A seroepidemiologic survey of canine visceral leishmaniosis among apparently healthy dogs in Croatia

www.elsevier.com/locate/vetpar

Veterinary Parasitology 131 (2005) 35–43

A seroepidemiologic survey of canine visceral leishmaniosis

among apparently healthy dogs in Croatia

T. Zivicnjak a,*, F. Martinkovic a, A. Marinculic a, V. Mrljak b, N. Kucer b,V. Matijatko b, Z. Mihaljevic c, R. Baric-Rafaj b

a Department for Parasitology and Parasitic Diseases, Veterinary Faculty, University of Zagreb, Heinzelova 55, 10000 Zagreb, Croatiab Clinic for Internal Diseases with Chair for Cynology, Veterinary Faculty, University of Zagreb,

Heinzelova 55, 10000 Zagreb, Croatiac Croatian Veterinary Institute, Savska 143, 10000 Zagreb, Croatia

Received 12 November 2004; received in revised form 12 April 2005; accepted 22 April 2005

Abstract

Cross-sectional investigation was done on seroprevalence of Leishmania sp. infection among apparently healthy dogs in an

area where canine leishmaniosis is endemic. Survey included 68 dogs living in the coastal city of Split, and 238 dogs living in 12

villages scattered in the hinterland. Each dog was clinically examined for the presence of some discrete signs compatible with

leishmaniosis and by dot-ELISA modification determined the presence of anti-Leishmania antibodies. The titre 1:600 and higher

was regarded as positive in the study. The seroprevalence ranged from 0 to 42.85%, depending on the location. 54.34% of the

seropositive dogs had moderately enlarged lymph nodes and/or some discrete changes on the skin. In our parasitological study,

Leishmania sp. was isolated from several seropositive animals that had some clinical signs and from a few which did not have

any.

Data analysis revealed that serological positivity to Leishmania sp. was not associated with a dog’s outdoor lifestyle and

utility, but was associated with the gender and age.

# 2005 Elsevier B.V. All rights reserved.

Keywords: Leishmania infantum; Dog; Epidemiology; Dot-ELISA; Croatia

1. Introduction

Canine leishmaniosis, caused by Leishmania

infantum, is an endemic in the Mediterranean basin

* Corresponding author. Tel.: +38 512 390 361;

fax: +38 512 390 362.

E-mail address: [email protected] (T. Zivicnjak).

0304-4017/$ – see front matter # 2005 Elsevier B.V. All rights reserved

doi:10.1016/j.vetpar.2005.04.036

and its seroprevalence ranges from 10 to 37% (Amela

et al., 1995; Fisa et al., 1999; Sideris et al., 1999).

Recent studies have demonstrated that in endemic

areas, a high percentage of dogs (60–80%) have come

into contact with the parasite without exhibiting any

signs of the disease (Ferrer, 1999). Since, clinical

manifestations including weight loss, elongated and

deformed nails, mouth ulcers, skin lesions, hair loss,

.

T. Zivicnjak et al. / Veterinary Parasitology 131 (2005) 35–4336

keratoconjuctivitis, dermatitis and lymphadenopathy

(Ferrer et al., 1988; Ciaramella et al., 1997; Koutinas

et al., 1999) are observed only in a low proportion of

the infected dogs, serodiagnosis has been considered

essential for evaluating the prevalence of the infection

(Gradoni et al., 1988; Ferrer, 1999). Seroepidemio-

logical studies of canine leishmaniosis have revealed a

large number of asymptomatic seropositive animals

(Portus et al., 1987; Sideris et al., 1999). The ability to

infect sand flies was similar in both asymptomatic

carriers and animals with different degrees of signs of

disease (Molina et al., 1994).

In the south littoral parts of Croatia, canine

leishmaniosis was recognized as a problem for the

first time in the first part of 20th century (Tartaglia,

1937). The medically relevant entomofauna of

Croatia has not yet been adequately investigated,

but some studies (Miscevic et al., 1998), clearly

indicate that Phlebotomus neglectus, P. perfiliewi

and P. tobii, which are proven vectors (Killick-

Kendrick, 1999) of protozoan parasite Leishmania

infantum, are to be found in the coastal region of

Dalmatia.

Since 1997, leishmaniosis in Croatia has been

proven parasitologically (demonstration of amasti-

gotes in stained smears of a lymph node aspirates), as

well as serologically (dot-ELISA, IIFAT) at the

Department for Parasitology and Parasitic Diseases

(Veterinary Faculty in Zagreb) in hundreds of

clinically ill dogs (Zivicnjak et al., 1998; Martinkovic

et al., 2001). Almost all the dogs lived in central and

south parts of Dalmatia (from the city of Trogir in the

west, Montenegro border in the east, Bosnia and

Herzegovina border in the north, the Adriatic sea in the

south, as well as on middle and south Dalmatian

islands). Although some dogs with proven leishma-

niosis lived in other parts of Croatia, they had, as a

rule, spent several summer weeks in the region.

Furthermore, the parasite has been successfully

isolated and cultivated in vitro. Electrophoresis of

four isolates from Dalmatia region was carried out in

starch gel according to previous protocols (Rioux

et al., 1990), using a panel of 13 enzymes (15

enzymatic loci) in WHO Collaborating Centre for

Leishmaniosis, Servicio de Parasitologia, Nacional de

Microbiologia, Instituto de Salud Carlos III; Madrid,

Spain by Carmen Chicharro Gonzalo. All of them

were Leishmania infantum zymodeme MON-1 (iso-

late no. 1: WHO code MCAN/HR/2003/LLM-1282;

no. 2: WHO code MCAN/HR/2003/LLM-1280; no. 3:

WHO code MCAN/HR/2003/LLM-1279; no. 4:

WHO code MCAN/HR/2003/LLM-1281.

Human visceral leishmaniosis has been sporadi-

cally reported in southern Croatia; however, as with

other diseases transmitted by arthropods, it is not a

major public health problem in Croatia (Mulic et al.,

2002).

The obligatory blood testing of all dogs exhibiting

evident clinical symptoms of leishmaniosis in Dalma-

tian counties is decreed by The Act on Veterinary

Care.

The aim of our study was to determine the

applicability of our dot-ELISA modification for

screening purposes on the seroprevalence of infection

among apparently healthy dogs which were not

included in the provisions of The Act on Veterinary

Care. The survey was performed in the endemic region

where a few hundred clinically sick dogs had been

passively detected (clinically, serologically and para-

sitologically confirmed) since 1997.

2. Materials and methods

2.1. Study area

The cross-sectional survey was carried out during

January and February 2003 in the central part of

Split-Dalmatia County in Croatia (Fig. 1). The county

is divided into three main parts: elevated hinterland

(Dalmatinska zagora), narrow coastal strip and the

islands. Parts of the Dinaric Alps, including Dinara

itself, form the border with Bosnia and Herzegovina,

while Kozjak, Mosor and Biokovo mountains separate

the coastal strip from the hinterland.

The county’s centre is Split (438300N, 168260E).

The population of the county is 463,676 (2001). Land

area is 4534 sq km. The climate and vegetation are

typically Mediterranean with temperate winters up to

600 m altitude. The mean annual air temperature

ranges from 13 to 17 8C. The yearly precipitation

pattern is maritime in character, with dry summers

and maximum precipitation during the cold months of

the year.

The survey included dogs from the coastal city

Split and twelve hinterland villages (latitudes 438340

T. Zivicnjak et al. / Veterinary Parasitology 131 (2005) 35–43 37

Fig. 1. A map of the study area. The numbers refer to the study sites, as numbered in Table 1. Squares (&) are indicating locations with at least

one positive dog. Dots (*) are indicating locations without positive dogs.

Table 1

Distribution of canine leishmaniosis in 13 locations in the Split-

Dalmatia County

Location Altitude

(m)

Positive/

studied

Frequency

(%)

1 Split 60 10/68 14.7

2 Kucine 130 1/11 9.09

3 Klis 241 8/29 27.58

4 Podgrape 132 3/14 21.42

5 Slime 457 0/10 0

6 Gornja Brela 538 1/14 7.14

7 Zagvozd 470 6/14 42.85

8 Zadvarje 231 6/58 10.34

9 Sestanovac 336 4/17 23.52

10 Zezevica 513 7/17 41.17

11 Lovrec 599 0/27 0

12 Studenci 699 0/12 0

13 Arzano 681 0/15 0

Total 46/306 15.03

The numbers refer to the study sites as numbered in Fig. 1.

and 438230N, longitude 168170 and 17830E) scattered

through the endemic region. The altitudes of the 12

villages included in this study varied from 130 to

699 m above sea level. The mean altitude of the city of

Split is 60 m above sea level (Table 1).

2.2. Animals

We focused on the dogs regarded as healthy by their

owners. The survey was carried out with 306 animals

aged six months or more (6–144 months; mean 35

months); 176 were males and 130 females; 68 were

from the city of Split and 238 dogs from the villages in

the region. The survey was carried out on hunting dogs

(N = 206) and guard dogs (N = 85) kept outdoors, and

a small number of pets (N = 15) kept indoors.

When the owners agreed with the protocol,

examination and peripheral blood collection were

carried out during the anti-rabies vaccination cam-

paign performed during January and February 2003

before the beginning of the phlebotominae season.

Information on age, sex, breed, and other character-

T. Zivicnjak et al. / Veterinary Parasitology 131 (2005) 35–4338

istics was gathered using a standardized questionnaire,

administered to the owners of each animal.

2.3. Clinical examination

Clinical status was evaluated according to criteria

suggested by Abranches et al. (1991) and modified by

Molina et al. (1994).

After clinical evaluation, dogs with obvious two or

more clinical signs of leishmaniosis (weight loss,

dermatitis, hair loss, mouth and skin ulcers, enlarged

lymph nodes, arthritis, and keratoconjuctivitis) were

excluded from the survey. In the survey were included

only those dogs without any signs (asymptomatic) and

those with moderately enlarged lymph nodes and/or

discrete skin changes, such as dull coat or exfoliative

dermatitis without hair loss.

2.4. Sample collection

Blood was collected by cephalic venipuncture; sera

were separated, transported to the laboratory and kept

at �20 8C until processing.

Popliteal lymph node aspirates were taken from the

dogs with enlarged lymph nodes and/or skin changes,

from the seropositive dogs and from 12 seronegative

asymptomatic dogs. Smears were prepared immedi-

ately after aspiration and fixed in methanol. In our

laboratory, we stained the smears with Giemsa and

examined under a light microscope for the presence of

amastigotes.

2.5. Parasite cultivation and antigen preparation

Leishmania infantum (WHO code MCAN/HR/

2003/LLM-1282) amastigotes isolated in 2002 from

popliteal lymph node of a seven-year-old, naturally

infected symptomatic German shepherd from Dalma-

tia were used to prepare antigen.

The amastigotes transformed in Evan’s modified

Tobie’s medium—EMTM (Evans, 1987) and promas-

tigotes for antigen production were cultivated in the

liquid medium as was previously described (Lımoncu

et al., 1997). Promastigotes were harvested during

stationary phase growth (5th day at 25 8C) and

characteristically contained >95% motile organisms.

These promastigotes were washed three times with

0.01 M phosphate-buffered saline solution (PBSS pH

7.2) followed by repeated centrifugation at 3000 rpm.

The sediment was resuspended in PBSS and adjusted

to a concentration of 20–30 promastigotes �107 ml�1. The suspension was three times frozen at

�20 8C and thawed at room temperature to disrupt the

cells. The protein concentration was adjusted (Brad-

ford, 1976) to 1 mg/ml. Aliquots of these proteins

were stored at �20 8C until required.

2.6. Dot-ELISA

In this survey, we used a rapid and economical dot-

ELISA to determine the presence of anti-Leishmania

antibodies, where 1 mg/ml of ‘‘crude’’ protozoan

antigen was ‘‘dotted’’ onto nitrocellulose strips

(3 mm � 25 mm); incubation took place in plastic

1.5 ml tubes (Eppendorff). Basically, the technique is

a combination of methods previously described

(Pappas et al., 1983; Mancianti et al., 1996, Vercam-

men et al., 1998) but modified and simplified.

Briefly, antigen was thawed at room temperature;

1 ml was dotted on each nitrocellulose membrane strip

and incubated for 30 min at 37 8C. The strips with

adsorbed antigen were placed in plastic 1.5 ml tubes

(Eppendorff) separately. Five hundred microliters of

blocking solution (5% BSA-PBSS) was added to

plastic tubes with antigen strips, and each strip was

incubated for 15 min at 37 8C.

Blocking solution was simply poured out, and

750 ml of diluted serum samples (1/600) was poured

into each tube. Incubation lasted for 30 min at 37 8C.

Diluted sera were poured out, and 1 ml 0.05% PBSS-

TWEEN 20 was added. After 10 min, the washing

solution was discarded; washing was repeated twice for

10 min. After the strips were rinsed three times, 1 ml of

a horseradish-labelled sheep anti-dog IgG conjugate

(Serotec, cat. no. AAI32P) diluted 1:5000 in PBSS was

added in each tube for 30 min with three washes in

succession. 1 ml of substrate solution (immediately

before the use 3 mg 4-chlor-1-naphtol dissolved in 1 ml

anhydrous methanol was mixed with 50 ml 9% H2O2,

than added to 25 ml PBSS) was added to each tube,

and incubated for 30 min at room temperature. The

chromogen solution was discarded and strips were

washed three times as described above. Colour

development was visually determined. The develop-

ment of a clear blue dot on strips when compared with

negative serum control was considered evidence of

T. Zivicnjak et al. / Veterinary Parasitology 131 (2005) 35–43 39

positivity. Those with indistinct or blurred stained area

were regarded negative in this study. Colour develop-

ment in negative controls was completely absent.

Two positive sera samples (titre � 1/1280) from

symptomatic dogs with parasitologically proven diag-

nosis and two negative control sera (titre � 1/40) from

healthy four-month-old puppies from endemic region

of Croatia (Dalmatia region) were included in every

assay. All sera were tested in duplicate and all were

retested at least once; at sera dilutions 1:600, the results

in each duplicate and retest were the same.

The sensitivity of the test was previously tested on

40 dogs with positive parasitological examination. All

40 sera samples from those dogs in serial dilutions

from 1:10 gave titres that were �1/1280. The

specificity was tested on 40 dogs from nonendemic

area (Slavonija region) and the highest positive

dilution was 1/160 in one dog, two dogs had positive

dilution 1/80, and 37 had �1/40 (unpublished data).

In order to separate clear positive and clear negative

results in the screening, we decided to test sera in

dilution 1/600. It was invariably a positive dilution in

dogs with positive parasitological examination, and an

invariably negative dilution in healthy control dogs

during sensitivity and specificity testing.

2.7. Data analysis

Epidemiological data on age, sex, dog utility and

location were recorded in a questionnaire filled out by

the owners and the participating veterinarians. Multi-

variate logistic regression models were used to

identify risk factors for Leishmania sp. seropositivity.

Each model was applied at the individual dog level

using epidemiological data as independent variables

and serological status as a dependent variable. For

assessing statistical significance of interaction, we

used likelihood ratio test (LRT). All statistical

analyses were performed using STATA 6.0 (Stata-

Corp. 2003. Stata Statistical Software: Release 6.

College Station, TX, USA).

Fig. 2. Age distribution. Lines are indicating sample size for each

age group, while bars are indicating prevalence of seropositive dogs

within each age group.

3. Results

A total of 46/306 samples analysed were positive

by dot-ELISA (titre 1/600) resulting with clear blue

dots on nitrocellulose strips.

The seropositive dogs were found in the city of

Split and in 8/12 villages surveyed. Mean seroposi-

tivity among the dog population in the city of Split

was 14.7%, and in the eight villages with canine

leishmaniosis it ranged from 7.1 to 42.8%. The

differences in prevalence among the different loca-

tions in which seropositive dogs were identified were

statistically significant ( p = 0.034). The highest odds

ratio (OR) for leismaniosis were noted with the dogs in

the villages Zagvozd (OR = 4.4; 95% confindence

interval/95% CI: 1.24–15.23), Zezevica (OR = 4.0;

95% CI: 1.25–13.16) and Klis (OR = 2.2; 95% CI:

0.77–5.04). The altitudes of the 12 villages included in

this study varied from 130 to 699 m above sea level.

The mean altitude of the city of Split is 60 m above sea

level. However, the differences in prevalence were not

associated to the altitude. Mean negative dogs’ age in

the survey was 34 months (range 6–144), while mean

seropositive dogs’ age was 43 months (range 12–120).

The significant differences ( p = 0.03) in the age of

seropositive dogs were found (Fig. 2). Leishmania

positive dogs were more likely to be aged 73–84 months

(OR = 7; 95% CI: 1.42–34.54) and 37–48 months

(OR = 5.9; 95% CI: 1.74–19.88). LRT test indicated

significant ( p < 0.01) interaction between location and

the dogs’ age. The highest OR was recorded in Zagvozd

(OR = 6.5; 95% CI: 1.65–25.43) and Zezevica

(OR = 5.8; 95% CI: 1.59–21.20), in the dogs aged

73–84 months (OR = 37.2; 95% CI: 4.14–331.1)

T. Zivicnjak et al. / Veterinary Parasitology 131 (2005) 35–4340

Fig. 3. Seroprevalence by sex. Seroprevalence (bares) among males

was higher ( p < 0.01) with 19.31% sero-positive animals (95% CI:

13.6–25.6) than in females, where 9.23% (95% CI: 4.08–13.96)

bitches were sero-positive (OR = 2.5; 95% CI: 1.22–5.0).

Fig. 4. Seroprevalence among utility groups. Bars are indicating the

seroprevalence among utility groups. Differences among utility

groups were not evident ( p > 0.05).

and 37–48 months (OR = 7.6; 95% CI: 1.98–28.83).

Seroprevalence (Fig. 3) among the males was higher

( p < 0.01) with 19.31% sero-positive animals (95%

CI: 13.6–25.6), than in the females, where 9.23% (95%

CI: 4.08–13.96) bitches were sero-positive (OR = 2.5;

95% CI: 1.22–5.0). The average age of the males did not

significantly differ from the age observed in the

females. Among utility groups (Fig. 4), the differences

were not evident ( p = 0.38); among hunting dogs

positive were 16.91% (95% CI: 11.75–22.06), guard

dogs 10.58% (95% CI: 3.96–17.46), and pets 13.33%

(95% CI: 6.15–32.81).

After clinical evaluation, 16 dogs exhibited

obvious, two or more clinical signs of leishmaniosis

(weight loss, dermatitis, hair loss, mouth and skin

ulcers, enlarged lymph nodes, arthritis, and kerato-

conjuctivitis). They were not included in the survey,

but their blood was tested in the county Veterinary

Institute, according to the provisions of The Act on

Veterinary Care (the results are not known to us).

During clinical examination of 306 dogs included

in the survey, 43 of them (14.5%) had some discrete

skin changes, such as exfoliative dermatitis without

hair loss and/or moderately enlarged lymph nodes, and

lymph node aspiration was done. 18/43 dogs were

seronegative and parasitologically negative. The

remaining 25/43 animals were seropositive, with 15

parasitologically positive dogs among them ((Fig. 5

group A). Moreover, 21 seropositive asymptomatic

dogs were also tested by lymph node biopsy, as were

12 seronegative dogs. In the seropositive asympto-

matic group (Fig. 5 group B), two dogs were

parasitologically positive. No dog was found serolo-

gically negative, and parasitologically positive.

Among the seropositive animals, 54.34% had

some signs compatible with leishmaniosis, whereas

45.7% were entirely asymptomatic. In the cohort of

seropositive dogs, 26.08% were parasitologically

positive as well (Fig. 5).

4. Discussion

The first cross-sectional sero-epidemiological

survey on canine leishmaniosis among apparently

healthy dogs was done in the enzootic region of

Croatia where a few hundred clinically sick dogs had

been passively detected since 1997. The obligatory

blood testing of all dogs exhibiting evident clinical

symptoms of leishmaniosis is decreed in Dalmatian

T. Zivicnjak et al. / Veterinary Parasitology 131 (2005) 35–43 41

Fig. 5. Clinical examination reliability. Group A: dogs with some

discrete skin changes, such as exfoliative dermatitis without hair loss

and/or moderately enlarged lymph nodes. Group B: asymptomatic

dogs, ser� par�: serologically and parasitologically negative; ser+ -

par+: serologically and parasitologically positive; ser+ par�: ser-

ologically positive and parasitologically negative.

counties by The Act on Veterinary Care. However,

following the usual pattern, the dog owners in this area

often euthanize the affected animals themseleves,

without ever consulting a veterinarian. Therefore, it is

impossible to estimate the number of ill dogs with any

precision or provide accurate figures. On the other

hand, the owners were unwilling to allow the testing

of dogs that did not appear ill for the ‘‘scientific

purposes’’; they often did not permit us to take their

blood. Consequently, we were able to obtain the

samples from only 306 asymptomatic dogs, out of

approximately 10,000 dogs living in the area. There-

fore, the dog samples should not be considered

representative of the whole dog population in the area

studied. We decided to use our dot-ELISA modifica-

tion as a simple and economical technique for field

screening of dog populations.

The survey was carried out on the dogs whose

owners regarded them healthy (not included in the

provisions of The Act on Veterinary Care) and agreed

with the protocol, but those dogs could not be regarded

as entirely asymptomatic. Although they worked well

and seemed well, and exhibited good appetite and

condition, some of them had some visible changes on

skin and/or moderate enlargement of lymph nodes.

Since, dogs considered healthy by their owners

(including asymptomatic infected dogs) will not be

taken to the veterinarian, these dogs will be a reservoir

for infection within the general population.

Similar to other countries in the Mediterranean

basin (Deplazes et al., 1998; Solano-Gallego et al.,

2001; Cardoso et al., 2004), the prevalence in the

endemic region of Croatia has been shown to widely

vary among the identified endemic areas. In the city of

Split, positive animals were scattered without any

grouping. Neither could we explain the differences in

prevalence among positive locations and high OR in

Zagvozd, Zezevica and Klis. These villages did not

differ significantly by the altitude. Three of four

villages without canine leishmaniosis are situated at

the altitude of 599 m or more. In Split-Dalmatia

County, 600 m altitude is the limit where the

Mediterranean climate switches to a cooler one,

associated with changes in ecological conditions. The

fourth village without positive dogs in the survey is

situated at an altitude of 457 m, in the close vicinity to

the three positive villages. According to Ashford

(2000), the distribution of leishmaniosis is determined

by its vector, reservoir host, and limiting factors, i.e.

specific environmental requirements that lead to focal

distribution of disease. In an entomological survey

performed in 1998 in the region (Miscevic et al.,

1998), the vectors were found in coastal cites of Split,

Omis and Makarska, but such survey was not

performed deeper in the hinterlands and at higher

altitudes. Further entomological investigations should

be done to investigate if the vector occurs at higher

altitudes. The distribution of seropositive dogs within

the canine population showed significant heterogene-

ity according to the age and sex; however, it was not

relevant whether the animal was kept mainly indoors

(pets) or outdoors (guard and hunting dogs). Our

prevalence results according to outdoors and indoors

lifestyles differ from the results published by

Zaffaroni et al., (1999) where serological positivity

to Leishmania sp. was significantly associated with a

dog’s outdoor lifestyle. The increase in the prevalence

of the seropositive dogs with age and the final decrease

in seropositivity in the older group of dogs (>84

T. Zivicnjak et al. / Veterinary Parasitology 131 (2005) 35–4342

months) is in agreement with other studies (Martinez

Cruz et al., 1990; Abranches et al., 1991; Fisa et al.,

1999). This could be related to the cumulative increase

of the time of exposure of dogs to phlebotomines and

the increase of the death rate in old animals.

As in some other Mediterranean foci (Martinez

Cruz et al., 1990; Fisa et al., 1999; Zaffaroni et al.,

1999), we found a higher prevalence among males

than in females. In our study, we could not find any

gender-associated differences in exposure to the

parasite; males and females were kept in the same

manner. One possible explanation could be an increase

in female mortality in which pregnancy and nursing

may play an important role (Fisa et al., 1999), but

gender-related differences in the host immune

response might play a role in the resistance and

susceptibility to infection. As it was shown in mice for

L. major (Mock and Nacy, 1988), hamsters for New

World leishmaniasis (Travi et al., 2002) and human

macrophages for L. donovani (Zhang et al., 2001)

possible testosterone immunomodulation in dogs

should be investigated.

Our dot-ELISA modification, used for screening

purposes, could detect subclinical and active leish-

maniosis at sera dilution 1/600, which was also

parasitologically proven in 26.08% cases. Lower titres

were not assayed in the screening to facilitate results

interpretation. Interpretation of results obtained by

detection of specific antibodies in dogs from endemic

areas is often difficult. When we deal with asympto-

matic animals in endemic areas, immunological

techniques do not discriminate between infected

and resistant animals (Cabral et al., 1998; Cardoso

et al., 1998).

We can conclude that we recorded a possible source

of infection in the area; we are also aware that there

might be a number of dogs with lower titre which were

not recorded in our cross-sectional study. There is a

need for the serological follow-up in a longitudinal

study, with serially diluted sera samples. Retests might

be helpful in distinguishing early phases of the disease

from resistant animals in those that have low antibody

titers.

Furthermore, the value of clinical examination

reliability that defines which dog has to be blood-

tested on leishmaniosis in the endemic area should be

re-evaluated. Although clinical examination is neces-

sary, our survey within asymptomatic dogs’ popula-

tion has shown how prevalence can be underestimated.

It is obvious that a diagnosis could be established only

after a combination of the clinical examination and

other diagnostic techniques.

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