Abstract: This study was made to investigate the seroprevalence of Zoonotic Visceral Leishmaniasis (ZVL) among the ownership dogs of villages in Sarab city and to find the possible relationship between to transmit of parasite from infected dogs to human beings. In this study blood sampling was made from 384 ownership dogs for recognizing seroprevalence of visceral Leishmaniasis with Indirect Immuno Fluorescence Antibody Test (IFAT). The serum was conjugated with anti leishmania infantom IgG antibodies and examined with fluorescent microscope. The seropervalence values among male and female animal were 9.2 and 6.5, respectively. Sero-Prevalence Rate (SPR) of CVL among ownership dogs in Sarab was 8.5% however, considering the cut off degree (1:80) from 384 dog 33 dogs of them was positive serum on the basis of anti L. infantom IgG antibodies. In this study, the antibody titer in male dogs was more than that of female dogs, however, it was not statistically significant (p = 0.023). The highest infection was found in dogs of jaldabakhan village 29.6% (8) and the lowest infection was found in khaki 3.3% (1). In this study only in Asbfroshan and Jaldabakhan areas 2 dogs had antibody 1:40, which was a negative serum antibody. There was statistically a significant difference between male and female infection to Visceral Leishmaniasis (p = 0.962). Considering the different animal reservoirs and various geographical animal conditions in this area and regarding the zooonotic parasite, controlling the animal reservoirs seems unavoidable and fighting against the vector sand fly and controlling plans are necessary.

INTRODUCTION

Visceral Leishmaniasis is one of the infected-systemic diseases whose importance can not be ignored from hygienic point of view. Visceral Leishmaniasis is greatly widespread in Middle East and its cause is Leishmania infantum (Handemir et al., 2004). It is transmitted to dogs and human beings via Phlebotomus sand fly and Lutzoumyia. Dogs are considered as the main domestic hosts and wolf and foxes as the main wilds hosts. Visceral Leishmaniasis was described for the first time in 1908 by Nicolle and Comet (Mohebali, 1996). From then on different reports from different parts of the world were published about that (Mohebali et al., 1998). So far at least four endemic foci of this disease from some areas of Ardabil, East Azerbaijan (Fallah et al., 2006), Fars, Boushehr and recently from Qom Provinces (Fakhar et al., 2004) have been investigated and approved. Every year sporadic cases of Leishmaniasis are reported from other parts of Iran (Mohebali et al., 2001a, b). In Ardabil and East Azarbaijan Provinces, the removed Leishmania from animal reservoir is recognized to be L. infantum lon 49 parasites after biochemical (Isoenzyme) experiments this parasite is exactly the same strain, which is in a variety of cases removed from the people infected to kala azar in the above-mentioned provinces (Mazlomi et al., 2000). Therefore, it can be absolutely said that the dogs infected to Leishmaniasis are the most important reservoir of this infection for human beings. In addition to veterinary importance, visceral Leishmaniasis dogs are important from medical and hygienic perspectives. It appears in dogs in acute, chronic and sub clinical forms and in a variety of cases without any clinical symptoms leading to the death of animal (Mohebali et al., 2001a). An exact method for diagnosing this disease in dogs suspicious to the infection is biopsy tissue) spleen, bone marrow, liver, lymphatic glands) and watching the amastigot form, or culturing the biopsy samples in nutritional environments and watching the promastigote forms of parasite (Rossi, 2004). However, it is not always possible to see these forms. Using a valid serology has a great importance in diagnosing this infection in dogs timely. IFAT method is one of the serological methods for which a high sensitivity and quality is reported. The main purpose of this study is recognizing the seropervalence of visceral Leishmaniasis in stray dogs of Sarab by IFAT and probable role of dogs in the transmission of this disease to human being and understanding its health importance.

MATERIALS AND METHODS

The method of this study was descriptive cross section and the sampling method was multi stage cluster random sampling (Moshfe et al., 2008). Out of 167 village in Sarab district, 30 villages (cluster) were selected randomly. For this study, on a previous accommodations with Veterinary organization of East Azerbaijan and the office of biological environment, 384 ownership dogs from 30 village of different areas of Sarab were collected randomly methods. Next, the blood samples were taken from the dogs. Five mililitre blood from cephalic or saphenous vein from each dog was taken. It is necessary to be said that before blood sampling all the information about age, sex, color and even the place and clinical examinations from the point of view of the presence of Visceral Leishmaniasis symptoms (exfoliative dermatitis, ulcerations, alopecia, thinness, largeness and complexities of the nails, lymphadenopathy, cachexia cataracts, size of the abdomen, ocular lesions and diarrhea (was recorded on especial forms which were designed for this purpose. After the samples were removed to lab, the serums were separated by experimental techniques and finally the serums were prepared for indirect Immunofluorescence test. From 384 dogs investigated, 306 dogs (79.7%) were male and 78 dogs (20.3%) were female. There was not any discrimination among dogs on the basis of races. For indirect Immuno fluorescence test L. infantum Promastigote antigen strain (Lon 49), which were cultured in RPMI 1640 environment were used accompanied with 10% serum of cow fetus (Fcs). Promastigote were removed from the culture environment and were washed 8 times in PBS. Parasites were focused on a micro slide with Acetone and the dogs serums were started to test by IFAT whit 1:20 tenderness. L. infantum promastigote were prepared from parasitology department of health faculty of Tabriz Medical University (Harith et al., 1986). In this study, anti dog IgG was conjugated with florescence Isothiocyanate (Sigma®, F4012). Titer of this conjugate was 1:50 Anti-leishmania specific antibodies, using the cut-off value of 1:80 and above were detected in male and female domestic dogs. Finally, the samples were investigated with a fluorescence microscope (Olympus, Bx50, Japan, 2004).

Data analysis: Chi-square and fisher exact tests were used to compare seropervalence values relative to gender. Analyses were conducted using SPSS software version 13.5 whit a probability (p<0.05) as statistically significant.

RESULTS

The IFAT test is highly sensitive and specialized and is used as a quality test in diagnosing leishmania (Semiao et al., 1996). The interpretation of the test is very important and that’s why those people and animals that have antibody against leishmania are considered as leishmania patients. This can be considered as a risk factor (Mancianti et al., 1995). In this study, only in Asbfroshan and Jaldabakhan areas 2 dogs had antibody 1:40, which was a negative antibody. The sero-pervalence values among male and female animal were 9.2 and 6.5, respectively. The Sero-Prevalence Rate (SPR) of CVL among ownership dogs in Sarab was 8.5% (Table 1). However, considering the cut off degree (1:80) from 384 dogs, 33 dog of them was positive serum on the basis of anti L. infantom IgG antibodies (Table 2). There was statistically a significant difference between male and female infection to Visceral Leishmaniasis (p = 0.962). The highest infection was found in dogs of Jaldabakhan village 29.6% (8) and the lowest infection was found in Khaki 3.3% (1) (Table 3).

Table 1:	Seropervalence of CVL infection by gender in Sarab district (2008-2009)
*Immuno Fluorescence Antibody

Table 2:	Distribution of titers of anti leishmania antibodies in dogs with IFA test

Table 3:	Distribution of CVL infection different villages of Sarab district

DISCUSSION

World Health Organization (WHO) considered the infection of L.infantom as one of the most important and common deceases. The Mediterranean kind of Visceral Leishmaniasis Is an important disease transmittable from animals to human beings (Gavgani et al., 2002; Garnham, 1971). This disease exists in >30 countries as an epidemic one. Dogs and wild canine (fox and jackals) are the main reservoirs of visceral Leishmaniasis in Iran (Mohebali et al., 2001b). Dogs are considered as an important resource of infection in endemic areas of Visceral Leishmaniasis in Iran. Because, first the population of dogs in Iran is high and second, the level of infection in dogs are higher and more importantly, the parasite concentrates easily in blood or under the skin of the dogs and becomes more accessible to Phlebotomus sand fly (Mohebali et al., 1998; Giraudoux et al., 2008).

A study made in Meshkin Shahr city, from 303 examined dog’s 14 and 20% of dogs with DAT and IFA methods, respectively were positive. In another study, in Firooz Aabad, Jahrom and Gir the level of infection of the examined dogs with DAT and IFA were reported to be 41.6 and 29.1%, respectively (Edrissian et al., 1993). In the other study, on the infection of dogs of Baft in Kerman Province, with IFA and ELISA methods, the level of infection of dogs was estimated to be 18 and 14.5%, respectively (Sharifi and Daneshvar, 1994). In the study of 1994 in Qort Tappeh village of Meshkin Shahr city, from 164 examined dogs with DAT and ELISA methods there were 12.2 and 16.4% positive serums, respectively. In the other studies by the same group in 2000 in Parikhan village of Meshkin Shahr city from 344 investigated dogs with DAT and ELISA tests there were 4.9 and 9.8% positive serums, respectively. In another study by the same group in 2000 in Dashti city from 105 investigated dogs with DAT and ELISA tests there were 53.8 and 1.9% positive serums, respectively. In other study on infection of dogs in Meshkin-Shar from Ardabil province with DAR methods the sero-pervalence rate was estimated to be 17.4% (Moshfe et al., 2008).

In a study on the dogs of Meshkin Shahr city, only 13.6% of dogs serums had positive clinical signs and on the basis of the results of this study, from 22 dogs whose antibody titer anti Leishmania with DAT method reached 1:2048, only 12 dogs (54.5%) had clinical symptoms (Bokaei et al., 1998). This issue has a great importance regarding epidemiology and the transmission of Visceral Leishmaniasis to human beings, because the dogs without any clinical symptoms similar to the dogs with clinical symptoms have the ability to transmit Visceral Leishmaniasis to human beings.

Therefore, in order to control Visceral Leishmaniasis in epidemic areas it is suggested that all the stray dogs be killed and the guard ones be monitored with a serological test and be killed in the cases of positive results by the above-mentioned test.

CONCLUSION

Finally, the infected people should be cured accompanying with controlling the vector mosquitoes provided that there is no damage to biological environment and no cause of a hygienic danger to human beings and Planning controlling programs is a proper step in the prevention of Visceral Leishmaniasis (Tesh, 1995; Mohebali et al., 2001a, b; Palatnik-de-Sousa et al., 2001).

ACKNOWLEDGEMENTS

I would like to express my best thanks to Dr. Ali Eslami, Dr. Nasser Hoghooghi, Rad, Dr. Mehdi mohebali, Dr. Homa Hajjaran and Dr. Mohammad Zakaria, Zadeh, the expert of veterinary Organization of East Azerbaijan, College of Medical science of Tabriz University and School of Public Health and Institute of Public Health Research, Tehran University of Medical Sciences and Emam Reza hospital and deputy ministry for Health of Tabriz university Medical science, Health centers in East Azerbaijan, Province and deputy ministry for research of Islamic Azad University of Marand.