Advantages of real-time PCR assay for diagnosis

and monitoring of canine leishmaniosis

O. Francino a,*, L. Altet a, E. Sanchez-Robert a, A. Rodriguez c,L. Solano-Gallego c, J. Alberola c, L. Ferrer d, A. Sanchez a, X. Roura b

a Servei Veterinari de Genetica Molecular, Facultat de Veterinaria, Universitat Autonoma de Barcelona,

08193 Bellaterra, Barcelona, Spainb Hospital Clınic Veterinari, Facultat de Veterinaria, Universitat Autonoma de Barcelona,

08193 Bellaterra, Barcelona, Spainc Departament de Farmacologia, Terapeutica i Toxicologia, Facultat de Veterinaria,

Universitat Autonoma de Barcelona, 08193 Bellaterra, Barcelona, Spaind Departament de Medicina i Cirurgia Animals, Facultat de Veterinaria,

Universitat Autonoma de Barcelona, 08193 Bellaterra, Barcelona, Spain

Received 13 October 2005; received in revised form 23 December 2005; accepted 10 January 2006

Abstract

The aim of the present study is to highlight the advantages of real-time quantitative PCR intended to aid in the diagnosis and

monitoring of canine leishmaniosis. Diagnosis of canine leishmaniosis is extremely challenging, especially in endemic areas,

due to the diverse and non-specific clinical manifestations, and due to the high seroprevalence rate in sub-clinical dogs.

Veterinarian clinicians are usually confronted with cases that are compatible with the disease, and with several diagnostic tests,

sometimes with contradictory results. We have developed a new TaqMan assay, targeting the kinetoplast, applied to 44 samples

of bone marrow aspirate or peripheral blood. The dynamic range of detection of Leishmania DNA was established in 7 logs and

the limit of detection is 0.001 parasites in the PCR reaction. At the time of diagnosis parasitemia ranges from less than 1 to

107 parasites/ml. The ability to quantify the parasite burden allowed: (i) to elucidate the status of positive dogs by conventional

PCR, although larger studies are necessary to clarify the dividing line between infection and disease, (ii) to estimate the kinetics

of the parasite load and the different response to the treatment in a follow-up and (iii) to validate blood as less invasive sample for

qPCR. The continuous data provided by real-time qPCR could solve the dilemma for the clinician managing cases of canine

leishmaniosis by differentiating between Leishmania-infected dogs or dogs with active disease of leishmaniosis.

# 2006 Elsevier B.V. All rights reserved.

Keywords: Leishmania infantum; Real-time PCR; Parasite quantification; Dog

www.elsevier.com/locate/vetpar

Veterinary Parasitology 137 (2006) 214–221

* Corresponding author. Tel.: +34 93 5811398; fax: +34 93 5812106.

E-mail address: Olga.Francino@uab.es (O. Francino).

0304-4017/$ – see front matter # 2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.vetpar.2006.01.011

O. Francino et al. / Veterinary Parasitology 137 (2006) 214–221 215

1. Introduction

Canine leishmaniosis (CL) is a severe systemic

infectious disease of the dog caused by protozoan

parasites of the genus Leishmania. It is a zoonosis with

the dog considered the main peridomestic reservoir of

the parasite (Slappendel, 1988; Molina et al., 1994;

Slappendel and Ferrer, 1998; Herwaldt, 1999; Moreno

and Alvar, 2002). CL is endemic in the Middle East,

South America and in the Mediterranean basin, where

the infection reaches an outstanding prevalence of

67%, although the prevalence of the disease is only

about 10% (Solano-Gallego et al., 2001). Clinical

features vary widely as a consequence of the numerous

pathogenic mechanisms involved in the disease

process and the diversity of the immune responses

exhibited in front of Leishmania infantum by

individual hosts either in humans or other mammals

(Solbach and Laskay, 2000). For that reason, diagnosis

of CL is extremely challenging due to the diverse and

non-specific clinical manifestations, and due to the

high seroprevalence rate in sub-clinical and asympto-

matic dogs in endemic areas (Berrahal et al., 1996;

Solano-Gallego et al., 2001).

In clinical practice, the veterinarian is usually

confronted with cases that are compatible with, or

suggestive of, CL according to the symptoms, and

with several diagnostic tests, sometimes with contra-

dictory results. This has lead to the development and

application of conventional and nested polymerase

chain reaction (PCR) to diagnose CL (Roura et al.,

1999; Fisa et al., 2001). However, since one of the

features of leishmaniosis is to have residual or latent

parasites after treatment, quantitative approaches are

necessary not only to elucidate the status of positive

PCR dogs in endemic areas, but also in the monitoring

of the parasitemia in post-treatment follow-up and in

the new vaccines development or drug trials.

Real-time PCR detection is replacing the conven-

tional PCR and nested PCR methods in the diagnosis

and follow-up of many diseases, providing the ability

to perform very sensitive, accurate and reproducible

measurements of specific DNA present in a sample,

even for Leishmania sp. parasites (Bretagne et al.,

2001; Bell and Ranford-Cartwright, 2002; Nicolas

et al., 2002; Bossolasco et al., 2003; Schulz et al.,

2003; Svobodova et al., 2003; Mary et al., 2004; Vitale

et al., 2004). The aim of the present study is to

highlight the advantages of a new real-time PCR assay

intended to aid in the diagnosis and monitoring of CL,

especially in endemic areas.

2. Materials and methods

2.1. Animal samples

The present study includes 44 dogs visiting

veterinarian hospitals with clinical signs compatible

with CL that have been evaluated at the Molecular

Genetics Veterinary Service by conventional PCR on

EDTA-bone marrow aspirate (Roura et al., 1999; see

below). Four of the dogs that were positive by

conventional PCR (26–29) were included in a post-

treatment follow-up. EDTA-bone marrow aspirate

were obtained every 3 months and were analyzed by

the Leishmania TaqMan assay to compare the results

to those obtained by conventional PCR. For 15 dogs

(30–44), EDTA-anti-coagulated blood was also

obtained at the same time than the EDTA-bone

marrow aspirate. In all the cases bone marrow

aspirates and/or peripheral blood samples were

obtained with the consent of the dog owners and

preserved at 4 8C until processed for a maximum of 1

week.

2.2. DNA isolation

DNA was obtained from 0.1 ml of bone marrow

aspirate or 0.5 ml of peripheral whole blood as

previously described (Roura et al., 1999). Briefly,

samples were washed in TE buffer pH 8.0 to disrupt

the erythrocyte membrane until the leukocyte pellet

was white. Leukocytes were then lysed by incubation

of the pellet in 0.1 ml of PK buffer (50 mM KCl,

10 mM Tris pH 8.0, 0.5% Tween-20 and 23 mg of

proteinase K) at 56 8C for 5 h. Before running the

PCR, proteinase K was inactivated by incubation of

the samples at 90 8C for 10 min. DNA was diluted in

milliQ water (1/10 for bone marrow aspirate and 1/5

for blood) and 5 ml were used for the PCR.

2.3. Conventional PCR

L. infantum specific oligonucleotide primers N13A

(50-AACTTTTCTGGTCCTCCGGG-30) and N13B

O. Francino et al. / Veterinary Parasitology 137 (2006) 214–221216

(50-CCCCCAGTTTCCCGCCC-30) were used to

amplify a 120-base-pair fragment of the Leishmania

kinetoplast DNA minicircle. PCR was conducted in a

20 ml final reaction mixture containing PCR buffer

�1, 0.150 mM dNTPs, 2 mM MgCl2, 0.2 mM of each

primer and 1U Taq Polymerase (EcoTaq, Ecogen).

The thermal cycling profile was as follows: 94 8C for

3 min, followed by 35 cycles at 94 8C for 30 s, 58 8Cfor 30 s and 72 8C for 30 s; with a final extension at

72 8C for 5 min. To ensure that negative results

corresponded to true negative samples rather than to a

problem with DNA loading, sample degradation, or

PCR inhibition, sample DNA was also amplified for

b-actin by using a forward primer (50-GACAGGATG-

CAGAAGGAGAT-30) and a reverse primer (50-TTG-

CTGATCCACATCTGCTG-30) at 0.3 mM of each

primer in the same conditions as above. Amplified

fragments were analyzed by electrophoresis in a 3%

agarose gel containing ethidium bromide (0.5 mg/ml).

2.4. Real-time quantitative PCR amplification

TaqMan-MGB probe and PCR primers were

designed to target conserved DNA regions of the

kinetoplast minicircle DNA from L. infantum. Primers

LEISH-1 (50-AACTTTTCTGGTCCTCCGGGTAG-

30) and LEISH-2 (50-ACCCCCAGTTTCCCGCC-30)are slight modifications of the N13A and N13B to be

run under universal conditions in the TaqMan assay.

The TaqMan-MGB probe (FAM-50-AAAAATGGGT-

GCAGAAAT-30-non-fluorescent quencher-MGB) was

designed to target a conserved region of the

kinetoplast with Primer Express 2.0 (Applied Bio-

systems, Foster City, CA). The eukaryotic 18S RNA

Pre-Developed TaqMan Assay Reagents (Applied

Biosystems, Foster City, CA) were used as internal

reference of canine genomic DNA.

Leishmania primers and probe were added at 900

and 200 nM, respectively. Duplicates were amplified

for each sample, both with the Leishmania and the 18S

RNA assays, in a 25 ml final volume reaction mixture

with the TaqMan Universal PCR Master Mix with

UNG Amperase to avoid carry-over contamination

(Applied Biosystems, Foster City, CA). The thermal

cycling profile was 50 8C for 2 min, 95 8C for 10 min,

40 cycles at 95 8C for 15 s and 60 8C for 1 min. Each

amplification run contained positive and negative

controls.

2.5. Sensitivity and efficiency of the Leishmania

TaqMan-MGB assay

Three replicates of 10-fold serial dilutions of

parasite DNA obtained from a culture of L. infantum

(MHOM/FR/78/LEM-75) were used to asses the

sensitivity and efficiency of the TaqMan Leishmania

assay. Efficiency was also tested in seeded samples

prepared with 10-fold serial dilutions of the parasite

DNA spiked over replicates of a negative Leishmania

blood sample.

2.6. Quantification of Leishmania parasites

The spiked samples with a known number of

parasites/well were used as calibrators by the

comparative Ct method (2�DDCt), allowing determin-

ing the number of parasites in any PCR sample,

independently of the amount of DNA added or the

presence of inhibitors (Livak and Schmittgen, 2001).

3. Results

3.1. Sensitivity and linearity of the Leishmania

TaqMan-MGB assay

The real-time quantitative PCR TaqMan assay

described here for L. infantum targets the conserved

region in the kinetoplast minicircle DNA (about

10,000 copies) (Rodgers et al., 1990; Weiss, 1995;

Wilson, 1995). The dynamic range of detection of

Leishmania DNA was established in 7 logs and the

limit of detection is 0.001 parasites in the PCR

reaction, with a correlation of 0.99.

3.2. Real-time qPCR versus conventional PCR in

CL

We have analyzed 25 samples of bone marrow

aspirates from dogs that have been previously

evaluated by conventional PCR, in order to compare

the results (Fig. 1). Parasite DNAwas detected in 21 of

the samples analyzed, in a range from 0.001 to 3400

parasites in the PCR, which corresponds to 0.2 para-

sites/ml of sample (dog 5) up to 6,800,000 parasites/

ml of sample (dog 25). Conventional PCR, routinely

used for the diagnosis of the disease, was negative for

O. Francino et al. / Veterinary Parasitology 137 (2006) 214–221 217

Fig. 1. Comparative results obtained in 25 bone marrow aspirates analyzed by conventional and real-time PCR. White bars correspond to

negative conventional PCR samples and black bars correspond to positive conventional PCR samples. y-Axis shows the number of parasites/ml

obtained by real-time PCR for each sample in a log-scale.

dogs with less than 30 parasites/ml of sample, and was

positive in all the cases above this range.

3.3. Evolution of the parasite load in the

treatment follow-up

Bone marrow aspirates of 4 dogs that were positive

to Leishmania by conventional PCR at the time of

diagnosis were obtained at different times in a post-

Fig. 2. Progression of parasitemia in four animals in the post-treatment fol

the x-axis. The number of parasites/ml of bone marrow aspirates obtained fo

scale. Bold line indicates negative or positive result for conventional PCR

treatment follow-up. Fig. 2 shows the results obtained

for each point both by conventional PCR and by the

real-time Leishmania qPCR assay. Dogs 26 and 29

showed a gradual decrease in parasite load by real-

time qPCR, and were negative by conventional PCR

after 3 (dog 29) or 6 (dog 26) months of treatment. On

the other hand, dogs 27 and 28, which were positive by

conventional PCR in each point of analysis, show

different kinetics of the parasite. Whereas dog 27

low-up. The different time-points of the follow-up are represented in

r each sample in each time-point is represented in the y-axis, in a log-

.

O. Francino et al. / Veterinary Parasitology 137 (2006) 214–221218

Fig. 3. Comparison between peripheral blood samples (white bars) and bone marrow aspirates (black bars) obtained at the same time from 15

dogs. The number of parasites/ml of sample is represented in the y-axis, in a log-scale. Bold line indicates negative or positive result for

conventional PCR.

showed a gradual decrease of the parasite load after 3

months of treatment, dog 28 initially responded to the

treatment with a drastic decrease of the parasite load,

followed by a drastic increase in the parasite burden,

which was concomitant with a relapse of the disease.

3.4. Detection of Leishmania in peripheral blood

samples

EDTA-anti-coagulated peripheral blood and bone

marrow aspirate samples from dogs 30 to 44 were

obtained at the same time point and were submitted for

conventional L. infantum PCR. All the samples were

also analyzed by the real-time qPCR assay in order to

compare the parasite load for each animal in both

tissues and to validate the peripheral blood as a

suitable sample for Leishmania detection. All the dogs

were positive for Leishmania qPCR (Fig. 3), with

parasite load ranging from 7 parasites/ml of sample

(dog 31) to 14,055 parasites/ml of sample (dog 44) in

peripheral blood samples and from 13 parasites/ml

(dog 30) to 12,287,840 parasites/ml (dog 44) in bone

marrow aspirates. The differences in the parasite load

obtained between bone marrow aspirate and periph-

eral blood of the same animal ranged from 4-fold (dog

31) up to 5000-fold higher (dogs 41 and 42), with the

exception of dogs 30 and 32, most likely due to these

animals were at the stage of parasite spread.

4. Discussion

The aim of the present study was to validate the

real-time quantitative PCR (qPCR) application in

order to address both the problem associated to

conventional PCR in the diagnosis of canine

leishmaniosis, and the monitoring of the parasitemia

in the post-treatment follow-up and in the new

vaccines development or drug trials.

In endemic areas the interpretation of the results

of some diagnostic test for Leishmania faced up

with the seroprevalence in sub-clinical dogs, and

clinicians in veterinary medicine are looking toward

PCR based methods as routine. On the other hand,

previous studies highlighted that a large part of the

dogs living in an area where canine leishmaniosis is

endemic (Mallorca, Balearic Islands) are infected by

Leishmania, and the prevalence of infection is

higher than the prevalence of the disease: 67% of

the animals were seropositive and/or positive by

conventional PCR whereas the prevalence of the

disease was 13% (Solano-Gallego et al., 2001).

Therefore, a positive result in conventional PCR can

also be obtained in sub-clinical and asymptomatic

dogs, especially in endemic areas, since canine

leishmaniosis is associated with tissue loads of

residual parasites (Berrahal et al., 1996; Solano-

Gallego et al., 2001).

O. Francino et al. / Veterinary Parasitology 137 (2006) 214–221 219

We have developed a real-time Leishmania Taq-

Man assay in order to overcome this situation, taking

advantage of the continuous data provided by real-

time qPCR, thus allowing quantifying the parasite

burden obtained for each sample as number of

parasites. We have chosen a qPCR design based on

TaqMan probe and targeting the minicircle kinetoplast

in order to maximize the sensitivity of the assay. The

sensitivity of 0.001 parasites per PCR reaction

obtained with the TaqMan assay is similar to that

recently described for Leishmania infection in humans

(Mary et al., 2004), and it is higher than that reported

in previous works due to both the target (kinetoplast)

and the TaqMan probe (Bretagne et al., 2001; Nicolas

et al., 2002; Bossolasco et al., 2003; Schulz et al.,

2003; Svobodova et al., 2003; Mary et al., 2004; Vitale

et al., 2004). Moreover, the 7 logs linear dynamic

range allows us to discriminate from 0.01 to 10,000

Leishmania parasites in a single reaction, which

corresponds to less than 1 parasite/ml to more than

107 parasites/ml of sample. Although parasitemia

below 1 parasite/ml would be inferior to the theore-

tical threshold corresponding to one parasite present in

the clinical sample submitted to extraction, similar

results have been reported in human samples. It could

be due to either an impaired extraction yield when

parasitemia is very low or to the detection of residual

parasite DNA that persist for a time inside the

macrophage after parasite destruction (Mary et al.,

2004).

The quantification of parasites by real-time qPCR

could be used to elucidate the status of dogs that are

positive for Leishmania by conventional PCR,

especially in endemic areas. It is important to remark

that the resulting data in conventional PCR is a

discrete variable with only two possible values:

positive or negative, and even samples with 5 log

differences in parasite load will be positive by

conventional PCR, as it is shown in Figs. 1 and 3.

Taking into account: (i) that all the samples submitted

to Leishmania PCR analysis are from dogs showing

any clinical sign compatible with the disease, (ii) that

leishmaniosis clinical signs are diverse, non-specific

and compatible with other diseases and (iii) that in an

endemic area you can find dogs with a dubtous

serology that can be infected with the parasite but

without an active disease, the ability to discriminate

from 0.001 to 10,000 Leishmania parasites in a single

reaction could help in the clinical decision. Based on

our preliminary results (data not shown), we could

suggest a number of parasites indicating infection

status but not disease. The results obtained for most of

the samples analyzed by qPCR in our diagnostic

service are clear: parasite is not detected or parasite

load is more than 1000 parasites/ml. However, we

have analyzed samples from two dogs with a parasite

load of 60–80 parasites/ml of bone marrow aspirate.

Taking into consideration that at the time of diagnosis

parasite load could vary by a wide range, the

veterinarian clinician decided that it was a low

parasite load and kept the dogs under supervision, but

without specific treatment for the disease. Six months

later qPCR analysis was performed in bone marrow

aspirates from the same dogs and the parasite load

decreased to less than 1 parasite/ml of sample. In these

cases qPCR could solve the dilemma of treating or not

treating an animal by differentiating between Leish-

mania-infected dogs or dogs with active disease of

leishmaniosis, although larger studies should be

necessary to better clarify the dividing line between

infection and disease.

The real-time qPCR turned out to be also very

useful to follow-up the parasite load in order to

estimate the efficacy of the treatment or the evolution

of the disease, as it is shown in Fig. 2. Parasite load

decreases with the treatment to less than 10 parasites/

ml of bone marrow aspirate for two of the dogs

(26 and 29), and both of them were negative to

Leishmania by conventional PCR after 3 or 6 months

of treatment. On the other hand, positive results were

obtained by conventional PCR in all the time points

for the other two dogs, but kinetic of the parasite were

completely different as revealed by the real-time

qPCR assay. The parasite load began to decrease after

3 months of treatment for dog 27, reaching a value of

40 parasites/ml of bone marrow aspirate after 12

months. Dog 28, with the highest parasite load at the

time of diagnosis, responded to the initial treatment

with a drastic decrease of the parasite load (230,000 to

50 parasites/ml), and followed by an increase in the

parasite load that was concomitant with a relapse of

the disease. Previous studies in human leishmaniosis

in immunocompromised patients indicate that a

clinical relapse is associated to the level of

parasitemia, and estimated that an increase above

10 parasites/ml of blood preceded a clinical relapse

O. Francino et al. / Veterinary Parasitology 137 (2006) 214–221220

(Pizzuto et al., 2001; Bossolasco et al., 2003; Mary

et al., 2004). In a similar way, the implementation of

the qPCR follow-up after therapy will aid in the

prognosis of CL and also in the prediction of infection

relapses in the survey of at-risk dogs. Another

advantage of the qPCR in front of conventional

PCR is that qPCR permitted to monitor the progres-

sion of infection in a more accurate way and to better

evaluate the efficacy of the treatment for each dog,

even in a short time-course follow-up (Alberola et al.,

2004).

Blood might be sufficient for the diagnosis of

infection due to the ability of quantifying extremely

low levels of parasitemia by qPCR, as it is shown in

Fig. 3, and it may save the need to perform more

invasive bone marrow aspirations. However, parasite

load is different among tissues due to the fact that

Leishmania infection could be tissue-dependent in

dogs, either by tissue tropism (Solano-Gallego et al.,

2001; Reithinger et al., 2002) or by organ-specific

immunity (Sanchez et al., 2004). Thus, the selection of

the tissue to be analyzed is a matter to consider for the

veterinarian clinician. This real-time qPCR assay has

also been applied for routine diagnostic in lymph node

aspirate, fresh or paraffin-embebbed biopsies from

different tissue origin, urine and conjunctival swabs

(data not shown).

The results presented here highlight the advantages

that real-time qPCR assay offers in the diagnostic and

follow-up of canine leishmaniosis, especially in

endemic areas, where a large part of the canine

population is exposed to the parasite but only a smaller

proportion of the dogs develop clinical disease. The

continuous data provided by real-time qPCR could

solve the dilemma for the clinician managing cases of

canine leishmaniosis by differentiating between

Leishmania-infected dogs or dogs with active disease

of leishmaniosis.

Acknowledgements

This work was supported by the Veterinary

Molecular Genetics Service (Universitat Autonoma

de Barcelona). We are thankful to the veterinarian

clinicians for providing the samples used in this study

and we are also thankful to the anonymous reviewers

for their helpful reviews.

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