Seroepidemiological Study On Canine Visceral Leishmaniasis In ...
Iranian J Parasitol: Vol. 3, No. 3, 2008, pp. 1-10
Original Article
Seroepidemiological Study on Canine Visceral Leishmaniasis in
Meshkin-Shahr District, Ardabil Province, Northwest of Iran
during 2006-2007
A Moshfe 1,2, * M Mohebali 1, GhH Edrissian 1, Z Zarei 3, B Akhoundi 1, B Kazemi 4, Sh Jamshidi 5, M Mah-
moodi 6
1Dept. of Medical Parasitology & Mycology, School of Public Health and Institute of Public Health Research,
Tehran University of Medical Sciences, Iran.
2Dept. of Parasitology, School of Medicine, Yasuj University of Medical Sciences, Iran
3Meshkin-Shahr Research Station, Meshkin-Shahr, Ardabil, Iran
4 Cellular and Molecular Biology Research Center, Shaid Beheshti University, M.C., Tehran, Iran
5Dept. of Internal Medicine of Small Animals, Veterinary hospital, Faculty of Veterinary Medicine, Tehran Uni-
versity of Medical Sciences, Iran
6Dept. of Epidemiology and Biostatistics, School of Public Health and Institute of Public Health Research, Tehran
University of Medical Sciences, Iran
(Received 5 Apr 2008; Accepted 22 Aug 2008)
Abstract
Background: This study aimed to determine the seroprevalence of canine visceral leishmaniasis in Meshkin-Shahr district
as endemic areas of human visceral leishmaniasis (HVL) for presenting control program of HVL to health authorities.
Methods: A seroepidemiological study to determine seroprevalence of canine visceral leishmaniasis (CVL) among owner-
ship dogs using direct agglutination tests (DAT) in 21 villages of Meshkin-Shahr district, Ardabil Province was carried out
from June 2006 to August 2007. Three hundred and eighty four ownership dogs were selected by multi-stage cluster sam-
pling. Chi-square and Fisher exact tests were used to compare seroprevalence values relative to gender, age and clinical
signs.
Results: Of the 384 serum samples tested by DAT, 17.4 %( 95%C.I, 13.2%-20.8%) were positive (1:320 and higher). No
statistical significant difference was found between male (16.5%) and female (20.2%) seroprevalence (P=0.416). The high-
est seroprevalence rate (64.2%) was observed among the ownership dogs of three years age and above. Only 25.4% of the
seropositive dogs had clinical signs and symptoms. The most clinical signs among symptomatic dogs were cachexia (75%)
and alopecia (36.5%).
Conclusion: The majority of seropositive dogs (74.6%) lived in endemic areas of Meshkin-Shahr district were asympto-
matic. It seems that all symptomatic and asymptomatic infected dogs are the most important risk factors for human infection in
VL endemic areas.
Keywords: Canine visceral leishmaniasis, Seroepidemiology, Direct agglutination test, Iran
Introduction
parts of the world (3) and incidence rate of the
very year, approximately 500,000 new
disease is increasing in some countries (1).
E cases of VL (1, 2) which cause 59,000 hu- Since 1980, annually more than 200 human
man deaths annually, are reported from various
cases have been diagnosed from North West of
* Corresponding author: Tel: +98-21-88951400,
1
E-mail: mmohebali@hotmail.com
A Moshfe et al: Seroepidemiological Study on Canine…
Iran, mostly, from Meshkin-Shahr areas (Fig.
This study aimed to determine the present
1). It seems, that kala-azar has being endemic in
status of seroprevalence of CVL in various
this area for a long time (4). Canine leishma-
parts of Meshkin-shahr district especially in
niasis is not only a veterinary problem but is
endemic foci of HVL to more identifying the
also a serious public health problem. Rapid de-
role of dog as natural reservoir of human kala-
tection of canine visceral leishmaniasis (CVL)
azar in these areas to presenting effective con-
is highly important for control of human vis-
trol programme of HVL to health authorities.
ceral leishmaniasis.
Domestic dogs (Canis familiaris) are the most
Materials and Methods
important animal reservoir hosts of CVL, which
is transmitted among canines and to humans by
Study area
phlebotomine sand flies (5-8). Dog ownership
Meshkin Shahr district is located in Ardabil
is considered as an important risk factor for
Province, north-west of Iran. It was calling
human infections in the endemic areas of the
"Khiav", "Orami", "Varavi" in the past. The
disease in Iran (9). In a seroepidemiological
city of Meshkin-Shahr is situated at an altitude
study, the seroprevalence of infection was ob-
of 1490m above sea level and is the nearest city
tained 14.8% in the studied dogs in Meshkin-
to the Sabalan high mountain (Fig.1). The
Shahr district (10).
weather of this district is moderate mountain-
The percentage of infected dogs living in an
ous. It covers an area of approximately 1530
area where canine visceral leishmaniasis is en-
km2 including 323 villages and its population is
demic has major public health implications. It
estimated to be 169967 among which 42% was
was demonstrated that infected, but asympto-
settled in urban areas and 58% in rural areas.
matic dogs were sources of the parasite for
Out of which a small part belongs to nomad
phlebotomine vector sandflies and consequently
tribes.
play an active role in the transmission of
Sampling
Leishmania infantum (11).
The method of this study was descriptive cross-
CVL caused by L. infantum is an endemic zoo-
section and the sampling method was multi
notic disease in the Mediterranean basin and
stage cluster random sampling. Out of 323 vil-
Middle East, including Iran where seropreva-
lages in Meshkin- Shahr district, 21 villages
lence rate of disease has been reported from 10
(cluster) were selected randomly and in each
to 37% (12-14).
cluster, serum samples were taken from 10 dogs
As the high proportion of infected dogs is as-
randomly. The investigation was carried out
ymptomatic, therefore, detection of specific an-
over a period of 15 months from June 2006 to
tibodies remains the method of choice for mass
August 2007 on 384 ownership dogs. All the
screening of dogs in epidemiological surveys
selected dogs were physically examined by a
and evaluation of prevalence (15-19).
doctor of veterinary medicine. Dog age was de-
Serological methods are highly sensitive and
termined by interviewing dog owners. Blood
non-invasive, so they are best suited for use in
samples were taken from the selected dogs by
field conditions (20, 21). Several diagnostic
venapuncture in villages of Meshkin- Shahr
tests are available to detect anti-Leishmania an-
where HVL is endemic, poured into 10 ml po-
tibodies in canine sera. In the present study, the
lypropylene tubes and processed 4-10 h after
direct agglutination test (DAT) was used as
collection. The collected blood samples were
sero-diagnostic tool, because it is a simple as
centrifuged at 800 ×g for 5-10 min, and the se-
well as valid test and does not require specia-
parated sera were stored at -20°C. All the serum
lized equipments (12, 22-25).
samples were tested by DAT in the Leish-
2
Iranian J Parasitol: Vol. 3, No. 3, 2008, pp. 1-10
maniasis Laboratory in the School of Public
Some Leishmania promastigotes, which had
Health, Tehran University of Medical Sciences.
been isolated from spleens of domestic dogs
Direct Agglutination Test
following mass production in RPMI1640 me-
The L. infantum antigens for this study were
dia, were analyzed by PCR technique. The pri-
prepared in the Leishmaniasis Laboratory, Pro-
mers, LeiRNAF (5'-CAC CAC GCC GCC TCC
tozoology Unit of the School of Public Health,
TCT CT-3') and LeiRNAR (5'-CCT CTC TTT
Tehran University of Medical Sciences. The
TTT CNC TGT GC-3') were used to amplify
principal phases of the procedure for making
the genes coding for internal transcribed spacer
DAT antigen were mass production of promas-
2 (ITS2) and compared the results with stan-
tigotes of L. infantum Lon49 (Iranian strain) in
dard species of L. infantum,
RPMI1640 plus 10% fetal bovine serum, tripsi-
(MCAN/IR/96/LON49),
L. tropica
nization of the parasites, staining with coomas-
(MHOM/IR102/Mash4) and L. major
sie brilliant blue and fixing with formaldehyde
(MRHO/IR/75/ER) in the School of Public
2% (24-26).
Health, Tehran University of Medical Sciences
The dog serum samples were tested by DAT,
(27,28).
initially, for screening purposes; dilutions were
Data analysis
made 1:80 and 1:320. Samples with titers 1:320
Chi-square and Fisher exact tests were used to
were diluted further to end-point titer 1:20480.
compare seroprevalence values relative to gen-
Negative control, wells (antigen only; on each
der, age and clinical signs. Analyses were con-
plate) and known negative and positive control
ducted using SPSS software version 13.5, with
serum samples were tested in each plate daily.
a probability (P) value of <0.05 as statistically
The cut off titer was defined as the highest dilu-
significant.
tion at which agglutination was still visible, as
blue dot, compared with negative control wells,
Results
which had clear blue dots. The positive stan-
dard control serum prepared from dogs with L.
The sero-prevalence rate (SPR) in titers 1: 320
infantum infection (at 1:20480) in an endemic
and above was 17.4 % (95% C.I, 13.2-20.8).
area and confirmed by microscopy, culture and
Seventy (25.4%) of the seropositive dogs showed
animal inoculation. Quantitative results ob-
at least one clinical sign including skin lesions,
tained with DAT are expressed as an antibody
such as exfoliative dermatitis and ulcerations, local
titer, i.e. the reciprocal of the highest dilution at
or generalized lymphadenopathy, cachexia, low
which agglutination (large diffuse blue mats) is
appetite, alopecia, ocular lesions, epistaxis and
still visible after 18 h incubation at room tem-
lameness. No clinical signs and symptoms were
perature (23). Two individuals read the tests
seen in 50 (74.6%) of seropositive dogs.
independently. The cut off was determined in
Anti-Leishmania specific antibodies, using the
previous studies by experimental infection (12,
cut-off value of 1:320 and above were detected
24).
in male and female domestic dogs. The sero-
We considered anti-Leishmania antibodies ti-
prevalence values among male and female ani-
ters at ≥ 1: 320 as canine Leishmania infection
mals were 16.5 % and 20.2%, respectively (Ta-
in this investigation.
ble 1). No statistically significant differences
Parasitological study
between canine Leishmania infection and gen-
To confirm Leishmania infection in dogs, ne-
der were observed.
cropsy was performed on two DAT highly pos-
Table 2 shows that 32.7% of symptomatic dogs
itive dogs, liver and spleen samples of these
were seropositive whereas 15.1% of asympto-
dogs cultured in specific media for Leishmania
matic dogs were Ab negative. The most symp-
such as NNN and RPMI 1640.
tomatic dog (No. 33) had 3 years old age and
3
A Moshfe et al: Seroepidemiological Study on Canine…
higher. Referring to animal age groups, the
among symptomatic dogs was cachexia (75%)
highest seroprevalence (39.4%) was found in
and alopecia (36.5%).
dogs greater than 8 years old and the lowest
Both two DAT positive dogs were parasitologi-
values (10%) in dogs under 3 years old (Table
cally positive and amastigotes were observed in
3). Strong statistical significance was observed
the viscera of them. Promastigotes were seen in
between ≥8 and <8 dog age groups.
NNN and RPMI1640 culture media after 2
Of the 384 dogs, 52(13.5%) dogs had at least
weeks. L. infantum was identified by PCR tech-
one clinical sign and 50% of them did not have
nique. Samples were scored as positive when a
titer of antibody while 50% had antibody titer
PCR product of 565 bp was detected (Fig.2).
detected by DAT (Table4).
The PCR products were sequenced at MWG
The titers of antibody in symptomatic dogs
Company, Germany. Nucleotide sequence data
were 1:320 to 1:20480. The most clinical sign
submitted to the GenBank database with Acces-
sion Number EU680963 and EU680962.
Table 1: Sero-prevalence of canine Leishmania infection by gender in Meshkin-Shahr district (2006-2007)
Gender
No. of dogs (%)
DAT* test Positive ( ≥1:320)
No.
Seroprevalence (%)
Male
290 (75.5)
48
16.5
Female
94 (24.5)
19
20.2
total
384 (100)
67
17.4
* Direct agglutination test
Table 2: Sero-prevalence of canine Leishmania infection by signs and symptoms in Meshkin-Shahr district (2006-2007)
Signs & Symp-
No. of dogs (%)
DAT test Positive (≥1:320)
toms
No. Seroprevalence (%)
Symptomatic 52
(13.5) 17
32.7
Asymptomatic 332
(86.5) 50
15.1
total 384
(100)
67
17.4
Table 3: Sero-prevalence of canine Leishmania infection by age in ownership dogs in Meshkin-Shahr district
(2006-2007)
Age group(years)
No. of dogs (%)
DAT test Positive ( ≥1:320)
No. Seroprevalence (%)
0-3 239
(62.2)
24
10.0
4-7 112
(29.2)
30
26.8
≥8 33
(8.6)
13 39.4
total 384
(100)
67
17.4
4
Iranian J Parasitol: Vol. 3, No. 3, 2008, pp. 1-10
Table 4: Distribution of titers of anti Leishmania antibodies in symptomatic and asymptomatic ownership dogs by
DAT in Meshkin-Shahr district (2006-2007)
Titer of Ab
No. of dogs (%)
Symptomatic
Asymptomatic
No.
%
No.
%
< 1:80
240 (62.5)
26
10.8
214
89.2
1:80 37
(9.6)
5
13.5
32
86.5
1:160 40
(10.4)
4 10.0
36
90.0
1:320 22
(5.7)
6
27.3
16
72.7
1:640 9
(2.3)
1
11.1
8
88.9
1:1280 13
(3.4)
4 30.8
9
69.2
1:2560 9
(2.3)
1
11.1
8
88.9
1:5120 6
(1.6)
0 0.0
6
100
1:20480 8
(2.1) 5 62.5
3
37.5
total 384
(100)
52
13.5
332
86.5
Fig 1: Geographical situation of Meshkin-Shahr district in Ardabil Province, northwest of Iran
5
A Moshfe et al: Seroepidemiological Study on Canine…
Fig. 2: Gel Electrophoresis of PCR products: A: Marker, 100 bp. BوE: Negative Control. C: Leishmania ma-
jor, 625 bp. D: Leishmania infantum, 565 bp
Discussion
technique and is well correlated with clinical
signs. According to previous studies (24, 26,
Dogs and wild canines are animal reservoir
35, 36) the performance of the DAT for detec-
hosts for L. infantum in both old and new
tion of L. infantum infection in humans and
worlds (3).Determination of the prevalence of
dogs was desirable. Therefore, we use DAT for
canine Leishmania infection is necessary to de-
the determination of sero-prevalence of canine
fine control measures for zoonotic visceral
Leishmania infection.
leishmaniasis (29).
With consideration to ecological and epidemi-
Our studies in the last decade showed that L.
ological changes in the studied areas during last
infantum Lon49 was the principal agent of the
decade, this study was designed.
disease in human and animal reservoirs in dif-
Based on our results, seroprevalence of CVL in
ferent parts of Iran (28, 30, 31). Dogs and wild
Meshkin-Shahr was determined 17.4% using
carnivores such as jackals, foxes and wolves,
the cut-off value of 1:320 and above. A seroe-
which have been found, infected with L. infan-
pidemiological study was carried out by Bokai
tum (32, 33) and domestic dogs are considered
et al. on 303 serum samples of ownership dogs
the most important source and reservoirs of L.
in Meshkin-Shahr district in 1998, the seropre-
infantum infection particularly in the endemic
valence of infection was obtained 14.8% in the
areas of Iran(9, 12).
studied dogs, and only 13.6% of the seroposi-
Visceral leishmaniasis, caused by L. infantum,
tive dogs were symptomatic (10). Based on two
is endemic in Meshkin-Shahr district where al-
studies that designed for CVL seroprevalence
most 40% of human VL cases in Iran were re-
determination in northwest of Iran, seropositiv-
ported in recent years (25, 31, 34).
ity rate was achieved 21.6% and 18.2% in the
Some scientists believe that nomadic from
northwest of Iran, respectively (9, 12).
Moghan district may have introduced the dis-
No statistical differences were found among
ease into the area from the northeast of Ardabil
Leishmania infection with regard to gender in
Province (12). A number of studies have carried
our study. Similar results were found by Ab-
out on diagnosis and epidemiologic surveys to
ranches et al. (1992) in Portugal; Poaio et
identify potential risk factors of the diseases in
al.,(1981) in Italy; Sideris et al.,(1996) in
the area (9, 10). For canine leishmaniasis, se-
Greece ,Bokai et al.,(1998) and Mohebali et
rology is considered a sensitive and useful
al.(2005) in Iran.(10, 12, 37-39).
6
Iranian J Parasitol: Vol. 3, No. 3, 2008, pp. 1-10
In the current study, we found canine Leishma-
seropositive dogs transmitted the parasite to the
nia infection mostly in older dogs (8 years and
sandfly vectors (11).
above). Statistical analysis revealed greater
In conclusion, the most important result was a
seroprevalence in groups of older dogs,
high proportion of seropositivity for leishma-
indicating that the probability of exposure to
niasis (15.1%) among dogs without clinical
the bite of sand flies infected with L. infantum
signs of canine leishmaniasis. These data are
increased with age of infected dogs (37, 40).
very important because ownership dogs can
The high prevalence of Leishmania infection in
play an important role in the epidemiology of
dogs appears to be due to high exposure with
this zoonotic disease. Furthermore, the domestic
Leishmania parasites both in villages and in the
dog population could be helpful sentinels to
wild areas.
follow the progress of the disease in endemic
Dogs from Meshkin-Shahr district seem to have
areas.
the most important role for the disease because
Control programs on infected dogs will be al-
of large dog populations (7 dogs/100 humans)
most impossible without taking effective meas-
and their heavy infections that sometimes
ures to determine the status of sero–positive in
reached to 20% seropositive in some of the vil-
asymptomatic dogs. Essentially, elimination of
lages (10, 41, 42).
infected animals has been recommended (48),
However, the role of asymptomatic but seropo-
but alternative control measures should be rec-
sitive dogs (50 out of 67) is difficult to explain
ommended for ethical and social reasons.
without a follow-up study. In a previous study,
Molina et al. (1994) found in Spain that asymp-
Acknowledgements
tomatic dogs as well as symptomatic cases
could be a cause when ability of sandflies to
Some parts of this study received financial sup-
pick up infection is not dependent in clinical
port from the Institute of Public Health Re-
manifestations (11). Undoubtedly, this condi-
search, Tehran University of Medical Sciences
tion indicates previous contact with the para-
(Projects No: 240/4659 and 130/6/10447).
site, but we do not know whether these dogs are
We thank Dr.H,Hajjaran and Mrs.S.Charehdar
immune resistant animals or whether they will
for helping us in laboratory techniques and
subsequently develop the disease (43). Thus,
Dr.R.Nikzad as well as Mr.A.Meiahipour for
our study and others confirm that the preva-
helping us to sampling, the staff of the District
lence of Leishmania infection in dogs has been
Health Centers in Ardabil Province and Mesh-
underestimated (43, 44).
kin- Shahr district particularly Dr. Gh. Hos-
The high proportions of asymptomatic to
seingholizadeh, Dr. A. Gangi and Mr.M.H Pa-
symptomatic in infected domestic dogs lacking
shaei. The authors declare that they have no
clinical signs may be related to development of
Conflict of Interests
protective immunity especially in older dogs
and their frequent exposure to Leishmania pa-
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