A survey on canine leishmaniasis in western Turkey by parasite, DNA and antibody detection assays

doi:10.1016/S0001-706X(99)00047-9

Acta Tropica

Volume 74, Issue 1, 5 January 2000, Pages 1-6

https://doi.org/10.1016/S0001-706X(99)00047-9 Get rights and content

Abstract

Human visceral leishmaniasis (VL) caused by Leishmania infantum is found throughout the Mediterranean Region, including Turkey, where dogs are considered to be the main reservoir host for this parasite. In the district of Manisa, western Turkey, 37 human VL cases were reported from June 1993–August 1997. Twenty-four villages in this district were chosen for a survey of disease prevalence in dogs. The dogs, 490 in total, were examined using either the indirect immunofluoresence assay (IFAT) or direct agglutination test (DAT). Anti-Leishmania antibodies were found by at least one test in 5.3% (26/490) of the dogs. Infections were confirmed by parasitological examination of or polymerase chain reaction (PCR) on lymph node aspirates in 65% (13/20) and 76.4% (13/17) of the seropositive dogs tested, respectively. The confirmation rate was 85% by combining the results of PCR and microscopy. Our results demonstrate that canine VL is wide spread in western Turkey where human VL is endemic, and that serodiagnosis is a valuable tool for monitoring the infection.

Introduction

In the Mediterranean region, visceral leishmaniasis (VL) is a zoonotic disease caused by Leishmania infantum. Dogs are believed to play an important role in human VL and the prevalence of canine infection rates ranges from 1.1% to 37% in different countries. However, no direct correlation has been reported between the incidence of canine and human infection (Harith et al., 1989). Since clinical manifestations including weight loss, elongated and deformed nails, mouth ulcers, skin lesions, hair loss, keratoconjuctivitis, dermatitis and lymphadenopathy are only observed in a low proportion of the infected dogs, serodiagnosis has been considered essential for evaluating the prevalence of the infection (Gradoni, 1995).

Several serodiagnostic tests, including the indirect immunofluoresence assay (IFAT), the direct agglutination test (DAT), enzyme-linked immunosorbent assays (ELISAs) and crossed immunoelectrophoresis (CIEP), have been used (Pozio et al., 1981), though direct microscopic examination and in vitro culture of tissue aspirates are still considered as the "gold standards". However, parasite detection in biopsy samples is influenced by several factors, including organ distribution, and culturing may be unsuccessful due to contamination. Therefore, molecular biological techniques such as the polymerase chain reaction (PCR) have been introduced to detect parasite DNA in tissue samples. PCR was found to be highly sensitive and specific using both bone marrow biopsies and lymph node aspirates (Ashford et al., 1995; Mathis and Deplazes, 1995; Osman et al., 1997).

CVL in Turkey was first described in 1951 in the cities of Bursa and Istanbul. However, the epidemiology of CVL has not been extensively studied and little is known about its distribution and prevalence. As such, there is a need for detailed surveys, especially in western Turkey where human VL occurs. The aim of this study was to survey the status of CVL in the Manisa region located in western Turkey using different serological and molecular biological methods.

Section snippets

Study area

Twenty-four villages in Manisa district of the western part of Turkey (Fig. 1), where 37 cases of human VL were reported between June 1993 and August 1997, were chosen for this study. All patients were chosen based on the presence of clinical symptoms typical of VL. VL was parasitologically confirmed by bone marrow aspiration. Household dogs, 4 to 48 in every village, representing 30–50% of the whole dog population were surveyed. The total number of dogs enrolled in this study was 490.

Blood sampling

Five ml

Results

The altitudes of the 24 villages included in this study varied from 33 to 850 meters above sea level. The main occupation of the villagers was farming and animal breeding. All 490 dogs sampled in the villages had owners and were used primarily for hunting and/or guarding. Dogs were kept outside the houses.

A total of 26 out of 490 dogs (5.3%) examined were found to be seropositive (IFAT titer = 1:128 and/or DAT titer =1:320). The remaining dogs were seronegative (IFAT titer <1:128 and DAT titer

Discussion

The successful implementation of control strategies for VL requires prior knowledge of the infection rates in dogs and localization of active disease foci. Epidemiological surveys of CVL are based on clinical symptoms of disease, antileishmanial antibody titers and/or detection of the parasite. Since a high proportion of infected dogs are asymptomatic, serodiagnosis is frequently used to evaluate the prevalence of leishmanial infections in large dog populations (Gradoni, 1995). However,

Acknowledgements

This study was financially supported by the European Community: AVICENNE Programme, grant AVI–CT92–0003 and INCO-DC grant IC18–CT95-0023. The authors wish to thank Nel Kroon and Gerard Schoone for technical assistance with the DAT and the PCR.

References (18)

P Abranches et al.
Canine leishmaniasis: pathological and ecological factors influencing transmission of infection
J. Parasitol.
(1991)
D.A Ashford et al.
Comparison of polymerase chain reaction and serology for the detection of canine visceral leishmaniasis
Am. J. Trop. Med. Hyg.
(1995)
S Bettini et al.
Canine leishmaniasis in the Mediterranean area and its implication for human leishmaniasis
Insect. Sci. Appl.
(1986)
M Boelaert et al.
Operational validation of the Direct Agglutination Test for diagnosis of visceral leishmaniasis
Am J Trop Med Hyg
(1999)
O Brandonisio et al.
Canine leishmaniasis in the Gargano promontory
Eur. J. Epidemiol.
(1992)
P.M De Korte et al.
Introduction of an improved DAT for the detection of Leishmania infantum infection in southern France
Parasitol. Res.
(1990)
T.G Evans et al.
Canine VL in northeast Brazil: assessment of serodiagnostic methods
Am. J. Trop. Med. Hyg.
(1990)
Gradoni L.M. (1995) Canine reservoir of zoonotic visceral leishmaniasis in the Mediterranean area: Epidemiology and...
A.E Harith et al.
Improvement of a direct agglutination test for field studies of visceral leishmaniasis
J. Clin. Microbiol.
(1988)

There are more references available in the full text version of this article.

Cited by (62)

Seroepidemiological and entomological survey in a new focus of zoonotic visceral leishmaniasis in Kars province, Northeastern Turkey
2015, Veterinary Parasitology
Visceral leishmaniasis (VL) has now been recorded from 38 provinces of Turkey. Twenty-one VL cases were reported within six years from settlements located in most northeastern Turkey and we therefore aimed to carry out an entomological and seroepidemiological survey in this new focus for clarifying risk factors. Blood samples from 290 children and 165 dogs were collected. Sera samples were investigated for anti-Leishmania antibodies using indirect fluorescent antibody test. Sand fly collection for determining the fauna and seasonal activity was performed in all settlements by CDC light traps between June and September 2006. Although no seropositive child was detected during the survey the overall seroprevalence rate of canine leishmaniasis was found as 7.2%. A total of 4154 sand flies were collected and 10 species of genus Phlebotomus were identified belonging to Adlerius, Larroussius, Paraphlebotomus and Phlebotomus subgenera. Among them Phlebotomus kandelakii s.l. (55.44%), Phlebotomus balcanicus (12.62%) and Phlebotomus neglectus (4.40%) was detected as probable vector species for this new focus. The poor sanitation, very high population size of sand flies, probably because of very short season, no control measures for sand flies as well as dogs, and presence of microclimate suitable for sand flies were considered as main risk factors in the area.
Canine visceral leishmaniasis: A comparative study of real-time PCR, conventional PCR, and direct agglutination on sera for the detection of Leishmania infantum infection
2013, Veterinary Parasitology
Citation Excerpt :
Serological tests such as the indirect immunofluorescence assay test (IFAT) is a routine method of diagnosing CVL, although asymptomatic dogs may yield false negative results (Almeida et al., 2005), and cross-reaction with other parasites and other Leishmania species can occur (Schulz et al., 2003; Ferreira et al., 2007). The direct agglutination test (DAT) is used as a serodiagnostic tool, because it is an appropriate and cost-effective test that does not require specialized equipment (Harith et al., 1989; Ozbel et al., 2000; Mohebali et al., 2005, 2006). Nevertheless, it has limited specificity in asymptomatic subjects, failing to distinguish between past and present
Canine visceral leishmaniasis (CVL) is endemic in northwestern Iran. This study aimed to compare real-time PCR, conventional PCR, and the direct agglutination test (DAT) for the diagnosis Leishmania infantum infection in 167 serum samples of domestic dog. Bone marrow was used for parasitological examination (smears and/or culture) in symptomatic visceral leishmaniasis, and serum was used for detection of L. infantum kinetoplast DNA (kDNA) by both conventional PCR and real-time PCR, while anti-L. infantum antibodies in sera were measured by DAT. The sera were collected from 37 symptomatic and 112 asymptomatic dogs during April to May 2011. Eighteen presumed negative samples were obtained from healthy dogs kept in non-endemic areas with no history of CVL and used as controls. All 18 samples were negative by DAT and Dipstick rK39. DAT confirmed previous exposure to L. infantum for all 149 serum samples collected from symptomatic and asymptomatic dogs in CVL endemic areas of Iran. Among the 37 symptomatic dogs, 20 (54%), 25 (67.6%), 36 (97.3%), and 37 (100%) showed L. infantum infection by parasitological methods, conventional PCR, real-time PCR, and DAT (≥1:80), respectively. Of 112 asymptomatic dogs, 79 (70.5%), 111 (99.1%), and 112 (100%) were shown to be positive by conventional PCR, and DAT (≥1:80), respectively. For ethical reasons, no asymptomatic or healthy control dogs were examined by parasitological methods. Three (16.7%) control dogs were positive by real-time PCR, but were negative by DAT, dipstick rK39, and conventional PCR methods. Parasitemia levels were measured by real-time PCR targeting kDNA using SYBR^® green assay. This quantitative technique detected infection in 89.9% (150/167) of the domestic dogs that harbored L. infantum kDNA, ranging from 0.01 49 to 310.1 parasites/ml. The average was 16.60 parasites/ml. A good agreement (0.97) was found between real-time PCR and DAT at ≥1:80 titer, used as cut-off value by Kappa analysis. Thus, real-time PCR as a quantitative PCR assay on serum samples represents a valuable tool for initial diagnosis of CVL when whole blood is not available.
A cross-sectional sero-epidemiological study of canine leishmaniasis in Greek mainland
2012, Acta Tropica
Canine leishmaniasis is endemic in Greece as in other countries of the Mediterranean basin. In this study, the regional prevalence of canine seropositivity to Leishmania spp. in Greek mainland was simultaneously assessed in 7 different regions. A total of 2620 serum samples were collected from clinically healthy dogs and were tested for anti-L. infantum antibodies by indirect immunofluorescence antibody test (IFAT) and enzyme linked immunosorbent assay (ELISA). A high degree of agreement (κ = 0.96) was observed between these two tests. The seroprevalence rate in the whole study population was nearly 20%, being highest among dogs living in Attiki (30.12%) and lowest for those living in Florina (2.05%). There was no difference in terms of the gender of the dogs, their length of hair coat or their utility; on the contrary, seroprevalence rates were significantly higher among dogs in the age groups of 1–3 years (23.39%) and 3–9 years (23.35%) than in younger (2.26%) or older (6.03%) dogs. Epidemiological data on the seroprevalence of canine leishmaniasis provide indirect information on the prevalence of the infection and the disease and are necessary to implement and then to evaluate the effectiveness of control measures.
Serological survey of Leishmania infantum in apparently healthy dogs in different areas of Spain
2023, Veterinary Medicine and Science
Evaluation of Cases with Suspected Canine Leishmaniasis History: A Five-year Retrospective Study (2016-2021)
2022, Turkiye Parazitoloji Dergisi
Determination of sand fly fauna and molecular detection of Leishmania in sand flies in Antalya Province, Southern Turkey
2021, Parasitology Research

View all citing articles on Scopus

View full text

A survey on canine leishmaniasis in western Turkey by parasite, DNA and antibody detection assays

Abstract

Introduction

Section snippets

Study area

Blood sampling

Results

Discussion

Acknowledgements

Canine leishmaniasis: pathological and ecological factors influencing transmission of infection

J. Parasitol.

Comparison of polymerase chain reaction and serology for the detection of canine visceral leishmaniasis

Am. J. Trop. Med. Hyg.

Canine leishmaniasis in the Mediterranean area and its implication for human leishmaniasis

Insect. Sci. Appl.

Operational validation of the Direct Agglutination Test for diagnosis of visceral leishmaniasis

Am J Trop Med Hyg

Canine leishmaniasis in the Gargano promontory

Eur. J. Epidemiol.

Introduction of an improved DAT for the detection of Leishmania infantum infection in southern France

Parasitol. Res.

Canine VL in northeast Brazil: assessment of serodiagnostic methods

Am. J. Trop. Med. Hyg.

Improvement of a direct agglutination test for field studies of visceral leishmaniasis

J. Clin. Microbiol.