J Arthropod-Borne Dis, December 2014, 8(2): 132–138 A Malmasi et al.: Seroepidemiologic Survey of … 132 Original Article Seroepidemiologic Survey of Canine Visceral Leishmaniasis in Tehran and Alborz Provinces of Iran *Abdolali Malmasi 1, Saeedeh Janitabar 1, Mehdi Mohebali 2, Behnaz Akhoundi 2, Nadi Maazi 1, Mehdi Aramoon 1, Narges Khorrami 1, Hesam A Seifi 3 1Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran 2Department of Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 3Department of Clinical Sciences, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran (Received 25 June 2012; accepted 2 Oct 2013) Abstract Background: A two year seroepidemiological study was designed to find out the seroprevalence and risk factors of canine visceral leishmaniasis (CVL) among stray and owned dogs in Tehran and Alborz Provinces of Iran. Methods: Blood samples of 602 dogs living in 11 counties of Tehran and Alborz Provinces were taken by venipuncture in 2008–2010. After separation of blood sera, anti-leishmanial antibodies were detected by direct agglutination test (DAT). Results: Overall, of the 408 and 194 serum samples collected randomly from dogs in 11 localities in Tehran and Alborz Provinces, 18/408 (4.41%) and 12/194 (6.18%) respectively were found positive. Among the localities, Shemiran in Tehran Province and Karaj In Alborz Province had the highest prevalence rates. No statistically significant differences were found between sex and living place but there was significant difference between living status (owned or stray) and CVL infection of dogs in Alborz Province (P= 0.018). The highest seroprevalence (7.5%) was found in dogs aged 3 to 5 years old. Only 20% of the seropositive dogs were symptomatic. Conclusion: Concerning possible human infections in Tehran and Alborz Provinces, both symptomatic and asymptomatic seropositive dogs should be considered as a risk. Keywords: Visceral leishmaniasis, Direct Agglutination Test, Dog, Iran Introduction Visceral leishmaniasis is one of the most important parasitic diseases affecting humans, domestic and wild canines worldwide. The infection is transmitted by sandflies. Reser- voir hosts vary within different geographic areas and can include domestic or wild ca- nines (Greene 2006). Domestic dogs (Canis familiaris) are principal reservoir hosts for Mediterranean type of visceral leishmaniasis caused by Leishmania infantum (Mohebali et al. 2005). The clinical manifestation of dis- ease vary from asymptomatic, self-limiting infections to fatal visceral leishmaniasis. Seroepidemiologic studies of canine leish- maniasis have shown a large number of asymp- tomatic seropositive animals in Meshkinshahr district of Ardabil Province (Moshfe et al. 2009). Canine leishmaniasis is not only a vet- erinary problem but also a serious public health concern. As the high proportion of infected dogs are asymptomatic, detection of specific antibodies remains the method of choice for mass screening of dogs in epidemiological surveys (Moshfe et al. 2008). Several diagnostic tests are available to detect anti-Leishmania antibodies in canine sera. In the present study, *Corresponding author: Dr Abdolali Malmasi, E- amalmasi@ut.ac.irmail: http://jad.tums.ac.ir Published Online: April 09, 2014 J Arthropod-Borne Dis, December 2014, 8(2): 132–138 A Malmasi et al.: Seroepidemiologic Survey of … 133 direct agglutination test (DAT) was used as it is a simple as well as a valid test and does not require specialized equipments (Moshfe et al. 2008). Up to now, Ardabil, East Azerbaijan, Fars, Bushehr and Qom Provinces have been recognized as endemic foci of canine vis- ceral leishmaniasis in Iran (Mohebali et al. 2001, Mohebali et al. 2005, Fakhar et al. 2005, Habibzadeh et al. 2007, Farzam et al. 2007, Moshfe et al. 2008, Khanmohammadi et al. 2010a, Khanmohammadi et al. 2010b). The objective of this first ever study in Tehran and Alborz Provinces of Iran was to determine the seroprevalence and distribu- tion of canine visceral leishmaniasis among dogs living in the urban and suburban areas of these highly populated regions with their new developing cities. Materials and Methods Study area This survey was conducted over a period of 2 years (from July 2008 to october 2010) in 11 counties from two provinces of Tehran and Alborz located across the north of the central plateau of Iran at 35° 40' 19" N, 51° 25' 28" E and 34° 43' 42" N, 50° 58' 19" E, respectively (Fig. 1). Samples were collected from 8 counties (Damavand, Eslam Shahr, Rey, Robat karim, Shahriar, Shemiran, Tehran, Varamin) of Tehran and 3 counties (Hasht- gerd, Karaj, Nazar Abad) of Alborz Prov- inces.The study population was selected by Convenience sampling. The geographical lo- cations, where this study was carried out are shown in Fig. 1. Sampling Totally, 602 dogs living in Tehran (n= 408) and Alborz (n= 194) Provinces were selected for this study. Ages of dogs were determined either by observing their teeth or asking from the owners, all dogs based on their ages put into three classes (0–3, 3–5 and >5 years). After the clinical examination of dogs, blood samples (2.5ml) were taken by venipuncture and put into 10ml polypropylene tubes and centrifuged at 800 x g for 5–10 minutes. Sera were separated and stored at -20 oC. All serum samples were tested by direct agglu- tination test (DAT) in leishmaniasis labora- tory of the School of Public Health, Tehran University of Medical Sciences. Direct Agglutination Test (DAT) The Direct Agglutination Test antigens were prepared from indigenous L. infantum [MCAN/IR/07/Moheb-gh. (GenBank acces- sion number FJ555210)] with the methods described by Harith et al. (1989) and Mohe- bali et al. (2005). Specific anti- Leishmania antibodies at a titer of ≥1: 320 in sera of indigenous dogs were considered as positive based on previous studies (Mohebali et al. 2011). Data analysis Chi-square (x2) analysis was used to com- pare seroprevalence values relative to gender, age, geographical zone, clinical signs and living status of dogs. Data regarding gender and age of some dogs from total population (30/602 and 36/602 respectively) were miss- ing ,thus we did not consider those data in the analysis. Analyses were conducted using SPSS software version 17 (SPSS Inc, Chi- cago, Illinois, USA), with a probability (P) value of <0.05 as statistically significant. Results Eighteen out of 408 (4.4%) of the canine blood samples collected from Tehran Province, and twelve out of 194 (6.1%) from Alborz Province, were seropositive (≥1: 320) by DAT. In general, no statistical significant dif- ferences were observed between seroprevalence values found in Tehran and Alborz Provinces http://jad.tums.ac.ir Published Online: April 09, 2014 J Arthropod-Borne Dis, December 2014, 8(2): 132–138 A Malmasi et al.: Seroepidemiologic Survey of … 134 (P= 0.35). Sero-prevalence (Titer ≥1: 320) of canine Leishmania infection by gender in Tehran and Alborz Provinces are shown in Table 1. There was no significant difference among two sexes in both Tehran and Alborz Provinces (P= 0.933 and P= 0.349 respective- ly). Referring to three age groups, the high- est seroprevalence (7.5%) was found in dogs aged 3 to 5 years old compared with other groups (P= 0.023), however there was no statistically significant difference among three age classes in each province (P= 0.134 and P= 0.082 in Alborz and Tehran Prov- inces respectively). Table 2 shows the sero- prevalence (Titer ≥1: 320) of canine Leish- mania infection regarding age classes in Tehran and Alborz Provinces. Only 6/30 (20%) of the seropositive dogs showed clinical signs of disease including lethargy, cachexia, skin lesions, alopecia, epistaxis and myopa- thy and the remaining 80% were asympto- matic. As shown in Table 3, 11/226 (4.6%) and 1/79 (1.2%) of stray dogs and 7/182 (5.7%) and 11/115 (8.7%) of owned dogs from Tehran and Alborz Provinces were se- ropositive respectively. There was statisti- cally significant difference between living status and canine VL infection of dogs in Alborz Province (P= 0.018 ). As shown in Table 4, Shemiran and Karaj counties in Teh- ran and Alborz Provinces showed the highest seroprevalence (14.2% and 10%, respectively). Fig. 1. Map of Iran shows 2 provinces and 11 localities (Damavand, Eslam Shahr, Rey, Robat karim, Shahriar, Shemiran, Tehran, Varamin, Hashtgerd, Karaj, Nazar Abad) where this survey was conducted http://jad.tums.ac.ir Published Online: April 09, 2014 J Arthropod-Borne Dis, December 2014, 8(2): 132–138 A Malmasi et al.: Seroepidemiologic Survey of … 135 Table 1. Sero-prevalence (Titer ≥1: 320) of canine Leishmani a infection by gender in Tehran and Alborz Provinces. Percentages were calculated in each group (male or female) totally and in each region FemaleMalesex P valuesNo. of infected dogs (Titer ≥1: 320) (%) No. of sam- pled dogs (%) No. of infected dogs (%) (Titer ≥1: 320) No. of sam- pled dogs (%) 0.9337 (4.6)146 (95.4)10 (4)232 (96)Tehran 0.3494 (4)90 (96)8 (7)104 (93)Alborz 0.85211 (4.5)236 (95.5)18 (5.7)336 (94.3)Total Table 2. Sero-prevalence (Titer ≥1: 320) of canine Leishmania infection regarding age groups in Tehran and Alborz Provinces of Iran. Percentages were calculated in each age group totally and in each region >53–50–3Age (yr) P valuesNo.of infected dogs (%) (Titer ≥1: 320) No. of sampled dogs (%) No. of infected dogs (%) (Titer ≥1: 320) No. of sampled dogs (%) No.of infected dogs (%) (Titer ≥1: 320) No. of sampled dogs (%) 0.0824 (6.4)59 (93.6)10 (6.8)138 (93.2)3 (1.6)179 (98.4)Tehran 0.1342 (9.5)19 (90.5)7 (7.4)88 (92.3)2 (0.4)83 (99.6)Alborz 0.0136 (7)78 (93)17 (7)226 (93)5 (2)262 (98)Total Table 3. Sero-prevalence (Titer ≥1: 320) of canine Leishmania infection regarding living status in Tehran and Alborz Provinces of Iran. Percentages were calculated in each group (stray or owned) totally and in each region OwnedStrayLiving status P valuesNo. of infected dogs (%) (Titer ≥1: 320) No. of sam- pled dogs (%) No. of infected dogs (%) (Titer ≥1: 320) No. of sampled dogs (%) 0.6187 (5.7)182 (96.3)11 (4.6)226 (95.4)Tehran 0.01811 (8.7)115 (91.3)1 (1.2)79 (98.8)Alborz 0.8218 (5.7)297 (94.3)12 (3.8)305 (96.2)Total Table 4. Distribution and seroprevalence of L.infantum infection (Titer ≥1: 320) among 602 dogs from 11 counties in Tehran and Alborz Provinces of Iran Province /Locality No. of sampled dogs No. / % of infected dogs(Titer ≥1: 320) Tehran Province 408 18 (4. 4) Damavand 62 4(6.4) Eslam Shahr 9 0(0) Rey 12 0(0) Robat karim 23 1(4.3) Shahriar 198 6(3) Shemiran 14 2(14.2) Tehran 79 5(6.3) Varamin 11 0(0) Alborz Province 194 12 (6.18) Hashtgerd 115 6(5.2) Karaj 60 6(10) Nazar Abad 19 0(0) Total 602 30(4.98) http://jad.tums.ac.ir Published Online: April 09, 2014 J Arthropod-Borne Dis, December 2014, 8(2): 132–138 A Malmasi et al.: Seroepidemiologic Survey of … 136 Discussion Dogs can remain infected by L. infantum without displaying apparent clinical signs of VL for years even for their entire life (More- no and Alvar 2002, Moshfe et al. 2009). Determination of the prevalence of canine Leishmania infection is necessary to define control measures for zoonotic visceral leish- maniasis (Mohebali et al. 2005). Reports from neighboring Turkey have shown the seroprevalences of 2.58% and 3.6% (Ozensoy et al. 1998, Aslantas et al. 2005), Jaffe showed that the disease was highly prevalent in central Israel and encroaching on urban ar- eas (Jaffe et al. 2004), seroprevalence of 18% and 26.6% with DAT and ELISA, respectively have been reported from Paki- stan (Rab et al. 1995). Based on our results, no statistically difference was found between genders. This finding is in consistent with other studies (Mohebali et al. 2001, Franca- silva et al. 2003, Mohebali et al. 2005, Farzam et al. 2007, Moshfe et al. 2008, Khan- mohammadi et al. 2010a) and in conflict with some others (Dantas-Torres et al. 2006, Khanmohammadi et al. 2010b, Zaffaroni et al. 1999). In the present study, the infection was more prevalent in dogs aged 3 to 5 years old. Simillarly, Bokai et al. (1998) showed the highest infection in middle-aged dogs. In the study of Mohebali et al. (2005) dogs aged 8 years or more, showed the highest seroprevalence. The results of the study of Dantas-Torres et al. (2006) showed that the seroprevalence rate was higher among juve- nile (≤1 year) dogs than adults (>1 years). In our study, only 20% of 30 seropositive dogs (titer ≥1: 320) showed the clinical fea- tures of disease and the remaining 80% were asymptomatic. Fourteen (46.6%) and ten (33.3 %) seropositive dogs in Tehran and alborz Provinces respectively were found asympto- matic which is in agreement with other studies reporting similar results (Mohebali et al. 2005, Moshfe et al. 2008, Dantas-Torres et al. 2006). There was statistically significant difference between living status and CVL infection of dogs in Alborz Province (P= 0.018). It seems that the owned dogs in this province, were much more likely to be exposed to Leish- mania infection than stray dogs. Owned dogs in Tehran Province also showed higher prevalence than stray dogs for CVL, but their difference was not significant (Table 3). Despite lower prevalnce for CVL in owned dogs population of Tehran Province, difference between two provinces in the same category was not significant (Table 3). Seroprevalence values of 6.1% and 4.4% for Leishmania infection among dogs in Alborz and Tehran Provinces, respectively, reflect the significant risk of exposure for both healthy dogs and human population in these regions. If lower cut-off value was considered (<1: 320) the prevalence should have been more than the current findings. Only 20% of the seropositive dogs were symptomatic. Since asymptomatic Leishmania-infected dogs as well as symptomatic ones, have a potential role in the maintenance of L. infantum infec- tion (Moshfe et al. 2009), finding and report- ing such number of infected asymptomatic dogs in these provinces of Iran for the first time should be considered as a risk from both veterinary and public health aspects. Among the localities, Shemiran (14.2 %) in Tehran Province and Karaj (10%) in Alborz Province had the highest prevalence rates. Conclusion Based on our survey these provinces es- pecially those localities and suburbs with high- er seroprevalence should be regarded as new endemic foci for CVL in Iran. The huge pop- ulation living inside these areas particularly in new developing cities around both provinces, should be considered as at risk population. http://jad.tums.ac.ir Published Online: April 09, 2014 J Arthropod-Borne Dis, December 2014, 8(2): 132–138 A Malmasi et al.: Seroepidemiologic Survey of … 137 Acknowledgements The authors would like to express their gratitude to all those who helped them run- ning this survey including A Aliari for his technical assistance in Small Animal Hos- pital, Faculty of Veterinary Medicine, Uni- versity of Tehran, P Nobakht the local pri- vate veterinarian in Hashtgerd county, S Tehranian for helping in statistical review of the manuscript and S Charehdar for her tech- nical assistance in Parasitology laboratory, School of Public Health, Tehran University of Medical Sciences. The authors declare that there is no conflict of interest. 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