Serological and molecular survey of zoonotic visceral leishmaniasis in stray dogs (Canis familiaris) from an endemic focus in Meshkin-Shahr district in Ardabil province, Iran

Fatemeh Tabatabaie; Yashar Nasirikaleybar; Mehdi Mohebali; Rahmat Solgi; Vahid Babaei; Zahra Heidari; Fariba Orujzadeh; Zabihollah Zarei

doi:10.4103/0972-9062.325636

RESEARCH ARTICLE

Year : 2021 | Volume : 58 | Issue : 3 | Page : 213-218

Serological and molecular survey of zoonotic visceral leishmaniasis in stray dogs (Canis familiaris) from an endemic focus in Meshkin-Shahr district in Ardabil province, Iran

Fatemeh Tabatabaie¹, Yashar Nasirikaleybar¹, Mehdi Mohebali², Rahmat Solgi³, Vahid Babaei⁴, Zahra Heidari⁵, Fariba Orujzadeh¹, Zabihollah Zarei²
¹ Department of Parasitology and Mycology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
² Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
³ Infectious Diseases Research Center, Birjand University of Medical Sciences, Birjand, Iran
⁴ Genetic department, Iran University of Medical Sciences, Tehran, Iran
⁵ Department of Medical Microbiology and Parasitology, School of Medicine, Ardabil University of Medical Sciences, Ardabil, Iran

Date of Submission	21-May-2019
Date of Decision	14-Jan-2020
Date of Web Publication	15-Feb-2022

Correspondence Address:
Zabihollah Zarei
Department of Parasitology andMycology, Faculty of Medicine, Iran University of Medical Sciences, Tehran
Iran

Source of Support: None, Conflict of Interest: None

Check

DOI: 10.4103/0972-9062.325636

Abstract

Background & objectives: Visceral leishmaniasis (VL),a protozoan disease caused by Leishmania infantum is a major public health problem and cause of death among infants aged under 1 year and the elderly in endemic foci of Iran. The aim of this study is to determine the status of L.infantum infection in stray dogs from Meshkin-Shahr, a typical endemic area of VL in Iran.
Methods: Sixty-eight randomly trapped stray dogs in Meshkin-Shahr area were tested for L. infantum infection using the direct agglutination test (DAT) from June to October 2016. The confirmation of seropositive samples was performed by Microscopic slides of spleen, culture and then PCR. The molecular methods performed by ITS1-PCR, RFLP-PCR and kDNA-PCR. The allof kDNA -PCR products were sequenced.
Results: Out of 68 examined stray dogs, 17 (25.0%) were positive for L. infantum by DAT (1:320 titers or higher). Parasite test showed that all of seropositive samples have amastigote forms in their spleens but only 3 out of them could be cultured. The kDNA-PCR confirmed all of seropositive samples but ITS1-PCR and RFLP-PCR only confirmed 3 out of 17 (17.6%) seropositive samples. The sequenced products showed 94% homology with L. infantum.
Interpretation & conclusion: The results showed a high prevalence of L. infantum infection in dogs in an endemic area of CVL and it provided key information for designing control programs against canine and human leishmaniasis.

Keywords: Visceral leishmaniasis; dogs; Seroprevalence; Polymerase Chain Reaction; Iran

How to cite this article:
Tabatabaie F, Nasirikaleybar Y, Mohebali M, Solgi R, Babaei V, Heidari Z, Orujzadeh F, Zarei Z. Serological and molecular survey of zoonotic visceral leishmaniasis in stray dogs (Canis familiaris) from an endemic focus in Meshkin-Shahr district in Ardabil province, Iran. J Vector Borne Dis 2021;58:213-8

How to cite this URL:
Tabatabaie F, Nasirikaleybar Y, Mohebali M, Solgi R, Babaei V, Heidari Z, Orujzadeh F, Zarei Z. Serological and molecular survey of zoonotic visceral leishmaniasis in stray dogs (Canis familiaris) from an endemic focus in Meshkin-Shahr district in Ardabil province, Iran. J Vector Borne Dis [serial online] 2021 [cited 2023 Mar 30];58:213-8. Available from: http://www.jvbd.org//text.asp?2021/58/3/213/325636

Introduction

Canine visceral leishmaniasis (CVL), caused by Leishmania infantum, is endemic in Meshkin-Shahr district of Ardabil province (Northwestern Iran). Dogs, as principal reservoir hosts, play an active role in transmission of CVL infection to humans^[1]. The dogs infected by VL have a wide range of clinical presentation, ranging from mild to fatal disease. Despite many studies in field of CVL, host factors related to clinical outcome as well as clinical symptoms include enlarged lymph nodes and hepato-splenomegaly are poorly understood^[2]. About 500000 cases of human visceral leishmaniosis (HVL) occur annually claiming 59000 deaths in different parts of the world^[3]. Sporadic occurrence of visceral leishmaniasis (VL) has been reported from distinctive parts of Iran, although the highest endemicities are recorded from northwestern and southern provinces of the country including Ardabil, Eastern Azerbaijan, Fars and Bushehr. The first case of human VL in Iran was reported by Pouya (1949) in a boy from the Caspian area in northern Iran^[4]. At present, more than 200 cases of VL, produced by L. infantum have been reported from Meshkin-shahr district^[5]. Different methods are used for laboratory diagnosis of VL such as microscopic examination of stained specimens or in vitro cultures of blood, bone-marrow, lymph nodes or spleen as well as determination of specific antibodies against Leishmania spp and performing polymerase chain reaction (PCR). Microscopic detection of parasites in clinical splenic materials is still considered the gold standard. Among serological tests, non-invasive direct agglutination test (DAT) is used extensively for seroprevalence evaluation of CVL in many regions. Moreover, in order to design appropriate control project, discriminate identification of Leishmania species in infected dogs by molecular methods is necessary^[6],[7].

Regarding limitations of traditional techniques for detection of leishmania in patients, vectors and reservoir hosts, researchers have developed specific PCR-based assays as sensitive and specific techniques^[8]. VL is still an important public health problem in Iran and currently occurs sporadically in all geographical zones of this country but in northwestern and southern parts of the country the disease is endemic. Domestic dogs and wild canines including jackals, foxes and wolves are the principal reservoir hosts for VL in all four distinct Iranian geographical zones. Determination of the prevalence of CVL is necessary for the epidemiological study and control of leishmaniasis^[4]. In this work, we study the current status of visceral leishmaniasis and provide the first description on the role of stray dogs as reservoir hosts of HVL in Meshkinshahr district. The factors associated with CVL were also assessed. This study was conducted in Meshkin-Shahr district of Ardabil province, located in northeastern Iran, which is considered an area of active transmission^[5].

Material & Methods

Study area

Meshkin-shahr district is located in Ardabil province, north-west of Iran (38° 44′ N, 47° 40′ E). The weather of this mountainous area is mild. The city of Meshkin-shahr is located at an altitude of 1625 m above sea level^[9].

Animals and sampling

This descriptive cross-sectional study was conducted in Meshkin-Shahr district over a period of 5 months from June to October 2016, during which 68 stray dogs (47 males and 21 females), statistically defined, were sampled. The general information on dog’s age, gender and symptoms as lymphadenopathy, alopecia, weight loss, and dermatitis were recorded by veterinarians. From each dog, a 4 ml-blood sample was collected via venipuncture and poured into a 10 ml-polypropylene tube. The collected blood samples were centrifuged at 3000 g for 15 min, and the sera were separated and stored at -20°C until tested by DAT. The sera were tested by the laboratory of the school of public health, Tehran University of Medical Sciences, Iran

DAT examination

The DAT antigen for this study was obtained from the Department of Parasitology, Faculty of Public Health, Tehran University of Medical Science and preserved at 4 °C until used. The main step of DAT antigen preparation was performed by culturing promastigotes of L. infantum (Iranian strain) in RPMI1640 media containing 10% fetal bovine serum, followed by trypsinization, fixation (2% formaldehyde) and staining of the parasites (Coomassie brilliant blue). The dogs’ sera were tested by DAT. For screening purposes, dilutions of 1:80 and 1:320 were prepared. Sera with antibody titers of 1:80 were diluted over titration end-point of 1:20480. Negative check antigen (just antigen) and known negative and positive check were done for each of the V shape microplate. In this study, we considered antibodies titers of ≥ 1: 320 as cut off sign for canine Leishmania infection^[10].

Parasitological study

The suspected dogs (anti-Leishmania antibodies ≥ 1: 320) were euthanized and smears were taken from the spleens. All prepared smears were fixed in methanol and then stained by Giemsa. Smears were examined for the presence of amastigotes under a light microscope with a magnification of 1000X. Furthermore, tissue samples were taken aseptically from the spleen of the captured and dissected dogs and cultured in two L. infantum special media: NNN (Novy- MacNeal -Nicolle) and RPMI 1640. The NNN media was prepared from nutrient agar including 10% whole rabbit blood over-laid by normal saline containing 100–200 IU/ml penicillin G and 1 mg/ml streptomycin. RPMI 1640 culture was made using RPMI 1640 powder, HEPES buffer and NaHCO3. The pH was set at 7.2–7.4 and DW added to the media up to 1000 ml. Finally, heat-inactivated FCS was added in order to enrich the media and accelerate the growth of parasites. The cultures were incubated at 23°C for up to six weeks and examined weekly for the growth of promastigotes. For mass production of promastigotes, RPMI1640 medium (Gibco, Germany) was used^[10]. Leishmania promastigotes that had been isolated from spleen following batch production in RPMI1640 media were poured into falcon tubes, and centrifuged at 3000 g for 15 min. The precipitates were then washed three times with phosphate buffered saline (PBS) and stored at -20°C until used for DNA extraction.

DNA extraction

The DNA was extracted from both promastigotes in culture media and amastigotes in Giemsa-stained smears of spleen samples. DNA extraction of promastigotes was carried out using GeNet Bio Kit, Korea. In order to extract DNA from Giemsa-stained smears of spleen, all the slides were rinsed with absolute ethanol containing 250 μl lysis buffer (50 mMNaCl, 50 Mm Tris, 10 mM EDTA, pH 7.4, 1% v/v Triton x-100 and 100 μg of proteinase k per ml). After a while, the smears were delivered to 1.5 ml micro tubes then DNA extraction was performed^[11].

ITS1-PCR

The ribosomal internal transcribed spacer 1 (ITS1) region was amplified using the primers LITSR (5′- CTGGATCATTTTCCGATG -3′) and L5.8S (5′- TGATACC ACTTATCGCACTT -3′) which separate the genes coding for the ssurRNA and 5.8S rRNA. The 50 μl reaction mixture contained 2 mM MgCl2 (Roche, Mannheim, Germany), 20 pmol of each primer, 200 μM dNTPs, 2 U of Taq polymerase and 5 μl of DNA in the PCR buffer (Roche Biotech). Reaction was over-laid with 50 μl of mineral oil and expanded in a thermocycler (Techne USA). All amplifications were programmed for an initial denaturation step of 5 min at 95°C, followed by 35 cycles of 94°C for 30 sec, 48°C for 30 s and 72°C for 1 min, with a final step of 10 min at 72°C. Three Leishmania standard species of L. major (MHOM/IR/75/ER), L. tropica (MHOM/IR/03/ Mash-878), and L. infantum (MCAN/IR/07/Moheb-gh) were used to monitor the reaction. A sample without template DNA was used as negative control. PCR products (15 μl) were run on 1% agarose gel stained with DNA safe and visualized under UV and sized by comparison with a 100 bp ladder. PCR products were kept at 4°C.

PCR-RFLP

For identification of Leishmania species, the ribosomal internal transcribed spacer 1 (ITS1) was amplified using particular primers then the non-purified PCR products (10.0 μl) were digested with 2 μl of the restriction enzyme HaeIII (Promega, Madison, WI, USA). After utilizing the restriction enzyme, banding patterns containing the fragments of 220 and 140 bp for L. major, the fragments of 200, 80, and 60 bp for L. infantum, and 3 fragments of 200, 60, and <60 bp for L. tropica were observed. The restriction fragments obtained were compared with the standard strains of L. infantum, L. tropica and L. major^{[11],[12],[13]}.

kDNA-PCR

For detection of L. infantum DNA, normal PCR was performed. A 145 bp fragment in Kinetoplast DNA was amplified by means of specific primers of the L. infantum species RV1 (5′- CTTTTCTGGTCCTCCGGGTAGG-3′) and RV2 (5′CCACCCGGCCCTATTTTACACCAA-3′). The 50 μl reaction mixture contained 3 mM MgCl2 (Roche, Mannheim, Germany), 50 pmol of each primer, 200 μM dNTPs, 1.5 U of Taq polymerase, 5 μl of DNA and 5 μl of 10×PCR buffer. The amplification conditions were 94°C for 4 min, followed by 40 cycles of denaturation at 94°C for 30 sec, annealing at 54°C for 30 sec, and extension at 72°C for 90 sec, with a final extension step at 72°C for 10 min. The PCR products were analyzed on 2% agarose gel electrophoresis and visualized under ultra-violet trans-illumination and sized by comparing with a 100 bp ladder. Three Leishmania standard species L. major (MHOM/IR/75/ER), L. tropica (MHOM/IR/03/ Mash-878) and L. infantum (MCAN/IR/07/Moheb-gh) were used to monitor the reaction as positive controls. A sample without template DNA was used as negative control.

DNA sequencing

The PCR products of all kDNA-PCR were sent to Pishgam Company, Tehran-Iran. Sequences were analyzed using NCBI BLAST program (www.ncbi.nlm.nih. gov)and aligned using Bioedit software (version 7.0.9, California)^[14]. Nucleotide sequence information described in this paper have been compare to the GenBank database with accession numbers, L. tropica AB901375, L. infantum Z35500, and L. major EU370905 as positive controls.

Statistical analysis

Data were analyzed by Chi-squared test using SPSS software version 22. Statistical significance degree was accepted at p<0.05 and 0.95 confidence interval.

Ethical statement

The procedures of this study were approved by the Ethical Committee of the Faculty of Medicine (Iran University of Medical Sciences, Code: IR.IUMS.REC 1394.9311554006) in accordance with Helsinki Declaration and Guidelines.

Results

Seroprevalence survey

In this subject, 68 dogs of the Meshkin-Shahr were surveyed. The dogs included 47 males and 21 females of at least 3 years old, 27 of which had clinical signs and 41 had no clinical signs. The maximum life span of dogs was 14 years and the average were 8.5 years. Out of 68 dogs, 17 cases (25%) showed anti- Leishmania specific antibodies with titers ≥ 1: 320. Thirteen (76%) of the seropositive dogs showed clinical signs [Table 1]. Infection with L. infantum (as detected by DAT) was associated with a clinical sign (OR=1.6, CI=1.19-2.1, P < 0.05). The most frequent clinical signs were lymphadenopathy, alopecia, weight loss, and dermatitis. The seroprevalence rate between male and female was 14 (83%) and 3 (17%), respectively. No significant correlations were observed between Leishmania infection and gender (OR=1.3, CI=0.9-1.9, P = 0.17). The highest seroprevalence was found in dogs that were 6 years old and the lowest value in dogs that were 9 years old [Table 1]. There was no significant correlation between age and antibodies titers (OR=1.29, CI=0.8 -1.7, P = 0.49).

Table 1: Seroprevalence of canine Leishmania infection among stray dogs by clinical sign and ages Meshkin-shahr district, Iran

Click here to view

Parasitological study

Amastigotes were observed in all of seropositive samples while, only three out of them could be cultured.

Molecular characterization

Seventeen seropositive samples containing three mass cultivated cultures and 17 splenic smears were examined with molecular methods. Three out of the 17 seropositive samples (17.6%) were positive to ITS1-PCR specific for Leishmania spp. All of them was related to genomic DNA of Leishmania spp. in mass cultivated media. In ITS1-PCR, the positive samples yielded a band of 340 bp. The pattern of digested PCR product by PCR- RFLP showed that the amplified sequences were related to L. infantum. The results of kDNA-PCR revealed that Leishmania DNA samples were positive in all of 17 sero-positive cases. All of them were successfully sequenced. Alignment of the obtained sequences showed that amplified sequences contained minor variations among the samples. Based on the sequence variation two classes of the sequence were divided. The main difference is that one stretches of sequence TTG in Class I were mutated to AAT in Class II [Figure 1]. The variation was confirmed with 70% of Class I and 30% of Class II. The two classes of isolates were submitted to GenBank (accession no: Class I: KY777592; Class II: MF449521). The PCR products of two classes of isolates were found to have 94% homology with L. infantum.

Figure 1: Alignment of two classes of sequences amplified from kDNA minicircles of L. infantum

Click here to view

Discussion

Outbreaks of CVL have been attributed to deforestation, environmental changes, population migrations, and individual risk factors such as malnutrition, HIV, genetic factors etc. Particular diagnostic techniques for canine leishmaniasis include microscopy, culture, serology, polymerase chain reaction and xenodiagnoses. In Iran, the two endemic foci of high endemicity for zoonotic visceral leishmaniasis are Fars province (southeast of Iran) and Meshkin-shahr district (northwest of Iran). Our research aimed at epidemiological investigation of CVL, which is considered as a neglected tropical disease. We intended to study the status of the disease in stray dogs using DAT and molecular assays in Meshkin-shahr district. DAT is a reliable test for serodiagnosis of visceral leishmaniosis in humans and dogs. DAT is easy, highly sensitive, specific and reproducible. Also, it does not require complex equipment. Thus, it has been a preferable method for CVL detection in this region^[7]. In this study, the seroprevalence of CVL was 25% with antibody titers of ≥ 1: 320. In other studies, the value of infection in dogs from Meshkin-shahr district was determined to be 17.4 and 23.4 by Moshfe et al. 2008^[9] and Barati et al. 2015^[15], respectively. The recorded increase in seroprevalence of visceral leishmaniasis in dogs in these districts may be attributed to various factors such as ecological variation and concomitant changes in activities of sandflies in this area. Previous studies showed that cold weather can affect increase of the intensity of the disease^[7],[15]. In the present study, similar to others’ finding, we could not establish any significant relation between gender and infection^[9],[15]. However, in contrast to studies undertaken in the same region, we could not find any significant association between age and infection^[10],[16]. This was probably due to smaller sample size, although some authors came up to same conclusion. In this study, 76% of seropositive dogs were symptomatic. This information is valuable and significant because dogs play a crucial role in the epidemiology and establishment of life cycle of this disease. Contrary to our finding, some studies reported a high proportion of asymptomatic dogs^{[9],[15],[16],[17]}. The presence of seropositive dogs emphasizes the importance of dogs without clinical implications in the epidemiology of zoonotic leishmaniasis. Hence, the urgency of conducting serological examination in dogs that have no signs as it is required for disease control planning. The high rate of seroprevalence in dogs of 4 and 6 years old is believed to be due to high exposure of stray dogs to sand fly bites at the first year of their life as postulated by some researchers^[15],[16]. This may be because of higher sensitivity of the younger animal compared to older dogs. There is inconsistency between researchers regarding the age, as a risk factor. Heidarpour et al. found high frequency of leishmaniasis in young dogs, Mohebali et al. found high frequency of leishmaniasis in older dogs and Miranda et al. reported bimodal distribution with a first peak formed in adult dogs and a second less evident peak in old dogs^{[10],[18],[19]}.

In this study, only 3 out of 17 positive amastigotes samples were cultured that it could be due to a low number of parasite in the tissue^[13]. Three mass cultivated cultures were positive by ITS1-PCR, PCR-PFLP and kDNA-PCR. Seventeen smears were positive by kDNA-PCR but negative by ITS1-PCR, PCR-PFLP. In comparison with different molecular methods used in this study, we found that kDNA-PCR was the most sensitive method for diagnosis of leishmaniasis. kDNA-PCR contains 10000 copies per cell with an approximate 200 bp conserved region and 600 bp variable region, while ITS1 target has 100 copies per cell. In addition to the copy number, PCR sensitivity is dependent on secondary structure and amplicon size. kDNA-PCR can detect 0.001 parasites/ml of sample, while ITS1-PCR can detect 1-6 parasites/ml^[20]. Therefore, in samples with low parasitemia, the use of kDNA-PCR is preferable. Based on previous studies for diagnosis of L. infantum in humans and dogs, kDNA-PCR were more sensitive than genomic-DNA-based, ITS1-PCR and parasitological methods for diagnosis of CVL in dogs^[12],[18]. In this study, only seropositive dogs have been evaluated by molecular methods, while some of the seronegative cases could be positive by molecular methods. Therefore, the molecular methods could not show the real prevalence of the infection in the evaluated dogs. Limitations of this study were expensive equipment and high cost per test, requirement of trained personnel, establishing completely equipped and staffed central laboratories in molecular methods and less specificity and sensitivity serology tests.

Conclusion

Our results show that ZVL is a problem in Meshkinshahr district. The results from present study demonstrated that around quarter of dogs living in endemic Meshkinshahr district, Iran, were infected by L. infantum. Control measures, such as treatment or eradication of infected dogs, must be taken to stop the transmission of the infection to vectors. Furthermore, DAT and kDNA-PCR can be used as effective methods for diagnosis of CVL in dogs to improve disease control strategies. To control zoonotic leishmaniasis, we propose periodic DAT examination of suspicious dogs, removing stray dogs, undertaking vector control; and improving public health education.

Acknowledgements

This study was technically supported by Meshkin- Shar Station, School of Public Health, Tehran University of Medical Sciences. The authors acknowledge the contribution of Tehran University of Medical Sciences in data collectors. We would like to acknowledge the financial support of Iran University of Medical Sciences (Code:1394.9311554006).

Conflict of interest: None

References

1.	Desjeux P. Leishmaniasis: current situation and new perspectives. Comparative immunology, microbiology and infectious diseases 2004; 27(5): 305–318.
2.	Wang J-Y, Ha Y, Gao C-H. The prevalence of canine Leishmania infantum infection in western China detected by PCR and serological tests. Parasites & vectors 2011; 4(1): 69.
3.	Van Griensven J, Diro E. Visceral Leishmaniasis: Recent Advances in Diagnostics and Treatment Regimens. Infectious Disease Clinics 2019; 33(1): 79–99.
4.	Mohebali M. Visceral leishmaniasis in Iran: review of the epidemiological and clinical features. Iranian journal of parasitology 2013; 8(3): 348.
5.	Moradiasl E, Rassi Y, Hanafi-Bojd AA. The Relationship between Climatic Factors and the Prevalence of Visceral Leishmaniasis in North West of Iran. International Journal of Pediatrics 2018; 6(2): 7169–7178.
6.	Hassanpour K, Aghamollaei H, Golpich M. Molecular epidemiological study of cutaneous leishmaniasis in the east north of Iran. Asian Pacific Journal of Tropical Disease 2014; 4: S540–S544.
7.	Mohebali M, Edrissian G, Nadim A. Application of direct agglutination test (DAT) for the diagnosis and seroepide-miological studies of visceral leishmaniasis in Iran. Iranian journal of parasitology 2006: 15–25.
8.	Davami MH, Motazedian MH, Kalantari M. Molecular survey on detection of Leishmania infection in rodent reservoirs in Jahrom District, Southern Iran. Journal of arthropod-borne diseases 2014; 8(2): 139.
9.	Moshfe A, Mohebali M, Edrissian G. Seroepidemiological study on canine visceral leishmaniasis in Meshkin-Shahr district, Ardabil province, northwest of Iran during 2006-2007. Iranian journal of parasitology 2008: 1–10.
10.	Mohebali M, Hajjaran H, Hamzavi Y. Epidemiological aspects of canine visceral leishmaniosis in the Islamic Republic of Iran. Veterinary parasitology 2005; 129(3-4): 243–251.
11.	Kazemi-Rad E, Mohebali M, Hajjaran H. Diagnosis and characterization of Leishmania species in Giemsa-stained slides by PCR-RFLP. Iranian Journal of Public Health 2008; 37(1): 54–60.
12.	Schönian G, Nasereddin A, Dinse N. PCR diagnosis and characterization of Leishmania in local and imported clinical samples. Diagnostic microbiology and infectious disease 2003; 47(1): 349–358.
13.	Mohammadiha A, Haghighi A, Mohebali M. Canine visceral leishmaniasis: a comparative study of real-time PCR, conven- tional PCR, and direct agglutination on sera for the detection of Leishmania infantum infection. Veterinary parasitology 2013; 192(1-3): 83–90.
14.	Solgi R, Dinparast Djadid N, Eslamifar A. Morphological and Molecular Characteristic of Megaselia scalaris (Diptera: Phoridae) Larvae as the Cause of Urinary Myiasis. Journal of Medical Entomology 2017; 54(3): 781–784.
15.	Barati M, Mohebali M, Alimohammadian MH. Canine visceral leishmaniasis: seroprevalence survey of asymptomatic dogs in an endemic area of northwestern Iran. Journal of Parasitic Diseases 2015; 39(2): 221–224.
16.	Hosseininejad M, Mohebali M, Hosseini F. Seroprevalence of canine visceral leishmaniasis in asymptomatic dogs in Iran. Iranian Journal of Veterinary Research 2012; 13(1): 54–57.
17.	Moshfe A, Mohebali M, Edrissian G. Canine visceral leishmaniasis: asymptomatic infected dogs as a source of L. infantum infection. Acta Trop 2009; 112(2): 101–5.
18.	Miranda S, Roura X, Picado A. Characterization of sex, age, and breed for a population of canine leishmaniosis diseased dogs. Research in veterinary science 2008; 85(1): 35–38.
19.	Heidarpour MR. Prevalence and risk factors for canine leishmaniasis in Mashhad, North-east of Iran. Iranian Journal of Veterinary Science and Technology 2012; 4(1): 37–46.
20.	E Fallah MK, S Rahbari, M Farshchian, S Farajnia, F Hamzavi, AM Asl. Serological survey and comparison of two polymerase chain reaction (PCR) assays with enzyme-linked immunosorbent assay (ELISA) for the diagnosis of canine visceral leishmaniasis in dogs. African Journal of Biotechnology 2011; 10(4): 648–56.