Veterinary Parasitology 186 (2012) 245–253

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Veterinary Parasitology
journal homepage: www.elsevier.com/locate/vetpar

One-year clinical and parasitological follow-up of dogs treated with

marbofloxacin for canine leishmaniosis
Sandrine Rougier a,∗ , Lilia Hasseine b , Pascal Delaunay b , Grégory Michel c , Pierre Marty b,c
a
Vétoquinol Laboratory, Magny Vernois, BP 189, 70204 Lure Cedex, France
b
Centre Hospitalier Universitaire de Nice, Parasitologie-Mycologie, Hôpital de l’Archet, 151 route de Saint Antoine de Ginestière, BP 3079,
06202 Nice Cedex 3, France
c
Equipe 6, Unité Inserm 895, Batiment Archimed, Université de Nice-Sophia Antipolis, Centre Méditerranéen de Médecine Moléculaire, BP 23194,
06204 Nice Cedex 3, France

a r t i c l e i n f o a b s t r a c t

Article history: The purpose of this international, multicentric, and non-comparative field trial was to
Received 1 February 2010 obtain complementary data on long-term clinical and parasitological follow-up of dogs
Received in revised form 19 October 2011 treated with marbofloxacin for canine leishmaniosis (CanL). Seventy-four dogs with clini-
Accepted 3 November 2011
cal signs of CanL and without severe renal failure were recruited in France, Spain and Italy,
and 61 of them were part of the analysis. Each dog was treated with palatable tablets of mar-
Keywords:
bofloxacin at 2 mg/kg once a day for 28 days. A clinical and parasitological follow-up was
Marbofloxacin
Canine leishmaniosis
performed regularly up to 12 months. Efficacy was demonstrated in 42 dogs (68.9%), within
Efficacy 51 days (mean value), 10 of them (23.8%) being clinically cured after 3 months. A decrease
Parasitic load of 61% in the sum of clinical scores was observed after 3 months. Haemato-biochemical
parameters improved in general, supporting the observed clinical efficacy. Relapse was
observed in 20/38 dogs (52.6%) approximately 5.5 months after treatment completion. The
blood parasite load generally developed in conformity with the clinical outcome, even if
exceptions were not rare. Lymph nodes remained positive by culture or PCR for a long
time, even in dogs for which a good clinical response was observed. Despite the incom-
plete parasite clearance, as is also the case with other anti-leishmanial drugs, these results
nevertheless confirm the relevance of marbofloxacin as a CanL treatment.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction treatment regimens have been tried for treatment of CanL,

Canine leishmaniosis (CanL) is a parasitic disease,
widespread in the Mediterranean basin. It is caused by a
protozoan parasite Leishmania infantum that is transmit-
ted by the bite of the sandfly of the genus Phlebotomus. It
affects both humans and animals, but the dog is the domes-
tic parasite reservoir.
The treatment of CanL still represents a challenge for
veterinarians. In past decades, many drugs and various
∗ Corresponding author. Tel.: +33 384625592; fax: +33 384625500.
E-mail address: sandrine.rougier@vetoquinol.com (S. Rougier).
with various efficacy results (Noli and Auxilia, 2005). Meg-
lumine antimoniate (Glucantime® , Mérial) is the oldest
registered product for this indication. However, due to
the decrease of sensitivity to antimonials (Gramiccia et al.,
1992; Faraut-Gambarelli et al., 1997; Carrio et al., 2001),
the registered dosage is no longer applied. Without any
consensus, numerous treatment regimens have been tried,
resulting in an inappropriate use of the product, possibly
at the origin of an increase in resistance already present
and an increased number of dogs in therapeutic failure.
Moreover, antimonials have side effects and can be toxic.
Lastly, a new product, miltefosine, has been registered in
many countries around the Mediterranean basin for the

0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetpar.2011.11.016
246 S. Rougier et al. / Veterinary Parasitology 186 (2012) 245–253
treatment of CanL but access to it is not yet possible in all
of them.
Marbofloxacin (Marbocyl® , Vétoquinol, Lure, France)
is a synthetic, third generation fluoroquinolone devel-
oped for veterinary use only. It presents potent activity
against several bacteria and is frequently used in the treat-
ment of a wide range of Gram-positive and Gram-negative
bacterial infections (Prescott et al., 2000; Meunier et al.,
2004). Like other quinolones, marbofloxacin inhibits the
bacterial enzyme DNA gyrase (or topoisomerase II), but
does not act on mammalian topoisomerases, due to their
structural differences from the bacterial topoisomerase
(Slunt et al., 1996; Prescott et al., 2000). Trypanosomatidae,
such as L. infantum, presents a genomic structure which
exhibits major similarities with bacteria (Slunt et al., 1996;
Chakraborty and Majumder, 1988).
Marbofloxacin previously demonstrated a direct and
indirect leishmanicidal activity via the TNF-␣ and NO
synthase pathways, and correlated with NO2 production
(Vouldoukis et al., 2006). Recent field studies have reported
encouraging results of a 28-day marbofloxacin treatment
for treating natural CanL which should, however, be con-
firmed on a larger population of dogs (Rougier et al., 2008).
The purpose of this study was to obtain complementary
data though a long-term (one year) clinical and parasito-
logical follow-up of dogs after they received a one-month
marbofloxacin treatment against CanL.
2. Materials and methods
2.1. Test population
Seventy-four dogs of both sexes and aged from 1 to
11 years were selected from veterinary clinics in France,
Spain and Italy between March and December 2006. All
of them presented with at least one clinical sign of canine
leishmaniosis such as cutaneous, gastro-intestinal or ocular
signs, weight loss or lymphadenomegaly. Moreover, to be
selected, dogs must not have received any antibiotic, corti-
costeroid or anti-leishmanial treatment within the 30 days
preceding inclusion or in the 2 months preceding inclu-
sion in the case of long-acting corticosteroids. In addition,
dogs with severe renal failure (defined as uraemia ≥ 1 g/L
and creatininaemia ≥ 20 mg/L) were not to be included.
Informed consent was obtained from the owners of all dogs
prior to their participation in this study.
The diagnosis had to be confirmed by parasitological
tests, by the presence of specific antileishmanial antibod-
ies evidenced by Western Blotting associated with positive
PCR or culture from blood, or also by direct examination
of amastigote forms or positive PCR from a lymph node
puncture. Therefore, as 4 dogs out of the selected popula-
tion did not present any positive parasitological tests, they
were excluded from the study after a few days. In addi-
tion, after blood analyses, 4 other dogs showed a positive
serology (≥1:200) for Ehrlichia canis and were then also
excluded from all analyses. Finally, 5 other dogs discontin-
ued follow-up during the first week for reasons unrelated
to treatment and were also excluded. Therefore, in total, 61
dogs were included and analysed in this study.
2.2. Treatments
All dogs received palatable tablets of marbofloxacin
(Marbocyl® P, Vétoquinol Laboratory, Lure, France) at the
dosage of 2 mg/kg once a day for 28 days.
Anti-inflammatory treatments (corticosteroids,
NSAIDs) and atropine, via local route only, were allowed
for managing ophthalmic lesions caused by Leishmania
infection. Vaccinations were allowed only after the first 3
months. In addition, use of repellent, insecticide spot-on
and collars against phlebotomine sandflies was recom-
mended during the one-year study in order to prevent a
leishmanial re-infection.
2.3. Schedule
All animals were examined at regular intervals during
the first 3 months of follow-up, on Days 0 (D0), 14 (D14),
28 (D28), 40 (D40), 56 (D56) and 84 (D84). Then, dogs were
re-examined at Months 6 (M6) and 12 (M12) in order to
assess a possible clinical relapse.
2.4. Clinical evaluations
At each examination, dogs were assessed for sys-
temic signs (weight loss, hyperthermia, listlessness
and lymphadenopathy), cutaneo-mucosal signs (alope-
cia/lunettes, squamosis/hyperkeratosis, seborrhoea, pyo-
dermatitis, ulcers, epistaxis, onychogryphosis and nod-
ule(s)), musculo-skeletal signs (limping/arthritis and amy-
otrophy), gastro-intestinal signs (diarrhoea, hepatomegaly,
splenomegaly, intestinal haemorrhage) and ocular signs
(conjunctivitis, keratitis, uveitis).
Each clinical parameter was classified according to its
severity on a numerical scale from 0 to 3, as follows:
0, absent; 1, mild; 2, moderate and 3, severe, except for
weight loss and hyperthermia which were numerical val-
ues and lymphadenomegaly, ulcers and nodules which
were scored as 0, absent; 1, local and 2, generalised.
The sum of the clinical scores was calculated by adding
the points given to each of the 19 listed clinical parameters
at each examination time.
2.5. Haemato-biochemical tests
All dogs were checked for complete blood count
(eosinophil and neutrophil count, lymphocyte count,
platelet count, haemoglobin concentration, mean corpus-
cular haemoglobin concentration (MCHC), mean corpus-
cular volume (MCV), packed cell volume (PCV), red and
white blood cell count (RBC and WBC respectively)), ALT,
AST, ALP, ␥-GT, urea, creatinine and protein electrophore-
sis (␣1-, ␣2-, ␤- and ␥-globulins, albumin, total proteins,
A/G ratio) on D0, D28, D56, D84, M6 and M12.
2.6. Parasitological examinations
In all animals included in this study, blood samples and
lymph node aspirates were taken for different parasitolog-
ical laboratory assays, each month during the first three
 S. Rougier et al. / Veterinary Parasitology 186 (2012) 245–253
months, and then at M12 or before in the case of clini-
cal relapse. Upon receipt, blood samples were divided into
two batches. One was stored at −20 ◦ C in order to perform
a kinetic serology and q-PCR. The second batch and lymph
node aspirates were analysed as soon as they were received
for serology, direct examination of amastigote forms, cul-
tures and conventional PCR.
2.6.1. Serologic tests
Serologic tests (Marty et al., 1994; Suffia et al., 1995)
consisted in:
- Western Blot (on D0 only): Immunoblots were scored as
positive when at least antibodies to the 14-kD and/or 18-
kD antigens of Leishmania were present.
- ELISA: Dog sera were analysed by enzyme-linked
immunosorbent assay (Leishmania ELISA IgG + IgM, Vir-
cell, T1000) using a lysate of L. infantum as antigen and
immunoblotting as previously described. The positive
cut-off level was fixed at 10 arbitrary units (AU).
2.6.2. Direct parasitological examinations
Appositions were made from lymph node aspiration and
stained with May-Grunwald Giemsa according to the con-
ventional method known.
2.6.3. Cultures
Cultures were made on NNN (Novy–McNeal–Nicolle)
medium and Schneider medium after cytocentrifugation
and stained with May-Grunwald Giemsa (Lima et al., 1997).
They were inoculated respectively with a cell suspension
equivalent to 2 ml of citrated blood and lymph node mate-
rial, and incubated at 25 ◦ C.
2.6.4. Polymerase chain reactions (PCRs)
PCR was performed on all blood samples, and on lymph
node samples only if the direct examination of amastigote
forms under microscopy was negative.
Total DNA was extracted from 0.5 to 5 ml of total blood
and lymph node aspiration using respectively QIAamp®
DNA maxi kit for DNA purification (Qiagen 51194) and
QIAamp® DNA mini kit for DNA purification (Qiagen
51304). The PCR technique used and the primers chosen
are those described by Le Fichoux et al. (1999).
The quantification of L. infantum DNA by q-PCR was per-
formed only from total blood samples on D0, D84, and M12
(or before in the case of early termination of follow-up),
according to a technique using QIAamp® DNA maxi kit for
DNA purification (Qiagen 51194). For accurate sensitivity,
kinetoplast DNA was chosen as the molecular target. The
sensitivity of this assay was 0.0001 parasite DNA equiva-
lents/reaction tube as sensitivity described by Mary et al.
(2004).
However, the conditions and the program of this q-PCR
were optimised using a LightCycler version 2.0 instru-
ment (Roche Applied Science). Light cycler Fast Start DNA
Master Hybrobes (cat. number 12 23 92 72001), Uracil
DNA glycosylase (Invitrogen 18054-015), 0.5 ␮M of direct
primer, 0.5 ␮M of reverse primer (MGW-Biotech France
SA) and 0.2 ␮M of TaqMan probe (TIB Molbiol) were
used. Assays were performed with a 20 ␮l final volume
247
using LightCycler glass capillaries (Roche 1909339) with a
5 ␮l sample of DNA. The standard curve was established
from Leishmania DNA extracted from 106 parasites; 5 ␮l
of serial dilutions, ranging from 10.000 to 0.001 parasites,
was introduced into reaction mixtures. TaqMan chemistry
allowed two-step temperature (94 and 60 ◦ C) cycling over
45 cycles.Canine glyceraldehydes-3-phosphate dehydro-
genase (GAPDH) gene in blood samples was also quantified.
GAPDH housekeeping gene was used to ensure that nega-
tive results corresponded to true negative samples rather
than to a problem with DNA loading, sample degrada-
tion, PCR inhibition or absence of canine cells. Reaction
mixtures contained LightCycler® Fast Start DNA Master
SYBRGreen (cat number 03 003 230 001), Uracil DNA gly-
cosylase (Invitrogen 18054-015), 1 ␮M each of forward and
reverse primers (Solano-Gallego et al., 2007). Assays were
performed with a 20 ␮l final volume using LightCycler glass
capillaries with 5 ␮l sample DNA. Thermal cycling was the
same as kinetoplast Leishmania q-PCR in order to perform
the two q-PCR on the same run.
2.7. Efficacy criteria
The therapeutic scheme was considered as effective if,
between D0 and M12:
- the dog showed a clinical improvement (i.e. the sum of
clinical scores was lower than before treatment),
- and the blood parasitic load decreased (q-PCR test find-
ings).
The treatment was classified as inefficacious if the ani-
mal did not clinically improve, or deteriorated, and/or the
blood parasitic load increased.
The relapse rate was also analysed. It was defined as an
initial therapeutic efficacy followed by the reappearance of
clinical signs (i.e. the sum of clinical scores was equal to, or
higher than, before treatment), and/or an increase of the
blood parasitic load reported by q-PCR.
2.8. Statistical analysis
The statistical analysis was conducted using SAS soft-
ware (SAS/STAT 9.1, SAS Institute). A descriptive analysis
was performed for all the variables related to the included
population: efficacy criteria on the one hand, and clini-
cal, haemato-biochemical and parasitological parameters
on the other hand. For comparisons of the two sub-
populations (naïve versus non-naïve dogs), a Fisher’s exact
test was applied. In addition, the relative difference of
the sum of scores between the two sub-populations was
adjusted for the leishmaniosis treatment status of dogs at
inclusion with a non parametric ANCOVA (transformation
on ranks and adjusted on the baseline value).
3. Results
3.1. Dog characteristics
Out of the 61 dogs analysed in the study, a major-
ity of males was represented (68.8%) as well as large
248 S. Rougier et al. / Veterinary Parasitology 186 (2012) 245–253
breeds: gundogs were predominant (32.8%), followed
by medium and large companion dogs (13.1%), and then
sheepdogs (11.5%). Cross breeds accounted for 24.6% of
the study population. Mean dog age was 5 years (±2.2).
Twenty dogs (32.8%) had previously presented with at
least one episode of clinical disease, treated either with
the association meglumine antimoniate (Glucantime® ,
Mérial) + allopurinol (Zyloric® , HAC Pharma) for 10 of
them or with meglumine antimoniate alone, at differ-
ent dosages, for 6. Other previous treatments consisted
in allopurinol alone (2 dogs), difloxacin (Dicural® , Fort
Dodge) + metronidazole (Flagyl® , Sanofi Aventis) (1 dog),
and allopurinol + metronidazole + ibafloxacin (Ibaflin® ,
Intervet-Schering Plough) (1 dog).
3.2. Physical examination on D0 (Fig. 1)
Cutaneous signs were regularly observed: squamo-
sis or hyperkeratosis (48/61 dogs), seborrhoea (44/61),
alopecia (35/61) or onychogryphosis (28/61). Thirty-seven
dogs presented with listlessness and 51 with lym-
phadenopathy. Amyotrophy was observed (27/61), as well
as splenomegaly (27/61) or conjunctivitis (25/61). Other
clinical signs were less often observed, such as pyoder-
matitis (22/61), ulcers (18/61), arthritis (14/61), diarrhoea
(11/61), hepatomegaly (10/61), epistaxis (9/61), keratitis
(8/61) or local nodules (2/61).
3.3. Laboratory findings and parasitological data on D0
(Table 1)
The most prominent haematological abnormalities
observed on D0 were anaemia (indicated by a decrease
in RBC count, haemoglobinaemia and PCV in 32.8%, 29.5%
and 27.9% of dogs, respectively), and thrombocytopaenia
(45.9%). In addition, 16.4% of the dog population presented
with leucocytosis.
According to biochemical parameters, a decrease in the
albumin/globulin ratio was observed in 73.8% of dogs. More
than half of the population showed a hyperproteinaemia
(52.5%); particularly, a ␤- and a ␥-globulinaemia were
present in 70.5% and 49.2% of the dog population, respec-
tively.
All dogs presented with at least the 18 kDa band and
44/61 (72.1%) with a full Western Blot profile with the
5 specific bands (14 + 18 + 21 + 23 + 31 kDa). 14 kDa and/or
18 kDa bands were considered as indicative of a previous
asymptomatic infection with L. infantum (Mary et al., 1992;
Marty et al., 1994).
Detection of anti-Leishmania antibodies by ELISA serol-
ogy showed a mean of 16.64 ± 3.58 AU.
Only 21.3% (13/61) of dogs showed amastigotes on
direct microscopic examination of lymph node material.
Among the other 48 dogs for which PCR should have been
performed on lymph node samples, only 43 were carried
out due to insufficient lymph node material for 5 dogs.
Among the 43 PCR performed, 22 (51.2%) were positive.
Therefore, 35/56 (62.5%) of lymph node samples appeared
parasitologically positive through either direct examina-
tion or PCR.
Promastigote cultures from blood samples were pos-
itive for 20.0% of dogs and from lymph node samples
for 35.0% of dogs. Thirty-four strains (obtained from 23
French, 7 Italian and 4 Spanish dogs) were isolated and
characterized by isoenzyme electrophoresis as L. infan-
tum MON-1 in all cases at the Montpellier WHO Reference
Centre.
According to real-time PCR performed on blood sam-
ples, parasite DNA was detected in 34/60 (56.7%) dogs, with
a mean value of 26.9 parasites/ml per dog.
3.4. Marbofloxacin treatment efficacy
Efficacy was observed in 42 dogs (68.9%), 10 of them
(23.8%) being clinically cured (i.e. no more clinical signs)
at M3. The efficacy rate increased progressively up to M3.
However, peak of efficacy was observed on D28 (52.4%).
Fourteen dogs (33.3%) responded after 2 months and 5
dogs (11.9%) after 3 months. One dog presented with a
progressive improvement during the one-year follow-up
and then showed a therapeutic response at M12. The mean
time to response was 51.3 ± 49.0 days. Twenty dogs out
of 38 (52.6%) were considered as relapsing after the third
month (M3). Relapse occurred about 5.5 months after mar-
bofloxacin treatment completion.
Depending on whether or not the dogs had experienced
previous episode(s) of leishmaniosis, a higher response
rate was observed when marbofloxacin was used as the
second or more anti-leishmanial treatment. Indeed, 85%
of dogs which had received at least one previous anti-
leishmanial treatment responded versus 61% responding
dogs among those for whom marbofloxacin was used as the
initial treatment (non-statistically significant, p = 0.079).
Similarly, time to efficacy was different between the two
sub-populations and was reached in a mean of 46 ± 17 days
for dogs already treated for leishmaniosis versus 55 ± 62
days for dogs for whom marbofloxacin was the initial anti-
leishmanial treatment.
3.5. Time-course evolution of clinical parameters
(Figs. 1 and 2)
Clinical improvement was observed during the first
3 months. The sum of clinical scores decreased by 61%
between D0 and the third month (D84), from a mean value
of 11/54 on D0 to 5/54 on D84. Forty-three percent of the
dog population showed a decrease of more than 80% of
the sum of clinical scores during the first 3 months, and
another 18% a decrease of 60–80%. Twenty-one percent of
dogs showed a decrease of the sum of clinical scores of
less than 40%. As previously observed, dogs in leishmanial
recurrence showed a higher decrease in percentage of the
sum of clinical scores than dogs for which marbofloxacin
was the initial treatment (77% versus 53% respectively,
non-statistically significant, p = 0.31). Sixty percent of dogs
previously treated showed a regression of their clinical
signs of more than 80% against 34% of naïve dogs (not sta-
tistically significant, p = 0.16).
 S. Rougier et al. / Veterinary Parasitology 186 (2012) 245–253 249

Lymphadenomegaly D0
D84
Squamosis / Hyperkeratosis
Seborrhea
Listlessness
Alopecia
Onychogryphosis
Splenomegaly
Amyotrophy
Conjunctivitis
Pyodermatitis
Ulcers
Arthritis
Diarrhoea
Hepatomegaly
Epistaxis
Keratitis
Nodules
Uveitis

0 10 20 30 40 50 60 70 80 90

Fig. 1. Percentages of dogs presenting with each clinical sign on D0 and D84.

3.6. Time course evolution of haemato-biochemical treatment. Consequently, hyperproteinaemia was less
parameters (Table 1) observed after 3 months.

During the first three months, a decrease in the number 3.7. Time course evolution of ELISA serology and
of dogs with leucocytosis was observed as well as in the parasitological exams during the first three months
number of dogs with thrombocytopaenia. Little improve-
ment was noted in dogs with anaemia. According to ELISA serology, promastigote cultures and
According to biochemical parameters, the A/G ratio conventional PCR, no global change was observed during
increased in general. An improvement was observed in the first three months of the study.
dogs with hypoalbuminaemia and with hyper ␤- and For ELISA serology, a slight but not significant decrease
␥-globulinaemia, with more dogs in the normal range was observed between D0 and D84 (from 16.6 ± 3.58 AU
for these parameters on D84 when compared to before to 16.1 ± 3.50 AU). Similarly, amastigote forms were less

14

All dogs
12 Naïve dogs
Sum of clinical scores (mean value)

Dogs previously treated

10

- 53%
6

4 - 61%

2
- 77%
Treatment

0
D0 D14 D28 D40 D56 D84

Fig. 2. Comparison of time-course evolution of sum of clinical scores (mean value): all analysed dogs, dogs previously treated for leishmaniosis and naïve
dogs.
250 S. Rougier et al. / Veterinary Parasitology 186 (2012) 245–253

Table 1
Evolution of haemato-biochemical parameters on inclusion day (D0), after three months (D84) and one-year follow-up (M12).

Parameter Normal reference If D0 (N = 61)b D28 (N = 59)b D56 (N = 51)b

rangea abnormal

m ± std Abnormal, n (%) m ± std Abnormal, n (%) m ± std Abnormal, n (%)

Haematology
WBC count 6–17 109 /L ↑ 12.1 ± 4.33 10 (16.4) 10.8 ± 3.57 3 (5.1) 10.2 ± 3.18 2 (3.9)
RBC count 5.5–8.5 1011 /L ↓ 5.9 ± 1.12 20 (32.8) 5.9 ± 1.17 22 (37.3) 5.7 ± 1.43 18 (35.3)
PCV 37–55% ↓ 42.0 ± 8.09 17 (27.9) 42.7 ± 8.96 17 (28.8) 41.3 ± 10.28 15 (29.4)
Haemoglobin 12–18 g/dL ↓ 13.5 ± 2.65 18 (29.5) 13.4 ± 2.89 17 (28.8) 12.9 ± 3.24 18 (35.3)
MCHC 32–36 g/dL ↓ 31.6 ± 1.68 27 (44.3) 30.9 ± 1.28 40 (67.8) 30.8 ± 1.89 37 (72.5)
MCV 60–77 fL ↑ 70.3 ± 4.62 3 (4.9) 71.9 ± 4.37 4 (6.8) 72.6 ± 4.94 5 (9.8)
Eosinophils 2–10% ↓ 4.7 ± 3.53 10 (16.4) 4.2 ± 3.30 13 (22.0) 5.1 ± 3.86 7 (13.7)
Lymphocytes 12–30% ↓ 21.3 ± 10.15 7 (11.5) 20.9 ± 9.51 8 (13.6) 21.6 ± 10.45 5 (9.8)
Neutrophils 60–77% ↑ 69.0 ± 11.20 21 (34.4) 70.4 ± 11.04 8 (13.6) 68.6 ± 11.79 10 (19.6)
Platelet count 2–5 1011 /L ↓ 2.2 ± 0.91 28 (45.9) 2.5 ± 1.14 20 (33.9) 2.1 ± 0.93 28 (54.9)
Biochemistry
Urea 3.34–8.3 mmol/L ↑ 6.7 ± 4.44 15 (24.6) 7.7 ± 5.59 14 (23.7) 7.1 ± 5.01 12 (23.5)
Creatinine 35–124 ␮mol/L ↑ 81.0 ± 29.48 4 (6.6) 89.2 ± 44.45 7 (11.9) 83.5 ± 39.85 3 (5.9)
ALP 6–90 U/L ↑ 51.1 ± 33.54 4 (6.8) 52.0 ± 45.48 4 (6.8) 61.3 ± 61.39 6 (11.8)
ALT 14–70 U/L ↑ 57.3 ± 59.81 8 (13.6) 51.6 ± 50.11 8 (13.6) 56.3 ± 65.33 7 (13.7)
AST 10–90 U/L ↑ 84.9 ± 175.75 13 (21.3) 63.2 ± 32.32 10 (16.9) 59.4 ± 32.86 7 (13.7)
␥-GT <20 U/L ↑ 2.5 ± 2.00 0 2.5 ± 1.78 0 2.5 ± 1.90 0
Total proteins 44–79 g/L ↑ 81.2 ± 13.41 32 (52.5) 81.8 ± 15.34 24 (40.7) 81.6 ± 16.70 25 (49.0)
Albumin 23–38 g/L ↓ 28.0 ± 7.18 14 (23.0) 27.4 ± 7.37 13 (22.0) 27.9 ± 6.62 12 (23.5)
A/G ratio ≥0.7 ↓ 0.58 ± 0.28 45 (73.8) 0.58 ± 0.29 40 (67.8) 0.58 ± 0.25 35 (68.6)
␣-1 globulin 2–3 g/L ↓ 2.7 ± 1.25 10 (16.4) 2.6 ± 0.58 5 (8.5) 2.6 ± 0.40 1 (2.0)
␣-2 globulin 9–15 g/L ↑ 11.6 ± 3.04 7 (11.5) 12.2 ± 3.28 9 (15.3) 12.4 ± 2.26 7 (13.7)
␤-Globulin 7–16 g/L ↑ 21.2 ± 7.68 43 (70.5) 20.0 ± 8.15 37 (62.7) 20.6 ± 8.55 36 (70.6)
␥-Globulin 3–7 g/L ↑ 17.9 ± 10.74 30 (49.2) 19.3 ± 12.79 28 (47.5) 17.9 ± 13.16 22 (43.1)

Parameter Normal reference If D84 (N = 55)b M6 (N = 29)b M12 (N = 26)b

rangea abnormal

m ± std Abnormal, n (%) m ± std Abnormal, n (%) m ± std Abnormal, n (%)

Haematology
WBC count 6–17 109 /L ↑ 11.0 ± 4.15 4 (7.3) 10.2 ± 3.56 1 (3.4) 10.7 ± 4.73 3 (11.5)
RBC count 5.5–8.5 1011 /L ↓ 5.9 ± 1.24 20 (36.4) 6.2 ± 1.01 7 (24.1) 6.0 ± 1.37 8 (30.8)
PCV 37–55% ↓ 42.3 ± 9.08 14 (25.5) 43.7 ± 7.95 6 (20.7) 43.9 ± 9.72 4 (15.4)
Haemoglobin 12–18 g/dL ↓ 13.3 ± 2.82 16 (29.1) 14.0 ± 2.51 6 (20.7) 14.0 ± 3.01 5 (19.2)
MCHC 32–36 g/dL ↓ 31.1 ± 1.57 39 (70.9) 31.6 ± 1.43 14 (48.3) 32.1 ± 1.81 7 (26.9)
MCV 60–77 fL ↑ 71.1 ± 4.44 7 (12.7) 70.3 ± 3.58 1 (3.4) 73.6 ± 4.72 4 (15.4)
Eosinophils 2–10% ↓ 4.5 ± 3.37 11 (20.0) 4.0 ± 3.02 5 (17.2) 5.2 ± 4.25 6 (23.1)
Lymphocytes 12–30% ↓ 20.8 ± 9.50 9 (16.4) 19.6 ± 9.83 2 (6.9) 15.5 ± 7.09 9 (34.6)
Neutrophils 60–77% ↑ 70.6 ± 9.87 12 (21.8) 72.4 ± 10.09 12 (41.4) 75.1 ± 8.59 11 (42.3)
Platelet count 2–5 1011 /L ↓ 2.4 ± 1.04 20 (36.4) 2.7 ± 1.47 13 (44.8) 2.0 ± 1.23 15 (57.7)
Biochemistry
Urea 3.34–8.3 mmol/L ↑ 7.3 ± 5.36 12 (21.8) 5.8 ± 3.71 4 (13.8) 7.4 ± 6.83 4 (15.4)
Creatinine 35–124 ␮mol/L ↑ 84.1 ± 75.14 3 (5.5) 71.4 ± 28.54 1 (3.5) 86.5 ± 73.29 2 (7.7)
ALP 6–90 U/L ↑ 69.0 ± 72.02 9 (16.4) 63.0 ± 44.06 6 (20.7) 72.3 ± 98.27 6 (23.1)
ALT 14–70 U/L ↑ 66.6 ± 99.08 9 (16.4) 59.9 ± 72.74 4 (13.8) 66.9 ± 61.91 7 (26.9)
AST 10–90 U/L ↑ 56.4 ± 25.49 6 (10.9) 54.2 ± 36.07 4 (13.8) 58.2 ± 33.30 2 (7.7)
␥-GT <20 U/L ↑ 2.9 ± 2.53 0 3.0 ± 2.63 0 3.6 ± 3.73 0
Total proteins 44–79 g/L ↑ 79.9 ± 14.94 23 (41.9) 73.5 ± 15.05 7 (24.1) 72.0 ± 17.13 5 (19.2)
Albumin 23–38 g/L ↓ 27.9 ± 6.92 12 (21.8) 28.4 ± 7.46 7 (24.1) 28.0 ± 7.63 5 (19.2)
A/G ratio ≥0.7 ↓ 0.61 ± 0.26 35 (63.6) 0.73 ± 0.35 12 (41.4) 0.73 ± 0.32 12 (46.2)
␣-1 globulin 2–3 g/L ↓ 2.5 ± 0.45 5 (9.1) 2.6 ± 0.45 3 (10.3) 2.5 ± 0.49 2 (7.7)
␣-2 globulin 9–15 g/L ↑ 12.3 ± 2.42 6 (10.9) 11.7 ± 1.65 0 11.4 ± 2.06 1 (3.8)
␤-Globulin 7–16 g/L ↑ 20.3 ± 8.78 35 (63.6) 18.3 ± 6.80 14 (48.3) 19.3 ± 13.33 10 (38.5)
␥-Globulin 3–7 g/L ↑ 15.9 ± 12.33 15 (27.3) 12.6 ± 11.96 6 (20.7) 10.7 ± 8.65 3 (11.5)
a
Reference ranges from the centralised laboratory.
b
Number of dogs with tubes usable for haemato-biochemical analysis.

observed under microscopic examination after three
months (21.7% positive on D0 versus 7.5% on D84), and the
number of positive cultures from lymph nodes decreased
(35.0% on D0 versus 22.6% on D84). But opposed to this,
no change was observed between the positivity rates of
blood cultures during 3 months (20.0% on D0 versus 19.2%
on D84).
Conventional PCR remained positive for the majority of
dogs for three months as well as from blood samples (44.4%
positive on D84) and from lymph node samples (62.5%).
 S. Rougier et al. / Veterinary Parasitology 186 (2012) 245–253
3.8. Real-time PCR kinetic results on D0, D84 and M12
(Table 2)
From all dogs included in the study, the mean
parasitic load varied from 26.9 parasites/ml on D0 to
93.5 parasites/ml on D84 and 21.6 parasites/ml at M12.
If we divide the population into two sub-groups
(response/no response), the parasitic load among dogs
in response (42/61) was 20.6 parasites/ml on D0 and
decreased to a mean of 8.1 parasites/ml on D84. Out of 42
dogs, 23 (54.8%) did not show any Leishmania DNA on D0
and 16 (69.6%) of them remained negative during the whole
study. Six dogs (14.3%) showed an increase of the para-
sitaemia; among them, 4 were in relapse and 2 remained in
a parasitaemia below 10 parasites/ml. Eleven dogs (26.2%)
had a parasitaemia over 10 parasites/ml on D0: 9 of them
showed a decrease of the parasitic load and 2 of them a stag-
nation of the parasitaemia close to 10 parasites/ml. Eight
dogs presented with less than 10 parasites/ml on D0 and
for 2 of them, no parasitic DNA could be detected at the
end of the follow-up.
In addition, the dogs in therapeutic failure (out of 19
dogs in failure, no kinetics were done for 7 dogs which
had stopped follow-up very early) showed a mean par-
asitic load of 46.6 parasites/ml at inclusion and a mean
increase up to 384.7 parasites/ml on D84. Among them, 1
dog did not evidence any parasitic DNA on D0. Four dogs
presented with less than 10 parasites/ml on D0 and a mean
of 138.5 parasites/ml was observed at the end of follow-
up. An increase of the parasitaemia was observed in these
5 dogs. Finally, 7 dogs showed more than 10 parasites/ml
on D0, and all of them also showed an increase of their
parasitaemia.
3.9. Safety considerations and assessment of
investigators’ satisfaction
Three adverse events likely to be related to mar-
bofloxacin treatment were reported: one consisted in a
decrease of vitality observed on D14, one dog presented
some vomiting and one dog had a decrease of appetite.
No specific treatment for these adverse events was admin-
istered and the follow-up of these 3 dogs progressed in
conformity with the study protocol.
In almost 77% of cases, veterinarians were satisfied with
the marbofloxacin treatment at M3, and 78.7% of them
remained satisfied at the end of the follow-up (between
M3 and M12).
4. Discussion
In a preliminary study (Rougier et al., 2008), a 28-day
marbofloxacin treatment seemed to be encouraging in the
treatment of CanL. The purpose of this study was to confirm
over a longer period the clinical results previously observed
as well as to generate additional data regarding product
efficacy on the parasites, by diversifying parasitological and
serological exams.
Marbofloxacin was efficacious in almost 70% of the
study population and allowed reaching clinical cure in
23.8%. Time to response was about 51 days but appeared
251
shortened in dogs in recurrence of CanL, as previously
observed (Rougier et al., 2008). A decrease of the sum
of clinical scores of 61% in three months was observed
with marbofloxacin. In addition, 61% of the dog population
revealed a reduction of the sum of their clinical scores of
more than 60% in 3 months. We confirm here the clinical
results previously observed as well as the persistence of
the therapeutic effect beyond the 28-day treatment period
(Rougier et al., 2008), both of which may be attributed
to the immunomodulatory properties of marbofloxacin
(Dalhoff and Shalit, 2003; Vouldoukis et al., 2006).
As regards haemato-biochemical disorders related to
CanL, we observed stabilization in CBC values during the
first 3 months, as previously described by several authors
(Koutinas et al., 2001; Valladares et al., 2001). However, an
improvement in 3 months was observed in dogs initially
presenting hypoalbuminaemia and/or with hyper ␤- and ␥-
globulinaemia, in parallel with an increase of the A/G ratio.
Combined with the clinical improvements, these observa-
tions allow confirming the efficacy of marbofloxacin in the
treatment of CanL (Kargin Kiral et al., 2004).
Miltefosine (Milteforan® , Virbac), a phosphatidyl-
choline analogue, was recently approved in several
European countries for the treatment of CanL. It seemed
interesting for us to compare the efficacy of marbofloxacin
with this new product because miltefosine is considered by
numerous veterinarians as a promising therapy, and both
products consist in an oral therapy, administered once a
day for 28 days. Clinical parameters followed were simi-
lar and a comparable scale system was used (Mateo et al.,
2009; Woerly et al., 2009). A similar decrease of the total
of clinical scores between D0 and M2 was observed for
both miltefosine and marbofloxacin (Mateo et al., 2009;
Woerly et al., 2009). About 60.7% versus 50.0% of dogs
treated with marbofloxacin and miltefosine respectively
showed a decrease of their clinical scores greater than
75% after 56 days (Woerly et al., 2009). Clinical cure
rate on D56 was similar between the 2 treatments (20%
and 19% for miltefosine and marbofloxacin, respectively)
(Woerly et al., 2009). A difference in efficacy seems, how-
ever, to concern sub-populations of dogs never treated
for CanL and sub-populations in recurrence. Indeed, it
appeared that miltefosine has similar efficacy on the two
sub-populations (Mateo et al., 2009; Woerly et al., 2009)
whereas marbofloxacin is better when administered to
dogs in recurrence (Rougier et al., 2008).
During the first three-month follow-up, we used par-
asitological methods, conventional PCR and serology to
follow the treatment efficacy on parasites. Upon inclusion,
before any treatment, only 21.3% of the dog population
showed amastigotes on direct microscopic examination of
lymph node aspirate. Direct examination is known to be
poorly sensitive particularly due to the low number of par-
asites in lymph node samples (Chulay and Bryceson, 1983).
In addition, lymph node puncture was reported by veteri-
narians as a difficult sampling technique, and most of them
collected little lymph node material, often combined with
blood. The poor quality of these samples may be one of the
reasons for the low identification rate.
As regards cultures performed at inclusion, 35% of them
from lymph node aspirate revealed leishmanial growth
252 S. Rougier et al. / Veterinary Parasitology 186 (2012) 245–253
Table 2
Mean blood parasitic load (number of parasites/ml) over time obtained from real-time PCR kinetics and according to sub-populations.
Sub-populations D0
N Parasitic load
All dogs 53
Dogs for which treatment was effective only 42
Dogs for which treatment was ineffective only 12
Among dogs for which treatment was effectivea
Dogs which relapsed (after D84) 20
Dogs which did not relapse (after D84) 18
a
4 dogs for which treatment was effective stopped the follow-up on D84; so, the relapse assessment has not been performed.
versus only 20% from blood samples. Although culture is
known to increase the sensitivity by almost 20% when com-
pared to direct examination sensitivity (Alvar et al., 2004),
the sensitivity increase proved to be, however, very lim-
ited in our study, undoubtedly due to a high number of
contaminated cultures, bacterial growth masking parasite
growth.
During the one-year follow-up, in contradiction with
the clinical improvements observed, no decrease in the
serological titres was demonstrated. The absence of a par-
allel between serology evolution and clinical improvement
has been previously published. Most authors consider that
serology cannot be used as a reliable criterion in the follow-
up of the clinical evolution of treated dogs, at least over
a short period (Solano-Gallego et al., 2001; Pennisi et al.,
2005). A longer follow-up of dogs in clinical improve-
ment for several years might have shown a significant titre
decrease.
As previously described, we observed dogs seronegative
for Leishmania but positive using the PCR technique (Oliva
et al., 2006), and, on the other hand, dogs PCR negative
for Leishmania while showing a positive serology (Solano-
Gallego et al., 2001). Blood can be considered as a medium
for parasite transport and not as a reservoir organ (Manna
et al., 2006). This could explain the fact that 16 out of 42
dogs studied never revealed any Leishmania DNA in blood
up to the end of the study, whereas all of them had a clin-
ical picture of leishmaniasis. But the lower the number of
parasites in the organism, the more difficult it is to reveal
Leishmania DNA in blood, and we can thus suppose that
these dogs were not highly infected. Blood is not, how-
ever, the best source of Leishmania, compared to skin or
lymph nodes (Strauss-Ayali et al., 2004). Consequently, a
single negative PCR from blood in Leishmanial-suspected
dogs with compatible clinical signs is not sufficient to rule
out infection. As is the case with other parasitological tests,
PCR does not permit a reliable diagnosis when considered
alone.
A clear parallel between parasitaemia development and
clinical outcome was generally observed, even if exceptions
were not unusual. The presence of severe clinical symp-
toms of leishmaniosis is now known not to be inevitably
correlated to a high parasitic load, and vice versa (Quinnell
et al., 2003; Alvar et al., 2004; Poot et al., 2005). Five dogs
showed discordant evolutions: among them, 4 showed
a one-year clinical improvement whereas parasitaemia
increased over time and 1 showed a clinical degradation
whereas parasitaemia significantly decreased. All these
D84 M12
N Parasitic load N Parasitic load
26.9 53 93.5 26 21.6
20.6 42 8.1 22 13.4
46.6 12 384.7 4 66.9
12.4 20 7.6 5 51.8
21.6 18 7.9 17 2.1
cases were considered as failures. Other cases which pre-
sented with a slight clinical improvement associated with
a low and stable parasitaemia were considered as respon-
dent to the marbofloxacin treatment; indeed, treatment
was considered to have controlled the infection in these
dogs.
In conclusion, the one-year follow-up of dogs treated
with marbofloxacin against canine leishmaniosis showed
that marbofloxacin allowed reaching clinical improvement
which was generally correlated with a decrease of the
blood parasitic load. Marbofloxacin showed an efficacy
in about 70% of treated dogs with a decrease of 61% of
the sum of clinical scores in 3 months. However, even
though dogs could be clinically improved, they remained
parasitologically positive and could harbour parasites for
a long time. Anti-leishmanial treatments are known for
not clearing the dogs and not eliminating all parasites
(Riera et al., 1999; Koutinas et al., 2001; Cortadellas, 2003;
Alvar et al., 2004; Manna et al., 2004; Pennisi et al., 2005;
Ikeda-Garcia et al., 2007; Gomes et al., 2008; Manna et al.,
2008). Veterinarians have to focus on the clinical status of
the dog, clinical improvement being the main criteria for
assessing treatment efficacy. As treated animals probably
remained carriers of parasites, they also remained suscepti-
ble to relapse. The contribution of this persistent subclinical
infection in the spreading of CanL however remains to be
demonstrated.
Acknowledgements
The authors would like to thank Elisabeth Bon-
net, Isabelle Franquet, Sophie Fornasero, Romy Knecht
and Stéphane Melhem for their technical assistance, Dr
Francine Pratlong (Montpellier WHO Reference Centre) for
the characterization of the strains and all veterinary inves-
tigators for their active contribution to this study.
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