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ARTICLE ORIGINAL
Epidemiology of canine leishmaniasis
by cross-sectional study in the French focus
of Cévennes
° KECK N. and °° DEREURE J.
° Laboratoire Départemental Vétérinaire de l’Hérault, 306, rue Croix de Las Cazes, B.P. 6070, F-34030 Montpellier.
Tel : 04.67.10.17.17, Fax : 04.67.54.32.02, E-mail : [email protected]
°° Laboratoire d’Écologie médicale et Pathologie parasitaire, Faculté de Médecine, 163, rue Auguste-Broussonet, F-34090 Montpellier
SUMMARY
RÉSUMÉ
A cross-sectional epidemiological survey was carried out on the distribution of canine leishmaniasis in the French focus of Cévennes. Animals were
sampled in two vegetation zones : Holm and White Oak zone (WO/HO) and
Holm Oak zone (HO) in which villages were chosen for collecting blood
from dogs as exhaustively as possible. Sera from 259 dogs were collected
and subjected to the indirect immunofluorescence antibodies test (IFAT) and
counter immuno-electrophoresis (CIE). Seroprevalence was 17.9 % in the
WO/HO level and 9.6 % in the HO level. Serological positivity to
Leishmania sp. was significantly associated with a life in the WO/HO level,
and sex, dog’s activity and age were found to be additional factors. Basic
reproductive number (R0) was 1.21 in the WO/HO level and 1.11 in the HO
level. Prevalence has moderately increased during the last ten years, especially in the peri-urban HO zone.
Etude épidémiologique de la leishmaniose canine en région cévenole.
Par N. KECK ET J. DEREURE
KEY-WORDS : leishmaniasis - dog - epidemiology France.
MOTS-CLÉS : leishmaniose - chien - epidémiologie France.
Introduction
emergence of resistant strains to pentavalent antimonial compounds which have led to a search for alternative drugs [28].
Canine leishmaniasis is also met in non-endemic areas,
concerning dogs which were exposed to the disease during
travels in other endemic areas. Limited outbreaks are sometimes reported by veterinary practitioners but not always
documented by epidemiological or entomological studies
[27]. Yet, the potential impact of warming on the establishment of new enzootic areas has to be evaluated [14].
Furthermore, dogs are considered as the main reservoir host
[10] even if other animals such as the European fox (Vulpes
vulpes) or the black rat (Rattus rattus) have also been identified as potential hosts of Leishmania sp. [30, 24].
Visceral leishmaniasis is a widespread disease, especially
in Mediterranean European countries, Africa, Middle East
Asia and South America. In south-western Europe, it is a
zoonotic disease due to Leishmania infantum.
The French focus of Cévennes is well known to be enzootic for leishmaniasis. The principal vector is Phlebotomus
ariasi which is anthropo-zoophilic in its behaviour [31].
Cross-sectional serological surveys have already been carried out in this region [5, 16, 23]. They show an increase of
the prevalence of canine leishmaniasis during the last ten
years and a wide range of values between different parts of
the focus.
Hence, this disease has become a significant veterinary
problem in this focus, which may be complicated by the
Revue Méd. Vét., 2003, 154, 10, 599-604
Une étude transversale évaluant la prévalence sérologique de la leishmaniose a été conduite dans le foyer des Cévennes. Les animaux ont été prélevés dans deux strates phyto-écologiques (chênaie mixte et chênaie d’yeuse),
à l’intérieur desquelles ont été choisis des villages où le recrutement s’est
fait de manière la plus exhaustive possible. Des échantillons de 259 chiens
ont été prélevés, puis analysés par immunofluorescence indirecte et électrosynérèse. La séroprévalence obtenue était de 17,9 % dans l’étage de la chênaie mixte et de 9,6 % dans l’étage de la chênaie d’Yeuse. Les animaux
vivant en chênaie mixte ont un risque plus élevé d’être séropositif qu’en
chênaie d’Yeuse (OR = 3,97, IC95% 1,33-12,71). Le taux de reproduction de
base est de 1,21 (chênaie mixte) et de 1,11 (chênaie d’Yeuse). La prévalence dans ces deux strates a modérément augmenté durant les dix dernières
années, principalement dans les zones péri-urbaines.
Human leishmaniasis usually occurs in young populations.
However, immunodepressed adults (consequently to HIV
infection or drugs used in organ transplants) are also greatly
600
exposed to the infection and very susceptible to the disease
which was shown in the focus of Cévennes [6]. This places
therefore leishmaniasis as an emerging human health problem.
Thus, it is important to estimate the prevalence of canine
leishmaniasis in order to evaluate the risk for human and animal health. The aim of this work was to estimate the prevalence of leishmaniasis with a cross-sectional study in two
areas of the french focus of Cévennes and to define epidemiological patterns and risk factors.
Material and methods
The Cévennes focus can be divided into five vegetation
zones with different prevalences of leishmaniasis [16]. The
main difference between the stages is vegetation, which is
correlated with the frequency of Phlebotomus ariasi and thus
the disease prevalence.
The data come from a cross-sectional serological survey of
the dog population carried out during the months of May and
June 1999. We used Lanotte’s assertion [16] for our work but
only carried out the survey in two zones :
- the holm and white oak (Quercus ilex and Q. pubescens)
zone (WO/HO zone) which is a rural stage where altitude is
generally high (500-700 m) and prevalence is expected to be
high.
- the holm oak (Q. ilex) zone (HO zone) which is more
peri-urban, where prevalence is expected to be lower.
In this area, transmission by Phlebotomus ariasi occurs
from late May to early September, but is usually concentrated
in July [7].
According to the results obtained by DEREURE [5] who
found that prevalence was 16,51 % in HO/WO zone and
5,51 % in HO zone, the number of dogs needed to be sampled
was 143 in each zone in order to compare prevalence using
statistical analysis with at least 80 % power.
In each zone, villages were chosen for collecting blood
from all native dogs as exhaustively as possible. Dogs younger than six months were excluded in order to sample dogs
which were exposed at least to one transmission season. Six
villages of the HO/WO zone and four villages of the HO zone
were studied.
Dog owners were informed of a free survey by announcements in the local press or in public places and by veterinary
surgeons working in the villages. Each dog owner provided
the age, breed, sex, activity, origin, nature of movements of
the dog. Activity was classified as pet dogs, guard dogs,
sheep-dogs and hunting dogs. Origin allowed to know whether the dog was recently living in the place or if it has been
there for a long time. Nature of movements was useful to
know if the dog had been in other countries where leishmaniasis is also enzootic.
SEROLOGICAL TESTS
Blood samples were collected by vene-puncture with the
agreement of the dog owner. Each sample was subjected to
KECK (N.) AND DEREURE (J.)
an immunofluorescent antibody test (IFAT) and counter
immuno-electrophoresis (CIE) as it has already been done in
other studies realized in the Cévennes focus [5, 16, 23]. Dogs
with titers ≥ 1/80 and positive CIE were considered positive.
Furthermore, in order to compare our results to previous studies in the focus, the prevalence rate was also estimated by
considering dogs with titers ≥ 1/80 as positive (with or
without positive CIE).
STATISTICAL ANALYSIS
The Woolf method was used for stratified analysis. Odds
Ratios (OR) for each risk factor, Khi2test, Khi2woolf for
interaction tests was calculated with Epi-Info®. The level of
significance was set at 5 %.
The model proposed by HASIBEDER et al. [12] in the
case of homogeneous transmission was used to calculate
basic case reproduction number R0. This considers two kinds
of dogs, type A and type B. After infection, type A dogs enter
a latent period during which they are serologically positive
but asymptomatic and not infectious to sandflies.
Afterwards, they become symptomatic and infectious. Type
B dogs are also serologically positive but are never infectious
to sandflies and can self-cure. This model was simplified by
DYE et al. [8] who considered that type B dogs were negligible [18] and that the proportion of infected sandflies was
very low [19]. The R0 value gives an idea of the number of
new infections occurring after the introduction of one infected animal into a safe host population.
Results
During the study, 259 sera were collected : 134 in the
HO/WO zone and 125 in the HO zone. A few dogs had been
imported to the Cévennes focus but at less than three months
of age.
The characteristics of dogs in each vegetation zone were
quite similar (table I) : sex ratio was approximately 50 % ;
cross-breed dogs and young dogs (less than three years) were
more numerous than other categories of breed and age.
Hunting dogs were more frequent in HO/WO zone which is
more rural and pet dogs were more frequent in the HO zone.
As they were few, sheepdogs were grouped with hunting
dogs for statistical analysis.
The prevalence obtained in the HO/WO stage was 17.2 %
and 9.6 % for HO when we considered dogs as positive if
IFAT was ≥ 1/80 (table II). It was lower if we considered
dogs as positive when IFAT was ≥ 1/80 and CIE positive
(14.2 % for HO/WO and 4 % for HO). All sera with positive
CIE had a titre of 1/80 or more. The frequency distribution of
antibody titre (figure 1) was unimodal. Low titres (1/20,
1/40) were more frequent in HO/WO zone than in HO.
The odds ratio measuring the risk of seropositivity (IFAT ≥
1/80, CIE +) in the HO/WO zone compared to HO zone was
3.97 (IC95% = 1.33 - 12.71). This indicates a strong association between seropositivity and the place where the dog lives.
In each zone, results were different between the villages
(table III). We obtained a wide range of prevalence rate
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EPIDEMIOLOGY OF CANINE LEISHMANIASIS BY CROSS-SECTIONAL STUDY IN THE FRENCH FOCUS OF CÉVENNES
WO : White Oak zone
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HO/WO : Holm Oak and White Oak zone
TABLE I. — Categories of dogs sampled. Characteristics of dogs in each vegetation zone concerning sex, race, age
and activity.
WO : White Oak zone
HO/WO : Holm Oak and White Oak zone
IFAT : immunofluorescent antibody test
CIE : counter immunoelectrophoresis
TABLE II. — Prevalence of canine leishmaniasis in two vegetation zones.
Prevalence rate was calculated when dogs were considered as positive
when IFAT was ≥ 1/80 and when it was ≥ 1/80 with a positive CIE.
values, especially in HO/WO zone (for example 33.3 % in
Monoblet and 0 % in Montoulieu). There was no statistical
difference between the villages of the HO/WO (Khi23 = 1.14,
p = 0.76) which was not the case between those of the HO
(Khi23 = 9.95, p = 0.08).
Stratified analysis calculating the OR for each class of sex,
activity and age shows that they are interacting factors (table
IV). Odds ratios are much higher for male dogs (7.61) and
watch dogs (10.29). In the population studied, the seroprevalence leishmaniasis is higher for watch dogs (20.5 %), than
for hunting dogs (9.3 %) or pet dogs (5.3 %). It is also higher
for males (12 %) than for females (6.5 %). Furthermore, there
was no association between the breed and seropositivity
(Khi22 = 0.76, p = 0.68).
Assuming the informations presented earlier, the basic
reproductive number if biting rates are homogeneous would
be 1.21 for the HO/WO zone and 1.11 for the HO zone.
Discussion
WO : White Oak zone
HO/WO : Holm Oak and White Oak zone
FIGURE 1. — Frequency distribution of antibody titre recorded with IFAT.
Revue Méd. Vét., 2003, 154, 10, 599-604
We have used in this study the same sampling strategies
and serological diagnosis than in previous studies already
done in the Cévennes. DEREURE (1993) found seroprevalence rates of 5.51 % in HO and 16.52 % in HO/WO.
Accoring to our results, canine leishmaniasis prevalence
seems to be stable, at least in HO/WO zone. This is illustrated by the low value of R0 (near 1) which is characteristic of
an endemic disease. Nevertheless, seroprevalence has increased in the HO zone (9.6 %) which is relevant to other observations showing that canine leishmaniasis may become a
more peri-urban disease. As human populations are more and
more present in peri-urban areas, this could represent a public
health problem. Prevalence values are different between the
two studied zones but we also find great disparities within
602
KECK (N.) AND DEREURE (J.)
those zones, especially in the HO/WO. This may be explained by the possible existence of micro foci where distinctive
local climatic conditions favour the development of
Phlebotomus ariasi. In fact, if the geographical classification
presented by LANOTTE [16] is confirmed, it is likely that
some differences, which are linked to the altitude or exposure
to the sun also exist within the vegetation zones. Thus, this
sandfly is more frequently found on the slopes of the hillsides
at altitude of 300-500 m, which is its preferred habitat and
also the place where villages are implanted [32].
WO : White Oak level
According to DYE et al. [8], the fact that antibody titre distribution is unimodal is characteristic of an endemic disease.
We also obtained a unimodal distribution but think that this
may also be due to the fact that more and more dogs are treated against leishmaniasis. This could lower the antibody titre
of infected dogs, as treatments markedly reduce antibody
titres [28]. Hence, 11 of the dogs that were serologically positive had already been treated or were receiving a treatment
when the study was done. Furthermore, we observed that low
IFAT antibody titres (1/20, 1/40) were more frequent in
HO/WO : Holm Oak and White Oak level
TABLE III. — Serological test results in each village of the two vegetation zones. Number of dogs tested and the
leishmaniasis seroprevalence results in each village of the two vegetation zones.
Table IV. — Results of stratified analysis. OR for each class of sex, activity and age are mentioned in dark characters (IC95%).
Revue Méd. Vét., 2003, 154, 10, 599-604
EPIDEMIOLOGY OF CANINE LEISHMANIASIS BY CROSS-SECTIONAL STUDY IN THE FRENCH FOCUS OF CÉVENNES
HO/WO zone. In this zone, dogs may be more frequently in
contact with the parasite, and respond to infection by producing low titres of antibodies but without becoming symptomatic. According to DYE et al. [8], it is possible that some
positive animals are included among them. This underlines
the question concerning the sensitivity of IFAT, although it
appears to be universally recognized as the standard.
LANOTTE et al. [17] evaluated IFAT sensitivity at 99 %
(± 1 %) and specificity at 68 %. The limitations of serology
are due to the long periods of serological latency, as antibodies are often detectable only several months after infection
[11]. It has also been investigated by DYE et al. [9] during a
two years cohort study showing that sensitivity and specificity are high during short period of 2-3 months (MarchApril) after a long incubation period. Furthermore, a proportion of seropositive dogs spontaneously convert to negative
[1].
Although we considered dogs as positive when IFAT was
≥ 1/80 for comparing our results to previous studies, we also
used the results of CIE to analyze the effect of risk factors.
This serological technique has already been used by REZAI
et al. [29] who found that this test correlated well with the
results of the IFAT. We also observed by operating field diagnosis serology for veterinary practitioners that it is a good
indicator of the evolution of the dog’s disease compared to
IFAT, which is consistent with observations made by
MANSUETO [20].
However, the major problem is to know wether dogs found
positive for leishmaniasis are also infective to phlebotomine
sandflies. This information would allow to evaluate the burden of infectiousness of a dog population in an endemic area.
There are proven connections between antibody titre and
infectiousness [9]. DYE et al. [9] concluded that the aim of
control programmes using serology should be to detect infectiousness rather than infection.
Other epidemiological studies conducted in France found
prevalence rates ranging from 3.5 to 20 % [13, 21, 26, 36]. In
other European countries, values are sometimes higher, especially in Spain and Italy where rates of 30 % were observed
[22, 25]. Despite the fact that leishmaniasis seroprevalence
seems to be stable, the absolute number of cases may be
higher due to the growth of the canine population (France :
7 million dogs in 1971 and 9 millions in 2000, FACCO
/SOFRES survey 2000 and Association Française
d’Information et de Recherche sur l’Animal de Compagnie,
personal communication). This increase of reservoir host
numbers could contribute to the spread of leishmaniasis.
The OR measuring the risk of infection in WO/HO zone
compared to HO (OR = 3.97 ; IC95% =1.33-12.71) is high.
This difference seems essentially linked to an unequal distribution of sandflies in these two ecological zones.
Concentration of P. ariasi is positively correlated with canine
leishmaniasis seroprevalence [16]. This difference between
different biotopes (altitude, rural/peri-urban) has also been
demonstrated by numerous authors. For example, OZON et
al. [26] found that seroprevalence varied with altitude (OR =
2,8, IC95% = 2.03-3.86) and COURTENAY et al. [4] showed
that the force of infection was 17 times higher in a rural than
Revue Méd. Vét., 2003, 154, 10, 599-604
603
in a periurban place. On the contrary, AMELA et al. [2] did
not find such difference.
Many authors consider that dog activity is an important
risk factor. It shows also an effect in our study since the risk
was higher for watch dogs, contrary to MILHAU [23] who
considered that hunting dogs were more exposed. In fact,
hunters are nowadays well informed about leishmaniasis and
know better how to prevent this disease. This may be different for watchdog owners, although the dog lives almost entirely outdoors and are bitten more frequently by the rather
exophilic P. ariasi. Pet dogs may be less affected by leishmaniasis because they spend more time inside the house.
Also, we showed that the age of dogs is a modifying factor.
Some authors found that prevalence was higher in old dogs
[16, 33]. In fact, old dogs may be more affected by this
disease since they have experienced more transmission seasons. Nevertheless, it seems that this effect is not so much
clear because other authors have observed an asymptotic distribution [8] or a bimodal distribution [2].
Sex is not often considered to be a risk factor [25, 26].
According to our results, the risk for males could be higher.
This has already been observed by CIARAMELLA et al. [3]
and ZAFFARONI et al. [37] and could be explained by an
increased out-door activity in males.
This study illustrates the difficulty to realize a representative epidemiological study. We used the voluntary recruitment of subjects, which does not permit the estimation of the
number of dog owners who may come. Random selection
from a clearly defined population would have been preferable, but was not possible because the French dog population is not completely registered.
Furthermore, as it was not possible to estimate the proportion of the dog population sampled, many of them may have
escaped to our study. This represents a partial source of bias.
Indeed, the population of dogs which were not sampled may
be different to those tested. We observed that dog owners preferred to bring young dogs and leave the old or ill dogs at
home. On the contrary, people who are interested by leishmaniasis are often those who have already been affected
by it.
Some of the problems described may be solved by prospective epidemiological studies, which could also be useful
to estimate the incidence of canine leishmaniasis. Repeated
investigations on the same dog cohorts coupled with the
study of the changes in vector populations could be a mean to
elaborate a mathematical model of the epidemiology of leishmaniasis. It could also be important to use other diagnosis
techniques as IFAT may be not enough sensitive [9]. For
example, PCR has already been widely studied for the diagnosis of leishmaniasis [15, 35]. The prognosis interest of
this technique for the diagnosis of canine leishmaniasis for
veterinary practictioners deserve to be evaluated
Acknoledgements
We thank Dr D. CARUSO, Mr P. LAMI, Mrs A. S. MERCIER-GUYON
for their valuable help in the field, Mrs I. GARRIS, MRS B. GERVAIS, Mrs
C. FOURRÉ for laboratory analysis, Dr M.P. PUECH, Dr M. RIOU for
announcements in the villlages. The authors are also grateful to Pr R.W.
ASHFORD for critical revision of the paper.
604
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