Molecular and Seroepidemiological Study of Leishmania Infantum Infection Among Humans, Dogs and Wild Canines from Azarshahr (New Endemic Focus), Iran

Full Length Research Paper

Molecular and seroepidemiological study of Leishmania infantum infection among humans, dogs and wild canines from Azarshahr (new endemic focus), Iran

Esmaeil Fallah1*, Mohammad Farshchian2 and Majid Khanmohammadi3

1Department of Parasitology Faculty of medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran. 2Department of health, Faculty of health and nutrition, Tabriz University of Medical Science, Tabriz, Iran. 3Department of Laboratory Sciences, Marand branch, Islamic Azad University, Marand, Iran.

Accepted 10 May, 2011

An epidemiological survey of visceral leishmaniosis among domestic and wild canines as well people was carried out in the new endemic focus of Azarshahr (northwest of Iran) to assess the distribution of the disease and the possible association between infection in dogs and people during 2008-2009. At first using direct agglutination test (DAT), an examination of visceral leishmaniosis was carried out among 1500 Azarshahr district residents children in the age from 6 month to 10 years old. Then, one hundred and twenty domestic dogs, ten jackals, ten foxes and ten wolves from the district were examined for clinical and serological signs of canine leishmaniosis. The sera were evaluated by DAT then parasitological study was performed for all seropositive animals. Human sera samples, the overall seropositive rate of disease was 1.3% (19/1500) (95% CI, 0.7 to 1.8) by DAT (1:3200 and above). Data analysis revealed that serological positivity was statistically associated with age groups (2 =7.46, P =0.023). Of the 120 serum sampled collected from 120 domestic and stray dogs, 19.2% (23/120) (95% CI, 12.1-26.2) were positive by DAT (1:320 and above). No statistically significant difference was found between male (17.9%) and female (37.5%) seroprevalence (P=0.172). Also, no statistically significant difference was found between serological positivity and age groups (P=0.107). Serology and parasitology tests that were performed in 30 wild canines showed 10% of these animals were infected by Leishmania infantum. Amastigotes of Leishmania were observed in 14 seropositive dogs after dissection and parasitological examinations. A polymerase chain reaction (PCR) genotyping tool was developed and used to identification of species of the seropositive dog isolates. This isolates were identified as L. infantum. The present study provides, for the first time, information on the distribution of the visceral leishmaniosis in Azarshahr county of Iran, as a new endemic focus of disease.

Key words: Visceral leishmaniosis, Seroprevalence, Polymerase chain reaction, Leishmania infantum, Azarshahr.

INTRODUCTION

Infection with the protozoan parasite Leishmania spp. complex species of Leishmania (L. donovani, Leishmania (Kinetoplastida: Trypanosomatidae) can lead to three donovani infantum, and Leishmania donovani chagasi) different forms of disease: cutaneous, mucocutaneous, (Farajnia et al; 2008) .Visceral leishmaniasis, is one of and visceral leishmaniasis (VL). Visceral leishmaniasis, the most important parasitic diseases worldwide and also called Kala-azar, is the most severe form of remains a challenge to public health in at least 65 leishmaniasis caused by the Leishmania donovani countries (Fallah et al., 2011; Khanmohammadi et al., 2010; Moreno et al., 2002; Desjeux et al., 2004). This disease is a zoonosis caused by Leishmania (Leishmania) infantum (Kinetoplastida: *Corresponding author. E-mail: [email protected] or Trypanosomatidae). This organism is an obligatory [email protected] Tel:/Fax: +98 411 3373745. intracellular protozoon that is transmitted to a 1238 Afr. J. Microbiol. Res.

Figure 1. Map of the northwest region of Iran, Azerbaijan.

susceptible vertebrate host an infected female (latitude 37° 43' 17'' N, longitude 46° 2' 59'' E, altitude 1367 m), a phlebotomine sand fly (Diptera: Psychodidae) is feeding municipality located on the south-east of Uromieh sea and have 45 (Fallah et al., 2011; Khanmohammadi et al., 2010). km distance from Tabriz city (East Azerbaijan's capital). Azarshahr had a population of about 114918 inhabitants (Figure 1). The Domestic dogs (Canis familiaris) are principal reservoir suspected vectors found in the focus include Phlebotomus hosts of Mediterranean type of visceral leishmaniasis. kandelakii and Phlebotomus perfiliewi (Rassi et al., 2005). These parasites cause a wide spectrum of clinical manifestations in humans and it is estimated that the annual occurrence of human visceral leishmaniosis (HVL) Sample and data collection cases worldwide is 500,000 (WHO, 2008). VL is dispersly This study was carried out over a period of 15 months during 2008 - endemic in several provinces of Iran including East 2009 on the 15 villages of Azarshahr. Blood sampling from 10% of Azerbaijan, Ardabil, Fars and Gom (Mohebali et al; children ≤10 years old was done. All study participants completed 2005). an epidemiological questionnaire and gave informed consent to In other Provinces of the Iran, the disease has been participate in the study. Basic demographic data, including age, reported in sporadic form (Edrissian et al., 1988; Fallah et sex, address, and altitude of the house, were obtained, as well as potential risks factors for kala-azar. A 5 ml blood was taken and al., 2001). Although, dogs are the main reservoirs for serum separated and stored at -20°C until serological analyses. human infection to VL (Mohebali et al., 2006), wild Also, a total of 120 domestic and stray dogs (males and females, carnivores such as jackals (Canis aureus) and foxes different ages) were selected by simple random sampling in the (Vulpes vulpes) have been also found infected with present study. Dogs were captured in fifteen areas of Azarshahr. Leishmania spp. These animals are assumed to be Feral dogs are abundant in the study areas, living close to man and reservoirs for parasites, particularly in regions where wandering around farm buildings and houses, where they are considered to be a nuisance by the local population. Obtained sporadic cases of disease have been reported. Dog’s age by dental formula and dental erosions should be (khanmohammadi et al., 201; Mohebali et al., 2006). The considered. The minimum sample size required (n=120) was objectives of this study were to determine the sero- calculated considering the dog population. 3 to 5 ml blood samples prevalence of human and canine visceral leishmaniosis in from all dogs were collected. Additionally, samples were taken from Azarshahr county of Iran as a new endemic focus of VL, 30 wild canines in this area: 10 jackals (C. aureus), 10 foxes (V. and to identify the natural reservoir of human kala-azar in vulpes) and 10 wolves (Canis lupus). Samples were centrifuged at 800 g for 5 to10 min. Then, the obtained sera were frozen at -20°C this area. Focus was particularly on dogs, with a view to until it could be tested for anti-Leishmania antibodies. After sample determining infection rates among them and their role in collection, a questionnaire was used to collect information regarding transmission of the disease to humans. the age, gender, and district of origin of each dog. All of the sera (human and dog samples) were tested for kala-azar using the DAT, according to the methods described by Harith et al. (1989). MATERIALS AND METHODS

Study area Serological test

The present cross-sectional study was carried out in Azarshahr Promastigotes of Leishmania were mass produced in RPMI Fallah et al. 1239

1640 (Sigma®) containing fetal calve serum (FCS) followed by taken under UV illumination (Farajnia et al., 2004; Noyes et al., trypsinization of the parasites, staining with coomassie brilliant blue 1996). and fixing by formaldehyde. The parasite source was L. donovani strain I-S, where kindly provided by Harith to the Pasteur Institute, Tehran. The live culture was maintained in NNN medium enriched Statistical analysis with liquid phase of liver infusion broth tryptose (LIT) and use for 2 antigen preparation (mohebali et al., 2005). L. donovani antigen Chi-squared ( ) was used to compare seroprevalence rates (SPR) prepared and used. The titer was defined as the highest dilution at relative to gender and age. The differences were considered which agglutination was still visible, as blue dot, compared with statistically significant when probability (P) value ≤0.05. The 95% negative control wells, which had clear blue dots. Specific confidence intervals (95% CI) of seroprevalence rates were Leishmania antibodies at a titer of 1:3200 and upper were calculated. Statistical analysis was performed using Epi Info considered as positive in previous studies for human and a titer of software, version 6. 1:320 and higher for domestic and wild canines too (Mohebali et al., 2006). Therefore, we considered anti-Leishmania antibodies titers at ≥1: 3200 and ≥1: 320 as human and canine Leishmania infection RESULTS in this investigation, respectively. Out of the 1500 human sera examined from the fifteen localities, 19 (1.3%) (95% confidence interval: 0.7-1.8) Parasitological study sera showed a reaction equal to or greater than 1:3200. The dogs were examined externally for signs of infection, and then All of the cases were children under 10 years. Data dissected for the following investigations: Smears were prepared analysis revealed that serological positivity was from any skin lesion detected, and from the livers and spleen of statistically associated with age groups (2 = 7.46, P = each dog. These were fixed with methanol, stained with Giemsa 0.023). The highest proportion of positive cases was in stain and examined microscopically for the presence of children < 1 year old and the rate among children amastigotes. Biopsy specimens were collected aseptically from spleen and liver, then cultured into biphasic culture media decreased with age. Seroprevalence rates in relation to (prepared from nutrient agar containing 10% whole rabbit blood data are shown in Table 1. About 1.3% of the overlail with liver infusion tryptose broth (LIT) containing 100 UI/ml seropositive individuals were males and 1.2% females, penicillin G and 1 g/ml streptomycin. The inoculation cultures were giving a male to female ratio of 1.08:1. One hundred and incubated at 21°C for up to 6 weeks and examined weekly for the twenty dogs from the municipality of Azarshahr were presence of promastigotes. examined. Anti-Leishmania specific antibodies, using the cut-off value of 1:320 and above were detected in male PCR amplification and female dogs. 112(93.3%) of the dogs were males and 8(6.6%) were females. The sero-prevalence values The promastigotes were isolated from the RPMI 1640 media and among male and female animals were 17.9 and 37.5%, tested using PCR. To prepare template DNA from Leishmania cultures, the pellet was suspended in 10 to 20 fold of disruption respectively (Table 2). No significant difference was buffer containing 100 mM Tris, 10 mM EDTA, pH 8.0, 2% SDS and found in the seroprevalence rates between canine 2 2% 2-mercaptoethanol which was added before use. The Leishmania infection and gender ( = 1.86, P = 0.172). suspension was incubated at 65°C for at least 30 min and cooled to Of the 120 samples, 23 tested positive to anti-Leishmania room temperature before precipitating with half volume of ice-cold antibodies, confirming previous exposure to Leishmania 3.0 M KAc, pH 5.5. The supernatant was further precipitated by an parasites. Referring to animal age groups, the highest equal volume of ice-cold Isopropanol at ~15k/15 min/4°C. The pellet was rinsed with 70% ethanol and air-dried before re suspending in sero-prevalence (44.4%) was found in dogs greater than sterile water. To prepare temple from biopsy samples of spleen, 50 8-year-old and the lowest values (15.3%) in dogs less to 500 mg of infected tissue was homogenized in a proper glass than 3-year-old. No significant difference was found in the homogenizer or ground in a micro fugue tube by a heat-sealed blue seroprevalence rates between canine Leishmania tip. After adding 2 to 3 fold disruption buffers, DNA preparation was infection and age (2 = 4.46, P = 0.107). Six of the 23 followed according to the above procedure. Specific primers were designed using a 778 bp partial sequence file of L. infantum sero-positive dogs showed at least one clinical sign minicircle DNA (gi|2598170|gb|AF027578.1|) through Blast search including lymphadenopathy, hair shedding, dermal and analyzed by Oligo Tech version 1.0. These included lesions, onychogriposis and cachexia which may be homologous 5'-CCC AAA CTT TTC TGG TCC TTC G-3', positioned regarded as symptomatic form of the disease, while the at 24-45 and complementary 5'-CCA CGA CGC ATC CAA TCC AA- other 17 dogs (73.9%) were asymptomatic. 3', positioned at 360-341 flanking a 337-bp fragment. Reaction The proportion of symptomatic infections in sero- cocktail contained 1.0x PCR buffer, 2.0 mM MgCl2, 0.2 mM dNTP’s, 0.5 mM each primers, 1-2 units of recombinant Taq DNA positive dogs was 26% (6/23) and overall prevalence of polymerase (Sina gen, Iran). CVL was 5% (6/120) (Table 3). Of 30 wild canines The final volume was adjusted to 20 or 25 µl per reaction captured in Azarshahr County, 5 cases were positive to including 2-5 µl of template. The reaction conditions included lid anti-Leishmania antibodies confirming previous exposure temperature of 105°C along with 4 min of initial denaturation at to Leishmania parasites. Results of parasitological and 95°C followed by 35 cycles of 95°C/30 s, 65°C/30 s and 72°C/1.0 serological tests in the wild canines that were captured min. The reactions were ended by additional extension at 72°C/10 min. PCR products were separated by horizontal electrophoresis in around the villages from endemic foci of HVL in 1 to 1.5% agarose gel, along with a molecular weight marker. After Azarshahr county of Iran are shown in Table 3. Visceral staining the gel in ethidium bromide solution, the photograph was leishmaniasis in a wolf from Azrashahr county of Iran was 1240 Afr. J. Microbiol. Res.

Table 1. Seroprevalence rates of anti-Leishmania antibodies among humans from Azarshahr in relation to epidemiological data.

Direct agglutination test Human’s data n Seroprevalence ratesd P-values Positivea Suspected b Negative c Age ≤1 176 6 2 168 3.4 (0.7-6) 2-5 790 7 16 767 0.8 (0.2-1.5) 6-10 534 6 14 514 1.1 (0.2-2) 0.023

Sex Male 757 10 16 731 1.3 (0.5-2.1) Female 743 9 16 718 1.2 (0.4-1.2) 0.849 Total 1500 19 32 1449 1.2 (0.7-1.8)

a Titer of antibody ≥ 1:3200 b Titer of antibody between 1:800-1:1600 c Titer of antibody lower than 1:800 d 95% confidence intervals are in brackets.

Table 2. Sero-prevalence of canine Leishmania infection by gender and age groups in Azarshahr district of Iran.

Dog’s data n Direct agglutination test a Sero-prevalence rates b P-values Age 0-3 72 11 15.3 (6.9-23.5) 4-7 39 8 20.5 (7.8-33.1) 0.107 ≥ 8 9 4 44.4 (11.9-76.9)

Sex Male 112 20 17.9 (10.7-24.9) 0.172 Female 8 3 37.5 (3.9-71.0) Total 120 23 19.2 (12.1-26.2)

a Titer of antibody ≥ 1:320 b 95% confidence intervals are in brackets.

Table 3. Results of parasitological and serological tests in wild canines captured around the villages from endemic foci of HVL in Azarshahr county of Iran.

Type of animal No. of animal Parasitological positive Serological positive Leishmania spp. Foxes 10 2 2 L. infantum Jackals 10 2 2 L. infantum Wolves 10 1 1 L. infantum Total 30 5 5 L. infantum

report for the first time. During this study, all of the specific serological tests, direct agglutination test (DAT) seropositive dogs (1:320 and higher) were necropsy after was found to be more specific (72 to 100%), sensitive (92 obtaining owner consent, all of them were to 100%), and practical particularly in endemic area of the parasitologically positive. According to the results of world (Edrissian et al., 1996). The results of the DAT for multiple PCR analyses, the isolates were identified as L. detection of L. infantum infection in humans and dogs infantum and those infected dogs are assumed to be were excellent. Therefore, we used of the DAT for the potential host reservoir for VL. determination of seroprevalence of human and canine Leishmania infection. In this study, serological surveys using DAT analysis showed 1.9% of the population in the DISCUSSION study areas had anti-Leishmania antibodies in titres of ≥ 1:3200, all of them children under 10 years old. These The prevalence of Kala-azar in this area, Among the were lower than those reported by Mohebali et al. (2001) Fallah et al. 1241

in Bushehr (3.4% n =1496). The peak number of cases following measures: one case finding, including a search was in children ≤ 1 year old and the seropositive rate for overt cases of kala-azar by spleen puncture and decreased with increasing of age of the children. Prior serology to detect recent infections which may or may not studies in Iran have shown a seropositive rate of about become overt; and control of the human reservoir, which 50% in the age group 1 to 2 years and 96% of is especially important in epidemic situations. Case seropositive cases in children up to 8 years old finding and treatment form the major component of any (Soleimanzadeh et al., 1993; Edrissian et al., 1999). control scheme. Also, the canine reservoir may be About 1.3% of seropositive individuals were males and controlled by reduction of the stray dog population (Tesh, 1.2% females. No statistically different (P<0.05) was 1995). observed between them. Previous DAT serological survey of VL in Azarshahr by Mirsamadi et al. (2003) was showed that (1.9% n = 1252) of the collected specimens ACKNOWLEDGEMENTS were anti-Leishmania antibody positive with ≥ 1:3200 titers and 1.2% with 1:1600 titters were suspicious. The This study was conducted in part by a research grant overall prevalence among dogs in our study (19.2%) was provided by the office of vice chancellor for research, higher than that reported in other endemic areas of East Tabriz University of Medical Sciences. We wish to Azerbaijan Province by Mohebali et al. (2005). Of the 120 express our thanks to Dr. Mohebali, for technical advice serum sampled collected from stray and domestic dogs, and critical comments as well as to Dr. Mahmoodpor, 19.2% were anti-Leishmania antibody positive. No Mirsamadi, and Alipour for technical assistance. We are statistical differences were found among canine grateful for the sincere collaboration of the directors and Leishmania infection with regard to gender in our study. staff members of the health and medical centers and Similar results were found by Abranches et al. (1992) in Azarshar environmental protection agencies. Portugal, Sideris et al. (1996); Bokai et al. (1998);

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