36 signs compatible with CanL at 47-wpi (Table 3). At 47-wpi, all dogs
× 10-3 parasites/ng of DNA (dog #1) in saliva samples. DNA quantities showed anti-Leishmania IgG2 values in serum and saliva above the
in bone marrow samples were higher than in saliva, ranging from 8.39 × established cut-off values (Table 3). Median levels of anti-Leishmania
10-3 parasites/ng of DNA (dog #2) up to 7,843.73 × 10-3 parasites/ng of IgG2 at 47-wpi were 1,481 UFL (25th-75th percentiles: 807.9-2,386
UFL) in serum and 235.3 UFL (25th-75th percentiles: 122.4-400.9
UFL) in saliva. Regarding levels of anti-Leishmania IgG2 in serum and
Table 2
saliva for CI and DI groups at 47-wpi, only a significant correlation be­
Ct values and parasite load by qPCR and clinical signs of the experimentally
infected dogs at 16-wpi. tween both groups was observed in serum (r = -0.866; p < 0.0001).
Concentrations of APPs are represented in Fig. 2. Median concentrations
Saliva (250 ng of DNA/ Bone marrow (250 ng of
of APPs above the reference values were observed in CRP at 47-wpi
reaction) DNA/reaction) Clinical
Dog (19.0 μg/mL; 25th-75th percentiles:10.1-50.8 μg/mL), with one dog
Ct Parasites/ng of Ct Parasites/ng of signs
above the reference values at 16-wpi and 6 dogs at 47-wpi; in ferritin at
value DNA ×103 value DNA ×103
16-wpi (207.8 μg/L; 25th-75th percentiles: 151-271.2 μg/L), with 7 dogs
1 34.88 0.10 26.59 13.07 - above the reference values, and at 47-wpi (483.2 μg/L; 25th-75th per­
2 NAC NAC NAC NAC -
3 33.82 0.19 24.50 44.33 -
centiles: 437.7-716.2 μg/L), with all dogs above the reference values; in
4 NAC NAC 27.01 10.23 - total proteins at 47-wpi (7.3 g/dL; 25th-75th percentiles: 6.4-7.9 g/dL),
5 34.80 0.11 26.56 13.31 - with 5 dogs above the reference values; and in globulins at 47-wpi (4.7
6 NAC NAC 28.59 4.06 - g/dL; 25th-75th percentiles: 3.6-5.5 g/dL), with 6 dogs above the
7 NAC NAC 37.90 0.02 -
reference values (Fig. 2). Significant differences in the concentration of
8 NAC NAC 37.34 0.02 -
9 NAC NAC 26.32 15.31 - APPs at 47-wpi between CI and DI groups were only observed for total
10 38.00 0.02 24.47 45.11 - proteins (p < 0.01) and globulins (p < 0.05).
11 NAC NAC 34.65 0.12 - For the entire data set, anti-Leishmania IgG2 antibodies were detec­
12 34.66 0.12 27.62 7.16 - ted in both serum and saliva of 10 out of 12 (83.3%) experimentally
UFL: Units of Fluorometry for Leishmania; NAC: no amplification curve detected; infected dogs at 47 wpi. At 47-wpi, the IgG2 UFL values between serum
In bold data of the CI group. and saliva samples had a significant correlation (r = 0.851, p < 0.001)

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A. Cantos-Barreda et al. Comparative Immunology, Microbiology and Infectious Diseases 73 (2020) 101542

Table 3
Parasite load by qPCR and anti-Leishmania IgG2 levels by time-resolved immunofluorometric assay (TR-IFMA) of the experimentally infected dogs at 47-wpi.
q-PCR TR-IFMA (UFL)

Dog Saliva (125 ng of DNA/reaction) Bone marrow (250 ng of DNA/reaction) Clinical signs
Saliva Serum
Ct value Parasites/ng of DNA ×103 Ct value Parasites/ng of DNA ×103

1 25.13 61.36 15.64 7843.73 113.31 1481.25 Weight loss

2 31.25 1.72 27.35 8.39 235.33 968.04 Weight loss
3 30.47 2.71 26.65 12.62 215.19 647.74 -
4 38.00 0.05 22.60 134.50 82.25 613.04 -
5 29.56 4.61 19.73 719.22 536.18 2202.44 -
6 30.87 2.15 15.84 6978.80 359.68 2568.67 -
7 NAC NAC NAC NAC 16.67 4.84 -
8 NAC NAC NAC NAC 17.22 4.84 -
9 35.42 0.15 21.27 292.52 442.08 2048.19 -
10 28.83 7.07 23.49 79.97 131.40 1055.84 -
11 NAC NAC NAC NAC 96.56 427.28 -
12 32.66 0.75 20.05 596.59 351.34 2737.93 Weight loss, exfoliative dermatitis < 10% body surface

UFL: Units of Fluorometry for Leishmania; NAC: no amplification curve detected; In bold data of the CI group.

Fig. 1. Linear regression and correlation plots.

(A) correlation between parasite load in bone
marrow and saliva, (B) correlation between UFL
values in saliva and serum, (C) correlation be­
tween parasite load in saliva and UFL values in
saliva, (D) correlation between parasite load in
saliva and UFL values in serum, (E) correlation
between parasite load in bone marrow and UFL
values in saliva, and (F) correlation between
parasite load in bone marrow and UFL values in
serum. r = correlation coefficient; p = signifi­
cance level. P < 0.05 was considered statisti­
cally significant.

5
A. Cantos-Barreda et al. Comparative Immunology, Microbiology and Infectious Diseases 73 (2020) 101542

Fig. 2. Concentrations of acute phase proteins in sera of the “control of infection” (CI) and “dissemination of infection” (DI) groups of dogs on the pre-infection time
(pre-i) and at 16- and 47-weeks post-infection (wpi). The plot shows median (line within box), 25th and 75th percentiles (box), minimum (5th percentiles) and
maximum (95th percentiles) (whiskers). Significant differences between sampling times and groups of animals are represented by asterisks (*p < 0.05, **p < 0.01,
****p < 0.0001). A horizontal dashed line represents the reference value for each APP [C-reactive protein (CRP): < 12μg/mL; ferritin: 90-160 μg/L; albumin: 2.5-3.6
g/dL; total proteins: 5.4-7.2 g/dL; globulins: 2.6-3.8 g/dL].

(Fig. 1B). No significant correlation between the parasite load in saliva (Fig. 1D) and also between the parasite load in bone marrow and the
and the IgG2 UFL values in saliva was found (r = 0.514, p = 0.09) IgG2 UFLs in saliva (r = 0.634, p < 0.05) (Fig. 1E) and serum (r = 0.854,
(Fig. 1C). However, significant correlations were observed for the p < 0.001) (Fig. 1F). Regarding the APPs concentrations, significant
parasite load in saliva and the IgG2 UFLs in serum (r = 0.607, p < 0.05) correlations between sampling times for each APP were observed for

6
A. Cantos-Barreda et al. Comparative Immunology, Microbiology and Infectious Diseases 73 (2020) 101542

CRP between pre-i and 16-wpi (r = -0.655, p < 0.05), for total proteins One strength of our study is the possibility of taking advantage of
between pre-i and 16-wpi (r = 0.715, p < 0.05), and for globulins be­ experimentally infected dogs, opening the possibility to monitor the
tween pre-i and 16-wpi (r = 0.606, p < 0.05). kinetics of the parasite load in the tissues analyzed during the course of
infection. The sampling points for evaluating the parasite load were set
4. Discussion at 16- and 47-wpi, in accordance with a previous report in which the
L. infantum parasite load was evaluated at 120 and 360 days post-
There are many qPCR assays developed for diagnosing Leishmania infection [23]. On 16-wpi, one dog from our study did not show
infection in dogs. However, most of them use sample sources obtained amplification in the bone marrow and saliva by qPCR, but amplification
by invasive procedures, such as blood, bone marrow, spleen, lymph was observed at 47-wpi, similarly to that observed in a previous
node, or skin [9,10,25]. This fact can difficult owners’ acceptance to experimental CanL study [44]. On 47-wpi, 3 out of the total 12 dogs did
perform the diagnosis, increase efforts by veterinarians for sample not show amplification in bone marrow and saliva by qPCR. These
collection, and therefore decrease the use of qPCR assays in the diag­ findings are in line with those of the latest referenced study, in which no
nostic routine. The presence of Leishmania DNA has previously been parasites were detected in bone marrow of 2 out of 6 dogs along the
described in non-invasive samples such as hair, conjunctival swabs, course of a one-year post-infection [44]. So, in this study, we detected
nasal swabs, vulvar swabs, or oral swabs of dogs [6,13,14,23]. Leishmania kDNA in paired bone marrow and saliva samples in 9/12
Regarding PCR on oral swabs, contradictory opinions have been re­ dogs on 47-wpi, which were classified as dogs with dissemination of
ported. Lombardo et al. [7] reported that qPCR on oral swab seems not infection. Whereas, the remaining 3 dogs with no Leishmania kDNA
to be a much sensitive method for the detection of Leishmania DNA in detection in both samples and levels of IgG2 in serum and saliva below
dogs without clinical leishmaniosis. However, de Almeida Ferreira et al. or near the cut-off were considered resistant to infection, according to
[14] reported that conventional PCR on oral swab showed a high po­ the LeishVet guidelines [3]. The fact that no parasites were detected
tential to diagnose Leishmania infection in dogs with clinical leishma­ after experimental infection could be associated with a control induced
niosis because of its high positive indices, equivalent to those obtained by the triggered immune response, probably associated with a Th1
from lymph node or bone marrow. Also, Aschar et al. [13] concluded profile [45]. Two of these three dogs, were also Leishmania-seronegative
that oral swab was a suitable sample for qPCR diagnosis of Leishmania in serum and saliva, while one dog still remains seropositive probably
infection in dogs with clinical leishmaniosis. In that study, oral swab due to anti-Leishmania antibody levels that are more persistent on time
showed high positive indices, even higher than those obtained from after infection. In addition, these Leishmania-negative dogs did not show
blood. Hernández et al. [23] performed qPCR on oral swabs from dogs clinical signs or clinicopathological abnormalities in the concentration
with clinical leishmaniosis and suggested that a combination of qPCR on of APPs. These proteins are components of the innate immune system
oral swab with a serological test can improve sensitivity and specificity response and change in concentration when tissue injury occurs, such as
for early diagnosis. The main challenges for the use of non-invasive inflammation [46], so we decided to include their measurement in the
samples, such as oral swab, in PCR assays seems to be related to the study. APPs concentrations in dogs from the CI group at 47-wpi were
lower sensitivity and lower parasite amount of these samples in com­ above the reference values of our laboratory in two dogs for CRP and one
parison with those obtained throught invasive procedures. However, dog for ferritin, in contrast to the dogs from the DI group, for which most
these reports [13,14,23] considered that oral swabs are suitable samples dogs showed high concentrations of CRP and ferritin. As a result, con­
for the detection of canine leishmaniosis. centrations of APPs in the CI group suggest that CanL was not active in
The present study aimed to analytically and clinically validate a these dogs.
TaqMan qPCR assay to quantify Leishmania kDNA in saliva samples of The TaqMan qPCR results derived from our study showed that
dogs. Saliva collection has the advantages of being non-invasive and sensitivity in the saliva samples could be equal to bone marrow aspirates
painless. Thus, simple, cheap, and repeated samplings at short-time in­ on 47-wpi (κ = 1; 95% CI: 1.000 – 1.000), although the parasite load
tervals following limited training levels could be performed, which fa­ detected in the saliva was lower than in bone marrow. These results are
cilitates the continuous monitoring of the animal [36,37]. Furthermore, in line with previous studies that found higher parasite load in bone
this procedure could have a big impact on screening programs, espe­ marrow than in the non-invasive samples analyzed, including oral swab
cially in areas where the infrastructure is poor or unavailable [22]. The [23] and found the same sensitivity in oral swab and bone marrow
potential of saliva as a non-invasive alternative sample to blood in the (79%) by conventional PCR [14]. Due to the lower parasite load found in
serological and molecular diagnosis of leishmaniosis has been reported saliva samples compared to bone marrow, we would recommend, as we
in the veterinary field as well as in human medicine [17,18,20]. In did in our study, to use the phenol/chloroform method for gDNA
addition, the detection of parasites in saliva could have an important extraction since this method provides higher DNA yield than commer­
epidemiological implication since it has been evidenced cases of parasite cial kits for nucleic acid purification.
transmission among infected dogs through bites and saliva [13,38–42]. Saliva samples have several advantages derived from its non-
The analytical validation of our assay showed a sensitivity of 0.0025 invasive collection, but limitations have also been pointed out when
parasites/reaction, similar to those previously reported to detect Leish­ using saliva as a sample source for diagnostic purposes, such as the lower
mania kDNA in humans [5], dogs [6], and wild Leporidae [24]. In sensitivity of the qPCR and the lower parasite load detected when
addition, performance characteristics of the qPCR obtained in this study compared to bone marrow. Our results suggest that saliva could be a
were considered acceptable [27], with a PCR efficiency between complementary sample to bone marrow for the diagnosis of Leishmania-
87.6%-93.8%, r2 between 0.99 and 0.996, and repeatability of 0.67, positive dogs by qPCR.
similarly to a previous study in which L. infantum DNA was found in the
hair of wild Leporidae [24]. The PCR efficiency of our assay is near the 5. Conclusion
lower limit of an acceptable E (90-110%). Our E results could be due to
several variables affecting the performance of the PCR, e.g. PCR in­ The qPCR used in this study is able to detect and quantify L. infantum
hibitors, PCR enhancers, DNA degradation, DNA concentration, length kDNA in the saliva of dogs. Our study reports that bone marrow is more
of the amplicon, secondary structure or primer quality [27]. The Leish­ sensitive than saliva to detect L. infantum kDNA in the early stages of the
mania kDNA minicircle (10,000 copies per parasite) was chosen as the infection. Although bone marrow shows higher parasite loads, saliva
molecular target due to its high copy number into the Leishmania samples have similar sensitivities in later stages of the infection.
genome [5]. This molecular target has been widely used for the diag­ Therefore, saliva could be used as a sample to detect and quantify
nosis of Leishmania infection due to being considered the most sensitive, L. infantum kDNA, taking into account its diagnostic limitations
offering detection of about 104 copies per cell [6,24,43]. compared to bone marrow.

7
A. Cantos-Barreda et al. Comparative Immunology, Microbiology and Infectious Diseases 73 (2020) 101542

Funding endemicity using PCR on several tissues and serology, J. Clin. Microbiol. 39 (2001)
560–563, https://doi.org/10.1128/JCM.39.2.560-563.2001.
[12] G. Miró, L. Cardoso, M.G. Pennisi, G. Oliva, G. Baneth, Canine leishmaniosis - new
This work was funded by the Seneca Foundation-Regional Agency for concepts and insights on an expanding zoonosis: part two, Trends Parasitol. 24
Science and Technology of the Government of Murcia, Spain [19894/ (2008) 371–377, https://doi.org/10.1016/j.pt.2008.05.003.
GERM/15]. ACB was supported by a research contract [21327/PDGI/ [13] M. Aschar, E.T.B. de Oliveira, M.D. Laurenti, M. Marcondes, J.E. Tolezano, R.
M. Hiramoto, C.E.P. Corbett, V.L.R. Da Matta, Value of the oral swab for the
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the Seneca Foundation-Regional Agency for Science and Technology of infantum, Vet. Parasitol. 225 (2016) 108–113, https://doi.org/10.1016/j.
the Government of Murcia, Spain and the European Social Fund. DE was vetpar.2016.06.005.
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supported by the Spanish Ministry of Economy, Industry, and Compet­ R. de Andrade, M.N. Melo, Nasal, Oral and Ear Swabs for Canine Visceral
itiveness [grant number IJC2018-035105-I]. MCT, RNAL, and MCL were Leishmaniasis Diagnosis: New Practical Approaches for Detection of Leishmania
supported by the Spanish Ministry of Economy, Industry, and Compet­ infantum DNA, PLoS Negl. Trop. Dis. 7 (2013) 1–8, https://doi.org/10.1371/
journal.pntd.0002150.
itiveness [grant numbers RTC-2016-50005-1, SAF2016-80998-R] and [15] F. Corvalan, R. Sampaio, Y. Brustoloni, A.R.M. Lima Júnior, DNA identification of
the ISCIII and FEDER [RD16/0027/0005]. Leishmania (Viannia) braziliensis in human saliva from a patient with American
cutaneous leishmaniasis, 17, 2011, pp. 98–102.
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Declaration of Competing Interest Short Report : Detection of Leishmania siamensis DNA in Saliva by Polymerase
Chain Reaction, Am. J. Trop. Med. Hyg. 89 (2013) 899–905, https://doi.org/
10.4269/ajtmh.12-0612.
The authors report no declarations of interest.
[17] P. Siriyasatien, S. Chusri, K. Kraivichian, N. Jariyapan, T. Hortiwakul,
K. Silpapojakul, A.M. Pym, A. Phumee, Early detection of novel Leishmania species
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tion of “Servicio de Apoyo a la Investigación”, University of Murcia, Leishmania DNA in saliva among patients with HIV/AIDS in Trang Province,
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